MED14-HY5 module orchestrates trade-off between cell-cycle activation and DNA damage response in Arabidopsis root

MED14-HY5 module orchestrates trade-off between cell-cycle activation and DNA damage response in Arabidopsis root | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article MED14-HY5 module orchestrates trade-off between cell-cycle activation and DNA damage response in Arabidopsis root Jitendra Thakur, Pallabi Thakur, Sourobh Maji, Apurva Gangal, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8222842/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Root system architecture is critical for plant growth and resilience. In this study, we uncovered a novel mechanism in Arabidopsis thaliana by which the Mediator complex subunit MED14 and the transcription factor ELONGATED HYPOCOTYL 5 (HY5) cooperatively regulate root development by maintaining redox and genomic stability. We found that the reduction in the level of AtMED14 causes mis-regulation of a large number of genes required for root development and hence affects the primary root development in Arabidopsis seedlings. Based on transcriptome data, flavonoid levels were examined and found to be deficient in the roots of the med14 mutant. Flavonoids are essential metabolites, and our findings suggest that they play key role in regulating ROS homeostasis in roots. The med14 roots have enhanced accumulation of ROS, leading to DNA damage and eventually activating the DNA damage response in the meristematic zone. This study unravels the coordination of MED14 with HY5 in regulating the cell-cycle genes. Under normal condition, MED14 facilitates HY5-mediated activation of cell-cycle genes, especially CYCD1;1 , however, during genotoxic stress, there is repression of these genes, thereby halting the cell-cycle. In contrast, under non-stress conditions, MED14 and HY5 suppress the expression of DNA damage response genes NAC044 and NAC085 , but this repression is relieved under oxidative stress. Conversely, the loss of function in NAC genes leads to increased expression of CYCD1;1 , but cannot effectively downregulate the expression of CYCD1;1 under genotoxic conditions. Such control over activation and repression of these genes is crucial for preventing unwarranted cell cycle arrest and ensuring proper root meristem function. Thus, our findings reveal a previously uncharacterized role of MED14-HY5 module in balancing ROS homeostasis and DNA damage response to safeguard root growth and development, providing a critical insight into how plants cope with environmental and endogenous genotoxic threats. Biological sciences/Molecular biology/Transcription/Transcriptional regulatory elements Biological sciences/Molecular biology/DNA damage and repair Mediator complex Transcription Transcriptional Regulation Gene Expression Reactive oxygen species DNA damage response DNA-protein interaction Flavonoids cell-cycle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Root is an essential organ for plant survival and productivity, serving both structural and physiological functions. It is required for anchoring and acquiring nutrients and water from the soil to the whole plant 1 , 2 . The development of roots is a complex multinetwork process that relies on genetic, biochemical, and environmental conditions to regulate temporal and spatial control of cell division and proliferation, particularly in the root apical meristem (RAM). DNA damage refers to structural alterations in the DNA molecule that compromise its integrity and function. It can arise from endogenous sources, like reactive oxygen species (ROS) generated during cellular metabolism, or through other exogenous agents, including ultraviolet (UV) and ionizing radiations (IR) such as X-ray and gamma ray, chemical mutagens, and also heavy metal toxicity and heat stress. Common types of DNA damage include base modifications, single-strand breaks (SSBs), double-strand breaks (DSBs), abasic sites, and DNA crosslinks 3 – 7 . These lesions can interfere with critical cellular processes like replication, transcription, and chromosome segregation. Plant root growth is severely affected by this type of DNA damage by activating the DNA damage response (DDR) pathways that halt the cell cycle in the root apical meristem to allow for repair. Persistent damage can trigger programmed cell death (PCD), reducing the population of dividing cells essential for root elongation 8 , 9 . DNA lesions further interfere with crucial hormonal signaling pathways, especially those involving auxin and cytokinin, leading to disrupted root patterning and impaired lateral root development 9 – 12 . Genome instability from unrepaired DNA further leads to abnormal cell division and differentiation. Collectively, these effects reduce root system efficiency, affecting water and nutrient uptake and overall plant growth. So, to cope with the DNA damage, plants need to activate their defence mechanism to repair the damaged DNA that helps to preserve genome stability by repairing lesions caused by UV light, ROS, and genotoxins. It regulates the cell cycle, particularly in meristematic tissues, ensuring proper cell division and development 13 – 15 . During seed germination, DDR restores DNA integrity essential for seedling establishment 16 , 17 . Additionally, it also enhances stress tolerance by mitigating DNA damage induced by abiotic stresses like heat and heavy metals 18 – 20 . DDR response is an intricate kinase-regulated network that is conserved in both plants and animals. In animals, the critical regulator of DDR is p53 Transcription Factor (TF) 21 . In plants, there are functional homolog of p53, identified as NAC-type TF NAC008/SOG1. Just like p53 in animals, NAC008/SOG1 is also phosphorylated by evolutionarily conserved kinases ATR and ATM and triggers the DDR response 22 – 24 . These ATR/ATM-mediated DDRs are partially dependent on dose-dependent ROS 25 , 26 . Phosphorylated NAC008/SOG1 binds to the promoter of NAC044 and NAC085 to induce the expression of DNA-repairing genes such as RAD17 , RAD51 , and BRCA1 genes 27 – 29 . Absence of NAC044 and NAC085 impedes Rep-MYB accumulation, thereby inhibiting G2/M progression 29 . Rep-MYBs suppress the expression of cyclins and thus hamper the activity of cyclin-associated CDKs, leading to cell-cycle arrest. Thus, Rep-MYBs play very important role as the repressors of cell-cycle genes in response to DNA damage 30 . As a part of the adaptation, plants have also developed specialized antioxidant systems to protect themselves from ROS. That is why plants have intricate mechanisms for ROS homeostasis 31 . The basal level ROS plays an important role in cell signaling and homeostasis, excessive ROS can damage DNA, proteins, lipids, and other macromolecules, thereby causing cellular dysfunction and genome instability 32 , 33 . In plants, ROS production can be driven by respiratory processes, photosynthesis, pathogenic infections, and the deficiency in antioxidant defences 34 – 36 . In addition, both biotic and abiotic stresses can also cause overproduction of ROS within the cells, leading to oxidative stress. Studies suggest that ROS can cause breaks in single-stranded (SSBs) and double-stranded (DSBs) DNAs, which leads to activation of the factors that are required for DNA damage responses (DDR) 37 , 38 . Though the number of TFs is known to regulate ROS-mediated gene expression, and different sets of TFs are involved in DDR induced due to high ROS accumulation, how these different sets of TFs coordinate with each other during normal and stress conditions for optimal levels of physiological responses remains elusive. There are reports that suggest exogenous cues can increase ROS production and can also induce the flavonol biosynthesis. In Arabidopsis, flavonols such as quercetin, kaempferol, and isorhamnetin are known to regulate root morphogenesis 39 . The ELONGATED HYPOCOTYL5 (HY5) transcription factor is a known regulator of flavonoid biosynthesis 40 , 41 . Mutation in HY5 reduces the primary root length and enhances the lateral root elongation by regulating cell division and cell elongation 41 , 42 . HY5 binds to the promoter of CHALCONE SYNTHASE ( CHS ) gene to regulate flavonoid biosynthesis 43 . HY5 also binds to the promoters of ROS detoxification genes to regulate ROS homeostasis under different light and nutrient stresses 44 – 46 . Thus, HY5 is very critical for controlling cell division in the root through flavonoid and ROS homeostasis. However, the role of HY5 in the regulation of the cell-cycle genes through ROS and its involvement in controlling DDR-responsive genes have not been explored. Transcription factors and chromatin regulators are also known to be involved in DDR in animals and plants. Studies in animals suggest that Mediator complex, the evolutionary conserved transcriptional cofactor, functions as an important link between transcription and DNA repair mechanism for maintaining genome integrity 47 . The Mediator complex is a large multiprotein complex that coordinates with different transcription factors and cofactors to regulate the transcription process in eukaryotes 48 . In plants, the Mediator complex typically consists of around 34–37 subunits 49 , 50 . These subunits are arranged in four modules- head, middle, and tail, making the core part and one dissociable kinase module 51 , 52 . Generally, the head and middle module subunits interact with RNA pol II and other components of the PIC, whereas tail module subunits interact with different TFs 48 . The Mediator subunits are involved in developmental processes including cell division and cell proliferation and also in the responses to environmental factors. Few Mediator subunits, such as MED2, MED5, MED16, and MED23 have been implicated in the phenylpropanoid pathway, essential for flavonoid biosynthesis 53 . In the analysis of protein-protein interaction network, some Mediator subunits were found to interact with the components of the DNA repair system 48 . Moreover, few Mediator subunit mutants are sensitive to UV radiation 54 , 55 . These studies indicate an important role of Mediator in stress-triggered DNA damage response. In our study, we have focused on MED14 subunit to understand the intricacies of the involvement of Mediator in DNA damage response in Arabidopsis root. While studying the structural topology of Arabidopsis Mediator complex, MED14 emerged as the most important subunit required for holding the complex 56 . MED14 was initially discovered as RGR1 in yeast, where it functions as a repressor of glucose-regulated genes 57 , 58 . In Arabidopsis, it was discovered as STRUWWELPETER/SWP 59 . MED14/SWP is a nuclear protein expressed in the meristematic region, promoting cell proliferation and elongation, particularly during root and leaf development 60 , 61 . MED14 also contributes to auxin-regulated processes leading to seed development, leaf formation, and lateral root formation in Oryza sativa 62 . Research revealed that MED14 is necessary to preserve repressive epigenetic marks and affect transcription in Arabidopsis. Additionally, MED14 regulates DNA methylation in non-CG contexts at certain RNA-directed DNA methylation targets 63 . Subsequently, the role of MED14 in managing heterochromatin transcription and DNA methylation has been emphasized 63 . Studies suggest that, any change in the epigenetic landscape of the chromatin also activates the DDR responses. In our study, whole genome transcriptome analysis of the med14 mutants revealed that MED14 could be involved in the transcriptional regulation of flavonoid genes, and metabolites required for chelating excess ROS from cells to maintain the ROS homeostasis in plants. All these genetic and physiological studies in med14 mutants suggest that MED14 could be an important integrator of ROS homeostasis, DDR responses, and cell division in plants. In this study, we focus on exploring the role of the Mediator complex in root development. While comparing the root phenotype in different Mediator mutants, we found that MED14 and MED17 are the most important subunits in all facets of root architectural development. We provide physiological and biochemical evidence confirming that the flavonoid biosynthesis pathway is disrupted due to the loss of the functional MED14. Our findings revealed that med14 mutants have increased ROS accumulation and trigger DNA damage-induced cell death at the root tips, even without exogenous stress. Further, we divulged that hy5 mutant is sensitive to DDR activation, and HY5 and MED14 together play critical roles in managing the ROS-induced DDR response necessary for maintaining the root cellular structure. Furthermore, we provided evidence that oxidative stress can inhibit CYCD1;1 expression by enhancing the expression of NACs and influencing primary root elongation in plants under stress. In this study, we elucidated the role of MED14 in the ROS-induced DDR activation, highlighting its coordination with HY5 to sustain DNA instability and ensure the persistence of the actual RSA. Results MED14 is critical for proper growth and development of the root In the last decade, several Mediator subunits have been shown to be involved in root development 64 . Despite their involvement, the precise molecular mechanism by which these subunits function in the process is still in its infancy. We still do not know how these subunits coordinate for optimal growth and architecture of roots in plants. In our earlier study, MED14 and MED17 emerged as the critical subunits to hold the whole Mediator complex together 56 . So, we decided to work on these two subunits to understand the molecular intricacies of the regulation of root development through Mediator complex. We compared the primary root length of mutants of different Mediator subunits ( med14 , med16 , med17 , med25 , cdk8 , and cycC ). We found that MED14 and MED17 showed robust effect on the overall root length, compared to other mediator mutants ( Supplemental Figure 1A and 1B ). In our previous study, we reported the requirement of MED17 for optimal root system architecture in Arabidopsis thaliana 65 . In this study, we have focused on the importance of MED14 in the growth of the primary root. We generated RNAi lines of MED14 in Col-0 background ( Supplemental Figure 2A ) and also screened the T-DNA insertion line (SAIL_373_07) in Col-3 background ( Supplemental Figure 2B ). The details of the primers used to amplify the region for generating the MED14 RNAi line are given in Supplemental Table 1 . The transcript level of MED14 was reduced to 85% in the MED14 RNAi line and 20% in the SAIL line of MED14 (hereafter referred to as med14-1 and med14-2, respectively) ( Supplemental Figure 2C ). When we compared the seedings, we found that the root part was majorly affected in med14-1 and med14-2 seedlings as compared to their respective wild-types ( Figure 1A-1C, Supplemental Figure 3E-3I ). Surprisingly, there was not much effect in the shoot tissue ( Supplemental Figure 3A-3C ). The fresh weight of the root was significantly reduced in both med14-1 and med14-2 as compared to the wild-types ( Supplemental Figure 3D ). So, we focused our study on the root development. We measured the length of primary roots on 5 th , 8 th , and 12 th days and found that the root growth was slower in these lines as compared to their respective wild-types ( Figure 1A-1C; Supplemental Figure 3E-3I ). In these med14 mutant seedlings, even the number of lateral roots was less with wider lateral root angles than that in wild-type seedlings ( Supplemental Figure 4A and 4B ). Observing propidium iodide-stained roots under microscope revealed shorter meristematic zones in med14 roots ( Figure 1D and 1E ). We counted the number of cells in the meristematic, transition, and elongation zones of roots, and found that in all three zones of med14 seedlings had lesser number of cells as compared to their corresponding wild-types ( Figure 1F-1H ). Moreover, cells in the meristematic zone and its neighbouring transition zone were slightly smaller in med14 roots ( Figure 1I and 1J ). There was no difference in the size of cells in the elongation zone ( Figure 1I and 1K ). These results indicate that cell division in the meristematic region of roots is affected. We confirmed this by staining the roots with 5-ethynyl 2’-deoxyuridine (EdU), a labeled nucleoside analog of thymine, that gets incorporated into replicating DNA 66 . Indeed, the number of EdU-labeled nuclei was significantly less in med14 roots as compared to wild-type ( Supplemental Figure 5A-5D, 5G ). Next, we checked the expression of CYCLIN B1 , a cell-cycle marker, in med14-1 and wild-type background. For this, pro CYCB1;1:GUS was expressed under the native promoter in med14 and wild-type seedlings. The number of GUS-stained nuclei was significantly less in the med14-1 roots ( Supplemental Figure 5H and 5I ). These results confirm that MED14 is required for cell division in the meristematic zone of primary root in Arabidopsis. Next, we made an attempt to recover the root phenotype by overexpressing MED14 in the med14-2 mutant background. The shortened primary root phenotype and lateral root number could be rescued in the MED14 complemented lines, further confirming that the root development defect was due to the mutation in the MED14 gene ( Supplemental Figure 6A-6D, 6G ). Even the reduced number of cells in the meristematic zone of med14 mutant roots was restored to a great extent in the roots of complementation lines ( Supplemental Figure 6E and 6F ). This suggests that an optimal growth level of MED14 expression is required for maintaining the proper growth of primary root in normal condition. Consistently, the number of EdU-incorporated nuclei in the complementation lines was same as wild type, suggesting defective cell division was rescued in these lines ( Supplemental Figure 5C-5F ). In addition to complementation lines, we also overexpressed MED14 in the wild-type background under its own native promoter ( Supplemental Figure 7A ). As expected, seedlings of overexpression lines had longer primary roots with longer meristematic zone as compared to wild-type seedlings ( Supplemental Figure 7A-7E ). These observations confirm that optimal level of MED14 is required for proper cell division in the root, regulating its growth and development. MED14 is involved in flavonoid biosynthesis to regulate primary root growth How does MED14 regulate root growth in Arabidopsis? In order to get this answer, we compared the transcriptome of med14 root with that of wild-type. Through RNA-sequencing, we identified 3909 differentially expressed genes in med14 roots. Among the differentially expressed genes, 2079 genes were significantly downregulated and 1830 genes were significantly upregulated in the mutant root as compared to the wild-type root, suggesting that MED14 is involved in both activation and repression of genes ( Supplemental Figure 8A ). Gene ontology (GO) analysis of the downregulated genes revealed that the secondary metabolite biosynthesis pathways, including phenylpropanoid and flavonoid biosynthesis pathways were significantly affected in the med14 mutant roots ( Supplemental Figure 8B ). We confirmed the downregulation of some of the main genes of the flavonoid biosynthesis pathway such as CHALCONE SYNTHASE ( CHS ), CHALCONE ISOMERASE ( CHI ), FLAVANONE 3-HYDROXYLASE ( F3H ), FLAVONOL SYNTHASE 1 ( FLS1 ) by using qRT-PCR analysis ( Figure 2A, 2B; Supplemental Figure 8A ). Driven by the expression profile of these genes, we investigated the distribution of flavonols in the med14 roots using DPBA (2-aminoethyl diphenylboric acid), a flavonol-specific staining dye 67 , and compared that with their respective wild-type roots. Indeed, there was less flavonol content in the roots of med14 mutants ( Figure 2C and 3D ). This was further confirmed by high-performance thin-layer chromatography (HPTLC). The HPTLC profile showed lower accumulation of quercetin (orange) and kaempferol (green) in the med14 roots as compared to the roots of wild-type seedlings ( Figure 2E ). All these results suggest that MED14 is involved in the regulation of flavonoid biosynthesis pathways in the roots of Arabidopsis. MED14 coordinates with HY5 to regulate the root growth HY5 is a well-known regulator of flavonoid-driven root growth in plants 42,68,69 . We confirmed the downregulation of HY5 in the roots of med14 mutants by qRT-PCR analysis ( Figure 3A ). To further confirm the regulation of HY5 transcription by MED14, we expressed HY5:GFP under its native promoter in wild-type and med14-1 mutant and checked the expression level. Indeed, the expression level of HY5:GFP driven by HY5 promoter was significantly reduced in med14 mutant ( Supplemental Figure 9A-9C) . This confirms that the activity of HY5 promoter is dependent on MED14 to some extent. To date, there is no report suggesting binding of Mediator subunits directly to the DNA; instead, transcription factors recruit the Mediator complex to their cognate binding sites on the DNA 70 . There is one report that indicates feedback regulation of HY5 71 . However, direct binding of HY5 to its own promoter has not been reported. So, we scanned the promoter of HY5 and found the probable HY5 binding sites in it. Indeed, HY5 was found to bind to its own promoter by ChIP analysis ( Figure 3B and 3C ). Interestingly, enrichment of HY5 on its promoter was substantially decreased in med14-1 lines ( Figure 3B and 3C ). To elucidate the presence of MED14, we checked the occupancy of MED14 on the promoter of HY5 , and indeed there was enrichment of MED14 on this promoter, which was diminished in the absence of HY5 ( Figure 3D ). These results suggest that MED14 regulates the expression of HY5 by modulating the binding of HY5 on its own promoter ( Figure 3B-3D ). Since there is less expression of HY5 in med14 mutant, there is low abundance of HY5 on its promoter. This suggests that MED14 is required for feedback loop of HY5 expression. Next, we explored whether there is direct interaction between MED14 and HY5. First, we predicted the probable binding sites of MED14 (i.e., Δ4 and Δ6) with full-length HY5 by molecular docking ( Supplemental Figure 10A and 10B ), and then confirmed this binding by BiFC. In the BiFC experiment, we could not find any interaction between full-length MED14 and HY5. It has been observed earlier that in ex-vivo experiments, full-length of some Mediator subunits are not able to interact with other proteins, probably due to regulatory regions involved in controlling their protein-protein interactions. So, we cloned fragments of MED14 and checked their interactions with full-length HY5. The regions from 531-769AA residues of MED14 (Δ4) and 1170-1704AA residues of MED14 (Δ6) were found to interact with HY5 ( Supplemental Figure 11A-11P). Thus, these results suggest that HY5 requires MED14 for its expression. Indeed, we observed a significant reduction in the transcript level of HY5 in both med14 mutants ( Figure 3A ), further supporting the role of MED14 as a transcriptional activator of HY5 . Next, we assessed the root phenotype of hy5 mutant and compared it with med14 . Indeed, just like med14 , hy5 seedlings also showed shorter primary roots ( Figure 3E and 3F ). In order to confirm the functioning of MED14 and HY5 in the same pathway, we generated med14-1 hy5 double mutant line. In the double mutant line, the primary root was as short as hy5 or med14 mutant seedlings ( Figure 3E and 3F ). We looked at the cellular organization in the roots of med14-1 , hy5, and med14-1 hy5 seedlings. Just like med14 mutant, the length of meristematic zone and the number of cells in the meristematic, transition, and elongation zones were found to be reduced in hy5, and med14-1 hy5 roots as compared to Col-0 roots ( Figure 3G, H, Supplemental Figure 12A-12C ). There was no additional effect in the double mutant ( Figure 3E-3H ). Also, the length of cells in the transition and elongation zone in both hy5 and med14-1 hy5 roots was similar to med14 roots ( Supplemental Figure 13A-13C ). Like med14-1 mutant, the EdU staining of roots of hy5 and med14-1 hy5 seedlings revealed compromised cell division as compared to Col-0 ( Figure 3I-3P, Supplemental Figure 13D ). Thus, the overall phenotype of hy5 mutant root is similar to that of med14 root, and there is no additional effect in the double mutant of med14 and hy5 together. These observations suggest that in the process of root development, MED14 and HY5 work together in the same pathway. To further dissect the possible regulatory pathway that has been affected by the functional loss of MED14 and HY5, we compared the transcriptome data of the hy5 seedlings published elsewhere 72 with our med14 transcriptome. A total of 301 genes were found to be downregulated in both hy5 and med14 mutants ( Supplemental Figure 14A ). In these downregulated genes, secondary metabolic pathways were highly represented, especially phenylpropanoid biosynthesis and flavonoid biosynthesis pathway genes such as CHS , CHI , CHIL , F3H , FLS1 , TT7 , and CoAOMTA1 ( Supplemental Figure 8A and 14B ). We confirmed this by qRT-PCR analysis of few important genes, namely CHS , CHI , F3H, and FLS1 ( Figure 2A, 2B, 4A, 4B ). Driven by such gene expression profile, we checked the effect of the flavonoid (quercetin) on root growth. Interestingly, exogenous application of quercetin increased the primary root length in all three mutants hy5 , med14, and the double mutant ( Figure 4C-4F ). There was no visible effect of 10 µM of quercetin on wild-type roots. Next, to understand the alteration in the cellular organization in roots, meristem cell numbers were counted in the PI-stained roots of Col-0, med14-1 , and hy5 seedlings. In response to quercetin treatment, there was a significant increase in the number of cells in the meristematic zone ( Figure 4G and 4H ). However, there was no change in cell size in response to quercetin ( Figure 4I and 4J ). These results suggest that MED14 and HY5 are both required for the flavonoid-driven cell division for the growth of primary root. MED14 balances the ROS homeostasis to facilitate the primary root growth Flavonoids play a crucial role in the root development as they help to regulate ROS homeostasis in the meristematic region 73,74 . In plants, a baseline level of ROS is essential for various signaling pathways required for proper growth. However, excessive ROS production can cause cellular damage, which can be detrimental to root growth and development. Since flavonoid level was affected in med14 mutants, we checked ROS levels in them by staining the roots with H 2 DCFDA, a common reagent used for investigating the production of ROS in living cells. The higher intensity of fluorescence in med14 roots revealed higher accumulation of ROS in them as compared to their respective wild-types ( Figure 5A-5E ). In the roots of the complementation line ( 35S:MED14-HA/med14 ), the fluorescence intensity was found to be reduced to the level of wild-type, validating the requirement of MED14 for ROS homeostasis ( Supplemental Figure 15A-15D ). Developmental events in root, especially cell division in the meristematic zone and transition from elongation to differentiation zone are regulated by ROS gradient mainly of H 2 O 2 and O 2 . - 75 . So, we made an attempt to evaluate the distribution of these specific ROS species, using NBT (Nitroblue tetrazolium) for superoxides (O 2 . - ) and DAB (3,3'-Diaminobenzidine) for hydrogen peroxide (H 2 O 2 ). The med14 mutants showed higher accumulation of both the O 2 . - and H 2 O 2 in the roots compared to the wild-type ( Figure 5F-5M ). To see if the increase in ROS affects the primary root growth, we exogenously applied different concentrations of H 2 O 2 to Col-0. We found an overall reduction in the primary root growth in wild-type seedlings under increasing concentration of H 2 O 2 ( Supplemental Figure 16A and 16B ). These results indicate that excess ROS can actually inhibit primary root growth. It suggests that the increased level of ROS could cause reduced growth of primary root in med14 mutants. To further confirm that, we designed a chemical complementation assay to chelate the availability of ROS in the seedlings. For that, we treated the 4-days-old seedlings with potassium iodide (KI), a well-known ROS scavenger 76 . There was no effect of KI on the growth of primary roots of wild-type seedlings. However, there was significant increase in the length of med14 roots ( Figure 5N and 5O ). These observations suggest that increased ROS accumulation is one of the major factors for defects in development in med14 mutants. Thus, MED14 is important for ROS homeostasis in the root, which is critical for its proper growth and development. MED14 and HY5 are involved in DNA damage response to control primary root elongation Excess ROS production is a common phenomenon in stress response 35,77,78 . The excess ROS production causes damage to DNA, which in turn activates DDR response. To investigate the impact of excessive ROS production on DDR response, we assessed the expression level of the DDR pathway genes in the med14 by qRT-PCR. We found that the transcript levels of GAMMA-IRRADIATION AND MITOMYCIN C INDUCED 1 / GMI1 , NAC044 , NAC085 , and NAC103 and Poly (ADP-ribose) polymerase ( PARP2 ) were increased in both med14 mutants, suggesting the activated DDR response in them ( Figure 6A and 6B ). So, to further clarify the potential role of MED14 in DDR, we checked the effect of different concentrations of HU (hydroxyurea), a well-known DNA-damaging agent, which blocks DNA replication during the S phase of the cell-cycle 79,80 , on the root growth in med14 and wild-type seedlings. The HU treatment inhibited the growth of primary root in these seedlings. However, the percentage of root growth inhibition was significantly more in med14 roots as compared to the wild-type ( Figure 6C-6E ). Next, we assessed cell death in the roots of HU-treated seedlings by PI staining, which is used as a marker for cell death regions 81 . We found that there were more PI-stained regions in the med14 roots as compared to the wild-type, revealing higher sensitivity of med14 mutants to the DNA-damaging agent ( Figure 6F). This sensitivity could be reversed to wild-type level in the complementation lines of 35S:MED14::HA / med14 ( Supplemental Figure 17A-17C ). All these results suggest that MED14 is required for optimal response to DNA damage. HY5 is one of the main regulators of ROS production in root 46 , and there is significant overlap in the genes regulated by both MED14 and HY5. So, we decided to conduct a thorough study to understand the relationship between the two in ROS-mediated root development. Therefore, the expression of ROS biosynthesis genes was compared from the transcriptomic data of hy5 72 and med14 mutant roots ( Supplemental Figure 18 ). A significant number of ROS biosynthesis genes were found to be affected in both mutants as compared to wild-type ( Supplemental Figure 18 ). As discussed earlier, increased ROS accumulation in med14 root activates the DDR. So, we proceeded to see if there is functional overlap between MED14 and HY5 in DDR responses. We checked the ROS accumulation in roots of single mutants of med14-1 and hy5 , and also in the double mutant of med14-1 hy5 by H 2 DCFDA staining. Just like in med14 , there was increased accumulation of ROS in hy5 and med14-1 hy5 roots ( Figure 7A-7D, 7F). In the roots of HY5 overexpression line, the ROS accumulation was reduced compared to wild-type ( Figure 7E and 7F ). Next, we checked the effect of genotoxic stress on the roots of these lines. As expected, the roots of single and double mutants were found to be more sensitive to exogenous HU ( Figure 7G and 7H ), whereas the HY5 overexpression line exhibited more tolerance to the genotoxic stress as compared to Col-0 ( Figure 7I and 7J ). Moreover, just like in med14 , treatment of seedlings with ROS scavenger KI could increase root length in hy5 ( Figure 7G and 7H ). This root sensitivity was further confirmed by cell death assay in the presence of exogenous HU. All med14-1 , hy5 and med14-1 hy5 mutants accumulated more cellular damage as compared to Col-0, while in the HY5 overexpression line, the cellular damage was significantly less as compared to Col-0 ( Figure 7K-7T ). All these results confirmed that there is an overlap between hy5 and med14 mutants in terms of root phenotype in response to genotoxic stress. Both MED14 and HY5 are involved in the DNA damage response triggered by higher ROS accumulation in the primary root. HY5 and MED14 regulate the transcript level of NAC044 and NAC085 and modulate root elongation under genotoxic stress HY5 is known to regulate ROS production and primary root phenotype under stress condition. However, its role in DDR remains unexplored. Therefore, we decided to investigate how MED14 and HY5 work together to regulate ROS-triggered DNA damage response in the root. It is already known that the DDR requires expression of ATAXIA-TELANGIECTASIA MUTATED ( ATM ) and ATAXIA TELANGIECTASIA-MUTATED AND RAD3-RELATED ( ATR) triggered by single and double-stranded DNA breaks caused by ROS 82 . In Arabidopsis, DNA damage triggers the activation of the ATM/ATR kinases, leading to phosphorylation and activation of the plant-specific transcription factor SOG1. SOG1 directly upregulates the transcription of NAC044 , NAC085, and NAC103 , which mediate downstream transcriptional responses, including the repression of G2/M-specific genes, thereby enforcing cell-cycle arrest during the DNA damage response 30,83 . So, we checked the expression of these genes in the roots of the seedlings treated with HU. We found the significant upregulation of NAC044 , NAC085 , and NAC103 in wild-type seedlings ( Supplemental Figure 19A ). Interestingly, there was no such upregulation was observed upon KI treatment, indicating that HU treatment can activate ROS-mediated DDR response in plants ( Supplemental Figure 19A) . Next, we checked the expression of these DDR-responsive genes ( NAC044 , NAC085 , and NAC103 ) in med14 roots upon KI treatment. Transcript levels of NAC044 , NAC085 , and NAC103 were comparatively reduced upon KI treatment ( Supplemental Figure 19B ). However, the transcript levels of ATR, ATM, and SOG1 in wild-type and med14 mutantremained unchanged ( Supplemental Figure 19A-19C ). Similarly, upregulation of NAC044 , NAC085 , and NAC103 genes was also observed in the hy5 root in normal condition ( Supplemental Figure 19D ), which was further reduced after KI treatment ( Supplemental Figure 19E ). These results confirm that hy5 and med14 mutants have elevated levels of ROS leading to the activation of DDR responsive pathway, suggesting that HY5 and MED14 are required for controlling this response. Flavonol is required for the suppression of ROS-triggered DDR response in plants Plants produce specialized secondary metabolites to neutralize the toxic effects of excess ROS. As discussed earlier, quercetin level in med14 seedlings is less than wild-type. So, we designed an experiment to see the effect of genotoxic agent HU in the presence and absence of quercetin and KI on the expression of DDR genes. Indeed, quercetin worked like a ROS scavenger ( Figure 4C and 4E) . In wild-type roots, there was significant elevation in the expression of NAC044 , NAC085 , and NAC103 in response to HU treatment ( Figure 8A-8C ), and in the presence of quercetin, just like KI, there was no such upregulation of these genes ( Figure 8A-8C ). In med14 roots, quercetin level is already low ( Figure 2E ). So, the expression of NAC044 , NAC085 , and NAC103 is already high ( Figure 8D ). When the seedlings are treated with exogenous quercetin, the expression level of NAC genes were significantly reduced to wild-type level ( Figure 8D ). Same effect was also observed in hy5 roots ( Figure 8E ). At cellular level also, the cell damage caused by HU could be protected by the exogenous application of quercetin and KI. Just like KI, the application of quercetin could effectively reduce the extent of HU-caused cell damage in Col-0 ( Figure 8F ). In med14 and hy5 mutants, quercetin reduced the HU-induced cell death area ( Figure 8G ). These findings confirmed that quercetin acts as a natural scavenger of ROS, helping protect plants from oxidative damage. Additionally, both MED14 and HY5 are required for quercetin to effectively safeguard the DNA. MED14 is required for NAC044 and NAC085-dependent restriction of cell division in root tip Considering the importance of NAC044, NAC085, and NAC103 in DDR response under genotoxic stress and their notable upregulation in both med14 and hy5 mutants, we decided to study genetic relationship between them in Arabidopsis. NAC044, NAC085, and NAC103 are involved in inhibiting the G2/M stage of cell-cycle in response to stress 28,29,84 . Therefore, nac044-1 , nac085-2 , and nac103 mutants are unresponsive to DDR signaling 28,29,84 . To check if the smaller root in med14 seedlings was due to higher expression of these NACs, we generated the double mutant lines of med14-1 nac044-1, hy5 nac044-1, and med14-1 nac085-2, hy5 nac085-2, and nac044-1 nac085-2 . Indeed, the short primary root length phenotype of med14-1 and hy5 was observed to be reversed in the double mutants and is comparable to wild-type seedling ( Supplemental Figure 20A and 20B) . This result confirms that the defect in the growth of primary root in med14 and hy5 mutants is due to ROS-triggered upregulation of NAC044 and NAC085 . Next, we observed the sensitivity of the roots in these mutant lines under genotoxic stress, and measured the cell death for the shortening of roots. For this, we performed cell death assay in the root under genotoxic stress. The med14 and hy5 roots exhibited increased cellular damage ( Supplemental Figure 21A-21T ). There was no such effect seen in the roots of nac044-1 , nac085-2 and nac044-1 nac085-2 doublemutants after HU treatment. However, there was some cell death observed in Col-0, med14-1 nac044-1, hy5 nac044-1, and med14-1 nac085-2, hy5 nac085-2 double mutants, but less than that in med14 and hy5 mutants ( Supplemental Figure 21A-21T ). Growth and elongation of primary roots are driven by cell division in the meristematic zone. So, we looked at the cell division in the roots of these mutants by EdU labeling followed by DAPI staining. As expected, there were more dividing cells in nac044-1 and nac085-2 than wild-type. Moreover, the reduced cell division phenotype in med14-1 root could be restored to wild-type level in med14-1 nac044-1 and med14-1 nac085-2 double mutants ( Supplemental Figure 20C-20I). These results suggest that the defect in root growth in med14 and hy5 are due to increased expression of NAC044 and NAC085, leading to hampered cell division. MED14 and HY5 promote activation of DDR response under oxidative stress HY5 is primarily known as an activator, as a positive regulator in light-dependent photomorphogenesis in plants 85,86 . However, HY5 also functions as a repressor in certain contexts 46,87,88 . MED14 was first discovered as a repressor in yeast and is also known to repress many genes in humans 57,58 . Under normal condition, DDR genes, especially NAC044 , NAC085 , and NAC103 , remain repressed but are upregulated under stress conditions. Here, we found that hy5 and med14 seedlings have activated DDR under normal conditions and are more sensitive to oxidative stress, which also activates DDR. The expression of NAC044 , NAC085, and NAC103 was higher even in non-stressed condition as compared to wild-type plants, suggesting that these NAC genes are de-repressed in hy5 and med14 mutants under normal condition. These findings indicate that MED14 and HY5 negatively regulate the transcription of NAC044 , NAC085 , and NAC103 in normal condition by either binding to their promoter, and this suppression is removed upon genotoxic stress. To comprehend the molecular mechanism of DDR regulation, we prima facie sought the binding of HY5 and MED14 on the promoter sites of DDR-responsive NAC genes. For this, we scanned the promoter regions of NAC044 and NAC085 and found HY5 binding elements in them. Indeed, through ChIP analysis, we found binding of HY5 to the promoters of NAC044 and NAC085 genes( Figure 9A and 9B ). However, the enrichment of HY5 in med14-1 was found to be reduced as compared to Col-0 background, suggesting that MED14 helps in the binding of HY5 on NAC044 and NAC085 promoters in normal condition ( Figure 9A and 9B ). Further to verify the coordination between HY5 and MED14, we checked the occupancy of MED14 on NAC044 and NAC085 promoters in Col-0 and hy5 backgrounds. In accordance, we found MED14 was also able to occupy the same regions of NAC044 and NAC085 , where the HY5 binds, and this occupancy was significantly reduced in the hy5 mutant background ( Figure 9C and 9D ). All these results suggest that HY5 recruits MED14 to the promoters of the DDR genes, NAC044 and NAC085, with the help of HY5 ( Figure 9A-9D ). To further elucidate the regulation of NAC genes through MED14 and HY5, we examined the expression of NAC044 , NAC085 , and NAC103 genes in the overexpression line of HY5 and compared them with wild-type under genotoxic stress. Genotoxic stress enhanced the transcript level of NAC044 , NAC085, and NAC103 in the wild-type roots, but not that much in the overexpression line ( Supplemental Figure 22A-22C ). Next, we checked the binding of HY5 and MED14 on the promoters of DDR genes under genotoxic stress. Interestingly, binding of HY5 to these promoterswas significantly reduced after HU treatment ( Figure 9E and 9F ). Similar pattern was also observed in the occupancy of MED14 on the promoters of NAC044 and NAC085 ( Figure 9G and 9H ). These results suggest that HY5 and MED14 play a crucial role in repressing the transcription of NAC044 and NAC085 genes under normal conditions, and this repression is lifted under genotoxic stress as the occupancy of HY5 and MED14 is reduced on the promoters of these genes. MED14 and HY5 positively regulate cell division Cell-cycle is essential for sustaining cell division in the root apical meristem, which drives continuous root growth in Arabidopsis 89,90 . Cyclins are essential regulators of the cell-cycle, controlling root meristem growth by promoting cell division. Amongst them, CYCDs drive the G1/S transition 91,92 , while CYCA and CYCB facilitate the G2/M transition, ensuring proper progression through the cell-cycle 93–96 . Reduced number of meristematic cells in the med14 and hy5 mutants ( Figure 3G; Supplemental Figure 12A ) indicates disturbed regulation of cell-cycle genes. To investigate this, we analyzed the transcriptomic data from med14 and hy5 roots and found CYCD1;1 , CYCD3;1 , CYCD3;2 , and KRP2 were commonly downregulated in both mutants ( Figure 10A ). We focused on CYCD1;1 as it was the most downregulated one ( Figure 10B ). To elucidate the regulatory mechanisms, we assessed the occupancy of HY5 and MED14 on the CYCD1;1 promoter region. ChIP-qPCR demonstrated significant enrichment of both HY5 and MED14 at the CYCD1;1 promoter, indicating their role in direct transcriptional regulation ( Figure 10C and 10D ). To gain deeper insight, we further conducted ChIP-qPCR to examine occupancy of MED14 at the CYCD1;1 promoter in the absence of HY5 ( Figure 10E ). The results showed a significant decrease in the occupancy of MED14 in the hy5 mutant ( Figure 10E ). All these data reveal that MED14 and HY5 positively regulates CYCD1;1 transcription in HY5-dependent manner. Oxidative stress negatively regulates CYCD1;1 expression by MED14-HY5 driven NAC044 and NAC085 Several studies have demonstrated that an excess level of ROS inhibits primary root growth by affecting the cell-cycle progression in Arabidopsis 97,98 . We observed that med14 and hy5 mutants have more accumulation of ROS and reduced expression of cell-cycle genes, including CYCD1;1 . So, to correlate whether oxidative stress affects the cell-cycle progression, we checked the transcript level of CYCD1;1 under genotoxic stress in wild-type. Genotoxic stress significantly reduced the expression of the CYCD1;1 ( Figure 10F ), which might be responsible for the cell-cycle arrest. Since MED14 and HY5 are the positive regulators of CYCD1;1 ( Figure 10A-10E ), we checked the effect of HU on the occupancy of HY5 and MED14 on the promoter of CYCD1;1 . Interestingly, occupancy of both HY5 and MED14 was significantly reduced upon genotoxicity ( Figure 10G and 10H ). This result suggests that genotoxic stress removes the activators like HY5 and MED14 from the promoter of CYCD1;1, and so there is a decrease in its expression level ( Figure 10F-10H ). We further validate these findings by the synthetic reporter assay in tobacco. For this luciferase gene was cloned under pro CYCD1;1 and pairwise co-transformed with either MED14 or HY5. Expression of luciferase enzyme revealed that MED14 and HY5 could activate CYCD1;1 promoter ( Supplemental Figure 23A-23D ). As expected, after HU treatment luciferase signal was low, suggesting repression of the CYCD1;1 promoter. These results explain the check on cell-cycle genes during stress condition. As NAC044 and NAC085 also regulate cell division ( Supplemental Figure 20C-20I ), we checked the expression of CYCD1;1 in nac mutant roots. Notably, there was a significant increase in CYCD1;1 expression in nac044-1 , nac085-2 , and the double mutants med14-1 nac044-1 and med14-1 nac085-2 . In contrast, CYCD1;1 expression was notably reduced in the med14-1 mutant ( Figure 10I ). These findings suggest that NAC transcription factors act as negative regulators of cell-cycle genes. Discussion A number of Mediator subunits have been implicated in root development (reviewed in 64 . However, it is still not clear how these subunits integrate internal and external cues to ultimately regulate root growth. In this study, we demonstrate that MED14 and HY5 form a crucial regulatory module that integrates cellular redox status with the DNA damage response to maintain root meristem integrity. MED14 acts as a backbone subunit of the Mediator complex required for maintaining the meristematic zone. In Arabidopsis thaliana , mutations in STRUWWELPETER (SWP)/MED14 disrupt shoot apical meristem (SAM) organization and shorten the period of cell proliferation, indicating its crucial role in regulating meristem patterning and the timing of cell-cycle arrest 60 . When we generated the knockout line of MED14 , none of the seeds survived, suggesting that MED14 is crucial for the survival of the plants. So, by using both RNAi-mediated knockdown ( med14-1 ) and T-DNA insertion mutant ( med14-2 ) lines, we observed significant defects in primary root length and overall root biomass (Fig. 1A; Supplemental Fig. 3D ). Cytological, histochemical, and phenotypic analyses showed that the reduced root growth was caused by the defect in meristematic zone, indicating that MED14 is mainly required for cell proliferation. The med14 seedlings also displayed increased cell death at the root meristem (Fig. 6F). We think that this cell death is critical for stopping the root growth during stress condition. So, the cell death actually represents a stress response, which is largely mediated by increased ROS production (Fig. 6F, 7A and 7B). The ROS level, including both superoxides and hydrogen peroxides, was increased in the med14 mutant (Fig. 5F-5M). Since proper ROS gradients are essential for balancing cell proliferation and differentiation in the roots, their imbalance likely contributes to the observed cell death in the root tip 99 , 100 . So, the treatment of med14 mutants with ROS scavenger KI partially rescued root length (Fig. 5N and 5O), supporting a link between redox imbalance and impaired root growth in med14 mutant. Transcriptome of med14 mutant roots revealed widespread transcriptional reprogramming, including downregulation of flavonoid and phenylpropanoid biosynthesis pathways ( Supplemental Fig. 8B ). Though the previous report indicates that flavonoids possess natural antioxidant properties, their specific role in plants is still not very clear. We have shown that indeed the flavonoid could function as a ROS scavenger in plants (Fig. 4C, 4D, 8F, 8G). In the med14 mutants, ROS was accumulated at a higher level, leading to cell death at the root tip (Fig. 5A-5D, and 6F ). Flavonoids such as quercetin are known to promote root meristem activity by modulating redox homeostasis and auxin transport 46 , 101 , 102 . However, how flavonoids are involved in meristem division is still not clear. We observed that exogenous application of quercetin partially rescued the root growth defects of med14 (Fig. 4C and 4D), suggesting that flavanol deficiency contributes significantly to the observed short root phenotype. Notably, quercetin treatment restored meristem cell number in med14 mutant without changing the cell size (Fig. 4G-4J). Flavonoid biosynthesis mutants also exhibit reduced root length 74 , highlighting the critical role of flavonoid-dependent redox regulation in maintaining the root meristem. HY5 is an important transcription factor that integrates light in flavonoid biosynthesis, and ROS signaling 41 , 46 . In med14 mutants, HY5 is significantly downregulated (Fig. 3A; Supplemental Fig. 9A-9C ). So, there are significant phenotypic similarities between hy5 and med14 mutants, including shorter primary root (Fig. 3E and 3F), shortened meristematic zones (Fig. 3G and 3H), and impaired cell division (Fig. 3I-3P). The med14-1 hy5 double mutant did not show any additive phenotype, confirming that MED14 and HY5 function in a shared genetic pathway. Mechanistically, this study establishes that MED14 facilitates transcription of HY5 and a feedback loop for its autoregulation (Fig. 11). Moreover, HY5 and MED14 modulate the flavonoid biosynthesis genes such as CHS, CHI, F3H, FLS1, TT7 , and CHIL for transcriptionally regulating the root development (Fig. 2A, 2B, and 4A, 4B) in both mutants. We found that in addition to co-regulation of flavonoid genes, HY5-MED14 modules have a larger significance. HY5 plays a key role in maintaining ROS homeostasis in Arabidopsis 46 , 103 . The transcriptomic comparison between med14 and hy5 roots revealed common upregulation of ROS biosynthesis genes, suggesting coordinated regulation. This also suggests that HY5 and MED14 function as repressors of ROS production genes in non-stressed conditions, thereby protecting the DNA from getting damaged to ensure proper growth and development of the root. An interaction between MED14 and HY5 ( Supplemental Fig. 11 ) and their co-occupancy on different promoters (Fig. 9A-9D, and 10E ) suggest that the HY5-MED14 module is critical for flavonoid-regulated gene expression for root development. The med14 and hy5 mutants exhibit elevated levels of ROS (Fig. 7A-7D). High ROS levels induce single and double-stranded DNA breakage and activate protein kinases like ATM and ATR present at the top of the DDR response pathway 104 , 105 . This activated ATM and ATR protein phosphorylates downstream TFs, a conserved DDR module. In plants, this TF is SOG1, phosphorylated by ATM and ARM upon DDR activation in Arabidopsis 23 , 24 . Further, the SOG1 transcriptionally activates a cascade of TFs like NAC044, NAC085 , and NAC103 and enzymes for cell-cycle arrest in the meristem and enhances the transition from cell division to endoreduplication 106 . We observed that higher ROS levels in med14 and hy5 activated DDR response in these mutants and also exhibited elevated levels of NAC044 , NAC085 , and NAC103 expression (Fig. 6A and 6B). We found that flavonoids, particularly quercetin, are critical in mitigating ROS-mediated DNA damage. The direct involvement of flavonoids in preventing cellular damage has not been explored to date. In this study, the exogenous application of quercetin effectively suppressed HU-induced NAC gene expression in wild-type roots. This suppression was normally observed in med14 and hy5 mutants, which naturally have lower quercetin levels. This observation demonstrated that under stress conditions, MED14 and HY5 play critical role in maintaining the ROS-triggered DNA damage by modulating the flavonoid biosynthesis. This study provides evidence that fine-tuned coordination between activation and repression of cell division under stressed and non-stressed situations is critical for sustaining meristem size and ensuring DNA stability in the RAM (Fig. 11). We have demonstrated that MED14 and HY5 function together to promote root meristem activity by activating key cell-cycle regulators (Fig. 10A), while simultaneously repressing stress-responsive NAC transcription factors under non-stressed conditions (Fig. 9A-9D). Studies have shown that both G1/S and G2/M stages can mediate cell-cycle arrest in response to DNA damage. Both hydroxyurea and zeocine trigger arrest at both the G2/M transition and the G1/S checkpoint 107 – 109 . We found that cell-cycle-related genes, including CYCD1;1 , were downregulated in med14 and hy5 mutants (Fig. 10A and 10B). CYCD1;1 is a D-type cyclin vital for the G1/S phase transition. Its downregulation likely disrupts cell-cycle progression and decreases the number of dividing cells in the root meristem. We confirmed that both HY5 and MED14 directly bind to the promoter of CYCD1;1 (Fig. 10C-10E). This cooperative action of HY5 and MED14 thus ensures the proper transcriptional activation of CYCD1;1 , which is essential for maintaining active cell division in the root tip (Fig. 11). Beyond their role in promoting cell-cycle progression, MED14 and HY5 also act as repressors of specific DDR genes under normal condition. In the absence of genotoxic stress, expression of the NAC transcription factors NAC044 , NAC085 , and NAC103 is low, thereby preventing unnecessary inhibition of the G2/M phase of the cell-cycle 28 , 110 , 111 . In med14 and hy5 mutants, we observed elevated expression of these NAC genes, indicating a loss of repression on cell-cycle genes (Fig. 6A, 6B, Supplemental Fig. 19D ). The resulting activation of the DDR pathway in these mutants leads to shortened roots and reduced meristematic cell numbers. This was further supported by the phenotyping of med14-1 nac044-1 , hy5 nac044-1 , and med14-1 nac085-2 , hy5 nac085-2 double mutants, in which the short-root phenotype was largely rescued due to reduced cell death at the root tips ( Supplemental Figs. 20 and 21 ). The modest increase in CYCD1;1 transcript level was also observed in the nac044-1 and nac085-2 single and double mutants with med14 (Fig. 10I), suggesting that these transcription factors are involved in regulating the cell-cycle at the transcriptional level and may function as potential repressors of cell cycle gene expression. This genetic evidence confirms that the growth defects in med14 and hy5 are at least partially attributable to the upregulation of NAC044 and NAC085 , which act to restrict cell division in response to stress. Mechanistically, we found that HY5 and MED14 occupy the promoters of NAC044 and NAC085 (Fig. 9A-9D). The occupancy of MED14 on these promoters was reduced in the hy5 mutant (Fig. 9C and 9D), indicating a cooperative repression mechanism. It is already reported that functional loss of NAC044 and NAC085 fails to repress G2/M-specific genes and shows continued cell division under genotoxic stress, resembling Rep-MYB mutants 29 . These NAC transcription factors likely regulate the stability or accumulation of Rep-MYBs (MYB3R3 and MYB3R5) 110 , ensuring sustained suppression of mitotic gene expression ( KNOLLE , CYCB1;2 , EPS15 HOMOLOGY DOMAIN 2 ( EHD2 ), and PLEIADE/MAP65-3 ) and proper cell-cycle arrest 111 , 112 . In our study under genotoxic stress, however, the binding of both HY5 and MED14 to the promoters of NAC044 and NAC085 , as well as CYCD1;1 , was significantly reduced, leading to the upregulation of NAC TFs and downregulation of CYCD1;1 . These data suggest that stress disrupts the MED14-HY5 repression complex, allowing DDR genes to be activated and halting cell division to permit DNA repair (Fig. 11). Together, these findings position the MED14-HY5 module as a critical modulator of growth and stress signaling pathways. While HY5 has long been known as a regulator of photomorphogenesis, our study expands its functionality in cell-cycle control. Similarly, MED14, a core component of the Mediator complex, emerges as a versatile co-regulator capable of supporting both transcriptional activation and repression depending on the context. By activating CYCD1;1 to promote cell division and repressing NAC044 and NAC085 to prevent premature DDR activation, the HY5-MED14 complex ensures that root growth proceeds optimally under favorable conditions. This discovery is particularly significant because it suggests that MED14 and HY5 are continuously required to prevent a baseline level of ROS-induced damage. This study also contrasts with a simple activation model and highlights the complex, context-dependent nature of transcriptional regulation for optimal growth and stress responses. Overall, our findings provide a new paradigm for understanding how plants manage the inevitable trade-off between growth and survival, offering a valuable model for engineering more resilient crop varieties. Material and Methods Plant materials and Growth condition To characterize the MED14, we used two types of mutant lines in our study. We made MED14 RNAi in the Columbia-0 (Col-0) background and another T-DNA insertion mutant (SAIL_373_C07) (Columbia-3 background), which has been obtained from ABRC. We have also used hy5 (SALK_056405), nac044-1 (SAIL_1286D02), nac085-2 (SALK_208662), med17 (SALK_102813), med16-2 (SALK_048091), med25/pft1-3 (SALK_059316), cdk8-1 (SALK_138675), cycC-b (SAIL_102_B02) mutants obtained from ABRC. To generate knockdown RNAi mutants of MED14, 600 bp of the long region was cloned into pHELLSGATE 12. That construct was transformed into Agrobacterium strain (GV3101). Then, wild-type (Col-0) plants were transformed using the Agrobacterium strain containing a recombinant plasmid via the floral dip method 113 . The transgenic plants were selected on kanamycin plates. After several attempts, we got four lines that had downregulation of the MED14 transcript as quantified by qRT-PCR using two sets of gene-specific primers. And only one line which had maximum downregulation, was used in this study. T-DNA mutants SAIL_373_C07 (ecotype Columbia-3 background) were obtained from ABRC. T-DNA was found to be inserted into the sixth exon of the MED14 gene. SAIL_373_C07 was first selected by BASTA (0.001%) spray. Insertion of T-DNA in SAIL_373_C07 was further checked by using T-DNA border and gene specific primers and homozygous (SAIL_373_C07) plants were found by using genotyping. Further, the transcript level of MED14 in T-DNA mutants by qRT-PCR with the help of gene-specific qRT-primers ( Supplemental Table 1 ). For the HY5 pro :HY5::GFP lines, a 3 kb promoter region along with the HY5 gene was amplified from genomic DNA and initially cloned into the pDONR207 vector. This construct was then transferred into the pGWB4 vector using Gateway cloning. The final recombinant construct was introduced into Agrobacterium cells for transformation into both the wild-type and med14-1 mutant backgrounds. Generation of transgenic plants by floral dip For the plant transformation, healthy flowering plants were taken. To avoid the mixture, wild-type siliques were cut and poured into the water for proper hydration one day before the floral dip. Prepared the 5mL of recombinant Agrobacterium inoculum overnight at 28℃ incubator shaker until the culture was saturated. The next day, poured the secondary inoculation of the Agrobacterium cells into 500 mL of LB broth at 28℃ incubator shaker until the OD reached 1.0. Meanwhile, the dipping solution was prepared with ½ strength of MS powder, 5% (w/v) sucrose, and 150 µL of Silwet L-77 (surfactant) in 500 mL of the solution, and the harvested Agrobacterium cells into the solution. Plants were dipped for 40 seconds, and covered the plants with black cloth to protect the plants from light for 24 hrs. Then, removed the cover and allow the plants to grow properly until the seeds are ready to harvest. Seed Sterilization, media preparation and treatment Seeds were surface sterilized for 1 min in 70% (v/v) ethanol and 12 mins in seed washing buffer containing 1.6% (v/v) Sodium hypochlorite, 0.01% SDS and milli Q water and then rinsed four times with sterile milli Q water. The sterilized seeds were kept for 2 days for stratification at 4ºC in dark and then plated those seeds onto solid ½ Murashige and Skoog (MS) medium and grown into a growth chamber with long-day conditions of 16 h light and 8 h dark at about 21ºC; 110 μmol. s-1.m-2 light intensity; and 60% relative humidity. The plants were grown till seedling stage on ½ MS medium with 1% sucrose, 0.05% MES Hydrate and 0.8% plant agar adjusted to pH 5.7. After 10 days the seeding was transferred to soil (Agropit: Vermiculite: Soilrite- 3:1:1 mixture). To prepare 0, 1, 1.5, and 2 mM concentrations of hydroxyurea (HU), a 1 M HU stock solution was made, and appropriate volumes were added to autoclaved ½ MS medium. Media containing 10 µM potassium iodide (KI) and 10 µM quercetin were prepared similarly to the HU media. For root phenotyping, seedlings were first grown on ½ MS medium for 4 days, then transferred to the treatment media for an additional 8 days before measuring root phenotypes. For staining experiments, 4-day-old seedlings were exposed to solid ½ MS medium containing HU, KI, or quercetin for 24–48 hours. For gene expression analysis, 6-day-old seedlings were transferred to treatment media containing 5 mM HU, 10 µM KI, and 10 µM quercetin for 24 hours. For ChIP-qPCR analysis, 6-day-old seedlings were transferred to HU-containing medium and treated for 24 hours. Phenotypic analysis of MED14 plants Arabidopsis primary roots and root tips were analysed by a confocal laser scanning microscope using a TCS SP8 microscope (Leica Camera). Cell length was measured from the confocal images. All the number of lateral roots was counted under compound microscope. Primary root length was analysed directly by using Image J software from digital images captured by a Nikon 3100 camera on the basis of using a ruler. Crossing First, selected the flower buds from mother plant which were expected to open next 2-3 days, and the rest the fertilized flowers and the siliques were removed from the inflorescence tip. Selected buds were emasculated with fine forceps. Emasculated buds with naked gynoecium were left for next 48 h with 60% humidity. After 48 h, mature pollens from healthy opened anthers were carefully sprayed over the stigma and marked the stigma properly. After crossing, plants were kept in proper light, temperature and humidity conditions so that the siliques could grow well. GUS staining Arabidopsis seedlings were taken into the GUS buffer solution and vacuum infiltrated the samples for about 30 mins. Then kept the samples for O/N at 37℃. Rinsed the plant tissue by using a washing solution (70% Ethanol and 30% Acetone) twice for about 2 hrs at 37℃ to remove the excess GUS buffer and chlorophyll. And observed under stereo microscope (Nikon SMZ 25 with DS-Fi3 camera). Propidium iodide (PI) staining For fluorescent propidium iodide (PI) staining, plants were taken from the growth medium to 2 µg/mL of PI solution for 5 mins and mounted in water on microscopic slides by coverslip. The samples were observed at the wavelength specific for the PI fluorescence with the fluorescence excitation maximum of PI was 535 nm and emission maximum were 617 nm by using a confocal laser scanning microscope using a TCS SP8 microscope (Leica Camera). Visualization of cell damage For genotoxic stress, 5-days-old wild-type and the mutant plants were transferred to MS plates containing 0, 0.5, 1mM, 1.5mM of HU. Seedlings were stained with PI (2 µg/mL) on a slide of 5 mins. Stained seedlings were observed by a confocal laser scanning microscope using a TCS SP8 microscope (Leica Camera). The fluorescence excitation maximum of PI was 535 nm and emission maximum was 617 nm. And the blotches were observed as a damage cells. Additionally, root length was measured from the image captured by a Nikon 3100 camera using Image J software. EdU staining EdU staining were performed using EdU detection kit (Invitrogen Alexa 647). In brief, wild-type, mutants and the transgenic seedlings were grown on ½ MS medium for 5 days. For EdU labelling, seedling was transferred into ½ MS broth containing 10 uM EdU for 3 h at 21ºC. Then the seedlings were fixed with 3.7% formaldehyde in phosphate buffer saline (PBS) solution for next 15 mins at room temperature. Fixed seedlings were washed with 3% BSA in PBS solution for twice and allowed to permeabilization into 0.1% triton X-100 in PBS for 20 mins at room temperature. Fixer was further washed with 3% BSA thrice and incubated with EdU detection cocktail for 30 mins at room temperature in dark followed by washing with PBS twice and allowed to further stained with DAPI for observation of fluorescence under confocal microscope. Fluorescence was recorded at wavelengths 520 excitation and 640 emission. DAPI staining DAPI were used to further stained the EdU labelled nuclei. To stained the roots with DAPI, 6-days-old EdU labelled seedlings were further stained with 20 µM of DAPI for 5 mins and observed under confocal microscope. Fluorescence was recorded at the wavelengths of 355 excitation and 465 emission. Detection of flavonoids DPBA staining- For flavonol staining, 6-days-old Arabidopsis seedlings were stained for 15 mins in 0.25% (w/v) diphenylboric acid 2-aminoethyl ester (DPBA; Sigma-Aldrich) and 0.005% (v/v) Triton X-100 114 . Fluorescence was visualized on a confocal laser scanning microscope using a TCS SP8 microscope (Leica Camera) (laser excitation 488 nm, emission 505-550 nm). High-performance thin-layer chromatography (HPTLC)- For the detection of flavonol glycosides, 6-days-old Arabidopsis root samples were harvested and extracted as described previously 115 . The extracted samples were run on Silica Gel 60F254 plates and developed by spraying 1% (w/v) DPBA and 5% (w/v) PEG and observed under UV detector at 365 nm. Developed green, oranges and blue bands were represented kaempferol, quercetin and sinapic acid derivative respectively 116 . Visualization of ROS by staining H2DCFDA staining- For the detection of overall ROS, 6-days-old Arabidopsis roots were stained with 10 µM H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) in potassium phosphate buffer (pH 7.4) for 10 mins and washed the excess dye with sterile milli Q thrice. And mounted the samples in water on microscopic slides by coverslip. Then fluorescence was visualized in GFP excitation wavelength of 488 nm and emission wavelength 500-525 nm on a confocal laser scanning microscope using a TCS SP8 microscope (Leica Camera). NBT staining- For detection of superoxides (O2-), 6-days-old roots were stained with 2 mM Nitroblue tetrazolium (NBT) solution in potassium phosphate buffer (pH 7.4) for 15 mins and washed the excess dye with sterile milli Q. Root samples were mounted in water and observed under stereo microscope (Nikon SMZ 25 with DS-Fi3 camera). DAB staining- For the detection of hydrogen peroxide (H2O2), 6-days-old roots were stained with 2 mg/mL Diamino benzidine (DAB) solution in potassium phosphate buffer (pH 7.4) for 15 mins and washed the excess dye with sterile milli Q. Root samples were mounted in water and observed under stereo microscope (Nikon SMZ 25 with DS-Fi3 camera). Gene expression analysis For analysing the expression of the genes, total RNA was extracted from 8-days-old seedlings using RNeasy plant mini kit (as per the protocol given in Qiagen manual), and cDNA was prepared using 1µg total RNA with Verso cDNA synthesis Kit (as per the Thermo Scientific kit manual protocol). Further, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed with SYBR Green I master mix using the Applied Biosystems QuantStudio 6 Flex Real-Time PCR systems. These qRT-PCR values were normalized with Ubiquitin and 18S as a control gene. qRT-PCR was performed with three biological replicates in triplicate for each gene. Bimolecular Fluorescence Complementation (BiFC) assay HY5 was cloned into the pDEST-VYCE vector, and MED14_Full length, MED14_Δ3, MED14_Δ4, and MED14_Δ6 were cloned into pDEST-VYNE . Recombinant plasmids along with empty vectors were transformed into Agrobacterium starin GV3101 and infiltrated into Nicotiana benthamiana leaves as described in 117 . Image was captured after 48 h light exposure using TCS SP8 confocal microscope (Leica Camera) with the YFP excitation 490 mm and emission 520 nm and for red fluorescence excitation maxima 550 nm and emission maxima 580 mm. Dual luciferase assay To assess CYCD1;1 transactivation, a 993 bp long promoter region with HY5-binding elements was cloned into the pGreenII 0800-LUC vector (containing 35S promoter). Transcription factor HY5 was cloned into pGWB4 under its native promoter, and MED14 was cloned into pGWB21. These constructs were co-expressed in N. benthamiana leaves alongside various effectors, with an empty vector as a control. After infiltration, plants were kept in the dark at 22°C for 24 h and then exposed to light for 48 hours. Luciferase activity was measured using a ChemiDoc imaging system. For HU treatment, after 48 h of light exposure, infiltrated leaves were excised at the petiole site and placed in ½ MS broth with mock and 2 mM HU treatments, which were allowed to proceed for the next 6 h. The excised leaves were then blotted with tissue paper before assessing luciferase activity. Chromatin immunoprecipitation (ChIP) Chromatin immunoprecipitation was performed according to the standard protocol with few modifications 118 . Eight-day-old Arabidopsis seedlings were fixed by vacuum infiltration in formaldehyde-containing crosslinking buffer (0.4 M sucrose, 10 mM Tris-HCl pH=8, 1 mM PMSF, 1 mM EDTA, and 1% formaldehyde) to preserve protein-DNA interactions. The crosslinking was quenched with glycine, and samples were thoroughly washed with water before freezing in liquid nitrogen. Frozen tissue was ground into a fine powder, and nuclei were isolated using a nucleus isolation buffer (0.25 M sucrose, 15 mM PIPES pH=6.8, 5 mM MgCl 2 , 60 mM KCl, 15 mM NaCl, 1 mM CaCl 2 , 0.9% Triton X-100, protease inhibitor SIGMA #lot 83612300) under cold conditions. The nuclei were lysed with nucleus lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, protease inhibitor), and chromatin was sonicated to shear DNA into fragments suitable for immunoprecipitation. The chromatin extract was pre-cleared with salmon sperm DNA–blocked Protein A agarose beads (Sigma-Aldrich 16-157) to reduce non-specific binding. A specific antibody against the target protein was then incubated overnight with the chromatin to allow formation of antibody-protein-DNA complexes. The immune complexes were captured using Protein A beads and subjected to a series of washes with buffers of increasing stringency to remove non-specifically bound material. Bound chromatin was eluted from the beads, and crosslinks were reversed by incubation at 65°C overnight. Proteins were digested with Proteinase K to purify DNA, followed by phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation to recover the DNA. The purified DNA pellet was washed with 70% ethanol, dried, resuspended in TE buffer, and stored at –20°C for downstream analyses such as qPCR or sequencing. Western blotting Approximately 100 mg of six-day-old Arabidopsis seedlings (or root and shoot independently) were frozen and ground into powder under liquid nitrogen. Proteins were extracted using a cold buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM DTT, 100 mM PMSF, 1 mM NaF, 20% glycerol, and protease inhibitor) and homogenized gently. The mixture was then centrifuged to remove debris. Protein concentration was measured via Bradford assay against a BSA standard curve. Equal amounts of protein were mixed with SDS loading dye, denatured at 95°C for 10 minutes, and loaded onto a freshly prepared 10-12% SDS-PAGE gel (as required). After electrophoresis, proteins were transferred to a nitrocellulose membrane at room temperature for 1.5 h. The membrane was further blocked with 5% BSA to prevent nonspecific binding, then incubated O/N with primary antibody at 4°C. Following washes, the membrane was incubated with HRP-conjugated secondary antibody for another 1 h. After further washes, protein bands were detected using a chemiluminescent substrate. Finally, bands were visualized and documented using a gel imaging system. RNA sequencing and data analysis For RNA-sequencing, 6-days-old roots of Col-0, med14-1 were collected in three biological replicates after germination. Total RNA was isolated using the Sigma Plant RNA isolation kit according to the manufacturer’s protocol. Library preparation was done using NEB kit, and RNA-sequencing was done on Illumina Hiseq X platform by Agrigenome, P.V. Ltd (Bengaluru, India). After removing the bad quality read by fastp, the clean reads were aligned to the Arabidopsis genome TAIR 10 (https://www.arabidopsis.org/) using HISAT 2 119 . Here, for RNA-seq of RNAi analysis, 2 nd exon of MED14 gene was trimmed out by the cut adapt sequence trimming tool from all samples (both for Col-0 and med14 samples). The expression level of each gene was analysed by the featureCount tool of Subread. For details of the libraries, read numbers, and alignments . For differential gene expression analyses, DEseq2 (v:1.22.2) tool was used. To determine the correlation among biological replicates, normalized read counts were analysed and volcano plot of log2Fold change with p-value was made. For up- and down-regulated genes, a cut-off value of |FC| ≥ 1.5 and p-value ≤ 0.05 was selected, respectively. Detailed pairwise comparisons for DEGs are provided . Heatmaps were constructed from log2 fold change values generated by DEGs analysis using the heatmap (Version-1.0.12) R tool. Scatter plots were constructed from the log2 fold change values generated by DEGs analysis using ggplot2 (version 3.5.1) R tool. For the heatmap and scatterplot, data with pvalue ≤ 0.05 were considered for analysis. Gene Ontology (GO) analysis was performed using ShinyGO 0.80 (http://bioinformatics.sdstate.edu/go/) with default parameters. Genes involved in cell cycle and ROS were manually selected from the Arabidopsis Information Resource website (TAIR). Protein-protein docking Protein–protein docking was carried out to investigate the potential interaction between AtHY5 and AtMED14. The three-dimensional structure of AtMED14 was predicted using the Phyre2 server 120 , while the crystal structure of AtHY5 (PDB ID: 2OQQ) was used for docking studies. Prior to docking, both protein structures were prepared using PyMOL 121 , where water molecules and heteroatoms were removed, and hydrogen atoms were added to facilitate proper modeling of hydrogen bonding. Initial docking poses were generated using the PatchDock server 122 , which identifies candidate binding conformations based on geometric shape complementarity. The resulting docking solutions were subsequently refined using FireDock 123 , which optimizes side-chain conformations and recalculates binding energies. Based on the refined energy scores, the top three docking solutions were selected to analyze the binding modes and interaction interfaces between HY5 and MED14. Quantification and statistical analysis All physiological, confocal, and stereomicroscopic experiments were considered as independent biological replicates containing at least 15-20 seedlings. Gene expression was analyzed using 2 -ΔΔCt values from RT-qPCR on three independent biological replicates. ChIP-qPCR experiments were conducted in three technical replicates (n=3) from a single representative experiment, with each experiment repeated two to three times independently. Physiological, confocal, and ChIP experiments with similar results/patterns were repeated as described in the figure legends. Statistical significance was determined using one-way ANOVA and Tukey's HSD test, with P ≤ 0.05 considered statistically significant and P > 0.05 considered non-significant. Some of the experiments used the t-test, with P ≤ 0.05 considered significant and P > 0.05 non-significant, and represented them as asterisks * (for P ≤ 0.05), ** (for P ≤ 0.005), and *** (for P ≤ 0.0005). Data analysis and graph creation were done using Microsoft Excel, and statistical analysis was performed with GraphPad Prism 8. Accession numbers The gene sequence data referenced in this article are available in the GenBank/EMBL databases under the following accession number: MED14 (AT3G04740), HY5 (AT5G11260), CYCD1;1 (AT1G70210), CYCB1;1 (AT4G37490), NAC044 (AT3G01600), NAC085 (AT5G14490), NAC103 (AT5G64060), SOG1 (AT1G25580), ATR (AT5G40820), ATM (AT3G48190), CHS (AT5G13930), CHI (AT3G55120), F3H (AT3G51240), and FLS1 (AT5G08640). Declarations Acknowledgements and Funding This study is partially funded by the grant BT/PR38349/GET/119/339/2020 and partially by grant BT/PR40169/BTIS/137/71/2023 both from the Department of Biotechnology, Government of India. The authors acknowledge core funding from the International Centre for Genetic Engineering and Biotechnology, New Delhi. PT acknowledges the Council of Scientific and Industrial Research (CSIR) for Junior and Senior Research Fellowships, Government of India. SM acknowledges fellowships from the Department of Biotechnology, Government of India (MK Bhan Fellow: BT/HRD/MK-YRFP/50/27/2021). AG acknowledges fellowships from the University Grants Commission (UGC) for the Junior Research Fellowship. Support from all the facilities of NIPGR and ICGEB is acknowledged. Authors are thankful to DeLCON for providing access to the literature. Authors are grateful to Prof. Nam-Hai Chua, Temasek Life Sciences Laboratory, National University of Singapore, Singapore, 117604, for providing the MED14 pro :MED14::YFP seeds, and also grateful to Prof. Yongjian Qiu, Department of Biology, University of Mississippi, Oxford, Mississippi 38677, USA, for providing 35S:MED14::HA/med14 seeds. Authors contribution PT planned and designed the experiments and further analyses, and wrote the first draft, figures with SM. 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Additional Declarations There is NO Competing Interest. Supplementary Files SupplementalTableS1.pdf Primers used in this study Supplementaryfigures.pdf Supplementary Figures Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8222842","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":555294044,"identity":"5e756f9c-a6c5-41a0-9255-23378336eb5a","order_by":0,"name":"Jitendra 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1","display":"","copyAsset":false,"role":"figure","size":163713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMED14 regulates root system architecture in Arabidopsis.\u003c/strong\u003e a, Representative images showing the 12th-day-old root phenotype of Col-0, med14-1, Col-3, and med14-2. b and c, Line graph showing the primary root length of wild-types and med14 mutants. d, Confocal images showing the 6th-day-old wild-type and med14 roots, stained with PI. Arrows showing the end of meristematic, transition, and elongation zones respectively. e, Graph showing the length of the meristematic zones in the primary roots of wild-type and med14 seedlings. f-h, Graphs representing the number of cells in meristematic, transition, and elongation zones in the roots of wild-type and med14 seedlings respectively. i, PIstained confocal images showing the cell length at the transition and elongation zone of wild-type and med14 mutants. Individual cells of transition and elongation zones were marked by yellow lines. j and k, Graphs represented the length of the cells in transition and elongation zones of wild-type and med14 roots respectively. Meristem length and cell length was measured from the confocal images of PI-stained roots by using the Image J software. Data shown in the graphs represents the average of three independent biological replicates containing at least 20 seedlings. Bar plots represent the mean value and the black circles represent the individual values. Statistical significance was determined using a oneway ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test. Statistical significance was determined by a P-value of 0.05 or lower (P ≤ 0.05), while Pvalues greater than 0.05 (P \u0026gt; 0.05) were considered non-significant (ns) and depicted with alphabets. Scale bar=100 mm (A), 100 μm (d and i).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/fec49988d7c4a91103418317.jpg"},{"id":97670034,"identity":"b202e5ca-230f-499e-b9a8-2a45cbf1a25a","added_by":"auto","created_at":"2025-12-08 09:29:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87692,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMED14 regulates flavonoid biosynthesis in Arabidopsis root. a and b, \u003c/strong\u003eScattered dot plots showing the expression of flavonoid biosynthesis genes in the root tissue of Col-0, \u003cem\u003emed14-1\u003c/em\u003e, Col-3, and \u003cem\u003emed14-2\u003c/em\u003e. \u003cstrong\u003ec, \u003c/strong\u003e6\u003csup\u003eth\u003c/sup\u003e-day-old roots of wild-type and \u003cem\u003emed14 \u003c/em\u003emutants were stained with DPBA to detect the flavonol glycosides and observed under the confocal microscope. \u003cstrong\u003ed, \u003c/strong\u003eGraph showing the fluorescence intensity of DPBA stained roots of wild-type and \u003cem\u003emed14 \u003c/em\u003emutants. \u003cstrong\u003ee, \u003c/strong\u003eHPTLC analysis showing the accumulation of flavonols in 6\u003csup\u003eth\u003c/sup\u003e-day-old roots of wild-type and \u003cem\u003emed14 \u003c/em\u003emutants. Different kaempferol (K) and quercetin (Q) derivatives were indicated by green and orange fluorescence respectively. The fluorescence intensity was measured by using Image J software. Gene expression values were calculated as fold change (2\u003csup\u003e-ΔΔCT\u003c/sup\u003e). qRT-PCR analysis was performed with three independent biological replicates (n=3) and the experiment was repeated twice. Scatter dot plots represent individual values of biological replicates and error bars denote standard deviation (SD). Data shown in the graphs represents the average of three independent biological replicates containing at least 12 seedlings. Bar plots represent the mean value and the black circles denote the individual values. Statistical significance was determined using a one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test. Statistical significance was determined by a P-value of 0.05 or lower (P ≤ 0.05), while P-values greater than 0.05 (P \u0026gt; 0.05) were considered non-significant (ns) and depicted with alphabets. Scale bar=100 μm.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/09f0feee1c31a0b4859cceb4.jpg"},{"id":97504095,"identity":"1a6cc40c-633f-42f0-bff8-bd33393e69fd","added_by":"auto","created_at":"2025-12-05 07:13:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":135670,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMED14 regulates the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHY5 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression to control the root architecture. a, \u003c/strong\u003eScattered dot plot showing the expression of HY5 in 6th-day-old root tissues independently. \u003cstrong\u003eb and c, \u003c/strong\u003eChIP-qPCR results showing the binding of HY5 at the promoter site of \u003cem\u003eHY5 \u003c/em\u003eand \u003cem\u003eTA3\u003c/em\u003e. \u003cstrong\u003ed, \u003c/strong\u003eChIP-qPCR results showing the occupancy of MED14 at the promoter sites of \u003cem\u003eHY5 \u003c/em\u003ein wild-type and \u003cem\u003ehy5 \u003c/em\u003emutant backgrounds. \u003cstrong\u003ee, \u003c/strong\u003ePhenotypic comparison of 12-days-old Col-0, \u003cem\u003emed14-1\u003c/em\u003e, \u003cem\u003ehy5\u003c/em\u003e, and \u003cem\u003emed14-1 hy5 \u003c/em\u003edouble mutants. \u003cstrong\u003ef, \u003c/strong\u003eGraphical representation showing the primary root length of 12-days-old seedlings of wild-type, \u003cem\u003emed14-1\u003c/em\u003e, \u003cem\u003ehy5\u003c/em\u003e, and double mutants. \u003cstrong\u003eg, \u003c/strong\u003eConfocal images showing the cellular organization of 6th-day-old roots of Col-0, \u003cem\u003emed14-1\u003c/em\u003e, \u003cem\u003ehy5\u003c/em\u003e, and \u003cem\u003emed14-1 hy5 \u003c/em\u003emutants, stained with PI. Arrows showing the end of meristematic, transition, and elongation zones respectively. \u003cstrong\u003eh, \u003c/strong\u003eGraph showing the meristem length. \u003cstrong\u003ei-p, \u003c/strong\u003eConfocal images showing the roots of Col-0, \u003cem\u003emed14-1\u003c/em\u003e, \u003cem\u003ehy5\u003c/em\u003e, and double mutants stained with EdU to detect the cell division. Gene expression values were calculated as Fold change (2\u003csup\u003e-ΔΔCT\u003c/sup\u003e). qRT-PCR analysis was performed with three independent biological replicates (n=3) and repeated the experiment twice. Scatter dot plots represent individual values of biological replicates and error bars denote standard deviation (SD). ChIP-qPCR was performed in 7-day-old seedlings. Untransformed Col-0 seedlings were taken as a negative control. \u003cem\u003eTA3pro \u003c/em\u003ewas taken as a negative control for the binding of HY5. For ChIP-qPCR, the position of the amplicon is relative to the ATG (start) site. Fold enrichment was calculated from no-antibody and anti-GFP antibody samples. Each ChIP-qPCR analysis was performed with three technical replicates (n=3) and repeated each experiment twice (n=2). Bar plots represent the mean value of three technical replicates. Each circle denotes a single value. Error bars denote SD. Statistical significance was determined using a one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test. Statistical significance was determined by a P-value of 0.05 or lower (P ≤ 0.05), while P-values greater than 0.05 (P \u0026gt; 0.05) were considered non-significant and depicted with alphabets. Bar=100 mm (e), Bar=100 μm (g).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/53e16fdb4a4750701c56f3be.jpg"},{"id":97504101,"identity":"e27508b6-f083-44b3-af80-93e024f5380c","added_by":"auto","created_at":"2025-12-05 07:13:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":185441,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMED14 regulates HY5-dependent flavonoid biosynthesis and facilitates root elongation. a and b, \u003c/strong\u003eScattered dot plots showing the expression of flavonoid biosynthesis genes in the roots of Col-0, \u003cem\u003ehy5\u003c/em\u003e, and \u003cem\u003emed14-1 hy5 \u003c/em\u003edouble mutants respectively. \u003cstrong\u003ec, \u003c/strong\u003eRepresentative images showing the root phenotype of Col-0, \u003cem\u003emed14-1\u003c/em\u003e, Col-3, and \u003cem\u003emed14-2 \u003c/em\u003eafter quercetin treatment. \u003cstrong\u003ed, \u003c/strong\u003eGraph showing the % root length increase after the quercetin treatment in wild-type and \u003cem\u003emed14 \u003c/em\u003emutants. \u003cstrong\u003ee, \u003c/strong\u003eRepresentative images showing root phenotype of Col-0, \u003cem\u003ehy5, \u003c/em\u003eand \u003cem\u003emed14-1 hy5 \u003c/em\u003emutants after quercetin treatment. \u003cstrong\u003ef, \u003c/strong\u003eGraph showing the % root length increase after the quercetin treatment in Col-0, \u003cem\u003ehy5\u003c/em\u003e, and \u003cem\u003emed14-1 hy5 \u003c/em\u003emutants. \u003cstrong\u003eg, \u003c/strong\u003eConfocal images highlighting the meristem zone in Col-0, \u003cem\u003emed14-1\u003c/em\u003e, and \u003cem\u003ehy5 \u003c/em\u003eroots after quercetin treatment, stained with PI. Yellow arrows showing the end of the meristematic zone in PI-stained roots. \u003cstrong\u003eh, \u003c/strong\u003eGraphical representation showing the number of meristem cells increased (%) in the Col-0, \u003cem\u003emed14-1\u003c/em\u003e, and \u003cem\u003ehy5 \u003c/em\u003eroots after quercetin treatment. \u003cstrong\u003ei, \u003c/strong\u003eRepresentative confocal images showing the elongated cell length marked by yellow lines in 6\u003csup\u003eth\u003c/sup\u003e-day-old roots of Col-0, \u003cem\u003emed14-1\u003c/em\u003e, and \u003cem\u003ehy5 \u003c/em\u003emutants after the quercetin treatment. \u003cstrong\u003ej, \u003c/strong\u003eGraph representing the change in the length of elongated cells (%) in wild-type, \u003cem\u003emed14-1 \u003c/em\u003eand \u003cem\u003ehy5 \u003c/em\u003emutants. Gene expression values were calculated as Fold change (2\u003csup\u003e-\u003c/sup\u003eΔΔCT). qRT-PCR analysis was performed with three independent biological replicates (n=3) and repeated the experiment twice. Scatter dot plots represent individual values of biological replicates and error bars denote standard deviation (SD). Data shown in the graphs represents the average of three independent biological replicates containing at least 20 seedlings. Bar plots represent the mean value and the black circles represent the individual values. Statistical significance was determined using a one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test. Statistical significance was determined by a P-value of 0.05 or lower (P ≤ 0.05), while P-values greater than 0.05 (P \u0026gt; 0.05) were considered non-significant (ns) and depicted with alphabets. Scale bar=100 mm (c), 100 μm (g).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/2f20804271254f1922076fa5.jpg"},{"id":97670056,"identity":"d3631c4f-5383-4173-aabe-5025a93d6edf","added_by":"auto","created_at":"2025-12-08 09:29:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":108270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMED14 regulates ROS homeostasis in Arabidopsis root. a-d, \u003c/strong\u003eConfocal images showing 6-days-old roots of Col-0, \u003cem\u003emed14-1\u003c/em\u003e, Col-3, and \u003cem\u003emed14-2 \u003c/em\u003estained with H\u003csub\u003e2\u003c/sub\u003eDCFDA. \u003cstrong\u003ee, \u003c/strong\u003eGraph showing the fluorescence intensity of H\u003csub\u003e2\u003c/sub\u003eDCFDA stain in the roots of wild-types and \u003cem\u003emed14 \u003c/em\u003emutants. The fluorescence intensity of H\u003csub\u003e2\u003c/sub\u003eDCFDA was measured by using Image J software. \u003cstrong\u003ef-i, \u003c/strong\u003eMicroscopic images showing the NBT-stained roots of wild-types and \u003cem\u003emed14 \u003c/em\u003emutants. Blue color denotes the superoxide accumulation in roots. \u003cstrong\u003ej-m, \u003c/strong\u003eMicroscopic images showing the DAB-stained roots of wild-types and \u003cem\u003emed14 \u003c/em\u003emutants. The brown color denotes the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation in the roots. \u003cstrong\u003en, \u003c/strong\u003eRepresentative images showing the root phenotype of wild-types and \u003cem\u003emed14 \u003c/em\u003emutants after KI treatment. \u003cstrong\u003eo, \u003c/strong\u003eGraph showing the % root length increase after the KI treatment in wild-types and \u003cem\u003emed14 \u003c/em\u003emutants. Data shown in the graphs represents the average of three independent biological replicates containing at least 20 seedlings. Bar graphs represent the mean value and the black circles represent the individual values. Error bars denote standard deviation (SD). Statistical significance was determined using a one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test. Statistical significance was determined by a P-value of 0.05 or lower (P ≤ 0.05), while P-values greater than 0.05 (P \u0026gt; 0.05) were considered non-significant and depicted with alphabets. Scale bar=100 μm (A-D), 500 μm (F-I), 200 μm (J-M), 100 mm (N).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/ff295c6352e428c8de712dbf.jpg"},{"id":97670934,"identity":"a00a9374-5493-4a0d-9ff1-857078a1c628","added_by":"auto","created_at":"2025-12-08 09:31:33","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":97591,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRole of MED14 in the DDR response to regulate the primary root elongation. a and b, \u003c/strong\u003eScattered dot plots showing the expression of DDR-responsive genes in roots of Col-0, \u003cem\u003emed14-1\u003c/em\u003e, Col-3, and \u003cem\u003emed14-2\u003c/em\u003e. \u003cstrong\u003ec, \u003c/strong\u003eRepresentative images showing root phenotype of Col-0 and \u003cem\u003emed14-1 \u003c/em\u003ein 1 mM concentration of HU. \u003cstrong\u003ed, \u003c/strong\u003eGraphical representation showing the primary root length of Col-0 and \u003cem\u003emed14-1 \u003c/em\u003ein 1 mM HU. \u003cstrong\u003ee, \u003c/strong\u003eGraph showing the % root length decrease in Col-0 and \u003cem\u003emed14-1 \u003c/em\u003ein HU. \u003cstrong\u003ef, \u003c/strong\u003eConfocal images showing the cellular damage in Col-0 and \u003cem\u003emed14-1 \u003c/em\u003eroots in different concentrations of HU. Red PI-stained blotches at the root tip denote cell death. Gene expression values were calculated as fold change (2\u003csup\u003e-ΔΔCT\u003c/sup\u003e). qRT-PCR analysis was performed with three independent biological replicates (n=3) and repeated the experiment twice. The scatter dot plot represents individual values of biological replicates and the graphs represents the average of three independent biological replicates containing at least 20 seedlings. Bar plots represent the mean value and the black circles represent the individual values. Error bars denote standard deviation (SD). Statistical significance was determined using a one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test. Statistical significance was determined by the P-value of 0.05 or lower (P ≤ 0.05), while P-values greater than 0.05 (P \u0026gt; 0.05) were considered non-significant (ns) and depicted with alphabets. Statistical differences have also been depicted by P value P\u0026lt;0.0005 represented with asterisks (***) as assessed by student’s t-test (e). Scale bar=100 mm (c), 100 μm (e).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/1f9474662a112453971c503b.jpg"},{"id":97670466,"identity":"58b47c64-d308-41f0-8be0-0c00481254d2","added_by":"auto","created_at":"2025-12-08 09:30:44","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":125137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHY5 regulates ROS-mediated primary root elongation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emed14\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a-e, \u003c/strong\u003eMicroscopic images representing the roots of Col-0, \u003cem\u003emed14-1\u003c/em\u003e, \u003cem\u003ehy5\u003c/em\u003e, \u003cem\u003emed14-1 hy5\u003c/em\u003e, and \u003cem\u003eHY5 prO:HY5::GFP \u003c/em\u003estained with H\u003csub\u003e2\u003c/sub\u003eDCFDA. \u003cstrong\u003ef, \u003c/strong\u003eGraph showing the relative fluorescence intensity of H\u003csub\u003e2\u003c/sub\u003eDCFDA stain in the roots of Col-0, \u003cem\u003emed14-1\u003c/em\u003e, \u003cem\u003ehy5\u003c/em\u003e, \u003cem\u003emed14-1 hy5\u003c/em\u003e, and \u003cem\u003eHY5 prO:HY5::GFP\u003c/em\u003e. \u003cstrong\u003eg, \u003c/strong\u003eRepresentative image showing the root phenotype of Col-0, \u003cem\u003ehy5\u003c/em\u003e, and \u003cem\u003emed14-1 hy5 \u003c/em\u003edouble mutant in HU and KI treatment. \u003cstrong\u003eh, \u003c/strong\u003eGraphical representation showing the change in % root length (decrease and increase) in Col-0, \u003cem\u003ehy5\u003c/em\u003e, and \u003cem\u003emed14-1 hy5 \u003c/em\u003edouble mutant after HU and KI treatment respectively. \u003cstrong\u003ei, \u003c/strong\u003eRepresentative images showing the root phenotype of Col-0 and \u003cem\u003eHY5\u003c/em\u003e\u003csub\u003e\u003cem\u003eprO\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:HY5::GFP \u003c/em\u003ein HU treatment. \u003cstrong\u003ej, \u003c/strong\u003eGraph showing the % root length decrease in Col-0 and \u003cem\u003eHY5\u003c/em\u003e\u003csub\u003e\u003cem\u003eprO\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:HY5::GFP \u003c/em\u003eafter HU treatment. \u003cstrong\u003ek-t, \u003c/strong\u003eConfocal images showing the cellular damage in Col-0, \u003cem\u003emed14-1\u003c/em\u003e, \u003cem\u003ehy5\u003c/em\u003e, \u003cem\u003emed14-1 hy5 \u003c/em\u003edouble mutant and \u003cem\u003eHY5 prO:HY5::GFP \u003c/em\u003eroots in HU treatment. Red PI-stained blotches at the root tip denote cell death. Data shown in the graphs represents the average of three independent biological replicates containing at least 20 seedlings. Bar plots represent the mean value and the black circles represent the individual values. Error bars denote standard deviation (SD). Statistical significance was determined using a one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test. Statistical significance was determined by the P-value of 0.05 or lower (P ≤ 0.05), while P-values greater than 0.05 (P \u0026gt; 0.05) were considered non-significant and depicted with alphabets. Statistical differences have also been depicted by P value P\u0026lt;0.0005 represented with asterisks (***) as assessed by student’s t-test (j). Scale bar=100 mm (g and i), 100 μm (a-e, k-t).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/460048dd0ab7f4ca1a06e6f1.jpg"},{"id":97670869,"identity":"79463e34-7736-4d10-8691-8910065c80fb","added_by":"auto","created_at":"2025-12-08 09:31:27","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":126811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlavonoids regulate the DDR response in Arabidopsis root by modulating ROS homeostasis. a-c, \u003c/strong\u003eScattered dot plot showing the transcript level of \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103 \u003c/em\u003ein Col-0 roots in response to Mock, HU, HU+quercetin, and HU+KI. \u003cstrong\u003ed and e, \u003c/strong\u003eScattered dot plots representing the expression of \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103 \u003c/em\u003ein Col-0, \u003cem\u003emed14-1\u003c/em\u003e, and \u003cem\u003ehy5 \u003c/em\u003eupon only quercetin treatment respectively. \u003cstrong\u003ef, \u003c/strong\u003eConfocal images showing the cell death in Col-0, \u003cem\u003emed14-1\u003c/em\u003e, and \u003cem\u003ehy5 \u003c/em\u003eroots in response to mock, HU, and HU along with quercetin and KI. PI-blotches indicate the cell death area. \u003cstrong\u003eg, \u003c/strong\u003eGraphical representation showing the measurement of cell death area in Col-0, \u003cem\u003emed14-1\u003c/em\u003e, and \u003cem\u003ehy5 \u003c/em\u003eroots upon mock, HU, and HU along with quercetin and KI from the confocal images. Gene expression values were calculated as fold change (2\u003csup\u003e-ΔΔCT\u003c/sup\u003e). qRT-PCR analysis was performed with three independent biological replicates (n=3) and repeated the experiment twice. The scatter dot plot represents individual values of biological replicates. CT values of figures a-c were normalized using 18S, and figures d and e were normalized with ubiquitin as an internal control. Data shown in the graphs represents the average of three independent biological replicates containing at least 15 seedlings. Bar plots represent the mean value and the black circles represent the individual values. Error bars denote standard deviation (SD). Statistical significance was determined using a one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test. Statistical significance was determined by the P-value of 0.05 or lower (P ≤ 0.05), while P-values greater than 0.05 (P \u0026gt; 0.05) were considered non-significant (ns) and depicted with alphabets. Scale bar=100 µm.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/23276ef9e0d2cfe450a31197.jpg"},{"id":97504109,"identity":"d00e8da2-2373-4c0d-aa58-791673342886","added_by":"auto","created_at":"2025-12-05 07:13:08","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":91998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding of HY5 and occupancy of MED14 at the promoter of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNAC044 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNAC085 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein genotoxic stress. a and b, \u003c/strong\u003eChIP-qPCR results showing the enrichment of HY5 at the promoter site of \u003cem\u003eNAC044 \u003c/em\u003eand \u003cem\u003eNAC085 \u003c/em\u003ein wild-type and \u003cem\u003emed14-1 \u003c/em\u003ebackground. \u003cstrong\u003ec and d, \u003c/strong\u003eChIP-qPCR results showing the occupancy of MED14 at the two different promoter sites of \u003cem\u003eNAC044 \u003c/em\u003eand \u003cem\u003eNAC085 \u003c/em\u003ein wild-type and \u003cem\u003ehy5 \u003c/em\u003emutant background. \u003cstrong\u003ee and f, \u003c/strong\u003eGraphs showing the binding of HY5 to the promoter of \u003cem\u003eNAC044 \u003c/em\u003eand \u003cem\u003eNAC085 \u003c/em\u003ein response to HU treatment respectively. \u003cstrong\u003eg and h, \u003c/strong\u003eGraphs showing the occupancy of MED14 to the promoter of \u003cem\u003eNAC044 \u003c/em\u003eand \u003cem\u003eNAC085 \u003c/em\u003ein response to HU treatment respectively. For checking the binding of HY5, \u003cem\u003eHY5\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:HY5::GFP \u003c/em\u003eand to check the occupancy of MED14, \u003cem\u003eMED14\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:MED14::YFP \u003c/em\u003elines were used. Untransformed Col-0 seedlings were taken as a negative control. ChIP-qPCR was performed in 7-day-old seedlings with 24 h HU treatment. For ChIP-qPCR, the position of the amplicon is relative to the ATG (start) site. Fold enrichment was calculated from no-antibody and anti-GFP antibody samples. Each ChIP-qPCR analysis was performed with three technical replicates (n=3) and repeated each experiment twice (n=2). Bar plots represent the mean value of three technical replicates. Each circle denotes a single value. Error bars denote SD. Statistical significance was determined using a one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test. Statistical significance was determined by a P-value of 0.05 or lower (P ≤ 0.05), while P-values greater than 0.05 (P \u0026gt; 0.05) were considered non-significant and depicted with alphabets.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/58b71bb2a2aae3d692218763.jpg"},{"id":97504106,"identity":"eb9436d8-a8af-4923-a3a8-786190d10d11","added_by":"auto","created_at":"2025-12-05 07:13:08","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":100431,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMED14 and HY5 transcriptionally regulate \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCYCD1;1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a, \u003c/strong\u003eTranscriptomic comparison of cell-cycle genes showing the commonly downregulated in both the \u003cem\u003emed14-1 \u003c/em\u003eand \u003cem\u003ehy5 \u003c/em\u003eroots. Blue-colored dots showing the downregulated genes in \u003cem\u003emed14-1\u003c/em\u003e, light green and black-colored dots showing the upregulated genes in \u003cem\u003emed14-1 \u003c/em\u003eand \u003cem\u003ehy5 \u003c/em\u003emutants respectively, red-colored dots showing the commonly downregulated genes and grey-colored dots showing the non-significant genes in both the mutants. \u003cstrong\u003eb, \u003c/strong\u003eScattered dot plot showing the transcript level of \u003cem\u003eCYCD1;1 \u003c/em\u003ein the roots of Col-0, \u003cem\u003emed14-1\u003c/em\u003e, and \u003cem\u003ehy5 \u003c/em\u003emutants. \u003cstrong\u003ec, \u003c/strong\u003eBar graph showing the binding of HY5 on the promoter of \u003cem\u003eCYCD1;1 \u003c/em\u003ein Col-0, and \u003cem\u003eHY5\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:HY5::GFP\u003c/em\u003e. \u003cstrong\u003ed, \u003c/strong\u003eBar graph showing the occupancy of MED14 on the promoter of \u003cem\u003eCYCD1;1 \u003c/em\u003ein 8-days-old seedlings of Col-0, and \u003cem\u003eMED14\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:MED14::YFP\u003c/em\u003e. \u003cstrong\u003ee, \u003c/strong\u003eBar graph showing the occupancy of MED14 on the promoter of \u003cem\u003eCYCD1;1 \u003c/em\u003ein Col-0, and \u003cem\u003eMED14pro:MED14::YFP \u003c/em\u003eand \u003cem\u003eMED14pro:MED14::YFP\u003c/em\u003e/\u003cem\u003ehy5 \u003c/em\u003elines. \u003cstrong\u003ef, \u003c/strong\u003eGraph showing the transcript level of \u003cem\u003eCYCD1;1 \u003c/em\u003ein response to genotoxic stress in wild-type root. \u003cstrong\u003eg, \u003c/strong\u003eGraph representing the binding of HY5 at the promoter site of \u003cem\u003eCYCD1;1 \u003c/em\u003ein Col-0 and \u003cem\u003eHY5\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:HY5::GFP \u003c/em\u003ein response to HU treatment. \u003cstrong\u003eh, \u003c/strong\u003eGraph showing the occupancy of MED14 at the promoter site of \u003cem\u003eCYCD1;1 \u003c/em\u003ein Col-0 and \u003cem\u003eMED14\u003c/em\u003e\u003csub\u003e\u003cem\u003epro\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e:MED14::YFP \u003c/em\u003ein HU treatment. \u003cstrong\u003ei, \u003c/strong\u003eScattered dot plot showing the transcript level of \u003cem\u003eCYCD1;1 \u003c/em\u003ein wild-type, \u003cem\u003emed14-1\u003c/em\u003e, \u003cem\u003enac044-1\u003c/em\u003e, \u003cem\u003enac085-2\u003c/em\u003e, \u003cem\u003emed14-1 nac044-1\u003c/em\u003e, and \u003cem\u003emed14-1 nac085-2 \u003c/em\u003emutant roots. Gene expression values were calculated as Fold change (2\u003csup\u003e-ΔΔCT\u003c/sup\u003e). qRT-PCR analysis was performed with three independent biological replicates (n=3). Scatter dot plots represent individual values of biological replicates and error bars denote standard deviation (SD). ChIP-qPCR was performed in 7-day-old seedlings with 24 h HU treatment. Untransformed Col-0 seedlings were taken as a negative control. The position of the amplicon is relative to the ATG (start) site. Fold enrichment was calculated from the no-antibody and anti-GFP antibody samples. Scattered line plots denote the scanning of the promoter sites of \u003cem\u003eCYCD1;1\u003c/em\u003e. Each ChIP-qPCR analysis was performed with three technical replicates (n=3) and repeated each experiment twice (n=2). Bar plots represent the mean value of three technical replicates. Each circle denotes single value. Line plots also showing the mean value and error bars denote SD. Statistical significance was determined by using a one-way ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test. Statistical significance was determined by a P-value of 0.05 or lower (P ≤ 0.05), while P-values greater than 0.05 (P \u0026gt; 0.05) were considered non-significant and depicted with alphabets. Statistical differences have also been depicted by P value P\u0026lt;0.0005 represented with asterisks (***) as assessed by student’s t-test (C, D and F).\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/45fdc7744296ee6bd144628e.jpg"},{"id":97504103,"identity":"5ac4c130-b158-4e9c-8e4d-e615276cadc4","added_by":"auto","created_at":"2025-12-05 07:13:08","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":70498,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation explaining the putative role of MED14 in HY5 driven root meristem division under oxidative stress conditions.\u003c/strong\u003e In normal plant cells, HY5 transcriptionally regulates its own expression by positive feedback mechanism and recruits MED14 at the promoter of HY5. After activation of HY5 binds on the promoter of flavonoid biosynthesis genes. Additionally, MED14 along with HY5 also occupy the CYCD1;1 promoter to positively regulate the cell division and suppresses the activation of DDR response by binding on the promoter of NAC044 and NAC085. HY5-targeted flavonoid biosynthesis transcriptionally regulates cell division in the root meristem. Flavonoids also inhibit ROS biosynthesis and protect it from oxidative damage. In oxidative stress conditions, HY5 and MED14 regulate ROS biosynthesis which eventually activates DDR response by the transcriptional activation of NAC044 and NAC085. The binding of HY5 and MED14 reduces the suppression of the promoter of NAC044 and NAC085 and repress the expression of CYCD1;1. This repression of CYCD1;1 further arrests the cell-cycle progression leading to inhibition of the primary root elongation. Moreover, MED14 and HY5 regulate the root meristem division by modulating the ROS-triggered DDR response under oxidative stress.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/02f1b28b58284f1c81157e73.jpg"},{"id":97677987,"identity":"b3857a33-29fd-4369-ba53-01ee8d162fe5","added_by":"auto","created_at":"2025-12-08 09:55:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4520388,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/470ab6d9-c3c7-4d5b-a4ce-19e223a6fb6b.pdf"},{"id":97671494,"identity":"ee417815-1560-4fd4-8708-fc81cefcc401","added_by":"auto","created_at":"2025-12-08 09:32:39","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":167102,"visible":true,"origin":"","legend":"Primers used in this study","description":"","filename":"SupplementalTableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/5042a21d4a9af70448e6aa32.pdf"},{"id":97504110,"identity":"baab3b07-50cc-4ebd-aeae-9a75c59ab243","added_by":"auto","created_at":"2025-12-05 07:13:08","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6828154,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"Supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8222842/v1/c9a85ed934c22fa0f9d01b84.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"MED14-HY5 module orchestrates trade-off between cell-cycle activation and DNA damage response in Arabidopsis root","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRoot is an essential organ for plant survival and productivity, serving both structural and physiological functions. It is required for anchoring and acquiring nutrients and water from the soil to the whole plant \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The development of roots is a complex multinetwork process that relies on genetic, biochemical, and environmental conditions to regulate temporal and spatial control of cell division and proliferation, particularly in the root apical meristem (RAM).\u003c/p\u003e\u003cp\u003eDNA damage refers to structural alterations in the DNA molecule that compromise its integrity and function. It can arise from endogenous sources, like reactive oxygen species (ROS) generated during cellular metabolism, or through other exogenous agents, including ultraviolet (UV) and ionizing radiations (IR) such as X-ray and gamma ray, chemical mutagens, and also heavy metal toxicity and heat stress. Common types of DNA damage include base modifications, single-strand breaks (SSBs), double-strand breaks (DSBs), abasic sites, and DNA crosslinks \u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. These lesions can interfere with critical cellular processes like replication, transcription, and chromosome segregation. Plant root growth is severely affected by this type of DNA damage by activating the DNA damage response (DDR) pathways that halt the cell cycle in the root apical meristem to allow for repair. Persistent damage can trigger programmed cell death (PCD), reducing the population of dividing cells essential for root elongation \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. DNA lesions further interfere with crucial hormonal signaling pathways, especially those involving auxin and cytokinin, leading to disrupted root patterning and impaired lateral root development \u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Genome instability from unrepaired DNA further leads to abnormal cell division and differentiation. Collectively, these effects reduce root system efficiency, affecting water and nutrient uptake and overall plant growth. So, to cope with the DNA damage, plants need to activate their defence mechanism to repair the damaged DNA that helps to preserve genome stability by repairing lesions caused by UV light, ROS, and genotoxins. It regulates the cell cycle, particularly in meristematic tissues, ensuring proper cell division and development \u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. During seed germination, DDR restores DNA integrity essential for seedling establishment \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Additionally, it also enhances stress tolerance by mitigating DNA damage induced by abiotic stresses like heat and heavy metals \u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDDR response is an intricate kinase-regulated network that is conserved in both plants and animals. In animals, the critical regulator of DDR is p53 Transcription Factor (TF) \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In plants, there are functional homolog of p53, identified as NAC-type TF NAC008/SOG1. Just like p53 in animals, NAC008/SOG1 is also phosphorylated by evolutionarily conserved kinases ATR and ATM and triggers the DDR response \u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. These ATR/ATM-mediated DDRs are partially dependent on dose-dependent ROS \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Phosphorylated NAC008/SOG1 binds to the promoter of \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u003c/em\u003e to induce the expression of DNA-repairing genes such as \u003cem\u003eRAD17\u003c/em\u003e, \u003cem\u003eRAD51\u003c/em\u003e, and \u003cem\u003eBRCA1\u003c/em\u003e genes \u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Absence of NAC044 and NAC085 impedes Rep-MYB accumulation, thereby inhibiting G2/M progression \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Rep-MYBs suppress the expression of cyclins and thus hamper the activity of cyclin-associated CDKs, leading to cell-cycle arrest. Thus, Rep-MYBs play very important role as the repressors of cell-cycle genes in response to DNA damage \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAs a part of the adaptation, plants have also developed specialized antioxidant systems to protect themselves from ROS. That is why plants have intricate mechanisms for ROS homeostasis \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The basal level ROS plays an important role in cell signaling and homeostasis, excessive ROS can damage DNA, proteins, lipids, and other macromolecules, thereby causing cellular dysfunction and genome instability \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In plants, ROS production can be driven by respiratory processes, photosynthesis, pathogenic infections, and the deficiency in antioxidant defences \u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In addition, both biotic and abiotic stresses can also cause overproduction of ROS within the cells, leading to oxidative stress. Studies suggest that ROS can cause breaks in single-stranded (SSBs) and double-stranded (DSBs) DNAs, which leads to activation of the factors that are required for DNA damage responses (DDR) \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Though the number of TFs is known to regulate ROS-mediated gene expression, and different sets of TFs are involved in DDR induced due to high ROS accumulation, how these different sets of TFs coordinate with each other during normal and stress conditions for optimal levels of physiological responses remains elusive. There are reports that suggest exogenous cues can increase ROS production and can also induce the flavonol biosynthesis. In Arabidopsis, flavonols such as quercetin, kaempferol, and isorhamnetin are known to regulate root morphogenesis \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The ELONGATED HYPOCOTYL5 (HY5) transcription factor is a known regulator of flavonoid biosynthesis \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Mutation in HY5 reduces the primary root length and enhances the lateral root elongation by regulating cell division and cell elongation \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. HY5 binds to the promoter of \u003cem\u003eCHALCONE SYNTHASE\u003c/em\u003e (\u003cem\u003eCHS\u003c/em\u003e) gene to regulate flavonoid biosynthesis \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. HY5 also binds to the promoters of ROS detoxification genes to regulate ROS homeostasis under different light and nutrient stresses \u003csup\u003e\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Thus, HY5 is very critical for controlling cell division in the root through flavonoid and ROS homeostasis. However, the role of HY5 in the regulation of the cell-cycle genes through ROS and its involvement in controlling DDR-responsive genes have not been explored.\u003c/p\u003e\u003cp\u003eTranscription factors and chromatin regulators are also known to be involved in DDR in animals and plants. Studies in animals suggest that Mediator complex, the evolutionary conserved transcriptional cofactor, functions as an important link between transcription and DNA repair mechanism for maintaining genome integrity \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The Mediator complex is a large multiprotein complex that coordinates with different transcription factors and cofactors to regulate the transcription process in eukaryotes \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In plants, the Mediator complex typically consists of around 34\u0026ndash;37 subunits \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. These subunits are arranged in four modules- head, middle, and tail, making the core part and one dissociable kinase module \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Generally, the head and middle module subunits interact with RNA pol II and other components of the PIC, whereas tail module subunits interact with different TFs \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The Mediator subunits are involved in developmental processes including cell division and cell proliferation and also in the responses to environmental factors. Few Mediator subunits, such as MED2, MED5, MED16, and MED23 have been implicated in the phenylpropanoid pathway, essential for flavonoid biosynthesis \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In the analysis of protein-protein interaction network, some Mediator subunits were found to interact with the components of the DNA repair system \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Moreover, few Mediator subunit mutants are sensitive to UV radiation \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. These studies indicate an important role of Mediator in stress-triggered DNA damage response. In our study, we have focused on MED14 subunit to understand the intricacies of the involvement of Mediator in DNA damage response in Arabidopsis root.\u003c/p\u003e\u003cp\u003eWhile studying the structural topology of Arabidopsis Mediator complex, MED14 emerged as the most important subunit required for holding the complex \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. MED14 was initially discovered as RGR1 in yeast, where it functions as a repressor of glucose-regulated genes \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. In Arabidopsis, it was discovered as STRUWWELPETER/SWP \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. MED14/SWP is a nuclear protein expressed in the meristematic region, promoting cell proliferation and elongation, particularly during root and leaf development \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. MED14 also contributes to auxin-regulated processes leading to seed development, leaf formation, and lateral root formation in \u003cem\u003eOryza sativa\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Research revealed that MED14 is necessary to preserve repressive epigenetic marks and affect transcription in Arabidopsis. Additionally, MED14 regulates DNA methylation in non-CG contexts at certain RNA-directed DNA methylation targets \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Subsequently, the role of MED14 in managing heterochromatin transcription and DNA methylation has been emphasized \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Studies suggest that, any change in the epigenetic landscape of the chromatin also activates the DDR responses. In our study, whole genome transcriptome analysis of the \u003cem\u003emed14\u003c/em\u003e mutants revealed that MED14 could be involved in the transcriptional regulation of flavonoid genes, and metabolites required for chelating excess ROS from cells to maintain the ROS homeostasis in plants. All these genetic and physiological studies in \u003cem\u003emed14\u003c/em\u003e mutants suggest that MED14 could be an important integrator of ROS homeostasis, DDR responses, and cell division in plants.\u003c/p\u003e\u003cp\u003eIn this study, we focus on exploring the role of the Mediator complex in root development. While comparing the root phenotype in different Mediator mutants, we found that MED14 and MED17 are the most important subunits in all facets of root architectural development. We provide physiological and biochemical evidence confirming that the flavonoid biosynthesis pathway is disrupted due to the loss of the functional MED14. Our findings revealed that \u003cem\u003emed14\u003c/em\u003e mutants have increased ROS accumulation and trigger DNA damage-induced cell death at the root tips, even without exogenous stress. Further, we divulged that \u003cem\u003ehy5\u003c/em\u003e mutant is sensitive to DDR activation, and HY5 and MED14 together play critical roles in managing the ROS-induced DDR response necessary for maintaining the root cellular structure. Furthermore, we provided evidence that oxidative stress can inhibit \u003cem\u003eCYCD1;1\u003c/em\u003e expression by enhancing the expression of \u003cem\u003eNACs\u003c/em\u003e and influencing primary root elongation in plants under stress. In this study, we elucidated the role of MED14 in the ROS-induced DDR activation, highlighting its coordination with HY5 to sustain DNA instability and ensure the persistence of the actual RSA.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMED14 is critical for proper growth and development of the root\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the last decade, several Mediator subunits have been shown to be involved in root development \u003csup\u003e64\u003c/sup\u003e. Despite their involvement, the precise molecular mechanism by which these subunits function in the process is still in its infancy. We still do not know how these subunits coordinate for optimal growth and architecture of roots in plants. In our earlier study, MED14 and MED17 emerged as the critical subunits to hold the whole Mediator complex together \u003csup\u003e56\u003c/sup\u003e. So, we decided to work on these two subunits to understand the molecular intricacies of the regulation of root development through Mediator complex. We compared the primary root length of mutants of different Mediator subunits (\u003cem\u003emed14\u003c/em\u003e, \u003cem\u003emed16\u003c/em\u003e, \u003cem\u003emed17\u003c/em\u003e, \u003cem\u003emed25\u003c/em\u003e, \u003cem\u003ecdk8\u003c/em\u003e, and \u003cem\u003ecycC\u003c/em\u003e). We found that MED14 and MED17 showed robust effect on the overall root length, compared to other mediator mutants (\u003cstrong\u003eSupplemental Figure 1A and 1B\u003c/strong\u003e). In our previous study, we reported the requirement of MED17 for optimal root system architecture in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e \u003csup\u003e65\u003c/sup\u003e. In this study, we have focused on the importance of MED14 in the growth of the primary root. We generated RNAi lines of MED14 in Col-0 background (\u003cstrong\u003eSupplemental Figure 2A\u003c/strong\u003e) and also screened the T-DNA insertion line (SAIL_373_07) in Col-3 background (\u003cstrong\u003eSupplemental Figure 2B\u003c/strong\u003e). The details of the primers used to amplify the region for generating the \u003cem\u003eMED14\u0026nbsp;\u003c/em\u003eRNAi line are given in \u003cstrong\u003eSupplemental Table 1\u003c/strong\u003e. The transcript level of \u003cem\u003eMED14\u003c/em\u003e was reduced to 85% in the \u003cem\u003eMED14\u0026nbsp;\u003c/em\u003eRNAi line and 20% in the SAIL line of \u003cem\u003eMED14\u003c/em\u003e (hereafter referred to as \u003cem\u003emed14-1\u003c/em\u003e and \u003cem\u003emed14-2,\u003c/em\u003e respectively) (\u003cstrong\u003eSupplemental Figure 2C\u003c/strong\u003e). When we compared the seedings, we found that the root part was majorly affected in \u003cem\u003emed14-1\u003c/em\u003e and \u003cem\u003emed14-2\u003c/em\u003e seedlings as compared to their respective wild-types (\u003cstrong\u003eFigure 1A-1C, Supplemental Figure 3E-3I\u003c/strong\u003e). Surprisingly, there was not much effect in the shoot tissue (\u003cstrong\u003eSupplemental Figure 3A-3C\u003c/strong\u003e). The fresh weight of the root was significantly reduced in both \u003cem\u003emed14-1\u003c/em\u003e and \u003cem\u003emed14-2\u003c/em\u003e as compared to the wild-types (\u003cstrong\u003eSupplemental Figure 3D\u003c/strong\u003e). So, we focused our study on the root development. \u0026nbsp;We measured the length of primary roots on 5\u003csup\u003eth\u003c/sup\u003e, 8\u003csup\u003eth\u003c/sup\u003e, and 12\u003csup\u003eth\u003c/sup\u003e days and found that the root growth was slower in these lines as compared to their respective wild-types (\u003cstrong\u003eFigure 1A-1C; Supplemental Figure 3E-3I\u003c/strong\u003e). In these \u003cem\u003emed14\u003c/em\u003e mutant seedlings, even the number of lateral roots was less with wider lateral root angles than that in wild-type seedlings (\u003cstrong\u003eSupplemental Figure 4A and 4B\u003c/strong\u003e). Observing propidium iodide-stained roots under microscope revealed shorter meristematic zones in \u003cem\u003emed14\u003c/em\u003e roots (\u003cstrong\u003eFigure 1D and 1E\u003c/strong\u003e). We counted the number of cells in the meristematic, transition, and elongation zones of roots, and found that in all three zones of \u003cem\u003emed14\u003c/em\u003e seedlings had lesser number of cells as compared to their corresponding wild-types (\u003cstrong\u003eFigure 1F-1H\u003c/strong\u003e). Moreover, cells in the meristematic zone and its neighbouring transition zone were slightly smaller in \u003cem\u003emed14\u003c/em\u003e roots (\u003cstrong\u003eFigure 1I and 1J\u003c/strong\u003e). There was no difference in the size of cells in the elongation zone (\u003cstrong\u003eFigure 1I and 1K\u003c/strong\u003e). \u0026nbsp; These results indicate that cell division in the meristematic region of roots is affected. We confirmed this by staining the roots with 5-ethynyl 2’-deoxyuridine (EdU), a labeled nucleoside analog of thymine, that gets incorporated into replicating DNA \u003csup\u003e66\u003c/sup\u003e. Indeed, the number of EdU-labeled nuclei was significantly less in \u003cem\u003emed14\u003c/em\u003e roots as compared to wild-type (\u003cstrong\u003eSupplemental Figure 5A-5D, 5G\u003c/strong\u003e). Next, we checked the expression of \u003cem\u003eCYCLIN B1\u003c/em\u003e, a cell-cycle marker, in \u003cem\u003emed14-1\u003c/em\u003e and wild-type background. For this, \u003cem\u003e\u003csub\u003epro\u003c/sub\u003eCYCB1;1:GUS\u0026nbsp;\u003c/em\u003ewas expressed under the native promoter in \u003cem\u003emed14\u003c/em\u003e and wild-type seedlings. The number of GUS-stained nuclei was significantly less in the\u003cem\u003e\u0026nbsp;med14-1\u0026nbsp;\u003c/em\u003eroots (\u003cstrong\u003eSupplemental Figure 5H and 5I\u003c/strong\u003e). These results confirm that MED14 is required for cell division in the meristematic zone of primary root in Arabidopsis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we made an attempt to recover the root phenotype by overexpressing \u003cem\u003eMED14\u003c/em\u003e in the \u003cem\u003emed14-2\u003c/em\u003e mutant background. The shortened primary root phenotype and lateral root number could be rescued in the MED14 complemented lines, further confirming that the root development defect was due to the mutation in the \u003cem\u003eMED14\u003c/em\u003e gene (\u003cstrong\u003eSupplemental Figure\u003c/strong\u003e \u003cstrong\u003e6A-6D, 6G\u003c/strong\u003e). Even the reduced number of cells in the meristematic zone of \u003cem\u003emed14\u0026nbsp;\u003c/em\u003emutant roots was restored to a great extent in the roots of complementation lines (\u003cstrong\u003eSupplemental Figure 6E and 6F\u003c/strong\u003e). This suggests that an optimal growth level of MED14 expression is required for maintaining the proper growth of primary root in normal condition. Consistently, the number of EdU-incorporated nuclei in the complementation lines was same as wild type, suggesting defective cell division was rescued in these lines (\u003cstrong\u003eSupplemental Figure 5C-5F\u003c/strong\u003e). In addition to complementation lines, we also overexpressed \u003cem\u003eMED14\u003c/em\u003e in the wild-type background under its own native promoter (\u003cstrong\u003eSupplemental Figure 7A\u003c/strong\u003e). As expected, seedlings of overexpression lines had longer primary roots with longer meristematic zone as compared to wild-type seedlings (\u003cstrong\u003eSupplemental Figure 7A-7E\u003c/strong\u003e). These observations confirm that optimal level of MED14 is required for proper cell division in the root, regulating its growth and development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMED14\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;is involved in flavonoid biosynthesis to regulate primary root growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHow does MED14 regulate root growth in Arabidopsis? In order to get this answer, we compared the transcriptome of \u003cem\u003emed14\u003c/em\u003e root with that of wild-type. Through RNA-sequencing, we identified 3909 differentially expressed genes in \u003cem\u003emed14\u003c/em\u003e roots. Among the differentially expressed genes, 2079 genes were significantly downregulated and 1830 genes were significantly upregulated in the mutant root as compared to the wild-type root, suggesting that MED14 is involved in both activation and repression of genes (\u003cstrong\u003eSupplemental Figure 8A\u003c/strong\u003e). Gene ontology (GO) analysis of the downregulated genes revealed that the secondary metabolite biosynthesis pathways, including phenylpropanoid and flavonoid biosynthesis pathways were significantly affected in the \u003cem\u003emed14\u003c/em\u003e mutant roots (\u003cstrong\u003eSupplemental Figure 8B\u003c/strong\u003e). We confirmed the downregulation of some of the main genes of the flavonoid biosynthesis pathway such as \u003cem\u003eCHALCONE SYNTHASE\u003c/em\u003e (\u003cem\u003eCHS\u003c/em\u003e), \u003cem\u003eCHALCONE ISOMERASE\u003c/em\u003e (\u003cem\u003eCHI\u003c/em\u003e), \u003cem\u003eFLAVANONE 3-HYDROXYLASE\u003c/em\u003e (\u003cem\u003eF3H\u003c/em\u003e), \u003cem\u003eFLAVONOL SYNTHASE 1\u003c/em\u003e (\u003cem\u003eFLS1\u003c/em\u003e) by using qRT-PCR analysis (\u003cstrong\u003eFigure 2A, 2B; Supplemental Figure 8A\u003c/strong\u003e). Driven by the expression profile of these genes, we investigated the distribution of flavonols in the \u003cem\u003emed14\u003c/em\u003e roots using DPBA (2-aminoethyl diphenylboric acid), a flavonol-specific staining dye \u003csup\u003e67\u003c/sup\u003e, and compared that with their respective wild-type roots. Indeed, there was less flavonol content in the roots of \u003cem\u003emed14\u003c/em\u003e mutants (\u003cstrong\u003eFigure 2C and 3D\u003c/strong\u003e). This was further confirmed by high-performance thin-layer chromatography (HPTLC). The HPTLC profile showed lower accumulation of quercetin (orange) and kaempferol (green) in the \u003cem\u003emed14\u0026nbsp;\u003c/em\u003eroots as compared to the roots of wild-type seedlings (\u003cstrong\u003eFigure 2E\u003c/strong\u003e). All these results suggest that MED14 is involved in the regulation of flavonoid biosynthesis pathways in the roots of Arabidopsis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMED14 coordinates with HY5 to regulate the root growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHY5 is a well-known regulator of flavonoid-driven root growth in plants \u003csup\u003e42,68,69\u003c/sup\u003e. We confirmed the downregulation of \u003cem\u003eHY5\u003c/em\u003e in the roots of \u003cem\u003emed14\u003c/em\u003e mutants by qRT-PCR analysis (\u003cstrong\u003eFigure 3A\u003c/strong\u003e).\u0026nbsp;To further confirm the regulation of \u003cem\u003eHY5\u003c/em\u003e transcription by MED14, we expressed HY5:GFP under its native promoter in wild-type and \u003cem\u003emed14-1\u003c/em\u003e mutant and checked the expression level. Indeed, the expression level of \u003cem\u003eHY5:GFP\u003c/em\u003e driven by \u003cem\u003eHY5\u003c/em\u003e promoter was significantly reduced in \u003cem\u003emed14\u003c/em\u003e mutant (\u003cstrong\u003eSupplemental Figure 9A-9C)\u003c/strong\u003e. This confirms that the activity of \u003cem\u003eHY5\u003c/em\u003e promoter is dependent on MED14 to some extent. To date, there is no report suggesting binding of Mediator subunits directly to the DNA; instead, transcription factors recruit the Mediator complex to their cognate binding sites on the DNA\u0026nbsp;\u003csup\u003e70\u003c/sup\u003e. There is one report that indicates feedback regulation of HY5\u0026nbsp;\u003csup\u003e71\u003c/sup\u003e. However, direct binding of HY5 to its own promoter has not been reported. So, we scanned the promoter of \u003cem\u003eHY5\u003c/em\u003e and found the probable HY5 binding sites in it. Indeed, HY5 was found to bind to its own promoter by ChIP analysis (\u003cstrong\u003eFigure 3B and 3C\u003c/strong\u003e). Interestingly, enrichment of HY5 on its promoter was substantially decreased in \u003cem\u003emed14-1\u003c/em\u003e lines (\u003cstrong\u003eFigure 3B and 3C\u003c/strong\u003e). To elucidate the presence of MED14, we checked the occupancy of MED14 on the promoter of \u003cem\u003eHY5\u003c/em\u003e, and indeed there was enrichment of MED14 on this promoter, which was diminished in the absence of HY5 (\u003cstrong\u003eFigure 3D\u003c/strong\u003e). These results suggest that MED14 regulates the expression of \u003cem\u003eHY5\u003c/em\u003e by modulating the binding of HY5 on its own promoter (\u003cstrong\u003eFigure 3B-3D\u003c/strong\u003e). Since there is less expression of \u003cem\u003eHY5\u003c/em\u003e in \u003cem\u003emed14\u003c/em\u003e mutant, there is low abundance of HY5 on its promoter. This suggests that MED14 is required for feedback loop of \u003cem\u003eHY5\u003c/em\u003e expression. Next, we explored whether there is direct interaction between MED14 and HY5. First, we predicted the probable binding sites of MED14 (i.e., Δ4 and Δ6) with full-length HY5 by molecular docking (\u003cstrong\u003eSupplemental Figure 10A and 10B\u003c/strong\u003e), and then confirmed this binding by BiFC. In the BiFC experiment, we could not find any interaction between full-length MED14 and HY5. It has been observed earlier that in ex-vivo experiments, full-length of some Mediator subunits are not able to interact with other proteins, probably due to regulatory regions involved in controlling their protein-protein interactions. So, we cloned fragments of MED14 and checked their interactions with full-length HY5. The regions from 531-769AA residues of MED14 (Δ4) and 1170-1704AA residues of MED14 (Δ6) were found to interact with HY5 (\u003cstrong\u003eSupplemental Figure 11A-11P).\u0026nbsp;\u003c/strong\u003eThus, these results suggest that HY5 requires MED14 for its expression. Indeed, we observed a significant reduction in the transcript level of \u003cem\u003eHY5\u003c/em\u003e in both \u003cem\u003emed14\u003c/em\u003e mutants (\u003cstrong\u003eFigure 3A\u003c/strong\u003e), further supporting the role of MED14 as a transcriptional activator of \u003cem\u003eHY5\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we assessed the root phenotype of \u003cem\u003ehy5\u003c/em\u003e mutant and compared it with \u003cem\u003emed14\u003c/em\u003e. Indeed, just like \u003cem\u003emed14\u003c/em\u003e, \u003cem\u003ehy5\u003c/em\u003e seedlings also showed shorter primary roots (\u003cstrong\u003eFigure 3E and 3F\u003c/strong\u003e). In order to confirm the functioning of MED14 and HY5 in the same pathway, we generated \u003cem\u003emed14-1\u003c/em\u003e \u003cem\u003ehy5\u003c/em\u003e double mutant line. In the double mutant line, the primary root was as short as \u003cem\u003ehy5\u003c/em\u003e or \u003cem\u003emed14\u003c/em\u003e mutant seedlings (\u003cstrong\u003eFigure 3E and 3F\u003c/strong\u003e). We looked at the cellular organization in the roots of \u003cem\u003emed14-1\u003c/em\u003e, \u003cem\u003ehy5,\u003c/em\u003e and \u003cem\u003emed14-1 hy5\u003c/em\u003e seedlings. Just like \u003cem\u003emed14\u003c/em\u003e mutant, the length of meristematic zone and the number of cells in the meristematic, transition, and elongation zones were found to be reduced in \u003cem\u003ehy5,\u003c/em\u003e and \u003cem\u003emed14-1\u003c/em\u003e \u003cem\u003ehy5\u003c/em\u003e roots as compared to Col-0 roots (\u003cstrong\u003eFigure 3G, H, Supplemental Figure 12A-12C\u003c/strong\u003e). There was no additional effect in the double mutant (\u003cstrong\u003eFigure 3E-3H\u003c/strong\u003e). Also, the length of cells in the transition and elongation zone in both \u003cem\u003ehy5\u003c/em\u003e and \u003cem\u003emed14-1 hy5\u003c/em\u003e roots was similar to \u003cem\u003emed14\u003c/em\u003e roots (\u003cstrong\u003eSupplemental Figure 13A-13C\u003c/strong\u003e). Like \u003cem\u003emed14-1\u003c/em\u003e mutant, the EdU staining of roots of \u003cem\u003ehy5\u003c/em\u003e and \u003cem\u003emed14-1\u003c/em\u003e \u003cem\u003ehy5\u003c/em\u003e seedlings revealed compromised cell division as compared to Col-0 (\u003cstrong\u003eFigure 3I-3P, Supplemental Figure 13D\u003c/strong\u003e). Thus, the overall phenotype of \u003cem\u003ehy5\u003c/em\u003e mutant root is similar to that of \u003cem\u003emed14\u003c/em\u003e root, and there is no additional effect in the double mutant of \u003cem\u003emed14\u0026nbsp;\u003c/em\u003eand \u003cem\u003ehy5\u003c/em\u003e together. These observations suggest that in the process of root development, MED14 and HY5 work together in the same pathway.\u003c/p\u003e\n\u003cp\u003eTo further dissect the possible regulatory pathway that has been affected by the functional loss of MED14 and HY5, we compared the transcriptome data of the \u003cem\u003ehy5\u003c/em\u003e seedlings published elsewhere \u003csup\u003e72\u003c/sup\u003e with\u0026nbsp;our \u003cem\u003emed14\u003c/em\u003e transcriptome. A total of 301 genes were found to be downregulated in both \u003cem\u003ehy5\u0026nbsp;\u003c/em\u003eand \u003cem\u003emed14\u003c/em\u003e mutants (\u003cstrong\u003eSupplemental Figure 14A\u003c/strong\u003e). In these downregulated genes, secondary metabolic pathways were highly represented, especially phenylpropanoid biosynthesis and flavonoid biosynthesis pathway genes such as \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eCHI\u003c/em\u003e, \u003cem\u003eCHIL\u003c/em\u003e,\u003cem\u003e\u0026nbsp;F3H\u003c/em\u003e, \u003cem\u003eFLS1\u003c/em\u003e, \u003cem\u003eTT7\u003c/em\u003e, and \u003cem\u003eCoAOMTA1\u003c/em\u003e (\u003cstrong\u003eSupplemental Figure 8A and 14B\u003c/strong\u003e). We confirmed this by qRT-PCR analysis of few important genes, namely \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eCHI\u003c/em\u003e, \u003cem\u003eF3H,\u003c/em\u003e and \u003cem\u003eFLS1\u003c/em\u003e (\u003cstrong\u003eFigure 2A, 2B, 4A, 4B\u003c/strong\u003e). Driven by such gene expression profile, we checked the effect of the flavonoid (quercetin) on root growth. Interestingly, exogenous application of quercetin increased the primary root length in all three mutants \u003cem\u003ehy5\u003c/em\u003e, \u003cem\u003emed14,\u0026nbsp;\u003c/em\u003eand the double mutant (\u003cstrong\u003eFigure 4C-4F\u003c/strong\u003e). There was no visible effect of 10 µM of quercetin on wild-type roots. Next, to understand the alteration in the cellular organization in roots, meristem cell numbers were counted in the PI-stained roots of Col-0, \u003cem\u003emed14-1\u003c/em\u003e, and \u003cem\u003ehy5\u003c/em\u003e seedlings. In response to quercetin treatment, there was a significant increase in the number of cells in the meristematic zone (\u003cstrong\u003eFigure 4G and 4H\u003c/strong\u003e). However, there was no change in cell size in response to quercetin (\u003cstrong\u003eFigure 4I and 4J\u003c/strong\u003e). These results suggest that MED14 and HY5 are both required for the flavonoid-driven cell division for the growth of primary root.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMED14 balances the ROS homeostasis to facilitate the primary root growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlavonoids play a crucial role in the root development as they help to regulate ROS homeostasis in the meristematic region \u003csup\u003e73,74\u003c/sup\u003e.\u0026nbsp;In plants, a baseline level of ROS is essential for various signaling pathways required for proper growth. However, excessive ROS production can cause cellular damage, which can be detrimental to root growth and development. Since flavonoid level was affected in\u003cem\u003e\u0026nbsp;med14\u0026nbsp;\u003c/em\u003emutants, we checked ROS levels in them by staining the roots with H\u003csub\u003e2\u003c/sub\u003eDCFDA, a common reagent used for investigating the production of ROS in living cells. The higher intensity of fluorescence in \u003cem\u003emed14\u003c/em\u003e roots revealed higher accumulation of ROS in them as compared to their respective wild-types (\u003cstrong\u003eFigure 5A-5E\u003c/strong\u003e). In the roots of the complementation line (\u003cem\u003e35S:MED14-HA/med14\u003c/em\u003e), the fluorescence intensity was found to be reduced to the level of wild-type, validating the requirement of MED14 for ROS homeostasis (\u003cstrong\u003eSupplemental Figure 15A-15D\u003c/strong\u003e). Developmental events in root, especially cell division in the meristematic zone and transition from elongation to differentiation zone are regulated by ROS gradient mainly of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003cstrong\u003e\u003csup\u003e.\u003c/sup\u003e\u003c/strong\u003e\u003csup\u003e-\u003c/sup\u003e \u003csup\u003e75\u003c/sup\u003e. So, we made an attempt to evaluate the distribution of these specific ROS species, using NBT (Nitroblue tetrazolium) for superoxides (O\u003csub\u003e2\u003c/sub\u003e\u003cstrong\u003e\u003csup\u003e.\u003c/sup\u003e\u003c/strong\u003e\u003csup\u003e-\u003c/sup\u003e) and DAB (3,3'-Diaminobenzidine) for hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). The\u003cem\u003e\u0026nbsp;med14\u0026nbsp;\u003c/em\u003emutants showed higher accumulation of both the O\u003csub\u003e2\u003c/sub\u003e\u003cstrong\u003e\u003csup\u003e.\u003c/sup\u003e\u003c/strong\u003e\u003csup\u003e-\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the roots compared to the wild-type (\u003cstrong\u003eFigure 5F-5M\u003c/strong\u003e). To see if the increase in ROS affects the primary root growth, we exogenously applied different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to Col-0. We found an overall reduction in the primary root growth in wild-type seedlings under increasing concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (\u003cstrong\u003eSupplemental Figure 16A and 16B\u003c/strong\u003e). These results indicate that excess ROS can actually inhibit primary root growth. It suggests that the increased level of ROS could cause reduced growth of primary root in \u003cem\u003emed14\u003c/em\u003e mutants. To further confirm that, we designed a chemical complementation assay to chelate the availability of ROS in the seedlings. For that, we treated the 4-days-old seedlings with potassium iodide (KI), a well-known ROS scavenger\u0026nbsp;\u003csup\u003e76\u003c/sup\u003e\u003cstrong\u003e.\u003c/strong\u003e There was no effect of KI on the growth of primary roots of wild-type seedlings. However, there was significant increase in the length of \u003cem\u003emed14\u0026nbsp;\u003c/em\u003eroots (\u003cstrong\u003eFigure 5N and 5O\u003c/strong\u003e). These observations suggest that increased ROS accumulation is one of the major factors for defects in development in \u003cem\u003emed14\u0026nbsp;\u003c/em\u003emutants. Thus, MED14 is important for ROS homeostasis in the root, which is critical for its proper growth and development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMED14 and HY5 are involved in DNA damage response to control primary root elongation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExcess ROS production is a common phenomenon in stress response \u003csup\u003e35,77,78\u003c/sup\u003e. The excess ROS production causes damage to DNA, which in turn activates DDR response. To investigate the impact of excessive ROS production on DDR response, we assessed the expression level of the DDR pathway genes in the \u003cem\u003emed14\u003c/em\u003e by qRT-PCR. We found that the transcript levels of\u003cem\u003eGAMMA-IRRADIATION AND MITOMYCIN C INDUCED 1\u003c/em\u003e/\u003cem\u003eGMI1\u003c/em\u003e, \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e and \u003cem\u003ePoly (ADP-ribose) polymerase\u0026nbsp;\u003c/em\u003e(\u003cem\u003ePARP2\u003c/em\u003e) were increased in both \u003cem\u003emed14\u003c/em\u003e mutants, suggesting the activated DDR response in them (\u003cstrong\u003eFigure 6A and 6B\u003c/strong\u003e). So, to further clarify the potential role of MED14 in DDR, we checked the effect of different concentrations of HU (hydroxyurea), a well-known DNA-damaging agent, which blocks DNA replication during the S phase of the cell-cycle\u0026nbsp;\u003csup\u003e79,80\u003c/sup\u003e, on the root growth in \u003cem\u003emed14\u003c/em\u003e and wild-type seedlings. The HU treatment inhibited the growth of primary root in these seedlings. However, the percentage of root growth inhibition was significantly more in \u003cem\u003emed14\u003c/em\u003e roots as compared to the wild-type (\u003cstrong\u003eFigure 6C-6E\u003c/strong\u003e). Next, we assessed cell death in the roots of HU-treated seedlings by PI staining, which is used as a marker for cell death regions\u0026nbsp;\u003csup\u003e81\u003c/sup\u003e. We found that there were more PI-stained regions in the\u003cem\u003e\u0026nbsp;med14\u0026nbsp;\u003c/em\u003eroots as compared to the wild-type, revealing higher sensitivity of \u003cem\u003emed14\u003c/em\u003e mutants to the DNA-damaging agent (\u003cstrong\u003eFigure 6F).\u0026nbsp;\u003c/strong\u003eThis sensitivity could be reversed to wild-type level in the complementation lines of\u0026nbsp; \u003cem\u003e35S:MED14::HA\u003c/em\u003e/\u003cem\u003emed14\u003c/em\u003e (\u003cstrong\u003eSupplemental Figure 17A-17C\u003c/strong\u003e). All these results suggest that MED14 is required for optimal response to DNA damage.\u003c/p\u003e\n\u003cp\u003eHY5 is one of the main regulators of ROS production in root \u003csup\u003e46\u003c/sup\u003e, and there is significant overlap in the genes regulated by both MED14 and HY5. So, we\u0026nbsp;decided to conduct a thorough study to understand the relationship between the two in ROS-mediated root development. Therefore, the expression of ROS biosynthesis genes was compared from the transcriptomic data of\u003cem\u003e\u0026nbsp;hy5\u0026nbsp;\u003c/em\u003e\u003csup\u003e72\u003c/sup\u003e and \u003cem\u003emed14\u003c/em\u003e mutant roots (\u003cstrong\u003eSupplemental Figure 18\u003c/strong\u003e). A significant number of ROS biosynthesis genes were found to be affected in both mutants as compared to wild-type (\u003cstrong\u003eSupplemental Figure 18\u003c/strong\u003e). As discussed earlier, increased ROS accumulation in \u003cem\u003emed14\u003c/em\u003e root activates the DDR. So, we proceeded to see if there is functional overlap between MED14 and HY5 in DDR responses. We checked the ROS accumulation in roots of single mutants of \u003cem\u003emed14-1\u0026nbsp;\u003c/em\u003eand \u003cem\u003ehy5\u003c/em\u003e, and also in the double mutant of \u003cem\u003emed14-1 hy5\u003c/em\u003e by H\u003csub\u003e2\u003c/sub\u003eDCFDA staining. Just like in \u003cem\u003emed14\u003c/em\u003e, there was increased accumulation of ROS in \u003cem\u003ehy5\u0026nbsp;\u003c/em\u003eand \u003cem\u003emed14-1 hy5\u003c/em\u003e roots (\u003cstrong\u003eFigure 7A-7D, 7F).\u003c/strong\u003e In the roots of HY5 overexpression line, the ROS accumulation was reduced compared to wild-type (\u003cstrong\u003eFigure 7E and 7F\u003c/strong\u003e). Next, we checked the effect of genotoxic stress on the roots of these lines. As expected, the roots of single and double mutants were found to be more sensitive to exogenous HU (\u003cstrong\u003eFigure 7G and 7H\u003c/strong\u003e), whereas the HY5 overexpression line exhibited more tolerance to the genotoxic stress as compared to Col-0 (\u003cstrong\u003eFigure 7I and 7J\u003c/strong\u003e). Moreover, just like in \u003cem\u003emed14\u003c/em\u003e, treatment of seedlings with ROS scavenger KI could increase root length in \u003cem\u003ehy5\u003c/em\u003e (\u003cstrong\u003eFigure 7G and 7H\u003c/strong\u003e). This root sensitivity was further confirmed by cell death assay in the presence of exogenous HU. All \u003cem\u003emed14-1\u003c/em\u003e, \u003cem\u003ehy5\u003c/em\u003e and \u003cem\u003emed14-1 hy5\u003c/em\u003e mutants accumulated more cellular damage as compared to Col-0, while in the HY5 overexpression line, the cellular damage was significantly less as compared to Col-0 (\u003cstrong\u003eFigure 7K-7T\u003c/strong\u003e). All these results confirmed that there is an overlap between \u003cem\u003ehy5\u003c/em\u003e and \u003cem\u003emed14\u0026nbsp;\u003c/em\u003emutants in terms of root phenotype in response to genotoxic stress. Both MED14 and HY5 are involved in the DNA damage response triggered by higher ROS accumulation in the primary root.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHY5 and MED14 regulate the transcript level of \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u003c/em\u003e and modulate root elongation under genotoxic stress\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHY5 is known to regulate ROS production and primary root phenotype under stress condition. However, its role in DDR remains unexplored. Therefore, we decided to investigate how MED14 and HY5 work together to regulate ROS-triggered DNA damage response in the root. It is already known that the DDR requires expression of \u003cem\u003eATAXIA-TELANGIECTASIA MUTATED\u003c/em\u003e (\u003cem\u003eATM\u003c/em\u003e) and \u003cem\u003eATAXIA TELANGIECTASIA-MUTATED AND RAD3-RELATED\u003c/em\u003e (\u003cem\u003eATR)\u003c/em\u003e triggered by single and double-stranded DNA breaks caused by ROS \u003csup\u003e82\u003c/sup\u003e. In Arabidopsis, DNA damage triggers the activation of the ATM/ATR kinases, leading to phosphorylation and activation of the plant-specific transcription factor SOG1. SOG1 directly upregulates the transcription of \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eNAC103\u003c/em\u003e, which mediate downstream transcriptional responses, including the repression of G2/M-specific genes, thereby enforcing cell-cycle arrest during the DNA damage response \u003csup\u003e30,83\u003c/sup\u003e. So, we checked the expression of these genes in the roots of the seedlings treated with HU. We found the significant upregulation of \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e in wild-type seedlings (\u003cstrong\u003eSupplemental Figure 19A\u003c/strong\u003e). Interestingly, there was no such upregulation was observed upon KI treatment, indicating that HU treatment can activate ROS-mediated DDR response in plants (\u003cstrong\u003eSupplemental Figure 19A)\u003c/strong\u003e. Next, we checked the expression of these DDR-responsive genes (\u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e) in \u003cem\u003emed14\u0026nbsp;\u003c/em\u003eroots upon\u0026nbsp;KI treatment. Transcript levels of \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e were comparatively reduced upon KI treatment (\u003cstrong\u003eSupplemental Figure 19B\u003c/strong\u003e). However, the transcript levels of \u003cem\u003eATR, ATM,\u003c/em\u003e and \u003cem\u003eSOG1\u0026nbsp;\u003c/em\u003ein wild-type and\u003cem\u003e\u0026nbsp;med14\u0026nbsp;\u003c/em\u003emutantremained unchanged (\u003cstrong\u003eSupplemental Figure 19A-19C\u003c/strong\u003e). Similarly, upregulation of \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e genes was also observed in the \u003cem\u003ehy5\u003c/em\u003e root in normal condition (\u003cstrong\u003eSupplemental Figure 19D\u003c/strong\u003e), which was further reduced after KI treatment (\u003cstrong\u003eSupplemental Figure 19E\u003c/strong\u003e). These results confirm that \u003cem\u003ehy5\u003c/em\u003e and \u003cem\u003emed14\u003c/em\u003e mutants have elevated levels of ROS leading to the activation of DDR responsive pathway, suggesting that HY5 and MED14 are required for controlling this response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlavonol is required for the suppression of ROS-triggered DDR response in plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlants produce specialized secondary metabolites to neutralize the toxic effects of excess ROS. As discussed earlier, quercetin level in \u003cem\u003emed14\u003c/em\u003e seedlings is less than wild-type. So, we designed an experiment to see the effect of genotoxic agent HU in the presence and absence of quercetin and KI on the expression of DDR genes. Indeed, quercetin worked like a ROS scavenger (\u003cstrong\u003eFigure 4C and 4E)\u003c/strong\u003e. In wild-type roots, there was significant elevation in the expression of \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e in response to HU treatment (\u003cstrong\u003eFigure 8A-8C\u003c/strong\u003e), and in the presence of quercetin, just like KI, there was no such upregulation of these genes (\u003cstrong\u003eFigure 8A-8C\u003c/strong\u003e). In \u003cem\u003emed14\u0026nbsp;\u003c/em\u003eroots, quercetin level is already low (\u003cstrong\u003eFigure 2E\u003c/strong\u003e). So, the expression of \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e is already high (\u003cstrong\u003eFigure 8D\u003c/strong\u003e). When the seedlings are treated with exogenous quercetin, the expression level of NAC genes were significantly reduced to wild-type level (\u003cstrong\u003eFigure 8D\u003c/strong\u003e). Same effect was also observed in \u003cem\u003ehy5\u003c/em\u003e roots (\u003cstrong\u003eFigure 8E\u003c/strong\u003e). At cellular level also, the cell damage caused by HU could be protected by the exogenous application of quercetin and KI. Just like KI, the application of quercetin could effectively reduce the extent of HU-caused cell damage in Col-0 (\u003cstrong\u003eFigure 8F\u003c/strong\u003e). \u0026nbsp;In \u003cem\u003emed14\u0026nbsp;\u003c/em\u003eand \u003cem\u003ehy5\u003c/em\u003e mutants, quercetin reduced the HU-induced cell death area (\u003cstrong\u003eFigure 8G\u003c/strong\u003e). These findings confirmed that quercetin acts as a natural scavenger of ROS, helping protect plants from oxidative damage. Additionally, both MED14 and HY5 are required for quercetin to effectively safeguard the DNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMED14 is required for NAC044 and NAC085-dependent restriction of cell division in root tip\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsidering the importance of NAC044, NAC085, and NAC103 in DDR response under genotoxic stress and their notable upregulation in both \u003cem\u003emed14\u0026nbsp;\u003c/em\u003eand \u003cem\u003ehy5\u003c/em\u003e mutants, we decided to study genetic relationship between them in Arabidopsis. NAC044, NAC085, and NAC103 are involved in inhibiting the G2/M stage of cell-cycle in response to stress \u003csup\u003e28,29,84\u003c/sup\u003e. Therefore, \u003cem\u003enac044-1\u003c/em\u003e, \u003cem\u003enac085-2\u003c/em\u003e, and \u003cem\u003enac103\u0026nbsp;\u003c/em\u003emutants are unresponsive to DDR signaling \u003csup\u003e28,29,84\u003c/sup\u003e. To check if the smaller root in \u003cem\u003emed14\u003c/em\u003e seedlings was due to higher expression of these NACs, we generated the double mutant lines of\u0026nbsp;\u003cem\u003emed14-1 nac044-1, hy5 nac044-1,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;med14-1 nac085-2, hy5 nac085-2,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;nac044-1 nac085-2\u003c/em\u003e. Indeed, the short primary root length phenotype of \u003cem\u003emed14-1 and hy5\u0026nbsp;\u003c/em\u003ewas observed to be reversed in the double mutants and is comparable to wild-type seedling (\u003cstrong\u003eSupplemental Figure 20A and 20B)\u003c/strong\u003e. This result confirms that the defect in the growth of primary root in \u003cem\u003emed14\u003c/em\u003e and \u003cem\u003ehy5\u003c/em\u003e mutants is due to ROS-triggered upregulation of \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u003c/em\u003e. Next, we observed the sensitivity of the roots in these mutant lines under genotoxic stress, and measured the cell death for the shortening of roots. For this, we performed cell death assay in the root under genotoxic stress. The \u003cem\u003emed14\u003c/em\u003e and \u003cem\u003ehy5\u003c/em\u003e roots exhibited increased cellular damage (\u003cstrong\u003eSupplemental Figure 21A-21T\u003c/strong\u003e). There was no such effect seen in the roots of \u003cem\u003enac044-1\u003c/em\u003e, \u003cem\u003enac085-2\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;nac044-1 nac085-2\u0026nbsp;\u003c/em\u003edoublemutants after HU treatment. However, there was some cell death observed in Col-0, \u003cem\u003emed14-1\u003c/em\u003e \u003cem\u003enac044-1, hy5 nac044-1,\u0026nbsp;\u003c/em\u003eand \u003cem\u003emed14-1\u003c/em\u003e \u003cem\u003enac085-2, hy5 nac085-2\u0026nbsp;\u003c/em\u003edouble mutants, but less than that in \u003cem\u003emed14\u003c/em\u003e and \u003cem\u003ehy5\u003c/em\u003e mutants (\u003cstrong\u003eSupplemental Figure 21A-21T\u003c/strong\u003e). Growth and elongation of primary roots are driven by cell division in the meristematic zone. So, we looked at the cell division in the roots of these mutants by EdU labeling followed by DAPI staining. As expected, there were more dividing cells in \u003cem\u003enac044-1\u003c/em\u003e and \u003cem\u003enac085-2\u003c/em\u003e than wild-type. Moreover, the reduced cell division phenotype in \u003cem\u003emed14-1\u003c/em\u003e root could be restored to wild-type level in \u003cem\u003emed14-1\u003c/em\u003e \u003cem\u003enac044-1\u003c/em\u003e and \u003cem\u003emed14-1\u003c/em\u003e \u003cem\u003enac085-2\u003c/em\u003e double mutants (\u003cstrong\u003eSupplemental Figure 20C-20I).\u0026nbsp;\u003c/strong\u003eThese results suggest that the defect in root growth in \u003cem\u003emed14\u003c/em\u003e and \u003cem\u003ehy5\u003c/em\u003e are due to increased expression of \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085,\u003c/em\u003e leading to hampered cell division.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMED14 and HY5 promote activation of DDR response under oxidative stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHY5 is primarily known as an activator, as a positive regulator in light-dependent photomorphogenesis in plants \u003csup\u003e85,86\u003c/sup\u003e. However, HY5 also functions as a repressor in certain contexts \u003csup\u003e46,87,88\u003c/sup\u003e. MED14 was first discovered as a repressor in yeast and is also known to repress many genes in humans \u003csup\u003e57,58\u003c/sup\u003e. Under normal condition, DDR genes, especially \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e, remain repressed but are upregulated under stress conditions. Here, we found that \u003cem\u003ehy5\u003c/em\u003e and \u003cem\u003emed14\u003c/em\u003e seedlings have activated DDR under normal conditions and are more sensitive to oxidative stress, which also activates DDR. The expression of \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085,\u003c/em\u003e and \u003cem\u003eNAC103\u003c/em\u003e was higher even in non-stressed condition as compared to wild-type plants, suggesting that these \u003cem\u003eNAC\u003c/em\u003e genes are de-repressed in \u003cem\u003ehy5\u003c/em\u003e and \u003cem\u003emed14\u003c/em\u003e mutants under normal condition. These findings indicate that MED14 and HY5 negatively regulate the transcription of \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e in normal condition by either binding to their promoter, and this suppression is removed upon genotoxic stress. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo comprehend the molecular mechanism of DDR regulation, we prima facie sought the binding of HY5 and MED14 on the promoter sites of DDR-responsive \u003cem\u003eNAC\u0026nbsp;\u003c/em\u003egenes. For this, we scanned the promoter regions of \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u0026nbsp;\u003c/em\u003eand found HY5 binding elements in them. Indeed, through ChIP analysis, we found binding of HY5 to the promoters of \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u0026nbsp;\u003c/em\u003egenes(\u003cstrong\u003eFigure 9A and 9B\u003c/strong\u003e). However, the enrichment of HY5 in \u003cem\u003emed14-1\u003c/em\u003e was found to be reduced as compared to Col-0 background, suggesting that MED14 helps in the binding of HY5 on \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u0026nbsp;\u003c/em\u003epromoters in normal condition (\u003cstrong\u003eFigure 9A and 9B\u003c/strong\u003e). Further to verify the coordination between HY5 and MED14, we checked the occupancy of MED14 on \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u0026nbsp;\u003c/em\u003epromoters in Col-0 and \u003cem\u003ehy5\u0026nbsp;\u003c/em\u003ebackgrounds. In accordance, we found MED14 was also able to occupy the same regions of \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u003c/em\u003e, where the HY5 binds, and this occupancy was significantly reduced in the \u003cem\u003ehy5\u003c/em\u003e mutant background (\u003cstrong\u003eFigure 9C and 9D\u003c/strong\u003e). All these results suggest that HY5 recruits MED14 to the promoters of the DDR genes, \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085,\u0026nbsp;\u003c/em\u003ewith the help of HY5 (\u003cstrong\u003eFigure 9A-9D\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further elucidate the regulation of NAC genes through MED14 and HY5, we examined the expression of \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e genes in the overexpression line of HY5 and compared them with wild-type under genotoxic stress. Genotoxic stress enhanced the transcript level of \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085,\u003c/em\u003e and \u003cem\u003eNAC103\u003c/em\u003e in the wild-type roots, but not that much in the overexpression line (\u003cstrong\u003eSupplemental Figure 22A-22C\u003c/strong\u003e). Next, we checked the binding of HY5 and MED14 on the promoters of DDR genes under genotoxic stress. Interestingly, binding of HY5 to these promoterswas significantly reduced after HU treatment (\u003cstrong\u003eFigure 9E and 9F\u003c/strong\u003e). Similar pattern was also observed in the occupancy of MED14 on the promoters of \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u003c/em\u003e (\u003cstrong\u003eFigure 9G and 9H\u003c/strong\u003e). These results suggest that HY5 and MED14 play a crucial role in repressing the transcription of \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u003c/em\u003e genes under normal conditions, and this repression is lifted under genotoxic stress as the occupancy of HY5 and MED14 is reduced on the promoters of these genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMED14 and HY5 positively regulate cell division\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell-cycle is essential for sustaining cell division in the root apical meristem, which drives continuous root growth in Arabidopsis \u003csup\u003e89,90\u003c/sup\u003e. Cyclins are essential regulators of the cell-cycle, controlling root meristem growth by promoting cell division. Amongst them, CYCDs drive the G1/S transition \u003csup\u003e91,92\u003c/sup\u003e, while CYCA and CYCB facilitate the G2/M transition, ensuring proper progression through the cell-cycle \u003csup\u003e93–96\u003c/sup\u003e. Reduced number of meristematic cells in the \u003cem\u003emed14\u0026nbsp;\u003c/em\u003eand \u003cem\u003ehy5\u003c/em\u003e mutants (\u003cstrong\u003eFigure 3G; Supplemental Figure 12A\u003c/strong\u003e) indicates disturbed regulation of cell-cycle genes. To investigate this, we analyzed the transcriptomic data from \u003cem\u003emed14\u003c/em\u003e and \u003cem\u003ehy5\u003c/em\u003e roots and found \u003cem\u003eCYCD1;1\u003c/em\u003e, \u003cem\u003eCYCD3;1\u003c/em\u003e, \u003cem\u003eCYCD3;2\u003c/em\u003e, and \u003cem\u003eKRP2\u003c/em\u003e were commonly downregulated in both mutants (\u003cstrong\u003eFigure 10A\u003c/strong\u003e). We focused on \u003cem\u003eCYCD1;1\u003c/em\u003e as it was the most downregulated one (\u003cstrong\u003eFigure 10B\u003c/strong\u003e). To elucidate the regulatory mechanisms, we assessed the occupancy of HY5 and MED14 on the \u003cem\u003eCYCD1;1\u0026nbsp;\u003c/em\u003epromoter region. ChIP-qPCR demonstrated significant enrichment of both HY5 and MED14 at the \u003cem\u003eCYCD1;1\u0026nbsp;\u003c/em\u003epromoter, indicating their role in direct transcriptional regulation (\u003cstrong\u003eFigure 10C and 10D\u003c/strong\u003e). To gain deeper insight, we further conducted ChIP-qPCR to examine occupancy of MED14 at the \u003cem\u003eCYCD1;1\u0026nbsp;\u003c/em\u003epromoter in the absence of HY5 (\u003cstrong\u003eFigure 10E\u003c/strong\u003e). The results showed a significant decrease in the occupancy of MED14 in the \u003cem\u003ehy5\u003c/em\u003e mutant (\u003cstrong\u003eFigure 10E\u003c/strong\u003e). All these data reveal that MED14 and HY5 positively regulates \u003cem\u003eCYCD1;1\u0026nbsp;\u003c/em\u003etranscription in HY5-dependent manner.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxidative stress negatively regulates \u003cem\u003eCYCD1;1\u003c/em\u003e expression by MED14-HY5 driven NAC044 and NAC085\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeveral studies have demonstrated that an excess level of ROS inhibits primary root growth by affecting the cell-cycle progression in Arabidopsis \u003csup\u003e97,98\u003c/sup\u003e. We observed that \u003cem\u003emed14\u003c/em\u003e and \u003cem\u003ehy5\u003c/em\u003e mutants have more accumulation of ROS and reduced expression of cell-cycle genes, including \u003cem\u003eCYCD1;1\u003c/em\u003e. So, to correlate whether oxidative stress affects the cell-cycle progression, we checked the transcript level of \u003cem\u003eCYCD1;1\u0026nbsp;\u003c/em\u003eunder genotoxic stress in wild-type. Genotoxic stress significantly reduced the expression of the \u003cem\u003eCYCD1;1\u003c/em\u003e (\u003cstrong\u003eFigure 10F\u003c/strong\u003e), which might be responsible for the cell-cycle arrest. Since MED14 and HY5 are the positive regulators of \u003cem\u003eCYCD1;1\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFigure 10A-10E\u003c/strong\u003e), we checked the effect of HU on the occupancy of HY5 and MED14 on the promoter of \u003cem\u003eCYCD1;1\u003c/em\u003e. Interestingly, occupancy of both HY5 and MED14 was significantly reduced upon genotoxicity (\u003cstrong\u003eFigure 10G and 10H\u003c/strong\u003e). This result suggests that genotoxic stress removes the activators like HY5 and MED14 from the promoter of \u003cem\u003eCYCD1;1,\u003c/em\u003e and so there is a decrease in its expression level (\u003cstrong\u003eFigure 10F-10H\u003c/strong\u003e). We further validate these findings by the synthetic reporter assay in tobacco. For this luciferase gene was cloned under \u003cem\u003e\u003csub\u003epro\u003c/sub\u003eCYCD1;1\u003c/em\u003e and pairwise co-transformed with either MED14 or HY5. Expression of luciferase enzyme revealed that MED14 and HY5 could activate \u003cem\u003eCYCD1;1\u003c/em\u003e promoter (\u003cstrong\u003eSupplemental Figure 23A-23D\u003c/strong\u003e). As expected, after HU treatment luciferase signal was low, suggesting repression of the \u003cem\u003eCYCD1;1\u003c/em\u003e promoter. These results explain the check on cell-cycle genes during stress condition. As NAC044 and NAC085 also regulate cell division (\u003cstrong\u003eSupplemental Figure 20C-20I\u003c/strong\u003e), we checked the expression of \u003cem\u003eCYCD1;1\u003c/em\u003e in \u003cem\u003enac\u003c/em\u003e mutant roots. Notably, there was a significant increase in \u003cem\u003eCYCD1;1\u003c/em\u003e expression in \u003cem\u003enac044-1\u003c/em\u003e, \u003cem\u003enac085-2\u003c/em\u003e, and the double mutants \u003cem\u003emed14-1 nac044-1\u003c/em\u003e and \u003cem\u003emed14-1 nac085-2\u003c/em\u003e. In contrast, \u003cem\u003eCYCD1;1\u003c/em\u003e expression was notably reduced in the \u003cem\u003emed14-1\u003c/em\u003e mutant (\u003cstrong\u003eFigure 10I\u003c/strong\u003e). These findings suggest that NAC transcription factors act as negative regulators of cell-cycle genes.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eA number of Mediator subunits have been implicated in root development (reviewed in \u003csup\u003e64\u003c/sup\u003e. However, it is still not clear how these subunits integrate internal and external cues to ultimately regulate root growth. In this study, we demonstrate that MED14 and HY5 form a crucial regulatory module that integrates cellular redox status with the DNA damage response to maintain root meristem integrity.\u003c/p\u003e\u003cp\u003eMED14 acts as a backbone subunit of the Mediator complex required for maintaining the meristematic zone. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, mutations in \u003cem\u003eSTRUWWELPETER (SWP)/MED14\u003c/em\u003e disrupt shoot apical meristem (SAM) organization and shorten the period of cell proliferation, indicating its crucial role in regulating meristem patterning and the timing of cell-cycle arrest \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. When we generated the knockout line of \u003cem\u003eMED14\u003c/em\u003e, none of the seeds survived, suggesting that MED14 is crucial for the survival of the plants. So, by using both RNAi-mediated knockdown (\u003cem\u003emed14-1\u003c/em\u003e) and T-DNA insertion mutant (\u003cem\u003emed14-2\u003c/em\u003e) lines, we observed significant defects in primary root length and overall root biomass (Fig.\u0026nbsp;1A; \u003cb\u003eSupplemental Fig.\u0026nbsp;3D\u003c/b\u003e). Cytological, histochemical, and phenotypic analyses showed that the reduced root growth was caused by the defect in meristematic zone, indicating that MED14 is mainly required for cell proliferation. The \u003cem\u003emed14\u003c/em\u003e seedlings also displayed increased cell death at the root meristem (Fig.\u0026nbsp;6F). We think that this cell death is critical for stopping the root growth during stress condition. So, the cell death actually represents a stress response, which is largely mediated by increased ROS production (Fig.\u0026nbsp;6F, 7A and 7B). The ROS level, including both superoxides and hydrogen peroxides, was increased in the \u003cem\u003emed14\u003c/em\u003e mutant (Fig.\u0026nbsp;5F-5M). Since proper ROS gradients are essential for balancing cell proliferation and differentiation in the roots, their imbalance likely contributes to the observed cell death in the root tip \u003csup\u003e\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e,\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e\u003c/sup\u003e. So, the treatment of \u003cem\u003emed14\u003c/em\u003e mutants with ROS scavenger KI partially rescued root length (Fig.\u0026nbsp;5N and 5O), supporting a link between redox imbalance and impaired root growth in \u003cem\u003emed14\u003c/em\u003e mutant.\u003c/p\u003e\u003cp\u003eTranscriptome of \u003cem\u003emed14\u003c/em\u003e mutant roots revealed widespread transcriptional reprogramming, including downregulation of flavonoid and phenylpropanoid biosynthesis pathways (\u003cb\u003eSupplemental Fig.\u0026nbsp;8B\u003c/b\u003e). Though the previous report indicates that flavonoids possess natural antioxidant properties, their specific role in plants is still not very clear. We have shown that indeed the flavonoid could function as a ROS scavenger in plants (Fig.\u0026nbsp;4C, 4D, 8F, 8G). In the \u003cem\u003emed14\u003c/em\u003e mutants, ROS was accumulated at a higher level, leading to cell death at the root tip (Fig.\u0026nbsp;5A-5D, \u003cb\u003eand 6F\u003c/b\u003e). Flavonoids such as quercetin are known to promote root meristem activity by modulating redox homeostasis and auxin transport \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e,\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e\u003c/sup\u003e. However, how flavonoids are involved in meristem division is still not clear. We observed that exogenous application of quercetin partially rescued the root growth defects of \u003cem\u003emed14\u003c/em\u003e (Fig.\u0026nbsp;4C and 4D), suggesting that flavanol deficiency contributes significantly to the observed short root phenotype. Notably, quercetin treatment restored meristem cell number in \u003cem\u003emed14\u003c/em\u003e mutant without changing the cell size (Fig.\u0026nbsp;4G-4J). Flavonoid biosynthesis mutants also exhibit reduced root length \u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e, highlighting the critical role of flavonoid-dependent redox regulation in maintaining the root meristem.\u003c/p\u003e\u003cp\u003eHY5 is an important transcription factor that integrates light in flavonoid biosynthesis, and ROS signaling \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003emed14\u003c/em\u003e mutants, HY5 is significantly downregulated (Fig.\u0026nbsp;3A; \u003cb\u003eSupplemental Fig.\u0026nbsp;9A-9C\u003c/b\u003e). So, there are significant phenotypic similarities between \u003cem\u003ehy5\u003c/em\u003e and \u003cem\u003emed14\u003c/em\u003e mutants, including shorter primary root (Fig.\u0026nbsp;3E and 3F), shortened meristematic zones (Fig.\u0026nbsp;3G and 3H), and impaired cell division (Fig.\u0026nbsp;3I-3P). The \u003cem\u003emed14-1 hy5\u003c/em\u003e double mutant did not show any additive phenotype, confirming that MED14 and HY5 function in a shared genetic pathway. Mechanistically, this study establishes that MED14 facilitates transcription of \u003cem\u003eHY5\u003c/em\u003e and a feedback loop for its autoregulation (Fig.\u0026nbsp;11). Moreover, HY5 and MED14 modulate the flavonoid biosynthesis genes such as \u003cem\u003eCHS, CHI, F3H, FLS1, TT7\u003c/em\u003e, and \u003cem\u003eCHIL\u003c/em\u003e for transcriptionally regulating the root development (Fig.\u0026nbsp;2A, 2B, and 4A, 4B) in both mutants. We found that in addition to co-regulation of flavonoid genes, HY5-MED14 modules have a larger significance. HY5 plays a key role in maintaining ROS homeostasis in Arabidopsis \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e\u003c/sup\u003e. The transcriptomic comparison between \u003cem\u003emed14\u003c/em\u003e and \u003cem\u003ehy5\u003c/em\u003e roots revealed common upregulation of ROS biosynthesis genes, suggesting coordinated regulation. This also suggests that HY5 and MED14 function as repressors of ROS production genes in non-stressed conditions, thereby protecting the DNA from getting damaged to ensure proper growth and development of the root. An interaction between MED14 and HY5 (\u003cb\u003eSupplemental Fig.\u0026nbsp;11\u003c/b\u003e) and their co-occupancy on different promoters (Fig.\u0026nbsp;9A-9D, \u003cb\u003eand 10E\u003c/b\u003e) suggest that the HY5-MED14 module is critical for flavonoid-regulated gene expression for root development.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003emed14\u003c/em\u003e and \u003cem\u003ehy5\u003c/em\u003e mutants exhibit elevated levels of ROS (Fig.\u0026nbsp;7A-7D). High ROS levels induce single and double-stranded DNA breakage and activate protein kinases like ATM and ATR present at the top of the DDR response pathway \u003csup\u003e\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e,\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u003c/sup\u003e. This activated ATM and ATR protein phosphorylates downstream TFs, a conserved DDR module. In plants, this TF is SOG1, phosphorylated by ATM and ARM upon DDR activation in Arabidopsis \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Further, the SOG1 transcriptionally activates a cascade of TFs like \u003cem\u003eNAC044, NAC085\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e and enzymes for cell-cycle arrest in the meristem and enhances the transition from cell division to endoreduplication \u003csup\u003e\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e\u003c/sup\u003e. We observed that higher ROS levels in \u003cem\u003emed14\u003c/em\u003e and \u003cem\u003ehy5\u003c/em\u003e activated DDR response in these mutants and also exhibited elevated levels of \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e expression (Fig.\u0026nbsp;6A and 6B). We found that flavonoids, particularly quercetin, are critical in mitigating ROS-mediated DNA damage. The direct involvement of flavonoids in preventing cellular damage has not been explored to date. In this study, the exogenous application of quercetin effectively suppressed HU-induced \u003cem\u003eNAC\u003c/em\u003e gene expression in wild-type roots. This suppression was normally observed in \u003cem\u003emed14\u003c/em\u003e and \u003cem\u003ehy5\u003c/em\u003e mutants, which naturally have lower quercetin levels. This observation demonstrated that under stress conditions, MED14 and HY5 play critical role in maintaining the ROS-triggered DNA damage by modulating the flavonoid biosynthesis.\u003c/p\u003e\u003cp\u003eThis study provides evidence that fine-tuned coordination between activation and repression of cell division under stressed and non-stressed situations is critical for sustaining meristem size and ensuring DNA stability in the RAM (Fig.\u0026nbsp;11). We have demonstrated that MED14 and HY5 function together to promote root meristem activity by activating key cell-cycle regulators (Fig.\u0026nbsp;10A), while simultaneously repressing stress-responsive NAC transcription factors under non-stressed conditions (Fig.\u0026nbsp;9A-9D). Studies have shown that both G1/S and G2/M stages can mediate cell-cycle arrest in response to DNA damage. Both hydroxyurea and zeocine trigger arrest at both the G2/M transition and the G1/S checkpoint \u003csup\u003e\u003cspan additionalcitationids=\"CR108\" citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e\u003c/sup\u003e. We found that cell-cycle-related genes, including \u003cem\u003eCYCD1;1\u003c/em\u003e, were downregulated in \u003cem\u003emed14\u003c/em\u003e and \u003cem\u003ehy5\u003c/em\u003e mutants (Fig.\u0026nbsp;10A and 10B). CYCD1;1 is a D-type cyclin vital for the G1/S phase transition. Its downregulation likely disrupts cell-cycle progression and decreases the number of dividing cells in the root meristem. We confirmed that both HY5 and MED14 directly bind to the promoter of \u003cem\u003eCYCD1;1\u003c/em\u003e (Fig.\u0026nbsp;10C-10E). This cooperative action of HY5 and MED14 thus ensures the proper transcriptional activation of \u003cem\u003eCYCD1;1\u003c/em\u003e, which is essential for maintaining active cell division in the root tip (Fig.\u0026nbsp;11).\u003c/p\u003e\u003cp\u003eBeyond their role in promoting cell-cycle progression, MED14 and HY5 also act as repressors of specific DDR genes under normal condition. In the absence of genotoxic stress, expression of the NAC transcription factors \u003cem\u003eNAC044\u003c/em\u003e, \u003cem\u003eNAC085\u003c/em\u003e, and \u003cem\u003eNAC103\u003c/em\u003e is low, thereby preventing unnecessary inhibition of the G2/M phase of the cell-cycle \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e,\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003emed14\u003c/em\u003e and \u003cem\u003ehy5\u003c/em\u003e mutants, we observed elevated expression of these \u003cem\u003eNAC\u003c/em\u003e genes, indicating a loss of repression on cell-cycle genes (Fig.\u0026nbsp;6A, 6B, \u003cb\u003eSupplemental Fig.\u0026nbsp;19D\u003c/b\u003e). The resulting activation of the DDR pathway in these mutants leads to shortened roots and reduced meristematic cell numbers. This was further supported by the phenotyping of \u003cem\u003emed14-1 nac044-1\u003c/em\u003e, \u003cem\u003ehy5 nac044-1\u003c/em\u003e, and \u003cem\u003emed14-1 nac085-2\u003c/em\u003e, \u003cem\u003ehy5 nac085-2\u003c/em\u003e double mutants, in which the short-root phenotype was largely rescued due to reduced cell death at the root tips (\u003cb\u003eSupplemental Figs.\u0026nbsp;20 and 21\u003c/b\u003e). The modest increase in \u003cem\u003eCYCD1;1\u003c/em\u003e transcript level was also observed in the \u003cem\u003enac044-1\u003c/em\u003e and \u003cem\u003enac085-2\u003c/em\u003e single and double mutants with \u003cem\u003emed14\u003c/em\u003e (Fig.\u0026nbsp;10I), suggesting that these transcription factors are involved in regulating the cell-cycle at the transcriptional level and may function as potential repressors of cell cycle gene expression. This genetic evidence confirms that the growth defects in \u003cem\u003emed14\u003c/em\u003e and \u003cem\u003ehy5\u003c/em\u003e are at least partially attributable to the upregulation of \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u003c/em\u003e, which act to restrict cell division in response to stress. Mechanistically, we found that HY5 and MED14 occupy the promoters of \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u003c/em\u003e (Fig.\u0026nbsp;9A-9D). The occupancy of MED14 on these promoters was reduced in the \u003cem\u003ehy5\u003c/em\u003e mutant (Fig.\u0026nbsp;9C and 9D), indicating a cooperative repression mechanism. It is already reported that functional loss of NAC044 and NAC085 fails to repress G2/M-specific genes and shows continued cell division under genotoxic stress, resembling Rep-MYB mutants \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. These NAC transcription factors likely regulate the stability or accumulation of Rep-MYBs (MYB3R3 and MYB3R5) \u003csup\u003e\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e\u003c/sup\u003e, ensuring sustained suppression of mitotic gene expression (\u003cem\u003eKNOLLE\u003c/em\u003e, \u003cem\u003eCYCB1;2\u003c/em\u003e, \u003cem\u003eEPS15 HOMOLOGY DOMAIN 2\u003c/em\u003e (\u003cem\u003eEHD2\u003c/em\u003e), and \u003cem\u003ePLEIADE/MAP65-3\u003c/em\u003e) and proper cell-cycle arrest \u003csup\u003e\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e,\u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e\u003c/sup\u003e. In our study under genotoxic stress, however, the binding of both HY5 and MED14 to the promoters of \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u003c/em\u003e, as well as \u003cem\u003eCYCD1;1\u003c/em\u003e, was significantly reduced, leading to the upregulation of NAC TFs and downregulation of \u003cem\u003eCYCD1;1\u003c/em\u003e. These data suggest that stress disrupts the MED14-HY5 repression complex, allowing DDR genes to be activated and halting cell division to permit DNA repair (Fig.\u0026nbsp;11).\u003c/p\u003e\u003cp\u003eTogether, these findings position the MED14-HY5 module as a critical modulator of growth and stress signaling pathways. While HY5 has long been known as a regulator of photomorphogenesis, our study expands its functionality in cell-cycle control. Similarly, MED14, a core component of the Mediator complex, emerges as a versatile co-regulator capable of supporting both transcriptional activation and repression depending on the context. By activating \u003cem\u003eCYCD1;1\u003c/em\u003e to promote cell division and repressing \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u003c/em\u003e to prevent premature DDR activation, the HY5-MED14 complex ensures that root growth proceeds optimally under favorable conditions. This discovery is particularly significant because it suggests that MED14 and HY5 are continuously required to prevent a baseline level of ROS-induced damage. This study also contrasts with a simple activation model and highlights the complex, context-dependent nature of transcriptional regulation for optimal growth and stress responses. Overall, our findings provide a new paradigm for understanding how plants manage the inevitable trade-off between growth and survival, offering a valuable model for engineering more resilient crop varieties.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and Growth condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo characterize the MED14, we used two types of mutant lines in our study. We made MED14 RNAi in the Columbia-0 (Col-0) background and another T-DNA insertion mutant (SAIL_373_C07) (Columbia-3 background), which has been obtained from ABRC. We have also used \u003cem\u003ehy5\u003c/em\u003e (SALK_056405), \u003cem\u003enac044-1\u0026nbsp;\u003c/em\u003e(SAIL_1286D02), \u003cem\u003enac085-2\u0026nbsp;\u003c/em\u003e(SALK_208662), \u003cem\u003emed17\u003c/em\u003e (SALK_102813), \u003cem\u003emed16-2\u0026nbsp;\u003c/em\u003e(SALK_048091), \u003cem\u003emed25/pft1-3\u003c/em\u003e (SALK_059316), \u003cem\u003ecdk8-1\u003c/em\u003e (SALK_138675), \u003cem\u003ecycC-b\u003c/em\u003e (SAIL_102_B02) mutants obtained from ABRC.\u003c/p\u003e\n\u003cp\u003eTo generate knockdown RNAi mutants of MED14, 600 bp of the long region was cloned into pHELLSGATE 12. That construct was transformed into Agrobacterium strain (GV3101). Then, wild-type (Col-0) plants were transformed using the Agrobacterium strain containing a recombinant plasmid via the floral dip method \u003csup\u003e113\u003c/sup\u003e. The transgenic plants were selected on kanamycin plates. After several attempts, we got four lines that had downregulation of the MED14 transcript as quantified by qRT-PCR using two sets of gene-specific primers. And only one line which had maximum downregulation, was used in this study. T-DNA mutants SAIL_373_C07 (ecotype Columbia-3 background) were obtained from ABRC. T-DNA was found to be inserted into the sixth exon of the\u003cem\u003e\u0026nbsp;MED14\u003c/em\u003e gene. SAIL_373_C07 was first selected by BASTA (0.001%) spray. Insertion of T-DNA in SAIL_373_C07 was further checked by using T-DNA border and gene specific primers and homozygous (SAIL_373_C07) plants were found by using genotyping. Further, the transcript level of \u003cem\u003eMED14\u003c/em\u003e in T-DNA mutants by qRT-PCR with the help of gene-specific qRT-primers (\u003cstrong\u003eSupplemental Table 1\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the \u003cem\u003eHY5\u003csub\u003epro\u003c/sub\u003e:HY5::GFP\u003c/em\u003e lines, a 3 kb promoter region along with the \u003cem\u003eHY5\u003c/em\u003e gene was amplified from genomic DNA and initially cloned into the pDONR207 vector. This construct was then transferred into the pGWB4 vector using Gateway cloning. The final recombinant construct was introduced into Agrobacterium cells for transformation into both the wild-type and \u003cem\u003emed14-1\u003c/em\u003e mutant backgrounds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of transgenic plants by floral dip\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the plant transformation, healthy flowering plants were taken. To avoid the mixture, wild-type siliques were cut and poured into the water for proper hydration one day before the floral dip. Prepared the 5mL of recombinant Agrobacterium inoculum overnight at 28℃ incubator shaker until the culture was saturated. The next day, poured the secondary inoculation of the Agrobacterium cells into 500 mL of LB broth at 28℃ incubator shaker until the OD reached 1.0. Meanwhile, the dipping solution was prepared with ½ strength of MS powder, 5% (w/v) sucrose, and 150 µL of Silwet L-77 (surfactant) in 500 mL of the solution, and the harvested Agrobacterium cells into the solution. Plants were dipped for 40 seconds, and covered the plants with black cloth to protect the plants from light for 24 hrs. Then, removed the cover and allow the plants to grow properly until the seeds are ready to harvest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeed Sterilization, media preparation and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeeds were surface sterilized for 1 min in 70% (v/v) ethanol and 12 mins in seed washing buffer containing 1.6% (v/v) Sodium hypochlorite, 0.01% SDS and milli Q water and then rinsed four times with sterile milli Q water. The sterilized seeds were kept for 2 days for stratification at 4ºC in dark and then plated those seeds onto solid ½ Murashige and Skoog (MS) medium and grown into a growth chamber with long-day conditions of 16 h light and 8 h dark at about 21ºC; 110 μmol. s-1.m-2 light intensity; and 60% relative humidity. The plants were grown till seedling stage on ½ MS medium with 1% sucrose, 0.05% MES Hydrate and 0.8% plant agar adjusted to pH 5.7. After 10 days the seeding was transferred to soil (Agropit: Vermiculite: Soilrite- 3:1:1 mixture).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo prepare 0, 1, 1.5, and 2 mM concentrations of hydroxyurea (HU), a 1 M HU stock solution was made, and appropriate volumes were added to autoclaved ½ MS medium. Media containing 10 µM potassium iodide (KI) and 10 µM quercetin were prepared similarly to the HU media. For root phenotyping, seedlings were first grown on ½ MS medium for 4 days, then transferred to the treatment media for an additional 8 days before measuring root phenotypes. For staining experiments, 4-day-old seedlings were exposed to solid ½ MS medium containing HU, KI, or quercetin for 24–48 hours. For gene expression analysis, 6-day-old seedlings were transferred to treatment media containing 5 mM HU, 10 µM KI, and 10 µM quercetin for 24 hours. For ChIP-qPCR analysis, 6-day-old seedlings were transferred to HU-containing medium and treated for 24 hours.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenotypic analysis of MED14 plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eArabidopsis primary roots and root tips were analysed by a confocal laser scanning microscope using a TCS SP8 microscope (Leica Camera). Cell length was measured from the confocal images. All the number of lateral roots was counted under compound microscope. Primary root length was analysed directly by using Image J software from digital images captured by a Nikon 3100 camera on the basis of using a ruler.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCrossing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, selected the flower buds from mother plant which were expected to open next 2-3 days, and the rest the fertilized flowers and the siliques were removed from the inflorescence tip. Selected buds were emasculated with fine forceps. Emasculated buds with naked gynoecium were left for next 48 h with 60% humidity. After 48 h, mature pollens from healthy opened anthers were carefully sprayed over the stigma and marked the stigma properly. After crossing, plants were kept in proper light, temperature and humidity conditions so that the siliques could grow well.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGUS staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eArabidopsis seedlings were taken into the GUS buffer solution and vacuum infiltrated the samples for about 30 mins. Then kept the samples for O/N at 37℃. Rinsed the plant tissue by using a washing solution (70% Ethanol and 30% Acetone) twice for about 2 hrs at 37℃ to remove the excess GUS buffer and chlorophyll. And observed under stereo microscope (Nikon SMZ 25 with DS-Fi3 camera).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePropidium iodide (PI) staining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor fluorescent propidium iodide (PI) staining, plants were taken from the growth medium to 2 µg/mL of PI solution for 5 mins and mounted in water on microscopic slides by coverslip. The samples were observed at the wavelength specific for the PI fluorescence with the fluorescence excitation maximum of PI was 535 nm and emission maximum were 617 nm by using a confocal laser scanning microscope using a TCS SP8 microscope (Leica Camera).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisualization of cell damage\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor genotoxic stress, 5-days-old wild-type and the mutant plants were transferred to MS plates containing 0, 0.5, 1mM, 1.5mM of HU. Seedlings were stained with PI (2 µg/mL) on a slide of 5 mins. Stained seedlings were observed by a confocal laser scanning microscope using a TCS SP8 microscope (Leica Camera). The fluorescence excitation maximum of PI was 535 nm and emission maximum was 617 nm. And the blotches were observed as a damage cells. Additionally, root length was measured from the image captured by a Nikon 3100 camera using Image J software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEdU staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEdU staining were performed using EdU detection kit (Invitrogen Alexa 647). In brief, wild-type, mutants and the transgenic seedlings were grown on ½ MS medium for 5 days. For EdU labelling, seedling was transferred into ½ MS broth containing 10 uM EdU for 3 h at 21ºC. Then the seedlings were fixed with 3.7% formaldehyde in phosphate buffer saline (PBS) solution for next 15 mins at room temperature. Fixed seedlings were washed with 3% BSA in PBS solution for twice and allowed to permeabilization into 0.1% triton X-100 in PBS for 20 mins at room temperature. Fixer was further washed with 3% BSA thrice and incubated with EdU detection cocktail for 30 mins at room temperature in dark followed by washing with PBS twice and allowed to further stained with DAPI for observation of fluorescence under confocal microscope. Fluorescence was recorded at wavelengths 520 excitation and 640 emission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDAPI staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDAPI were used to further stained the EdU labelled nuclei. To stained the roots with DAPI, 6-days-old EdU labelled seedlings were further stained with 20 µM of DAPI for 5 mins and observed under confocal microscope. Fluorescence was recorded at the wavelengths of 355 excitation and 465 emission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of flavonoids\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDPBA staining-\u003c/em\u003e For flavonol staining, 6-days-old Arabidopsis seedlings were stained for 15 mins in 0.25% (w/v) diphenylboric acid 2-aminoethyl ester (DPBA; Sigma-Aldrich) and 0.005% (v/v) Triton X-100 \u003csup\u003e114\u003c/sup\u003e. Fluorescence was visualized on a confocal laser scanning microscope using a TCS SP8 microscope (Leica Camera) (laser excitation 488 nm, emission 505-550 nm).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHigh-performance thin-layer chromatography (HPTLC)-\u003c/em\u003e For the detection of flavonol glycosides, 6-days-old Arabidopsis root samples were harvested and extracted as described previously \u003csup\u003e115\u003c/sup\u003e. \u0026nbsp;The extracted samples were run on Silica Gel 60F254 plates and developed by spraying 1% (w/v) DPBA and 5% (w/v) PEG and observed under UV detector at 365 nm. Developed green, oranges and blue bands were represented kaempferol, quercetin and sinapic acid derivative respectively \u003csup\u003e116\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisualization of ROS by staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eH2DCFDA staining-\u003c/em\u003e For the detection of overall ROS, 6-days-old Arabidopsis roots were stained with 10 µM H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) in potassium phosphate buffer (pH 7.4) for 10 mins and washed the excess dye with sterile milli Q thrice. And mounted the samples in water on microscopic slides by coverslip. Then fluorescence was visualized in GFP excitation wavelength of 488 nm and emission wavelength 500-525 nm on a confocal laser scanning microscope using a TCS SP8 microscope (Leica Camera).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNBT staining-\u003c/em\u003e For detection of superoxides (O2-), 6-days-old roots were stained with 2 mM Nitroblue tetrazolium (NBT) solution in potassium phosphate buffer (pH 7.4) for 15 mins and washed the excess dye with sterile milli Q. Root samples were mounted in water and observed under stereo microscope (Nikon SMZ 25 with DS-Fi3 camera).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDAB staining-\u003c/em\u003e For the detection of hydrogen peroxide (H2O2), 6-days-old roots were stained with 2 mg/mL Diamino benzidine (DAB) solution in potassium phosphate buffer (pH 7.4) for 15 mins and washed the excess dye with sterile milli Q. Root samples were mounted in water and observed under stereo microscope (Nikon SMZ 25 with DS-Fi3 camera).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor analysing the expression of the genes, total RNA was extracted from 8-days-old seedlings using RNeasy plant mini kit (as per the protocol given in Qiagen manual), and cDNA was prepared using 1µg total RNA with Verso cDNA synthesis Kit (as per the Thermo Scientific kit manual protocol). Further, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed with SYBR Green I master mix using the Applied Biosystems QuantStudio 6 Flex Real-Time PCR systems. These qRT-PCR values were normalized with Ubiquitin and 18S as a control gene. qRT-PCR was performed with three biological replicates in triplicate for each gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBimolecular Fluorescence Complementation (BiFC) assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHY5 was cloned into the pDEST-VYCE vector, and MED14_Full length, MED14_Δ3, MED14_Δ4, and MED14_Δ6 were cloned into pDEST-VYNE . Recombinant plasmids along with empty vectors were transformed into Agrobacterium starin GV3101 and infiltrated into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves as described in \u003csup\u003e117\u003c/sup\u003e. Image was captured after 48 h light exposure using TCS SP8 confocal microscope (Leica Camera) with the YFP excitation 490 mm and emission 520 nm and for red fluorescence excitation maxima 550 nm and emission maxima 580 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDual luciferase assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess \u003cem\u003eCYCD1;1\u003c/em\u003e transactivation, a 993 bp long promoter region with HY5-binding elements was cloned into the pGreenII 0800-LUC vector (containing 35S promoter). Transcription factor HY5 was cloned into pGWB4 under its native promoter, and MED14 was cloned into pGWB21. These constructs were co-expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves alongside various effectors, with an empty vector as a control. After infiltration, plants were kept in the dark at 22°C for 24 h and then exposed to light for 48 hours. Luciferase activity was measured using a ChemiDoc imaging system. For HU treatment, after 48 h of light exposure, infiltrated leaves were excised at the petiole site and placed in ½ MS broth with mock and 2 mM HU treatments, which were allowed to proceed for the next 6 h. The excised leaves were then blotted with tissue paper before assessing luciferase activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatin immunoprecipitation (ChIP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromatin immunoprecipitation was performed according to the standard protocol with few modifications \u003csup\u003e118\u003c/sup\u003e. Eight-day-old Arabidopsis seedlings were fixed by vacuum infiltration in formaldehyde-containing crosslinking buffer (0.4 M sucrose, 10 mM Tris-HCl pH=8, 1 mM PMSF, 1 mM EDTA, and 1% formaldehyde) to preserve protein-DNA interactions. The crosslinking was quenched with glycine, and samples were thoroughly washed with water before freezing in liquid nitrogen. Frozen tissue was ground into a fine powder, and nuclei were isolated using a nucleus isolation buffer (0.25 M sucrose, 15 mM PIPES pH=6.8, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 60 mM KCl, 15 mM NaCl, 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.9% Triton X-100, protease inhibitor SIGMA #lot 83612300) under cold conditions. The nuclei were lysed with nucleus lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, protease inhibitor), and chromatin was sonicated to shear DNA into fragments suitable for immunoprecipitation. The chromatin extract was pre-cleared with salmon sperm DNA–blocked Protein A agarose beads (Sigma-Aldrich 16-157) to reduce non-specific binding. A specific antibody against the target protein was then incubated overnight with the chromatin to allow formation of antibody-protein-DNA complexes. The immune complexes were captured using Protein A beads and subjected to a series of washes with buffers of increasing stringency to remove non-specifically bound material. Bound chromatin was eluted from the beads, and crosslinks were reversed by incubation at 65°C overnight. Proteins were digested with Proteinase K to purify DNA, followed by phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation to recover the DNA. The purified DNA pellet was washed with 70% ethanol, dried, resuspended in TE buffer, and stored at –20°C for downstream analyses such as qPCR or sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 100 mg of six-day-old Arabidopsis seedlings (or root and shoot independently) were frozen and ground into powder under liquid nitrogen. Proteins were extracted using a cold buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM DTT, 100 mM PMSF, 1 mM NaF, 20% glycerol, and protease inhibitor) and homogenized gently. The mixture was then centrifuged to remove debris. Protein concentration was measured via Bradford assay against a BSA standard curve. Equal amounts of protein were mixed with SDS loading dye, denatured at 95°C for 10 minutes, and loaded onto a freshly prepared 10-12% SDS-PAGE gel (as required). After electrophoresis, proteins were transferred to a nitrocellulose membrane at room temperature for 1.5 h. The membrane was further blocked with 5% BSA to prevent nonspecific binding, then incubated O/N with primary antibody at 4°C. Following washes, the membrane was incubated with HRP-conjugated secondary antibody for another 1 h. After further washes, protein bands were detected using a chemiluminescent substrate. Finally, bands were visualized and documented using a gel imaging system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA sequencing and data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor RNA-sequencing, 6-days-old roots of Col-0, \u003cem\u003emed14-1\u003c/em\u003e were collected in three biological replicates after germination. Total RNA was isolated using the Sigma Plant RNA isolation kit according to the manufacturer’s protocol. Library preparation was done using NEB kit, and RNA-sequencing was done on Illumina Hiseq X platform by Agrigenome, P.V. Ltd (Bengaluru, India). After removing the bad quality read by fastp, the clean reads were aligned to the Arabidopsis genome TAIR 10 (https://www.arabidopsis.org/) using HISAT 2 \u003csup\u003e119\u003c/sup\u003e. Here, for RNA-seq of RNAi analysis, 2\u003csup\u003end\u0026nbsp;\u003c/sup\u003eexon of \u003cem\u003eMED14\u003c/em\u003e gene was trimmed out by the cut adapt sequence trimming tool from all samples (both for Col-0 and \u003cem\u003emed14\u003c/em\u003e samples). The expression level of each gene was analysed by the featureCount tool of Subread. For details of the libraries, read numbers, and alignments\u003cstrong\u003e.\u003c/strong\u003e For differential gene expression analyses, DEseq2 (v:1.22.2) tool was used. To determine the correlation among biological replicates, normalized read counts were analysed and volcano plot of log2Fold change with p-value was made. For up- and down-regulated genes, a cut-off value of |FC| ≥ 1.5 and p-value ≤ 0.05 was selected, respectively. Detailed pairwise comparisons for DEGs are provided\u003cstrong\u003e.\u003c/strong\u003e Heatmaps were constructed from log2 fold change values generated by DEGs analysis using the heatmap (Version-1.0.12) R tool. Scatter plots were constructed from the log2 fold change values generated by DEGs analysis using ggplot2 (version 3.5.1) R tool. For the heatmap and scatterplot, data with pvalue ≤ 0.05 were considered for analysis. Gene Ontology (GO) analysis was performed using ShinyGO 0.80 (http://bioinformatics.sdstate.edu/go/) with default parameters. Genes involved in cell cycle and ROS were manually selected from the Arabidopsis Information Resource website (TAIR).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein-protein docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein–protein docking was carried out to investigate the potential interaction between AtHY5 and AtMED14. The three-dimensional structure of AtMED14 was predicted using the Phyre2 server \u003csup\u003e120\u003c/sup\u003e, while the crystal structure of AtHY5 (PDB ID: 2OQQ) was used for docking studies. Prior to docking, both protein structures were prepared using PyMOL \u003csup\u003e121\u003c/sup\u003e, where water molecules and heteroatoms were removed, and hydrogen atoms were added to facilitate proper modeling of hydrogen bonding.\u003c/p\u003e\n\u003cp\u003eInitial docking poses were generated using the PatchDock server \u003csup\u003e122\u003c/sup\u003e, which identifies candidate binding conformations based on geometric shape complementarity. The resulting docking solutions were subsequently refined using FireDock \u003csup\u003e123\u003c/sup\u003e, which optimizes side-chain conformations and recalculates binding energies. Based on the refined energy scores, the top three docking solutions were selected to analyze the binding modes and interaction interfaces between HY5 and MED14.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification and statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll physiological, confocal, and stereomicroscopic experiments were considered as independent biological replicates containing at least 15-20 seedlings. Gene expression was analyzed using 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e values from RT-qPCR on three independent biological replicates. ChIP-qPCR experiments were conducted in three technical replicates (n=3) from a single representative experiment, with each experiment repeated two to three times independently. Physiological, confocal, and ChIP experiments with similar results/patterns were repeated as described in the figure legends. Statistical significance was determined using one-way ANOVA and Tukey's HSD test, with P ≤ 0.05 considered statistically significant and P \u0026gt; 0.05 considered non-significant. Some of the experiments used the t-test, with P ≤ 0.05 considered significant and P \u0026gt; 0.05 non-significant, and represented them as asterisks * (for P ≤ 0.05), ** (for P ≤ 0.005), and *** (for P ≤ 0.0005). Data analysis and graph creation were done using Microsoft Excel, and statistical analysis was performed with GraphPad Prism 8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccession numbers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe gene sequence data referenced in this article are available in the GenBank/EMBL databases under the following accession number: MED14 (AT3G04740), HY5 (AT5G11260), CYCD1;1 (AT1G70210), CYCB1;1 (AT4G37490), NAC044 (AT3G01600), NAC085 (AT5G14490), NAC103 (AT5G64060), SOG1 (AT1G25580), ATR (AT5G40820), ATM (AT3G48190), CHS (AT5G13930), CHI (AT3G55120), F3H (AT3G51240), and FLS1 (AT5G08640).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements and Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is partially funded by the grant BT/PR38349/GET/119/339/2020 and partially by grant BT/PR40169/BTIS/137/71/2023 both from the Department of Biotechnology, Government of India. The authors acknowledge core funding from the International Centre for Genetic Engineering and Biotechnology, New Delhi. PT acknowledges the Council of Scientific and Industrial Research (CSIR) for Junior and Senior Research Fellowships, Government of India. SM acknowledges fellowships from the Department of Biotechnology, Government of India (MK Bhan Fellow: BT/HRD/MK-YRFP/50/27/2021). AG acknowledges fellowships from the University Grants Commission (UGC) for the Junior Research Fellowship. Support from all the facilities of NIPGR and ICGEB is acknowledged. \u0026nbsp;Authors are thankful to DeLCON for providing access to the literature. Authors are grateful to Prof. Nam-Hai Chua, Temasek Life Sciences Laboratory, National University of Singapore, Singapore, 117604, for providing the \u003cem\u003eMED14\u003csub\u003epro\u003c/sub\u003e:MED14::YFP\u003c/em\u003e seeds, and also grateful to Prof. Yongjian Qiu, Department of Biology, University of Mississippi, Oxford, Mississippi 38677, USA, for providing \u003cem\u003e35S:MED14::HA/med14\u003c/em\u003e seeds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePT planned and designed the experiments and further analyses, and wrote the first draft, figures with SM. AG, AP, and ND helped to perform the experiments. SD analysed the RNA-sequencing data. MW performed the in-silico analyses. SM and AR helped in revising the draft and critically reviewed the content, and provided feedback and valuable suggestions. JKT and SM conceived the project, planned the study, arranged for funding, corrected the manuscript, and gave final approval of the published work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available in the supplementary material of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eIngram, P. A. \u0026amp; Malamy, J. E. Root System Architecture. in 75\u0026ndash;117 (2010). doi:10.1016/B978-0-12-380868-4.00002-8.\u003c/li\u003e\n\u003cli\u003eSmith, S. \u0026amp; De Smet, I. 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In this study, we uncovered a novel mechanism in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e by which the Mediator complex subunit MED14 and the transcription factor ELONGATED HYPOCOTYL 5 (HY5) cooperatively regulate root development by maintaining redox and genomic stability. We found that the reduction in the level of AtMED14 causes mis-regulation of a large number of genes required for root development and hence affects the primary root development in Arabidopsis seedlings. Based on transcriptome data, flavonoid levels were examined and found to be deficient in the roots of the \u003cem\u003emed14\u003c/em\u003e mutant. Flavonoids are essential metabolites, and our findings suggest that they play key role in regulating ROS homeostasis in roots. The \u003cem\u003emed14\u003c/em\u003e roots have enhanced accumulation of ROS, leading to DNA damage and eventually activating the DNA damage response in the meristematic zone. This study unravels the coordination of MED14 with HY5 in regulating the cell-cycle genes. Under normal condition, MED14 facilitates HY5-mediated activation of cell-cycle genes, especially \u003cem\u003eCYCD1;1\u003c/em\u003e, however, during genotoxic stress, there is repression of these genes, thereby halting the cell-cycle. In contrast, under non-stress conditions, MED14 and HY5 suppress the expression of DNA damage response genes \u003cem\u003eNAC044\u003c/em\u003e and \u003cem\u003eNAC085\u003c/em\u003e, but this repression is relieved under oxidative stress. Conversely, the loss of function in \u003cem\u003eNAC\u003c/em\u003e genes leads to increased expression of \u003cem\u003eCYCD1;1\u003c/em\u003e, but cannot effectively downregulate the expression of \u003cem\u003eCYCD1;1\u003c/em\u003e under genotoxic conditions. Such control over activation and repression of these genes is crucial for preventing unwarranted cell cycle arrest and ensuring proper root meristem function. Thus, our findings reveal a previously uncharacterized role of MED14-HY5 module in balancing ROS homeostasis and DNA damage response to safeguard root growth and development, providing a critical insight into how plants cope with environmental and endogenous genotoxic threats.\u003c/p\u003e","manuscriptTitle":"MED14-HY5 module orchestrates trade-off between cell-cycle activation and DNA damage response in Arabidopsis root","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-05 07:13:03","doi":"10.21203/rs.3.rs-8222842/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f62be4d7-849a-4474-ab41-657e98768d9e","owner":[],"postedDate":"December 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":59099948,"name":"Biological sciences/Molecular biology/Transcription/Transcriptional regulatory elements"},{"id":59099949,"name":"Biological sciences/Molecular biology/DNA damage and repair"}],"tags":[],"updatedAt":"2026-01-15T13:55:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-05 07:13:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8222842","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8222842","identity":"rs-8222842","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}