The function and regulatory mechanism of tea plant transcription factor CsMYB116 in drought stress response

The function and regulatory mechanism of tea plant transcription factor CsMYB116 in drought stress response | 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 Research Article The function and regulatory mechanism of tea plant transcription factor CsMYB116 in drought stress response Xiaoxia Zhao, Xinhan You, Wenjuan Ma, Xueying Xie, Jian Hou, Xiaoyang Han This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8519228/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract The MYB transcription factor family plays a crucial regulatory role in the growth and quality development of tea plants. Building upon previous drought-related transcriptome data, this study systematically investigated the function and regulatory mechanisms of the tea plant transcription factor CsMYB116 in response to drought stress, employing physiological and molecular biology methods. Through heterologous overexpression and gene silencing experiments, CsMYB116 was demonstrated to positively regulate drought resistance in tea plants. Under drought conditions, overexpression lines exhibited increased lateral root development, enhanced photosystem stability, elevated antioxidant enzyme activity, higher proline content, and a significant reduction in membrane lipid peroxidation. Conversely, gene silencing resulted in photosystem damage, diminished antioxidant capacity, and disrupted hormone signaling in tea seedlings. Transcriptome sequencing revealed significant alterations in key metabolic pathways-including hormone signal transduction, cell wall synthesis, phenylpropanoid metabolism, and ABC transporter activity—in silenced plants subjected to drought stress. Furthermore, yeast two-hybrid assays confirmed interactions between CsMYB116 and both ABC transporters and IAA-related proteins. This study is the first to demonstrate that CsMYB116 enhances drought adaptability in tea plants by positively regulating gene expression. These findings contribute to a deeper understanding of the molecular regulatory mechanisms underlying tea plant responses to drought and identify CsMYB116 as a promising target for molecular breeding aimed at improving drought tolerance in tea plants. tea plant drought stress functional verification MYB Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Drought represents a major abiotic stress factor that constrains plant growth, development, and yield formation (Sato et al. 2024). With the ongoing trend of global warming, the frequency and intensity of drought events are projected to increase (Gebrechorkos et al. 2025). Tea plants ( Camellia sinensis L.), which are characteristically moisture-dependent, exhibit high sensitivity to water availability. Their growth and development (Zheng et al. 2025), secondary metabolism (Shao et al. 2025), and flavor quality (Zhou et al. 2014) are all contingent upon sufficient and stable water supply. Drought conditions not only markedly diminish tea yield and quality but also elevate plant mortality rates (Upadhyaya and Panda 2013). Consequently, a comprehensive understanding of the mechanisms governing tea plants’ responses to drought stress is essential for maintaining stable tea production and for the development of drought-tolerant cultivars. Within the intricate regulatory network governing plant responses to drought and other environmental stresses, transcriptional regulation assumes a pivotal role. The MYB transcription factor family represents one of the largest and most functionally diverse groups in plants (Yuan et al. 2021), with the R2R3-MYB subfamily being particularly prominent due to its extensive involvement in plant growth and development (Dubos et al. 2010), regulation of secondary metabolism (Chen et al. 2019), and responses to various abiotic stresses (Wang et al. 2021). Empirical studies have demonstrated that MYB transcription factors enhance plant drought tolerance by modulating root architecture, hormone signaling pathways, reactive oxygen species (ROS) scavenging systems, and osmotic adjustment mechanisms (Wang et al. 2025). Regarding root development, MYB transcription factors augment water uptake capacity through the regulation of root architecture. For instance, overexpression of the soybean gene GmMYB84 promotes primary root elongation and increases plant survival rates under drought conditions (Wang et al. 2017). In Arabidopsis, AtMYB96 mediates abscisic acid (ABA) signaling and interacts with auxin pathways to regulate root architecture, thereby enhancing drought tolerance (Seo et al. 2009). Comparable findings have been reported in tea plants, where tea polyphenols mediate the regulation of CsPOD44 by CsMYB77 , contributing to improved root drought resistance (Xu et al. 2025). Furthermore, MYB transcription factors are extensively involved in hormone signaling pathways that enhance plant drought tolerance. In soybean, 43 MYB genes are induced by ABA and participate in drought response mechanisms (Liao et al. 2008); similarly, in Arabidopsis, AtMYB2 and AtMYB52 positively regulate drought tolerance via the ABA pathway (Abe et al. 2003). Notably, different hormone signaling pathways often interact, forming a coordinated regulatory network. For example, under drought stress, overexpression of IbMYB48 in sweet potato leads to significant upregulation of genes related to both ABA and jasmonic acid (JA), exemplifying a multi-hormonal coordinated response (Zhao et al. 2022b). Additionally, hormone regulation mediated by MYB transcription factors is frequently associated with ROS scavenging systems. Under drought conditions, silencing of XsMYB44 in Xanthoceras sorbifolia results in decreased expression of antioxidant genes such as XsPOD and XsSOD , elevated levels of ROS and malondialdehyde (MDA), and diminished drought tolerance (Li et al. 2021a). Similarly, FtMYB22 participates in drought responses through the ABA signaling pathway, accompanied by dynamic fluctuations in MDA and ROS levels (Zhao et al. 2022a), underscoring the critical role of MYB transcription factors in coordinating hormone signaling and oxidative homeostasis. The role of the MYB family in plant drought resistance has attracted considerable attention. This study employs molecular biology, physiology, and transcriptomics to analyze and elucidate the critical regulatory mechanisms of CsMYB116 in tea plants under drought stress for the first time. The research aims to advance our understanding of the molecular regulatory pathways involved in tea plants' responses to drought and is expected to identify novel targets for improving drought tolerance in tea plants. Furthermore, this study seeks to offer strategies and insights to facilitate the selection of drought-tolerant tea cultivars. Materials and methods Experimental materials and Treatments The plant materials utilized in this study were one-year-old tea seedlings 'ShuchaZao' purchased from Nanjing Yarun Tea Industry Co., Ltd. (36°28' N, 116°82' E). In the drought treatment experiment, 8% PEG6000 solution was used to simulate drought stress, and normal nutrient solution culture was used as the control. Additionally, CsMYB116 overexpression lines and wild-type Arabidopsis thaliana were subjected to natural drought conditions, involving a 10-day period without watering followed by 3 days of rewatering, after which their phenotypic responses were assessed. Bioinformatic Analysis of CsMYB116 Homologous protein sequences of CsMYB116 were retrieved from the NCBI database. A phylogenetic tree was constructed using MEGA11.0, and multiple sequence alignments were conducted with DNAMAN. The amino acid composition, molecular weight, isoelectric point, physicochemical properties, and hydrophobicity of CsMYB116 were analyzed utilizing the ProtParam tool available on the ExPASy platform. The transmembrane regions were predicted using DeepTMHMM, while the secondary structure was inferred through SOPMA. The tertiary structure was modeled via homology using SWISS-MODEL. Furthermore, the presence of a signal peptide was assessed with SignalP4.1, and cis-acting elements within the promoter region were identified based on the Plant CARE database. Visualization of these elements was performed using TBtools software (version 2.210). Determination of Physiological Indices The activities of the enzymes proline oxidase (PRO), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in tea plants and Arabidopsis thaliana were quantified using enzyme activity assay kits (Grish Biotech Co., Ltd., Suzhou, China). Malondialdehyde (MDA) content was determined via the thiobarbituric acid (TBA) colorimetric method (Alatawi et al. 2023). Chlorophyll fluorescence parameters were assessed using a pulse amplitude-modulated fluorometer (FMS-2, Hansatech, UK). Endogenous plant hormones were analyzed by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS, Sciex 4500, USA) (Chen et al. 2023a). Each physiological parameter was measured in triplicate biological replicates. RNA Extraction and Real-time Quantitative PCR (RT-qPCR) Analysis Total RNA was extracted from tea plant leaves utilizing the RNAprep Pure Plant Plus Kit (Tiangen Biotech (Beijing) Co., Ltd., China). The concentration and quality of the RNA were assessed using a NanoDrop 2000 spectrophotometer (Shanghai Yuanxi Instrument Co., Ltd., China) and agarose gel electrophoresis. First-strand complementary DNA (cDNA) was synthesized using total RNA as the template. Gene expression was quantified via real-time fluorescence PCR using the LightCycler 480 II system (Roche, Switzerland). qPCR amplification was performed in a 20 µL reaction volume utilizing ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China). The reaction mixture comprised 10 µL of SYBR Green premix, 0.4 µL each of forward and reverse primers (10 µmol/L), 1 µL of cDNA template, with the remaining volume adjusted using nuclease-free water. The thermal cycling conditions were as follows: initial denaturation at 95°C for 30 seconds; followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing/extension at 60°C for 30 seconds. Relative expression levels of CsMYB116 were calculated using the 2 −ΔΔCt method, with β-Actin serving as the internal reference gene. Each treatment included three biological replicates. Subcellular Localization Analysis of CsMYB116 The coding sequence (CDS) of the CsMYB116 gene was cloned into the plant expression vector pRI101-AN-GFP to generate the recombinant vector 35S:: CsMYB116 -GFP. Both the recombinant plasmid and the empty vector 35S::GFP were introduced into Agrobacterium tumefaciens strain GV3101, which was subsequently used to infiltrate the leaves of robust, four-week-old tobacco plants. Following a 12-hour incubation period in darkness and two days of normal growth, the localization of the fluorescence signal in the transformed tobacco cells was observed using a two-photon laser scanning confocal microscope (LSM880NLO, Carl Zeiss, Germany). Heterologous Overexpression in Arabidopsis The full-length CDS of CsMYB116 was cloned into the pRI101-AN vector to generate the overexpression vector 35S:: CsMYB116 , with the empty vector serving as a negative control. The recombinant plasmid was introduced into Agrobacterium tumefaciens strain GV3101 via the freeze-thaw method and subsequently used to transform Arabidopsis thaliana (Col-0) wild-type (WT) plants through the floral dip technique. T1 seeds were harvested, surface-sterilized, and sown on Murashige and Skoog (MS) medium containing kanamycin to select for positive transformants. Selected plants were transplanted into soil to obtain T2 and T3 generations. Homozygous T3 lines overexpressing CsMYB116 were confirmed by DNA analysis and RT-qPCR analysis, and lines exhibiting high expression levels were chosen for further validation and analysis. CsMYB116 Gene Silencing A specific fragment of CsMYB116 , approximately 300 bp was amplified and inserted into the virus-induced gene silencing (VIGS) vector pTRV2 to generate the silencing vector pTRV2- CsMYB116 . The empty pTRV2 vector served as a negative control. The pTRV1 vector was co-transformed with either the empty pTRV2 vector or pTRV2- CsMYB116 into Agrobacterium tumefaciens strain GV3101. The activated bacterial suspensions were mixed in a 1:1 ratio. Healthy tea seedlings exhibiting robust axillary buds and two to three true leaves were selected for vacuum infiltration. Following inoculation, the plants were maintained in darkness for four hours before being transferred to light conditions for continued growth. Four weeks post-inoculation, gene silencing efficiency was assessed by RT-qPCR, followed by subsequent validation analyses. Transcriptome analysis The drought stress treatment was administered to CsMYB116 gene-silenced lines, followed by the extraction of total RNA from tea plant leaves. The RNA samples were subsequently submitted to Oebiotech Co., Ltd. (Shanghai, China) for transcriptome sequencing. Transcriptome assembly was carried out using the genome of 'ShuchaZao' as a reference, and all subsequent analyses were based on this assembly. Yeast two-hybrid (Y2H) assay The full-length CDS of CsMYB116 was cloned into the pGBKT7 vector to generate the bait plasmid pGBKT7- CsMYB116 . This plasmid was subsequently transformed into Y2HGold yeast competent cells, which were then plated on selective media, including SD/-Trp, SD/-Trp supplemented with X-α-Gal, and SD/-Trp supplemented with both X-α-Gal and 200 ng/mL Aureobasidin A (AbA). The plates were incubated at 30°C for 2 to 3 days to assess the development of blue coloration. To validate protein–protein interactions, the bait plasmid pGBKT7- CsMYB116 was co-transformed with prey plasmids into the Y2HGold yeast strain. The transformants were initially selected on SD/-Trp/-Leu double dropout (DDO) medium. Subsequently, positive clones were streaked onto quadruple dropout (QDO) medium (SD/-Trp/-Leu/-His/-Ade) supplemented with X-α-gal and AbA to confirm the interactions. All plates were incubated at 30°C for 3 to 5 days, after which the results were documented through observation and photography. Statistical analysis Physiological data were analyzed using SPSS v23.0, and significant differences between treatments were determined by one-way analysis of variance (ANOVA) ( p < 0.05). GraphPad Prism 8.0 software was employed for graphical representation, and all data were derived from three biological replicates. Transcriptome data were analyzed to identify differentially expressed genes (DEGs) using DESeq2 v1.22.1, applying a corrected p-value < 0.05 and |log2FoldChange| ≥ 1 as criteria for significance. Principal component analysis (PCA) plots, volcano plots, as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment maps were generated using the OE Biotech cloud platform ( https://cloud.oebiotech.cn ). Results Screening and bioinformatics analysis of drought resistance genes In this study, eighteen genes exhibiting significant upregulation were selected from a previously established transcriptome database for RT-qPCR validation. The results demonstrated that the expression level of CsMYB116 was markedly higher than that of the other genes analyzed (Fig. 1 a). Consequently, CsMYB116 was selected for further functional characterization. Physicochemical analysis of the CsMYB116 protein indicated that it comprises 273 amino acids, with a molecular weight of 31.04 kDa and a theoretical isoelectric point (pI) of 8.59. The grand average of hydropathy (GRAVY) value is -0.542, suggesting that the protein is hydrophilic, containing a greater proportion of hydrophilic amino acids relative to hydrophobic ones (Fig. 1 b). The instability index of CsMYB116 is 56.84, classifying it as an unstable protein. Multiple sequence alignment of homologous proteins revealed that CsMYB116 contains two conserved SANT/MYB domains, thereby categorizing it as an R2R3-MYB transcription factor (Fig. S1 ). Phylogenetic analysis further indicated that CsMYB116 shares high sequence homology with R2R3-MYB transcription factors from various plant species, exhibiting the closest relationship to Vitis vinifera (Fig. 1 c). Moreover, prediction analyses of signal peptides and transmembrane domains within the CsMYB116 amino acid sequence identified the absence of both transmembrane helices (Fig. 1 d) and signal peptides (Fig. 1 e). Secondary structure prediction revealed that the CsMYB116 protein predominantly comprises random coils and extended strands, alongside α-helices (Fig. 1 f). The predicted tertiary structure corresponded well with the secondary structure model (Fig. 1 g). Analysis of cis-acting elements within the 2000 bp upstream promoter region identified motifs associated with defense and stress responses, light responsiveness, anaerobic induction, as well as auxin- and jasmonate-related regulatory elements (Fig. 1 h). Expression characteristics and subcellular localization of CsMYB116 gene The expression of CsMYB116 was analyzed at various time points under drought stress, revealing an initial increase followed by a decrease, with peak expression observed at 48 hours (Fig. 2 a). To further elucidate the subcellular localization of CsMYB116 , GFP-tagged CsMYB116 constructs were transiently expressed in tobacco epidermal cells. The 35S:: CsMYB116 -GFP fusion protein was exclusively localized to the nucleus of tobacco mesophyll cells. In addition, the strong colocalization with DAPI staining signals corroborated these findings (Fig. 2 b). Effects of heterologous overexpression of CsMYB116 on plants under drought stress To elucidate the regulatory function of CsMYB116 in plant responses to drought stress, this study generated Arabidopsis thaliana lines overexpressing CsMYB116 . Integration of the exogenous CsMYB116 gene into the transgenic Arabidopsis genome was confirmed by DNA gel electrophoresis (Fig. S2 ). Subsequently, RNA was extracted from the overexpressing plants for qPCR validation (Fig. 2 c), and two overexpression lines (OE2 and OE7) were selected for drought tolerance assays. Under standard MS medium conditions (Fig. 2 d), no significant differences in primary root length were observed between wild-type (WT) and transgenic plants. However, under drought-mimicking conditions induced by 100 mM and 200 mM mannitol, the primary roots of OE2 and OE7 were significantly shorter than those of WT ( p < 0.05) (Fig. 2 e), whereas the number of lateral roots was significantly increased ( p < 0.05) (Fig. 2 f). These findings indicate that CsMYB116 enhances drought tolerance in plants by modulating root system architecture. After 10 days of drought treatment, wild-type (WT) plants exhibited pronounced wilting and leaf chlorosis, whereas OE2 and OE7 transgenic lines maintained relatively robust growth. Three days post-rehydration, the growth performance of OE2 and OE7 plants surpassed that of WT plants (Fig. 2 g). Moreover, after drought stress, the Fv/Fm ratio (Fig. 2 h) and PSII efficiency (Fig. 2 i) in OE2 and OE7 plants were significantly higher than those observed in WT plants, with the electron transport rate (ETR) remaining elevated (Fig. 2 j). Additionally, the MDA content was significantly lower in OE2 and OE7 plants compared with WT plants (Fig. 2 k). Enzyme activity assays showed that the activities of CAT (Fig. 2 l), POD (Fig. 2 m), and SOD (Fig. 2 n) in OE2 and OE7 plants were significantly increased under drought stress. Both the Pro (Proline) content (Fig. 2 o) and RWC (Relative Water Content) (Fig. 2 p) were significantly higher in OE2 and OE7 plants than in WT plants. Moreover, the chlorophyll content of OE2 and OE7 plants remained at a high level after drought treatment (Fig. 2 q). Collectively, these results indicate that overexpression of CsMYB116 confers enhanced drought tolerance by improving light conversion efficiency, increasing the activity of the antioxidant enzyme system and osmotic adjustment capacity, and sustaining leaf water potential over an extended period. Effects of transient silencing CsMYB116 on physiological metabolism of tea seedlings under drought stress To further investigate the role of CsMYB116 under drought stress, a TRV-mediated transient gene silencing system was utilized to suppress CsMYB116 expression in tea plants, resulting in a significant decrease in CsMYB116 expression levels ( p < 0.05) (Fig. 3 a). Compared to the control group, CsMYB116 silencing markedly impaired drought tolerance, as demonstrated by leaf wilting and pronounced growth inhibition in TRV-treated plants subjected to drought conditions (Fig. 3 b). Furthermore, the silenced plants exhibited significantly reduced Fv/Fm, PSII efficiency, and ETR relative to controls (Fig. 3 c- 3 e) ( p < 0.05). Activities of the antioxidant enzymes CAT, POD, and SOD were also significantly diminished in silenced plants under drought stress (Fig. 3 f- 3 h) ( p < 0.05). Collectively, these findings suggest that CsMYB116 silencing compromises photosystem protection and antioxidant defense mechanisms in tea seedlings, thereby diminishing their drought tolerance. The results of hormone analysis revealed that silencing CsMYB116 significantly altered the levels of various endogenous hormones. Compared to CK, the concentrations of jasmonic acid (JA) (Fig. 3 i), salicylic acid (SA) (Fig. 3 j), jasmonic acid-isoleucine (JA-Ile) (Fig. 3 k), abscisic acid (ABA) (Fig. 3 l), 12-oxo-phytodienoic acid (OPDA) (Fig. 3 m), and indoleacetic acid (IAA) (Fig. 3 n) were markedly elevated in TRV plants. These hormones typically accumulate in substantial amounts under severe stress conditions, suggesting that CsMYB116 -silenced plants experience increased stress during drought. Regarding gibberellin metabolism, the levels of GA3 (Fig. 3 o), GA7 (Fig. 3 p), and GA19 (Fig. 3 q) were significantly increased in the silenced plants. Conversely, the concentrations of biologically active gibberellins GA1 (Fig. 3 r) and GA4 (Fig. 3 s) were significantly reduced, with GA44 (Fig. 3 t) levels also notably decreased. These findings suggest that CsMYB116 silencing does not inhibit the early stages of gibberellin biosynthesis but may impair the conversion efficiency from gibberellin precursors to their active forms, resulting in insufficient accumulation of active gibberellins. This disruption in gibberellin metabolism corresponds with the observed growth inhibition phenotype of silenced plants under drought stress. In summary, the hormone profile of plants following CsMYB116 silencing is characterized by generally elevated stress-related hormones and reduced levels of active gibberellins, which likely compromises the maintenance of normal physiological homeostasis during drought and consequently diminishes drought resistance. Effects of transient silencing of CsMYB116 on transcription of tea seedlings under drought stress Transcriptome sequencing was conducted to investigate TRV-silenced tea seedlings. RT-qPCR validation was conducted on 15 randomly selected differentially expressed genes (DEGs) (Fig. 4 a). The results demonstrated a strong correlation between the expression patterns of the validated genes and the FPKM values derived from the RNA sequencing data (R 2 = 0.85377) (Fig. 4 b), thereby confirming the reliability of the transcriptomic dataset. Principal component analysis (PCA) revealed a clear distinction between CK (control) and TRV-treated samples, indicating pronounced alterations in gene expression within leaves following CsMYB116 silencing (Fig. 4 c). Differentially expressed genes (DEGs) were depicted using a volcano plot, revealing 4,042 upregulated and 1,876 downregulated genes in the silenced seedlings (Fig. 4 d; Table S1 ). GO enrichment analysis indicated that these DEGs were significantly overrepresented across the Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) (Fig. 4 e). In total, the DEGs corresponded to 2,341 GO terms, with upregulated genes associated with 1,925 terms and downregulated genes with 1,254 terms (Table S2 ). Notably, within the BP category, pathways such as cell wall organization, plant secondary cell wall biogenesis, and xylan biosynthetic processes were significantly enriched. In the CC category, the extracellular region, plasma membrane, and plant cell wall were prominently represented. Within the MF category, DEGs were enriched in activities including O-acetyltransferase activity, microtubule binding, and endo-1,3-beta-D-glucanase activity (Table S3). These results suggest that CsMYB116 may directly or indirectly influence cell wall-related pathways, thereby modulating plant drought resistance. KEGG enrichment analysis revealed that differentially expressed genes (DEGs) were predominantly enriched in pathways related to ABC transporters (ko02010), plant hormone signal transduction (ko04075), starch and sucrose metabolism (ko00500), phenylpropanoid biosynthesis (ko00940), and motor protein-related pathways (ko03070) (Fig. 4 f; Table S4-S6). Among them, the most differentially expressed genes were starch and sucrose metabolism pathways. Some enzyme genes involved in the regulation of glucose metabolism (such as TPS1 , TPPA , TPPJ and fructose / hexokinase) were significantly up-regulated, and a variety of key hydrolases involved in starch and sucrose degradation (including AMY1.1 , BGLU42 , BGLU12 , DPEP and some BAM and INV genes) were significantly down-regulated. Additionally, the expression of WAXY and certain members of the AGPS gene family, which are involved in starch synthesis, was also suppressed. In this experiment, we identified significant alterations in 45 genes associated with the plant hormone signal transduction pathway, of which 35 were up-regulated and 10 were down-regulated. These genes are involved in the signaling pathways of ABA, auxin, cytokinin, gibberellin, brassinolide, and jasmonic acid. Within the abscisic acid-mediated signaling pathway, the receptor gene PYL4 and the downstream transcription factor ABI5 were down-regulated, whereas DPBF2 was up-regulated. In the auxin-mediated signal transduction pathway, 25 genes-including IAA/Aux , SAUR50 , LAX , AUX22 , and ARG7 -were predominantly up-regulated, indicating overall activation of auxin signaling. Regarding the cytokinin pathway, CYCD3-1 and AHP4 exhibited up-regulation, while members of the ARR gene family showed both up- and down-regulation. In the gibberellin signaling pathway, regulatory factors SLRL1 and GAI1 were up-regulated, whereas the GA receptor GID1B was down-regulated. Key genes involved in jasmonic acid signaling, COI1 and JAR6 , were down-regulated, while components of the brassinolide signaling pathway, including BZR1 , kinase SRK2A , and XTH1 , were up-regulated. Additionally, the down-regulation of the TGA1 transcription factor family may influence the cross-regulation among multiple hormone signaling pathways. Within the motor protein pathway, the majority of differentially expressed genes were up-regulated, notably including numerous members of the tubulin family ( TUBA , TUBB1 , TUBB2 ) and the kinesin family ( KIN5 , KIN7 , KIN12 , KIN14 ). Conversely, the actin gene ACT7 and kinesin gene KIN7O exhibited down-regulation. In the phenylpropanoid metabolic pathway, which is associated with structural defense, a total of 36 genes displayed altered expression. Although several peroxidases (e.g., PER12 , PER47 , PER72 ), involved in the oxidation and polymerization of lignin monomers and aromatic compounds, as well as genes related to ester synthesis, were significantly up-regulated, the expression of the rate-limiting enzyme PAL and the branch-regulating enzyme HCT1 in the pathway, was down-regulated. This down-regulation likely constrained the synthesis of pathway precursors. Within the ABC transporter pathway, a total of 32 genes exhibited significant differential expression, with 20 genes upregulated and 12 genes downregulated. Specifically, in the ABCB subfamily, the genes ABCB1 , ABCB2 , ABCB11 , ABCB13 , and ABCB19 were significantly upregulated. In the ABCG subfamily, sterol transporter genes ABCG8 , ABCG11 , ABCG12 , ABCG14 , and the defense-related gene ABCG32 were upregulated; however, both upregulated and downregulated genes were observed in ABCG7 and ABCG34 , while the stratum corneum lipid secretion gene ABCG31 was downregulated. All genes within the ABCC subfamily, including ABCC10 and ABCC3 , were downregulated. Additionally, the PDR2 gene in the PDR subfamily was significantly downregulated. The bidirectional regulation of ABCG7 , coupled with the downregulation of PDR2 , may further influence the drought adaptability of tea plants, underscoring the complex and dynamic regulation of ABC transporters in transmembrane material transport. In summary, CsMYB116 functions as a regulatory factor that is essential for coordinating energy distribution, hormone signaling, cell wall reinforcement, and the optimization of material transport by directly or indirectly modulating these critical pathways. The absence of CsMYB116 expression results in the disruption of the entire drought response network, thereby markedly diminishing the drought tolerance of tea plants. Screening and verification of interacting proteins Self-activation verification revealed that Y2H yeast cells transfected with the BD- CsMYB116 vector were able to grow normally on SD/-Trp medium and exhibited growth with blue coloration on SD/-Trp supplemented with X-α-gal medium. This observation indicates that CsMYB116 fused to the BD vector exhibits self-activation activity. Furthermore, the addition of 200 ng/mL AbA to the medium effectively inhibited yeast growth, demonstrating that this concentration of AbA can suppress the self-activation of BD- CsMYB116 (Fig. 5 a). Yeast library screening was conducted using the BD- CsMYB116 vector following the suppression of self-activation. Subsequent colony PCR and sequencing analyses identified 38 candidate proteins. To further validate the interactions between these candidates and CsMYB116 , proteins associated with drought resistance functions, including ABC transporters and auxin-related proteins, were selected for yeast two-hybrid point-to-point verification. The results demonstrated that yeast strains co-transformed with CsMYB116 -BD and each candidate protein-AD grew robustly on selective media lacking Trp, Leu, His, and Ade, and exhibited a pronounced blue coloration on X-α-gal plates. In contrast, the negative controls failed to grow on selective media and showed no color development (Fig. 5 b), confirming the specific interaction between CsMYB116 and these proteins in yeast. These findings suggest that CsMYB116 may modulate the drought response in tea plants through interactions with transporters and proteins involved in auxin metabolism. Discussion Plant adaptation to drought stress constituted a complex physiological process governed by multiple regulatory networks. In the present study, the pivotal role of the tea plant transcription factor CsMYB116 in mediating drought stress responses was systematically elucidated for the first time through an integrative approach combining physiological assessments, molecular biology techniques, and transcriptomic analyses. Our findings consistently demonstrated that CsMYB116 , a nuclear-localized positive regulatory transcription factor, enhanced drought tolerance in tea plants by modulating root architecture, antioxidant defense mechanisms, photosynthetic protection, and hormone signaling pathways. Root systems constituted the principal organs through which plants detected and responded to soil drought stress (Yang et al. 2024). Prior studies had revealed that, under conditions of heterogeneous soil moisture or drought stress, many plant species adopted a strategy characterized by a reduction in lateral root branching coupled with the promotion of primary root elongation into deeper soil strata, thereby enhancing drought tolerance (Hazman and Brown 2018). For example, maize demonstrated diminished lateral root branching to facilitate deeper penetration of the primary root (Zhan et al. 2015). In contrast, certain species improved water uptake and drought resilience by suppressing longitudinal growth of the primary root while stimulating lateral root proliferation (Karlova et al. 2021; Shoaib et al. 2022). Specifically, overexpression of PtrYY1 ( PtrYY1 -OE) under drought conditions had been shown to promote lateral root growth and development in poplar (Sun et al. 2023a). Moreover, an increase in lateral root density had been correlated with enhanced plant responses to water deficit and alterations in yield components (Placido et al. 2020). Under drought stress, transgenic Arabidopsis plants overexpressing PvMLP19 exhibited a significant augmentation in lateral root number and root system remodeling, culminating in improved stress tolerance (Yerlikaya et al. 2025). In the current investigation, Arabidopsis plants overexpressing CsMYB116 displayed inhibited primary root elongation concomitant with a pronounced increase in lateral root number under drought conditions. Furthermore, an interaction between CsMYB116 and auxin (IAA)-related proteins was identified, with IAA recognized as a pivotal hormone directly regulating root system development. These results implied that CsMYB116 enhanced drought tolerance by modulating auxin metabolism to regulate lateral root formation. Collectively, the data indicated that CsMYB116 mediated root system remodeling and augmented drought resistance by increasing lateral root density, thereby expanding the absorptive surface area within the upper soil layers. Drought stress frequently resulted in an accumulation of excess light energy within plants, leading to PSII and the generation of reactive oxygen species (ROS), which subsequently induced oxidative damage (Sami et al. 2021). To mitigate the detrimental effects of drought, numerous plant species enhanced the biosynthesis of antioxidant enzymes, thereby reducing oxidative stress and alleviating drought-induced damage (Kosar et al. 2021). In the present study, transgenic plants overexpressing CsMYB116 exhibited significantly higher values of Fv/Fm, PSII efficiency, and ETR following drought exposure, concomitant with a marked reduction in MDA content relative to wild-type controls. These findings demonstrated that CsMYB116 conferred substantial protection to the photosynthetic apparatus and cellular membranes under drought conditions. Crucially, this protective effect was closely associated with the upregulation of a robust antioxidant defense system, as evidenced by the pronounced increases in CAT, POD, and SOD activities observed in the overexpressing lines. This mechanism aligned with the observations of Sun et al. (2025), who reported that AtMYB37 mitigated drought-induced photosynthetic inhibition in Arabidopsis by modulating the expression of genes involved in ROS metabolism, thereby enhancing drought tolerance. Such evidence suggested that MYB transcription factors across diverse species might share conserved roles in protecting the photosynthetic machinery from oxidative damage. Similarly, MYB transcription factors such as IbMYB116 in sweet potato (Zhou et al. 2019) and AgMYB5 in celery (Sun et al. 2023b) had been shown to regulate antioxidant and ROS scavenging-related genes, reducing ROS accumulation under drought stress and improving drought resilience. Conversely, CsMYB116 -silenced plants in this study displayed significantly diminished antioxidant enzyme activities and reduced drought tolerance. Collectively, these results implied that CsMYB116 might directly or indirectly orchestrate an effective ROS scavenging system, thereby preserving the structural integrity and functional capacity of photosynthetic membranes, which constituted a fundamental physiological basis for enhanced drought tolerance. Plant hormones served as fundamental signaling molecules that modulated plant responses to drought stress (Liao et al. 2025). Notably, ABA, JA along with its bioactive conjugate JA-Ile, SA, as well as auxins and gibberellins, collectively constituted a complex and highly interactive hormonal regulatory network (Gao et al. 2024; Musazade et al. 2025). Prior research had demonstrated that elevated hormone concentrations under drought conditions did not inherently confer enhanced drought tolerance; rather, their functional efficacy was contingent upon the maintenance of hormonal homeostasis and the proper activation of downstream signaling cascades (Xiao et al. 2025). In the present study, however, we observed that CsMYB116 -silenced plants exhibited significant accumulation of ABA, JA, JA-Ile, SA, and OPDA under drought stress, yet these plants manifested pronounced wilting, damage to the photosynthetic apparatus, and diminished antioxidant capacity. This ostensibly paradoxical outcome contrasted with the established role of CsMYB116 as a positive regulator. This phenomenon was attributed to the excessive accumulation of endogenous hormones resulting from a heightened stress burden, rather than being an indication of improved stress adaptability (Fatma et al. 2022; Lamarque et al. 2020; Saleem et al. 2021). Specifically, although ABA was recognized as a central hormone in drought response, its overaccumulation or dysregulation of signaling pathways frequently led to growth inhibition and attenuated drought tolerance (Finkelstein, 2013). Supporting this, transcriptomic analyses revealed downregulation of the ABA receptor gene PYL4 and the key transcription factor ABI5 following CsMYB116 silencing, which suggested impaired ABA signal perception and transduction. Consequently, the elevated ABA levels exerted minimal protective effects, thereby compromising the hormone’s role in drought resistance. Furthermore, within the JA and SA metabolic pathways, despite a marked accumulation of JA, JA-Ile, and their precursor OPDA following the silencing of CsMYB116 , the observed downregulation of key JA signaling components, namely COI1 and JAR6, indicated a constrained JA signal transduction. This limitation may subsequently have influenced JA metabolism. Prior research had demonstrated that excessive or aberrant activation of JA signaling could disrupt abscisic acid (ABA)-mediated drought responses and potentially heighten stress sensitivity (Fu et al. 2017). Concurrently, the pronounced elevation in SA levels may further have antagonized both ABA and JA signaling pathways, resulting in the over accumulation of ROS and consequent impairment of cellular homeostasis (Elsisi et al. 2024; Moeder et al. 2010). Notably, CsMYB116 silencing also led to a significant increase in auxin concentrations, accompanied by substantial upregulation of genes within the IAA/Aux, SAUR, and LAX families. The activation of these auxin-related compounds and gene families may have intensified energy expenditure and water requirements, thereby diminishing drought tolerance (Li et al. 2021b). This observation suggested that CsMYB116 may have functioned as a negative regulator of auxin expression and accumulation. Simultaneously, alterations in gibberellin metabolism and signaling were evident, characterized by reduced levels of bioactive gibberellins alongside upregulated expression of signaling repressors, indicative of a disrupted balance between growth and stress response mechanisms (Liao et al. 2023). Collectively, these findings demonstrated that silencing CsMYB116 induced a state of “high accumulation but low efficiency” across multiple phytohormones, thereby perturbing the hormonal regulatory network and culminating in a comprehensive decline in antioxidant defenses, photosystem stability, and drought resistance. MYB transcription factors played a critical role in regulating secondary metabolic pathways, including phenylpropanoid metabolism (Pratyusha and Sarada 2022). Transcriptomic analysis conducted in this study demonstrated that silencing of CsMYB116 within the phenylpropanoid biosynthesis pathway resulted in the upregulation of downstream peroxidase genes, accompanied by the downregulation of key rate-limiting enzymes, PAL and HCT1. This regulatory shift constrained the biosynthesis of lignin and other stress-associated secondary metabolites (Aluko et al. 2025), thereby compromising the structural defense mechanisms of tea plants. Furthermore, under drought stress, plant starch reserves could be hydrolyzed into soluble sugars, which served dual functions as growth substrates and as osmotic regulators and antioxidants that alleviated drought-induced damage (Moran et al. 2017; Khan et al. 2021). Within the starch and sucrose metabolism pathway, CsMYB116 silencing led to a marked upregulation of genes encoding key enzymes involved in sugar signaling and metabolic regulation, while several hydrolases responsible for starch and sucrose degradation were significantly downregulated. Concurrently, the expression of WAXY and certain members of the AGPS family, which were implicated in starch biosynthesis, was also suppressed. These findings suggested a disruption in the dynamic balance of carbon allocation among synthesis, degradation, and signaling processes, potentially resulting in an inadequate supply of energy and osmotic regulators (Chen et al. 2024). Consequently, this imbalance impaired the plant’s capacity to sustain metabolic homeostasis under drought conditions. Furthermore, this investigation identified a potential functional relationship between CsMYB116 and ABC transporters. ABC transporters were extensively implicated in the transmembrane movement of hormones (such as ABA), lipids, and secondary metabolites, serving as critical components in maintaining cellular compartmentalization and facilitating the targeted distribution of substances (Huang et al. 2021; Lefevre and Boutry 2018). Transcriptomic analyses demonstrated a significant enrichment of differentially expressed genes within the ABC transporter pathway, with pronounced alterations observed in defense-associated members of the ABCB and ABCG subfamilies. These findings suggested that CsMYB116 modulated the transmembrane transport and allocation of lipids, hormones, or secondary metabolites by influencing ABC transporter activity (Wang et al. 2020; Zhang et al. 2025). Notably, the bidirectional regulation of ABCG7 alongside the downregulation of PDR2 constrained the effective intercellular or intertissue transport of key defense compounds (Gupta et al. 2019). Concurrently, the pervasive upregulation of cytoskeleton-related genes coupled with the downregulation of ACT7 indicated a perturbation in cytoskeletal dynamics, which impacted cellular expansion, material transport, and mechanical integrity (Chun et al. 2021). Moreover, yeast two-hybrid assays provided direct evidence of physical interactions between CsMYB116 and ABC transporters, implying that the regulatory influence of CsMYB116 extended beyond transcriptional control of ABC transporter genes to potentially modulating transporter activity or stability via protein-protein interactions. Collectively, these results proposed that CsMYB116 exerted a nuanced regulatory function in the transmembrane transport of hormones, secondary metabolites, or ions, thereby introducing a novel regulatory dimension to its role in plant drought stress responses. Conclusion This study systematically demonstrated, for the first time, that CsMYB116 regulated drought resistance in tea plants by coordinating multiple biological processes, including photosystem protection, reactive oxygen species scavenging, hormone homeostasis restoration, cell wall modification, and ABC transporter-mediated transmembrane transport. These findings not only enhanced the molecular-level understanding of the ‘growth-defense’ trade-off mechanism in tea plant stress adaptation but also identified a pleiotropic key target for molecular breeding aimed at improving tea plant stress resistance. Base on these results, future research will focus on constructing a genome-wide target map of CsMYB116 , analyzing its transcriptional regulatory network, and validating its interactions and functions with key factors to further elucidate the regulatory mechanisms underlying drought resistance. Declarations Acknowledgements This work was supported by the National Natural Science Foundation of China (32573088; 32302437). Author contributions Xiaoyang Han designed this study. Xiaoxia Zhao wrote the main manuscript text guided by Xiaoyang Han and prepared Figures and Talbes. Xinhan You, Wenjuan Ma, Xueying Xie performed the data analysis. Jian Hou provided test materials. Data availability The database will be provided on request. Conflict of interest The authors have no conflicts of interest to disclose. References Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. 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08:10:34","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107757,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8519228/v1/86536dc744f2398260898d94.html"},{"id":100248910,"identity":"15ca7685-26ce-4171-8874-edc340e9fa21","added_by":"auto","created_at":"2026-01-14 14:41:09","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":400429,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of drought-resistant genes and bioinformatics analysis of \u003cem\u003eCsMYB116\u003c/em\u003e. \u003cstrong\u003ea\u003c/strong\u003e, Relative expression levels of 18 up-regulated genes selected from previous transcriptome data under drought stress, with \u003cem\u003eCsMYB116\u003c/em\u003e exhibiting the highest expression level; \u003cstrong\u003eb\u003c/strong\u003e, Analysis of protein physicochemical properties (hydrophilicity/hydrophobicity); \u003cstrong\u003ec\u003c/strong\u003e, Phylogenetic tree of \u003cem\u003eCsMYB116\u003c/em\u003ewith R2R3-MYB transcription factors from different plant species; \u003cstrong\u003ed\u003c/strong\u003e, Prediction analysis of transmembrane domains; \u003cstrong\u003ee\u003c/strong\u003e, Prediction analysis of signal peptides; f, Prediction of the secondary structure of the \u003cem\u003eCsMYB116\u003c/em\u003eprotein; g, Tertiary structure of the \u003cem\u003eCsMYB116\u003c/em\u003e protein obtained through homology modeling; \u003cstrong\u003eh\u003c/strong\u003e, Distribution of cis-acting elements in the promoter region (2000 bp upstream) of the \u003cem\u003eCsMYB116\u003c/em\u003egene.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8519228/v1/e6876edc5e6b73800a60ae37.jpeg"},{"id":100248911,"identity":"700247ed-bc20-4c9f-a8e3-7644284a0a67","added_by":"auto","created_at":"2026-01-14 14:41:09","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":463800,"visible":true,"origin":"","legend":"\u003cp\u003eExpression pattern and subcellular localization of \u003cem\u003eCsMYB116\u003c/em\u003e, and the effects of its heterologous overexpression on drought resistance in Arabidopsis thaliana. \u003cstrong\u003ea\u003c/strong\u003e, Relative gene expression levels of \u003cem\u003eCsMYB116\u003c/em\u003ein tea plants at different time points under drought stress; \u003cstrong\u003eb\u003c/strong\u003e, Subcellular localization of the \u003cem\u003eCsMYB116\u003c/em\u003e-GFP fusion protein in tobacco epidermal cells (co-localization with the nuclear dye DAPI); \u003cstrong\u003ec\u003c/strong\u003e, Relative expression levels of \u003cem\u003eCsMYB116\u003c/em\u003e in wild-type (WT) and two \u003cem\u003eCsMYB116\u003c/em\u003e-overexpressing lines (OE2, OE7); d-f, Root phenotype differences between WT and overexpressing Arabidopsis lines under normal conditions and mannitol-simulated drought stress: \u003cstrong\u003ed\u003c/strong\u003e, representative root phenotypes; \u003cstrong\u003ee\u003c/strong\u003e, primary root length; \u003cstrong\u003ef\u003c/strong\u003e, number of lateral roots; \u003cstrong\u003eg\u003c/strong\u003e, Phenotypes of WT and overexpressing lines after 10 days of natural drought treatment followed by 3 days of re-watering; \u003cstrong\u003eh\u003c/strong\u003e, Fv/Fm; \u003cstrong\u003ei\u003c/strong\u003e, PSII; \u003cstrong\u003ej\u003c/strong\u003e, ETR; \u003cstrong\u003ek\u003c/strong\u003e, Malondialdehyde (MDA) content; \u003cstrong\u003el\u003c/strong\u003e, Catalase (CAT) activity; \u003cstrong\u003em\u003c/strong\u003e, Peroxidase (POD) activity; \u003cstrong\u003en\u003c/strong\u003e, Superoxide dismutase (SOD) activity; \u003cstrong\u003eo\u003c/strong\u003e, Proline (Pro) content; \u003cstrong\u003ep\u003c/strong\u003e, Leaf relative water content (RWC); \u003cstrong\u003eq\u003c/strong\u003e, Chlorophyll content.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8519228/v1/07339fc828b04d6303b78eb9.jpeg"},{"id":100248914,"identity":"71f408d2-fd94-4200-919e-bf9218dd0d0d","added_by":"auto","created_at":"2026-01-14 14:41:09","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":338524,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of transient silencing of \u003cem\u003eCsMYB116\u003c/em\u003eon the physiological metabolism of tea seedlings under drought stress. \u003cstrong\u003ea\u003c/strong\u003e, Relative expression levels of \u003cem\u003eCsMYB116\u003c/em\u003ein the empty vector control group (CK) and the \u003cem\u003eCsMYB116 \u003c/em\u003esilencing group (TRV); \u003cstrong\u003eb\u003c/strong\u003e, Phenotypes of CK and TRV plants after drought treatment; c-e, Photosynthetic parameters of CK and TRV plants under drought stress: \u003cstrong\u003ec\u003c/strong\u003e, Fv/Fm; \u003cstrong\u003ed\u003c/strong\u003e, PSII; \u003cstrong\u003ee\u003c/strong\u003e, ETR; f-h, Antioxidant enzyme activities of CK and TRV plants under drought stress: \u003cstrong\u003ef\u003c/strong\u003e, CAT; \u003cstrong\u003eg\u003c/strong\u003e, POD; \u003cstrong\u003eh\u003c/strong\u003e, SOD; i-t, Changes in endogenous hormone contents in CK and TRV plants under drought stress: \u003cstrong\u003ei\u003c/strong\u003e, JA; \u003cstrong\u003ej\u003c/strong\u003e, SA; \u003cstrong\u003ek\u003c/strong\u003e, JA‑Ile; \u003cstrong\u003el\u003c/strong\u003e, ABA; \u003cstrong\u003em\u003c/strong\u003e, OPDA; \u003cstrong\u003en\u003c/strong\u003e, IAA; \u003cstrong\u003eo\u003c/strong\u003e, GA3; \u003cstrong\u003ep\u003c/strong\u003e, GA7; \u003cstrong\u003eq\u003c/strong\u003e, GA19; \u003cstrong\u003er\u003c/strong\u003e, GA1; \u003cstrong\u003es\u003c/strong\u003e, GA4; \u003cstrong\u003et\u003c/strong\u003e, GA4.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8519228/v1/2c4ec3d4163ccf2d0f09c46b.jpeg"},{"id":100372407,"identity":"5a329f5b-82bd-49a9-8190-e96dd2cdbe57","added_by":"auto","created_at":"2026-01-16 08:12:16","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":365338,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of transient silencing of \u003cem\u003eCsMYB116\u003c/em\u003eon the transcriptome of tea seedlings under drought stress. \u003cstrong\u003ea\u003c/strong\u003e, RT‑qPCR validation of 15 randomly selected DEGs; \u003cstrong\u003eb\u003c/strong\u003e, Correlation between RT‑qPCR and RNA‑seq (FPKM) fold‑changes (R²=0.85377), confirming transcriptome data reliability; \u003cstrong\u003ec\u003c/strong\u003e, Principal component analysis (PCA) showing the separation between the control group and the silencing group samples; \u003cstrong\u003ed\u003c/strong\u003e, Volcano plot of differentially expressed genes (DEGs) in the CK \u003cem\u003evs.\u003c/em\u003e TRV comparison (up‑regulated: 4,042; down‑regulated: 1,876); \u003cstrong\u003ee\u003c/strong\u003e, GO enrichment analysis (Biological Process, Cellular Component, Molecular Function); \u003cstrong\u003ef\u003c/strong\u003e, KEGG pathway enrichment analysis of DEGs.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8519228/v1/694344b907d40c063349bf81.jpeg"},{"id":100371614,"identity":"cc18bcc9-f1cb-49be-84b5-0da504cab0eb","added_by":"auto","created_at":"2026-01-16 08:10:36","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":158581,"visible":true,"origin":"","legend":"\u003cp\u003eScreening and validation of \u003cem\u003eCsMYB116\u003c/em\u003e-interacting proteins. \u003cstrong\u003ea\u003c/strong\u003e, Autoactivation test of the \u003cem\u003eCsMYB116\u003c/em\u003e-BD fusion protein (SD/-Trp+X-α-gal±AbA); \u003cstrong\u003eb\u003c/strong\u003e, Yeast two-hybrid point-to-point validation of the interaction between CsMYB116 and ABC transporters or auxin-related proteins (SD/-Trp/-Leu/-His/-Ade+X-α-gal).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8519228/v1/e0590e6c20ea22a15bbac086.jpeg"},{"id":100383851,"identity":"a4459441-1b9a-4342-9611-4dcc102cd063","added_by":"auto","created_at":"2026-01-16 10:48:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2727461,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8519228/v1/84dde8ef-21a7-4c86-8d43-a33f1bb9730e.pdf"},{"id":100371384,"identity":"6842448c-c1d1-4b99-a9c7-23619407e501","added_by":"auto","created_at":"2026-01-16 08:09:58","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3656076,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8519228/v1/44324fd357bf5c77ff381e2d.xlsx"},{"id":100248915,"identity":"184205c6-97f1-4529-9eff-1e95b4fbbda3","added_by":"auto","created_at":"2026-01-14 14:41:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":555106,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-8519228/v1/0e2e3c94620fdc69bf4789f3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The function and regulatory mechanism of tea plant transcription factor CsMYB116 in drought stress response","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDrought represents a major abiotic stress factor that constrains plant growth, development, and yield formation (Sato et al. 2024). With the ongoing trend of global warming, the frequency and intensity of drought events are projected to increase (Gebrechorkos et al. 2025). Tea plants (\u003cem\u003eCamellia sinensis\u003c/em\u003e L.), which are characteristically moisture-dependent, exhibit high sensitivity to water availability. Their growth and development (Zheng et al. 2025), secondary metabolism (Shao et al. 2025), and flavor quality (Zhou et al. 2014) are all contingent upon sufficient and stable water supply. Drought conditions not only markedly diminish tea yield and quality but also elevate plant mortality rates (Upadhyaya and Panda 2013). Consequently, a comprehensive understanding of the mechanisms governing tea plants\u0026rsquo; responses to drought stress is essential for maintaining stable tea production and for the development of drought-tolerant cultivars.\u003c/p\u003e \u003cp\u003eWithin the intricate regulatory network governing plant responses to drought and other environmental stresses, transcriptional regulation assumes a pivotal role. The MYB transcription factor family represents one of the largest and most functionally diverse groups in plants (Yuan et al. 2021), with the R2R3-MYB subfamily being particularly prominent due to its extensive involvement in plant growth and development (Dubos et al. 2010), regulation of secondary metabolism (Chen et al. 2019), and responses to various abiotic stresses (Wang et al. 2021). Empirical studies have demonstrated that MYB transcription factors enhance plant drought tolerance by modulating root architecture, hormone signaling pathways, reactive oxygen species (ROS) scavenging systems, and osmotic adjustment mechanisms (Wang et al. 2025). Regarding root development, MYB transcription factors augment water uptake capacity through the regulation of root architecture. For instance, overexpression of the soybean gene \u003cem\u003eGmMYB84\u003c/em\u003e promotes primary root elongation and increases plant survival rates under drought conditions (Wang et al. 2017). In Arabidopsis, \u003cem\u003eAtMYB96\u003c/em\u003e mediates abscisic acid (ABA) signaling and interacts with auxin pathways to regulate root architecture, thereby enhancing drought tolerance (Seo et al. 2009). Comparable findings have been reported in tea plants, where tea polyphenols mediate the regulation of \u003cem\u003eCsPOD44\u003c/em\u003e by \u003cem\u003eCsMYB77\u003c/em\u003e, contributing to improved root drought resistance (Xu et al. 2025). Furthermore, MYB transcription factors are extensively involved in hormone signaling pathways that enhance plant drought tolerance. In soybean, 43 MYB genes are induced by ABA and participate in drought response mechanisms (Liao et al. 2008); similarly, in Arabidopsis, \u003cem\u003eAtMYB2\u003c/em\u003e and \u003cem\u003eAtMYB52\u003c/em\u003e positively regulate drought tolerance via the ABA pathway (Abe et al. 2003). Notably, different hormone signaling pathways often interact, forming a coordinated regulatory network. For example, under drought stress, overexpression of \u003cem\u003eIbMYB48\u003c/em\u003e in sweet potato leads to significant upregulation of genes related to both ABA and jasmonic acid (JA), exemplifying a multi-hormonal coordinated response (Zhao et al. 2022b). Additionally, hormone regulation mediated by MYB transcription factors is frequently associated with ROS scavenging systems. Under drought conditions, silencing of \u003cem\u003eXsMYB44\u003c/em\u003e in Xanthoceras sorbifolia results in decreased expression of antioxidant genes such as \u003cem\u003eXsPOD\u003c/em\u003e and \u003cem\u003eXsSOD\u003c/em\u003e, elevated levels of ROS and malondialdehyde (MDA), and diminished drought tolerance (Li et al. 2021a). Similarly, \u003cem\u003eFtMYB22\u003c/em\u003e participates in drought responses through the ABA signaling pathway, accompanied by dynamic fluctuations in MDA and ROS levels (Zhao et al. 2022a), underscoring the critical role of MYB transcription factors in coordinating hormone signaling and oxidative homeostasis.\u003c/p\u003e \u003cp\u003eThe role of the MYB family in plant drought resistance has attracted considerable attention. This study employs molecular biology, physiology, and transcriptomics to analyze and elucidate the critical regulatory mechanisms of \u003cem\u003eCsMYB116\u003c/em\u003e in tea plants under drought stress for the first time. The research aims to advance our understanding of the molecular regulatory pathways involved in tea plants' responses to drought and is expected to identify novel targets for improving drought tolerance in tea plants. Furthermore, this study seeks to offer strategies and insights to facilitate the selection of drought-tolerant tea cultivars.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental materials and Treatments\u003c/h2\u003e \u003cp\u003eThe plant materials utilized in this study were one-year-old tea seedlings 'ShuchaZao' purchased from Nanjing Yarun Tea Industry Co., Ltd. (36\u0026deg;28' N, 116\u0026deg;82' E). In the drought treatment experiment, 8% PEG6000 solution was used to simulate drought stress, and normal nutrient solution culture was used as the control. Additionally, \u003cem\u003eCsMYB116\u003c/em\u003e overexpression lines and wild-type \u003cem\u003eArabidopsis thaliana\u003c/em\u003e were subjected to natural drought conditions, involving a 10-day period without watering followed by 3 days of rewatering, after which their phenotypic responses were assessed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBioinformatic Analysis of\u003c/b\u003e \u003cb\u003eCsMYB116\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHomologous protein sequences of \u003cem\u003eCsMYB116\u003c/em\u003e were retrieved from the NCBI database. A phylogenetic tree was constructed using MEGA11.0, and multiple sequence alignments were conducted with DNAMAN. The amino acid composition, molecular weight, isoelectric point, physicochemical properties, and hydrophobicity of \u003cem\u003eCsMYB116\u003c/em\u003e were analyzed utilizing the ProtParam tool available on the ExPASy platform. The transmembrane regions were predicted using DeepTMHMM, while the secondary structure was inferred through SOPMA. The tertiary structure was modeled via homology using SWISS-MODEL. Furthermore, the presence of a signal peptide was assessed with SignalP4.1, and cis-acting elements within the promoter region were identified based on the Plant CARE database. Visualization of these elements was performed using TBtools software (version 2.210).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of Physiological Indices\u003c/h3\u003e\n\u003cp\u003eThe activities of the enzymes proline oxidase (PRO), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in tea plants and Arabidopsis thaliana were quantified using enzyme activity assay kits (Grish Biotech Co., Ltd., Suzhou, China). Malondialdehyde (MDA) content was determined via the thiobarbituric acid (TBA) colorimetric method (Alatawi et al. 2023). Chlorophyll fluorescence parameters were assessed using a pulse amplitude-modulated fluorometer (FMS-2, Hansatech, UK). Endogenous plant hormones were analyzed by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS, Sciex 4500, USA) (Chen et al. 2023a). Each physiological parameter was measured in triplicate biological replicates.\u003c/p\u003e\n\u003ch3\u003eRNA Extraction and Real-time Quantitative PCR (RT-qPCR) Analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from tea plant leaves utilizing the RNAprep Pure Plant Plus Kit (Tiangen Biotech (Beijing) Co., Ltd., China). The concentration and quality of the RNA were assessed using a NanoDrop 2000 spectrophotometer (Shanghai Yuanxi Instrument Co., Ltd., China) and agarose gel electrophoresis.\u003c/p\u003e \u003cp\u003eFirst-strand complementary DNA (cDNA) was synthesized using total RNA as the template. Gene expression was quantified via real-time fluorescence PCR using the LightCycler 480 II system (Roche, Switzerland). qPCR amplification was performed in a 20 \u0026micro;L reaction volume utilizing ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China). The reaction mixture comprised 10 \u0026micro;L of SYBR Green premix, 0.4 \u0026micro;L each of forward and reverse primers (10 \u0026micro;mol/L), 1 \u0026micro;L of cDNA template, with the remaining volume adjusted using nuclease-free water. The thermal cycling conditions were as follows: initial denaturation at 95\u0026deg;C for 30 seconds; followed by 40 cycles of denaturation at 95\u0026deg;C for 10 seconds and annealing/extension at 60\u0026deg;C for 30 seconds. Relative expression levels of \u003cem\u003eCsMYB116\u003c/em\u003e were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method, with β-Actin serving as the internal reference gene. Each treatment included three biological replicates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSubcellular Localization Analysis of\u003c/b\u003e \u003cb\u003eCsMYB116\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe coding sequence (CDS) of the \u003cem\u003eCsMYB116\u003c/em\u003e gene was cloned into the plant expression vector pRI101-AN-GFP to generate the recombinant vector 35S::\u003cem\u003eCsMYB116\u003c/em\u003e-GFP. Both the recombinant plasmid and the empty vector 35S::GFP were introduced into Agrobacterium tumefaciens strain GV3101, which was subsequently used to infiltrate the leaves of robust, four-week-old tobacco plants. Following a 12-hour incubation period in darkness and two days of normal growth, the localization of the fluorescence signal in the transformed tobacco cells was observed using a two-photon laser scanning confocal microscope (LSM880NLO, Carl Zeiss, Germany).\u003c/p\u003e\n\u003ch3\u003eHeterologous Overexpression in Arabidopsis\u003c/h3\u003e\n\u003cp\u003eThe full-length CDS of \u003cem\u003eCsMYB116\u003c/em\u003e was cloned into the pRI101-AN vector to generate the overexpression vector 35S::\u003cem\u003eCsMYB116\u003c/em\u003e, with the empty vector serving as a negative control. The recombinant plasmid was introduced into Agrobacterium tumefaciens strain GV3101 via the freeze-thaw method and subsequently used to transform \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Col-0) wild-type (WT) plants through the floral dip technique. T1 seeds were harvested, surface-sterilized, and sown on Murashige and Skoog (MS) medium containing kanamycin to select for positive transformants. Selected plants were transplanted into soil to obtain T2 and T3 generations. Homozygous T3 lines overexpressing \u003cem\u003eCsMYB116\u003c/em\u003e were confirmed by DNA analysis and RT-qPCR analysis, and lines exhibiting high expression levels were chosen for further validation and analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCsMYB116\u003c/b\u003e \u003cb\u003eGene Silencing\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA specific fragment of \u003cem\u003eCsMYB116\u003c/em\u003e, approximately 300 bp was amplified and inserted into the virus-induced gene silencing (VIGS) vector pTRV2 to generate the silencing vector pTRV2-\u003cem\u003eCsMYB116\u003c/em\u003e. The empty pTRV2 vector served as a negative control. The pTRV1 vector was co-transformed with either the empty pTRV2 vector or pTRV2-\u003cem\u003eCsMYB116\u003c/em\u003e into Agrobacterium tumefaciens strain GV3101. The activated bacterial suspensions were mixed in a 1:1 ratio. Healthy tea seedlings exhibiting robust axillary buds and two to three true leaves were selected for vacuum infiltration. Following inoculation, the plants were maintained in darkness for four hours before being transferred to light conditions for continued growth. Four weeks post-inoculation, gene silencing efficiency was assessed by RT-qPCR, followed by subsequent validation analyses.\u003c/p\u003e\n\u003ch3\u003eTranscriptome analysis\u003c/h3\u003e\n\u003cp\u003eThe drought stress treatment was administered to \u003cem\u003eCsMYB116\u003c/em\u003e gene-silenced lines, followed by the extraction of total RNA from tea plant leaves. The RNA samples were subsequently submitted to Oebiotech Co., Ltd. (Shanghai, China) for transcriptome sequencing. Transcriptome assembly was carried out using the genome of 'ShuchaZao' as a reference, and all subsequent analyses were based on this assembly.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eYeast two-hybrid (Y2H) assay\u003c/h2\u003e \u003cp\u003eThe full-length CDS of \u003cem\u003eCsMYB116\u003c/em\u003e was cloned into the pGBKT7 vector to generate the bait plasmid pGBKT7-\u003cem\u003eCsMYB116\u003c/em\u003e. This plasmid was subsequently transformed into Y2HGold yeast competent cells, which were then plated on selective media, including SD/-Trp, SD/-Trp supplemented with X-α-Gal, and SD/-Trp supplemented with both X-α-Gal and 200 ng/mL Aureobasidin A (AbA). The plates were incubated at 30\u0026deg;C for 2 to 3 days to assess the development of blue coloration.\u003c/p\u003e \u003cp\u003eTo validate protein\u0026ndash;protein interactions, the bait plasmid pGBKT7-\u003cem\u003eCsMYB116\u003c/em\u003e was co-transformed with prey plasmids into the Y2HGold yeast strain. The transformants were initially selected on SD/-Trp/-Leu double dropout (DDO) medium. Subsequently, positive clones were streaked onto quadruple dropout (QDO) medium (SD/-Trp/-Leu/-His/-Ade) supplemented with X-α-gal and AbA to confirm the interactions. All plates were incubated at 30\u0026deg;C for 3 to 5 days, after which the results were documented through observation and photography.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003ePhysiological data were analyzed using SPSS v23.0, and significant differences between treatments were determined by one-way analysis of variance (ANOVA) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). GraphPad Prism 8.0 software was employed for graphical representation, and all data were derived from three biological replicates. Transcriptome data were analyzed to identify differentially expressed genes (DEGs) using DESeq2 v1.22.1, applying a corrected p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2FoldChange| \u0026ge; 1 as criteria for significance. Principal component analysis (PCA) plots, volcano plots, as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment maps were generated using the OE Biotech cloud platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.oebiotech.cn\u003c/span\u003e\u003cspan address=\"https://cloud.oebiotech.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eScreening and bioinformatics analysis of drought resistance genes\u003c/h2\u003e \u003cp\u003eIn this study, eighteen genes exhibiting significant upregulation were selected from a previously established transcriptome database for RT-qPCR validation. The results demonstrated that the expression level of \u003cem\u003eCsMYB116\u003c/em\u003e was markedly higher than that of the other genes analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Consequently, \u003cem\u003eCsMYB116\u003c/em\u003e was selected for further functional characterization. Physicochemical analysis of the \u003cem\u003eCsMYB116\u003c/em\u003e protein indicated that it comprises 273 amino acids, with a molecular weight of 31.04 kDa and a theoretical isoelectric point (pI) of 8.59. The grand average of hydropathy (GRAVY) value is -0.542, suggesting that the protein is hydrophilic, containing a greater proportion of hydrophilic amino acids relative to hydrophobic ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The instability index of \u003cem\u003eCsMYB116\u003c/em\u003e is 56.84, classifying it as an unstable protein. Multiple sequence alignment of homologous proteins revealed that \u003cem\u003eCsMYB116\u003c/em\u003e contains two conserved SANT/MYB domains, thereby categorizing it as an R2R3-MYB transcription factor (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Phylogenetic analysis further indicated that \u003cem\u003eCsMYB116\u003c/em\u003e shares high sequence homology with R2R3-MYB transcription factors from various plant species, exhibiting the closest relationship to Vitis vinifera (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Moreover, prediction analyses of signal peptides and transmembrane domains within the \u003cem\u003eCsMYB116\u003c/em\u003e amino acid sequence identified the absence of both transmembrane helices (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) and signal peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Secondary structure prediction revealed that the \u003cem\u003eCsMYB116\u003c/em\u003e protein predominantly comprises random coils and extended strands, alongside α-helices (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). The predicted tertiary structure corresponded well with the secondary structure model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Analysis of cis-acting elements within the 2000 bp upstream promoter region identified motifs associated with defense and stress responses, light responsiveness, anaerobic induction, as well as auxin- and jasmonate-related regulatory elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression characteristics and subcellular localization of\u003c/b\u003e \u003cb\u003eCsMYB116\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe expression of \u003cem\u003eCsMYB116\u003c/em\u003e was analyzed at various time points under drought stress, revealing an initial increase followed by a decrease, with peak expression observed at 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). To further elucidate the subcellular localization of \u003cem\u003eCsMYB116\u003c/em\u003e, GFP-tagged \u003cem\u003eCsMYB116\u003c/em\u003e constructs were transiently expressed in tobacco epidermal cells. The 35S::\u003cem\u003eCsMYB116\u003c/em\u003e-GFP fusion protein was exclusively localized to the nucleus of tobacco mesophyll cells. In addition, the strong colocalization with DAPI staining signals corroborated these findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of heterologous overexpression of\u003c/b\u003e \u003cb\u003eCsMYB116\u003c/b\u003e \u003cb\u003eon plants under drought stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the regulatory function of \u003cem\u003eCsMYB116\u003c/em\u003e in plant responses to drought stress, this study generated Arabidopsis thaliana lines overexpressing \u003cem\u003eCsMYB116\u003c/em\u003e. Integration of the exogenous \u003cem\u003eCsMYB116\u003c/em\u003e gene into the transgenic Arabidopsis genome was confirmed by DNA gel electrophoresis (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Subsequently, RNA was extracted from the overexpressing plants for qPCR validation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), and two overexpression lines (OE2 and OE7) were selected for drought tolerance assays. Under standard MS medium conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), no significant differences in primary root length were observed between wild-type (WT) and transgenic plants. However, under drought-mimicking conditions induced by 100 mM and 200 mM mannitol, the primary roots of OE2 and OE7 were significantly shorter than those of WT (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), whereas the number of lateral roots was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). These findings indicate that \u003cem\u003eCsMYB116\u003c/em\u003e enhances drought tolerance in plants by modulating root system architecture.\u003c/p\u003e \u003cp\u003eAfter 10 days of drought treatment, wild-type (WT) plants exhibited pronounced wilting and leaf chlorosis, whereas OE2 and OE7 transgenic lines maintained relatively robust growth. Three days post-rehydration, the growth performance of OE2 and OE7 plants surpassed that of WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Moreover, after drought stress, the Fv/Fm ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh) and PSII efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei) in OE2 and OE7 plants were significantly higher than those observed in WT plants, with the electron transport rate (ETR) remaining elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). Additionally, the MDA content was significantly lower in OE2 and OE7 plants compared with WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek). Enzyme activity assays showed that the activities of CAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el), POD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003em), and SOD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003en) in OE2 and OE7 plants were significantly increased under drought stress. Both the Pro (Proline) content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eo) and RWC (Relative Water Content) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ep) were significantly higher in OE2 and OE7 plants than in WT plants. Moreover, the chlorophyll content of OE2 and OE7 plants remained at a high level after drought treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eq). Collectively, these results indicate that overexpression of \u003cem\u003eCsMYB116\u003c/em\u003e confers enhanced drought tolerance by improving light conversion efficiency, increasing the activity of the antioxidant enzyme system and osmotic adjustment capacity, and sustaining leaf water potential over an extended period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of transient silencing\u003c/b\u003e \u003cb\u003eCsMYB116\u003c/b\u003e \u003cb\u003eon physiological metabolism of tea seedlings under drought stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further investigate the role of \u003cem\u003eCsMYB116\u003c/em\u003e under drought stress, a TRV-mediated transient gene silencing system was utilized to suppress \u003cem\u003eCsMYB116\u003c/em\u003e expression in tea plants, resulting in a significant decrease in \u003cem\u003eCsMYB116\u003c/em\u003e expression levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Compared to the control group, \u003cem\u003eCsMYB116\u003c/em\u003e silencing markedly impaired drought tolerance, as demonstrated by leaf wilting and pronounced growth inhibition in TRV-treated plants subjected to drought conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Furthermore, the silenced plants exhibited significantly reduced Fv/Fm, PSII efficiency, and ETR relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Activities of the antioxidant enzymes CAT, POD, and SOD were also significantly diminished in silenced plants under drought stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Collectively, these findings suggest that \u003cem\u003eCsMYB116\u003c/em\u003e silencing compromises photosystem protection and antioxidant defense mechanisms in tea seedlings, thereby diminishing their drought tolerance.\u003c/p\u003e \u003cp\u003eThe results of hormone analysis revealed that silencing \u003cem\u003eCsMYB116\u003c/em\u003e significantly altered the levels of various endogenous hormones. Compared to CK, the concentrations of jasmonic acid (JA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei), salicylic acid (SA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej), jasmonic acid-isoleucine (JA-Ile) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek), abscisic acid (ABA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el), 12-oxo-phytodienoic acid (OPDA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em), and indoleacetic acid (IAA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003en) were markedly elevated in TRV plants. These hormones typically accumulate in substantial amounts under severe stress conditions, suggesting that \u003cem\u003eCsMYB116\u003c/em\u003e-silenced plants experience increased stress during drought. Regarding gibberellin metabolism, the levels of GA3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eo), GA7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ep), and GA19 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eq) were significantly increased in the silenced plants. Conversely, the concentrations of biologically active gibberellins GA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003er) and GA4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003es) were significantly reduced, with GA44 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003et) levels also notably decreased. These findings suggest that \u003cem\u003eCsMYB116\u003c/em\u003e silencing does not inhibit the early stages of gibberellin biosynthesis but may impair the conversion efficiency from gibberellin precursors to their active forms, resulting in insufficient accumulation of active gibberellins. This disruption in gibberellin metabolism corresponds with the observed growth inhibition phenotype of silenced plants under drought stress. In summary, the hormone profile of plants following \u003cem\u003eCsMYB116\u003c/em\u003e silencing is characterized by generally elevated stress-related hormones and reduced levels of active gibberellins, which likely compromises the maintenance of normal physiological homeostasis during drought and consequently diminishes drought resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of transient silencing of\u003c/b\u003e \u003cb\u003eCsMYB116\u003c/b\u003e \u003cb\u003eon transcription of tea seedlings under drought stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTranscriptome sequencing was conducted to investigate TRV-silenced tea seedlings. RT-qPCR validation was conducted on 15 randomly selected differentially expressed genes (DEGs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The results demonstrated a strong correlation between the expression patterns of the validated genes and the FPKM values derived from the RNA sequencing data (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.85377) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), thereby confirming the reliability of the transcriptomic dataset. Principal component analysis (PCA) revealed a clear distinction between CK (control) and TRV-treated samples, indicating pronounced alterations in gene expression within leaves following \u003cem\u003eCsMYB116\u003c/em\u003e silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Differentially expressed genes (DEGs) were depicted using a volcano plot, revealing 4,042 upregulated and 1,876 downregulated genes in the silenced seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). GO enrichment analysis indicated that these DEGs were significantly overrepresented across the Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). In total, the DEGs corresponded to 2,341 GO terms, with upregulated genes associated with 1,925 terms and downregulated genes with 1,254 terms (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Notably, within the BP category, pathways such as cell wall organization, plant secondary cell wall biogenesis, and xylan biosynthetic processes were significantly enriched. In the CC category, the extracellular region, plasma membrane, and plant cell wall were prominently represented. Within the MF category, DEGs were enriched in activities including O-acetyltransferase activity, microtubule binding, and endo-1,3-beta-D-glucanase activity (Table S3). These results suggest that \u003cem\u003eCsMYB116\u003c/em\u003e may directly or indirectly influence cell wall-related pathways, thereby modulating plant drought resistance.\u003c/p\u003e \u003cp\u003eKEGG enrichment analysis revealed that differentially expressed genes (DEGs) were predominantly enriched in pathways related to ABC transporters (ko02010), plant hormone signal transduction (ko04075), starch and sucrose metabolism (ko00500), phenylpropanoid biosynthesis (ko00940), and motor protein-related pathways (ko03070) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef; Table S4-S6). Among them, the most differentially expressed genes were starch and sucrose metabolism pathways. Some enzyme genes involved in the regulation of glucose metabolism (such as \u003cem\u003eTPS1\u003c/em\u003e, \u003cem\u003eTPPA\u003c/em\u003e, \u003cem\u003eTPPJ\u003c/em\u003e and fructose / hexokinase) were significantly up-regulated, and a variety of key hydrolases involved in starch and sucrose degradation (including \u003cem\u003eAMY1.1\u003c/em\u003e, \u003cem\u003eBGLU42\u003c/em\u003e, \u003cem\u003eBGLU12\u003c/em\u003e, \u003cem\u003eDPEP\u003c/em\u003e and some \u003cem\u003eBAM\u003c/em\u003e and \u003cem\u003eINV\u003c/em\u003e genes) were significantly down-regulated. Additionally, the expression of \u003cem\u003eWAXY\u003c/em\u003e and certain members of the \u003cem\u003eAGPS\u003c/em\u003e gene family, which are involved in starch synthesis, was also suppressed.\u003c/p\u003e \u003cp\u003eIn this experiment, we identified significant alterations in 45 genes associated with the plant hormone signal transduction pathway, of which 35 were up-regulated and 10 were down-regulated. These genes are involved in the signaling pathways of ABA, auxin, cytokinin, gibberellin, brassinolide, and jasmonic acid. Within the abscisic acid-mediated signaling pathway, the receptor gene \u003cem\u003ePYL4\u003c/em\u003e and the downstream transcription factor \u003cem\u003eABI5\u003c/em\u003e were down-regulated, whereas \u003cem\u003eDPBF2\u003c/em\u003e was up-regulated. In the auxin-mediated signal transduction pathway, 25 genes-including \u003cem\u003eIAA/Aux\u003c/em\u003e, \u003cem\u003eSAUR50\u003c/em\u003e, \u003cem\u003eLAX\u003c/em\u003e, \u003cem\u003eAUX22\u003c/em\u003e, and \u003cem\u003eARG7\u003c/em\u003e-were predominantly up-regulated, indicating overall activation of auxin signaling. Regarding the cytokinin pathway, \u003cem\u003eCYCD3-1\u003c/em\u003e and \u003cem\u003eAHP4\u003c/em\u003e exhibited up-regulation, while members of the \u003cem\u003eARR\u003c/em\u003e gene family showed both up- and down-regulation. In the gibberellin signaling pathway, regulatory factors \u003cem\u003eSLRL1\u003c/em\u003e and \u003cem\u003eGAI1\u003c/em\u003e were up-regulated, whereas the GA receptor \u003cem\u003eGID1B\u003c/em\u003e was down-regulated. Key genes involved in jasmonic acid signaling, \u003cem\u003eCOI1\u003c/em\u003e and \u003cem\u003eJAR6\u003c/em\u003e, were down-regulated, while components of the brassinolide signaling pathway, including \u003cem\u003eBZR1\u003c/em\u003e, kinase \u003cem\u003eSRK2A\u003c/em\u003e, and \u003cem\u003eXTH1\u003c/em\u003e, were up-regulated. Additionally, the down-regulation of the \u003cem\u003eTGA1\u003c/em\u003e transcription factor family may influence the cross-regulation among multiple hormone signaling pathways.\u003c/p\u003e \u003cp\u003eWithin the motor protein pathway, the majority of differentially expressed genes were up-regulated, notably including numerous members of the tubulin family (\u003cem\u003eTUBA\u003c/em\u003e, \u003cem\u003eTUBB1\u003c/em\u003e, \u003cem\u003eTUBB2\u003c/em\u003e) and the kinesin family (\u003cem\u003eKIN5\u003c/em\u003e, \u003cem\u003eKIN7\u003c/em\u003e, \u003cem\u003eKIN12\u003c/em\u003e, \u003cem\u003eKIN14\u003c/em\u003e). Conversely, the actin gene \u003cem\u003eACT7\u003c/em\u003e and kinesin gene \u003cem\u003eKIN7O\u003c/em\u003e exhibited down-regulation. In the phenylpropanoid metabolic pathway, which is associated with structural defense, a total of 36 genes displayed altered expression. Although several peroxidases (e.g., \u003cem\u003ePER12\u003c/em\u003e, \u003cem\u003ePER47\u003c/em\u003e, \u003cem\u003ePER72\u003c/em\u003e), involved in the oxidation and polymerization of lignin monomers and aromatic compounds, as well as genes related to ester synthesis, were significantly up-regulated, the expression of the rate-limiting enzyme \u003cem\u003ePAL\u003c/em\u003e and the branch-regulating enzyme \u003cem\u003eHCT1\u003c/em\u003e in the pathway, was down-regulated. This down-regulation likely constrained the synthesis of pathway precursors.\u003c/p\u003e \u003cp\u003eWithin the ABC transporter pathway, a total of 32 genes exhibited significant differential expression, with 20 genes upregulated and 12 genes downregulated. Specifically, in the \u003cem\u003eABCB\u003c/em\u003e subfamily, the genes \u003cem\u003eABCB1\u003c/em\u003e, \u003cem\u003eABCB2\u003c/em\u003e, \u003cem\u003eABCB11\u003c/em\u003e, \u003cem\u003eABCB13\u003c/em\u003e, and \u003cem\u003eABCB19\u003c/em\u003e were significantly upregulated. In the \u003cem\u003eABCG\u003c/em\u003e subfamily, sterol transporter genes \u003cem\u003eABCG8\u003c/em\u003e, \u003cem\u003eABCG11\u003c/em\u003e, \u003cem\u003eABCG12\u003c/em\u003e, \u003cem\u003eABCG14\u003c/em\u003e, and the defense-related gene \u003cem\u003eABCG32\u003c/em\u003e were upregulated; however, both upregulated and downregulated genes were observed in \u003cem\u003eABCG7\u003c/em\u003e and \u003cem\u003eABCG34\u003c/em\u003e, while the stratum corneum lipid secretion gene \u003cem\u003eABCG31\u003c/em\u003e was downregulated. All genes within the \u003cem\u003eABCC\u003c/em\u003e subfamily, including \u003cem\u003eABCC10\u003c/em\u003e and \u003cem\u003eABCC3\u003c/em\u003e, were downregulated. Additionally, the \u003cem\u003ePDR2\u003c/em\u003e gene in the \u003cem\u003ePDR\u003c/em\u003e subfamily was significantly downregulated. The bidirectional regulation of \u003cem\u003eABCG7\u003c/em\u003e, coupled with the downregulation of \u003cem\u003ePDR2\u003c/em\u003e, may further influence the drought adaptability of tea plants, underscoring the complex and dynamic regulation of ABC transporters in transmembrane material transport.\u003c/p\u003e \u003cp\u003eIn summary, \u003cem\u003eCsMYB116\u003c/em\u003e functions as a regulatory factor that is essential for coordinating energy distribution, hormone signaling, cell wall reinforcement, and the optimization of material transport by directly or indirectly modulating these critical pathways. The absence of \u003cem\u003eCsMYB116\u003c/em\u003e expression results in the disruption of the entire drought response network, thereby markedly diminishing the drought tolerance of tea plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eScreening and verification of interacting proteins\u003c/h2\u003e \u003cp\u003eSelf-activation verification revealed that Y2H yeast cells transfected with the BD-\u003cem\u003eCsMYB116\u003c/em\u003e vector were able to grow normally on SD/-Trp medium and exhibited growth with blue coloration on SD/-Trp supplemented with X-α-gal medium. This observation indicates that \u003cem\u003eCsMYB116\u003c/em\u003e fused to the BD vector exhibits self-activation activity. Furthermore, the addition of 200 ng/mL AbA to the medium effectively inhibited yeast growth, demonstrating that this concentration of AbA can suppress the self-activation of BD-\u003cem\u003eCsMYB116\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eYeast library screening was conducted using the BD-\u003cem\u003eCsMYB116\u003c/em\u003e vector following the suppression of self-activation. Subsequent colony PCR and sequencing analyses identified 38 candidate proteins. To further validate the interactions between these candidates and \u003cem\u003eCsMYB116\u003c/em\u003e, proteins associated with drought resistance functions, including ABC transporters and auxin-related proteins, were selected for yeast two-hybrid point-to-point verification. The results demonstrated that yeast strains co-transformed with \u003cem\u003eCsMYB116\u003c/em\u003e-BD and each candidate protein-AD grew robustly on selective media lacking Trp, Leu, His, and Ade, and exhibited a pronounced blue coloration on X-α-gal plates. In contrast, the negative controls failed to grow on selective media and showed no color development (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), confirming the specific interaction between \u003cem\u003eCsMYB116\u003c/em\u003e and these proteins in yeast. These findings suggest that \u003cem\u003eCsMYB116\u003c/em\u003e may modulate the drought response in tea plants through interactions with transporters and proteins involved in auxin metabolism.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlant adaptation to drought stress constituted a complex physiological process governed by multiple regulatory networks. In the present study, the pivotal role of the tea plant transcription factor \u003cem\u003eCsMYB116\u003c/em\u003e in mediating drought stress responses was systematically elucidated for the first time through an integrative approach combining physiological assessments, molecular biology techniques, and transcriptomic analyses. Our findings consistently demonstrated that \u003cem\u003eCsMYB116\u003c/em\u003e, a nuclear-localized positive regulatory transcription factor, enhanced drought tolerance in tea plants by modulating root architecture, antioxidant defense mechanisms, photosynthetic protection, and hormone signaling pathways.\u003c/p\u003e \u003cp\u003eRoot systems constituted the principal organs through which plants detected and responded to soil drought stress (Yang et al. 2024). Prior studies had revealed that, under conditions of heterogeneous soil moisture or drought stress, many plant species adopted a strategy characterized by a reduction in lateral root branching coupled with the promotion of primary root elongation into deeper soil strata, thereby enhancing drought tolerance (Hazman and Brown 2018). For example, maize demonstrated diminished lateral root branching to facilitate deeper penetration of the primary root (Zhan et al. 2015). In contrast, certain species improved water uptake and drought resilience by suppressing longitudinal growth of the primary root while stimulating lateral root proliferation (Karlova et al. 2021; Shoaib et al. 2022). Specifically, overexpression of \u003cem\u003ePtrYY1\u003c/em\u003e (\u003cem\u003ePtrYY1\u003c/em\u003e-OE) under drought conditions had been shown to promote lateral root growth and development in poplar (Sun et al. 2023a). Moreover, an increase in lateral root density had been correlated with enhanced plant responses to water deficit and alterations in yield components (Placido et al. 2020). Under drought stress, transgenic Arabidopsis plants overexpressing \u003cem\u003ePvMLP19\u003c/em\u003e exhibited a significant augmentation in lateral root number and root system remodeling, culminating in improved stress tolerance (Yerlikaya et al. 2025). In the current investigation, Arabidopsis plants overexpressing \u003cem\u003eCsMYB116\u003c/em\u003e displayed inhibited primary root elongation concomitant with a pronounced increase in lateral root number under drought conditions. Furthermore, an interaction between \u003cem\u003eCsMYB116\u003c/em\u003e and auxin (IAA)-related proteins was identified, with IAA recognized as a pivotal hormone directly regulating root system development. These results implied that \u003cem\u003eCsMYB116\u003c/em\u003e enhanced drought tolerance by modulating auxin metabolism to regulate lateral root formation. Collectively, the data indicated that \u003cem\u003eCsMYB116\u003c/em\u003e mediated root system remodeling and augmented drought resistance by increasing lateral root density, thereby expanding the absorptive surface area within the upper soil layers.\u003c/p\u003e \u003cp\u003eDrought stress frequently resulted in an accumulation of excess light energy within plants, leading to PSII and the generation of reactive oxygen species (ROS), which subsequently induced oxidative damage (Sami et al. 2021). To mitigate the detrimental effects of drought, numerous plant species enhanced the biosynthesis of antioxidant enzymes, thereby reducing oxidative stress and alleviating drought-induced damage (Kosar et al. 2021). In the present study, transgenic plants overexpressing \u003cem\u003eCsMYB116\u003c/em\u003e exhibited significantly higher values of Fv/Fm, PSII efficiency, and ETR following drought exposure, concomitant with a marked reduction in MDA content relative to wild-type controls. These findings demonstrated that \u003cem\u003eCsMYB116\u003c/em\u003e conferred substantial protection to the photosynthetic apparatus and cellular membranes under drought conditions. Crucially, this protective effect was closely associated with the upregulation of a robust antioxidant defense system, as evidenced by the pronounced increases in CAT, POD, and SOD activities observed in the overexpressing lines. This mechanism aligned with the observations of Sun et al. (2025), who reported that \u003cem\u003eAtMYB37\u003c/em\u003e mitigated drought-induced photosynthetic inhibition in Arabidopsis by modulating the expression of genes involved in ROS metabolism, thereby enhancing drought tolerance. Such evidence suggested that MYB transcription factors across diverse species might share conserved roles in protecting the photosynthetic machinery from oxidative damage. Similarly, MYB transcription factors such as \u003cem\u003eIbMYB116\u003c/em\u003e in sweet potato (Zhou et al. 2019) and \u003cem\u003eAgMYB5\u003c/em\u003e in celery (Sun et al. 2023b) had been shown to regulate antioxidant and ROS scavenging-related genes, reducing ROS accumulation under drought stress and improving drought resilience. Conversely, \u003cem\u003eCsMYB116\u003c/em\u003e-silenced plants in this study displayed significantly diminished antioxidant enzyme activities and reduced drought tolerance. Collectively, these results implied that \u003cem\u003eCsMYB116\u003c/em\u003e might directly or indirectly orchestrate an effective ROS scavenging system, thereby preserving the structural integrity and functional capacity of photosynthetic membranes, which constituted a fundamental physiological basis for enhanced drought tolerance.\u003c/p\u003e \u003cp\u003ePlant hormones served as fundamental signaling molecules that modulated plant responses to drought stress (Liao et al. 2025). Notably, ABA, JA along with its bioactive conjugate JA-Ile, SA, as well as auxins and gibberellins, collectively constituted a complex and highly interactive hormonal regulatory network (Gao et al. 2024; Musazade et al. 2025). Prior research had demonstrated that elevated hormone concentrations under drought conditions did not inherently confer enhanced drought tolerance; rather, their functional efficacy was contingent upon the maintenance of hormonal homeostasis and the proper activation of downstream signaling cascades (Xiao et al. 2025). In the present study, however, we observed that \u003cem\u003eCsMYB116\u003c/em\u003e-silenced plants exhibited significant accumulation of ABA, JA, JA-Ile, SA, and OPDA under drought stress, yet these plants manifested pronounced wilting, damage to the photosynthetic apparatus, and diminished antioxidant capacity. This ostensibly paradoxical outcome contrasted with the established role of \u003cem\u003eCsMYB116\u003c/em\u003e as a positive regulator. This phenomenon was attributed to the excessive accumulation of endogenous hormones resulting from a heightened stress burden, rather than being an indication of improved stress adaptability (Fatma et al. 2022; Lamarque et al. 2020; Saleem et al. 2021). Specifically, although ABA was recognized as a central hormone in drought response, its overaccumulation or dysregulation of signaling pathways frequently led to growth inhibition and attenuated drought tolerance (Finkelstein, 2013). Supporting this, transcriptomic analyses revealed downregulation of the ABA receptor gene PYL4 and the key transcription factor ABI5 following \u003cem\u003eCsMYB116\u003c/em\u003e silencing, which suggested impaired ABA signal perception and transduction. Consequently, the elevated ABA levels exerted minimal protective effects, thereby compromising the hormone\u0026rsquo;s role in drought resistance.\u003c/p\u003e \u003cp\u003eFurthermore, within the JA and SA metabolic pathways, despite a marked accumulation of JA, JA-Ile, and their precursor OPDA following the silencing of \u003cem\u003eCsMYB116\u003c/em\u003e, the observed downregulation of key JA signaling components, namely COI1 and JAR6, indicated a constrained JA signal transduction. This limitation may subsequently have influenced JA metabolism. Prior research had demonstrated that excessive or aberrant activation of JA signaling could disrupt abscisic acid (ABA)-mediated drought responses and potentially heighten stress sensitivity (Fu et al. 2017). Concurrently, the pronounced elevation in SA levels may further have antagonized both ABA and JA signaling pathways, resulting in the over accumulation of ROS and consequent impairment of cellular homeostasis (Elsisi et al. 2024; Moeder et al. 2010). Notably, \u003cem\u003eCsMYB116\u003c/em\u003e silencing also led to a significant increase in auxin concentrations, accompanied by substantial upregulation of genes within the IAA/Aux, SAUR, and LAX families. The activation of these auxin-related compounds and gene families may have intensified energy expenditure and water requirements, thereby diminishing drought tolerance (Li et al. 2021b). This observation suggested that \u003cem\u003eCsMYB116\u003c/em\u003e may have functioned as a negative regulator of auxin expression and accumulation. Simultaneously, alterations in gibberellin metabolism and signaling were evident, characterized by reduced levels of bioactive gibberellins alongside upregulated expression of signaling repressors, indicative of a disrupted balance between growth and stress response mechanisms (Liao et al. 2023). Collectively, these findings demonstrated that silencing \u003cem\u003eCsMYB116\u003c/em\u003e induced a state of \u0026ldquo;high accumulation but low efficiency\u0026rdquo; across multiple phytohormones, thereby perturbing the hormonal regulatory network and culminating in a comprehensive decline in antioxidant defenses, photosystem stability, and drought resistance.\u003c/p\u003e \u003cp\u003eMYB transcription factors played a critical role in regulating secondary metabolic pathways, including phenylpropanoid metabolism (Pratyusha and Sarada 2022). Transcriptomic analysis conducted in this study demonstrated that silencing of \u003cem\u003eCsMYB116\u003c/em\u003e within the phenylpropanoid biosynthesis pathway resulted in the upregulation of downstream peroxidase genes, accompanied by the downregulation of key rate-limiting enzymes, PAL and HCT1. This regulatory shift constrained the biosynthesis of lignin and other stress-associated secondary metabolites (Aluko et al. 2025), thereby compromising the structural defense mechanisms of tea plants. Furthermore, under drought stress, plant starch reserves could be hydrolyzed into soluble sugars, which served dual functions as growth substrates and as osmotic regulators and antioxidants that alleviated drought-induced damage (Moran et al. 2017; Khan et al. 2021). Within the starch and sucrose metabolism pathway, \u003cem\u003eCsMYB116\u003c/em\u003e silencing led to a marked upregulation of genes encoding key enzymes involved in sugar signaling and metabolic regulation, while several hydrolases responsible for starch and sucrose degradation were significantly downregulated. Concurrently, the expression of WAXY and certain members of the AGPS family, which were implicated in starch biosynthesis, was also suppressed. These findings suggested a disruption in the dynamic balance of carbon allocation among synthesis, degradation, and signaling processes, potentially resulting in an inadequate supply of energy and osmotic regulators (Chen et al. 2024). Consequently, this imbalance impaired the plant\u0026rsquo;s capacity to sustain metabolic homeostasis under drought conditions.\u003c/p\u003e \u003cp\u003eFurthermore, this investigation identified a potential functional relationship between \u003cem\u003eCsMYB116\u003c/em\u003e and ABC transporters. ABC transporters were extensively implicated in the transmembrane movement of hormones (such as ABA), lipids, and secondary metabolites, serving as critical components in maintaining cellular compartmentalization and facilitating the targeted distribution of substances (Huang et al. 2021; Lefevre and Boutry 2018). Transcriptomic analyses demonstrated a significant enrichment of differentially expressed genes within the ABC transporter pathway, with pronounced alterations observed in defense-associated members of the ABCB and ABCG subfamilies. These findings suggested that \u003cem\u003eCsMYB116\u003c/em\u003e modulated the transmembrane transport and allocation of lipids, hormones, or secondary metabolites by influencing ABC transporter activity (Wang et al. 2020; Zhang et al. 2025). Notably, the bidirectional regulation of ABCG7 alongside the downregulation of PDR2 constrained the effective intercellular or intertissue transport of key defense compounds (Gupta et al. 2019). Concurrently, the pervasive upregulation of cytoskeleton-related genes coupled with the downregulation of ACT7 indicated a perturbation in cytoskeletal dynamics, which impacted cellular expansion, material transport, and mechanical integrity (Chun et al. 2021). Moreover, yeast two-hybrid assays provided direct evidence of physical interactions between \u003cem\u003eCsMYB116\u003c/em\u003e and ABC transporters, implying that the regulatory influence of \u003cem\u003eCsMYB116\u003c/em\u003e extended beyond transcriptional control of ABC transporter genes to potentially modulating transporter activity or stability via protein-protein interactions. Collectively, these results proposed that \u003cem\u003eCsMYB116\u003c/em\u003e exerted a nuanced regulatory function in the transmembrane transport of hormones, secondary metabolites, or ions, thereby introducing a novel regulatory dimension to its role in plant drought stress responses.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study systematically demonstrated, for the first time, that \u003cem\u003eCsMYB116\u003c/em\u003e regulated drought resistance in tea plants by coordinating multiple biological processes, including photosystem protection, reactive oxygen species scavenging, hormone homeostasis restoration, cell wall modification, and ABC transporter-mediated transmembrane transport. These findings not only enhanced the molecular-level understanding of the \u0026lsquo;growth-defense\u0026rsquo; trade-off mechanism in tea plant stress adaptation but also identified a pleiotropic key target for molecular breeding aimed at improving tea plant stress resistance. Base on these results, future research will focus on constructing a genome-wide target map of \u003cem\u003eCsMYB116\u003c/em\u003e, analyzing its transcriptional regulatory network, and validating its interactions and functions with key factors to further elucidate the regulatory mechanisms underlying drought resistance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32573088; 32302437).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaoyang Han designed this study. Xiaoxia Zhao wrote the main manuscript text guided by Xiaoyang Han and prepared Figures and Talbes. Xinhan You, Wenjuan Ma, Xueying Xie performed the data analysis. Jian Hou provided test materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe database will be provided on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. 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Genes (Basel) 13(10): 1883. https://doi.org/10.3390/genes13101883\u003c/li\u003e\n \u003cli\u003eZheng D, Fu M, Sun C, Yang Q, Zhang X, Lu J, Chang M, Liu L, Wan X, Chen Q (2025) CsABF8 mediates drought-induced ABA signaling in the regulation of raffinose biosynthesis in \u003cem\u003eCamellia sinensis\u003c/em\u003e leaves. Int J Biol Macromol 311:143521. https://doi.org/ 10.1016/j.ijbiomac.2025.143521.\u003c/li\u003e\n \u003cli\u003eZhou L, Xu H, Mischke S, Meinhardt LW, Zhang D, Zhu X, Li X, Fang W (2014) Exogenous abscisic acid significantly affects proteome in tea plant (\u003cem\u003eCamellia sinensis\u003c/em\u003e) exposed to drought stress. Hortic Res 1:14029. https://doi.org/ 10.1038/hortres. 2014.29.\u003c/li\u003e\n \u003cli\u003eZhou Y, Zhu H, He S, Zhai H, Zhao N, Xing S, Wei Z, Liu Q (2019) A novel sweetpotato transcription factor gene \u003cem\u003eIbMYB116\u003c/em\u003e enhances drought tolerance in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e. Front Plant Sci 10:1025. https://doi.org/ 10.3389/fpls.2019.01025.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"tea plant, drought stress, functional verification, MYB","lastPublishedDoi":"10.21203/rs.3.rs-8519228/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8519228/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe MYB transcription factor family plays a crucial regulatory role in the growth and quality development of tea plants. Building upon previous drought-related transcriptome data, this study systematically investigated the function and regulatory mechanisms of the tea plant transcription factor \u003cem\u003eCsMYB116\u003c/em\u003e in response to drought stress, employing physiological and molecular biology methods. Through heterologous overexpression and gene silencing experiments, \u003cem\u003eCsMYB116\u003c/em\u003e was demonstrated to positively regulate drought resistance in tea plants. Under drought conditions, overexpression lines exhibited increased lateral root development, enhanced photosystem stability, elevated antioxidant enzyme activity, higher proline content, and a significant reduction in membrane lipid peroxidation. Conversely, gene silencing resulted in photosystem damage, diminished antioxidant capacity, and disrupted hormone signaling in tea seedlings. Transcriptome sequencing revealed significant alterations in key metabolic pathways-including hormone signal transduction, cell wall synthesis, phenylpropanoid metabolism, and ABC transporter activity\u0026mdash;in silenced plants subjected to drought stress. Furthermore, yeast two-hybrid assays confirmed interactions between \u003cem\u003eCsMYB116\u003c/em\u003e and both ABC transporters and IAA-related proteins. This study is the first to demonstrate that \u003cem\u003eCsMYB116\u003c/em\u003e enhances drought adaptability in tea plants by positively regulating gene expression. These findings contribute to a deeper understanding of the molecular regulatory mechanisms underlying tea plant responses to drought and identify \u003cem\u003eCsMYB116\u003c/em\u003e as a promising target for molecular breeding aimed at improving drought tolerance in tea plants.\u003c/p\u003e","manuscriptTitle":"The function and regulatory mechanism of tea plant transcription factor CsMYB116 in drought stress response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-14 14:40:58","doi":"10.21203/rs.3.rs-8519228/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-17T13:38:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46003105456363960104050411652335881031","date":"2026-03-31T04:29:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201604553397606248424333687814483467323","date":"2026-01-13T12:02:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-13T02:43:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-05T11:38:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-05T11:36:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Growth Regulation","date":"2026-01-05T08:52:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"182a5497-5db8-47bb-96c3-2ca7998b4100","owner":[],"postedDate":"January 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-14T14:40:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-14 14:40:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8519228","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8519228","identity":"rs-8519228","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}