SmMYC2 mediates ROS homeostasis to confer drought resistance in Salvia miltiorrhiza through an ABA-independent pathway

SmMYC2 mediates ROS homeostasis to confer drought resistance in Salvia miltiorrhiza through an ABA-independent pathway | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 5 January 2025 V1 Latest version Share on SmMYC2 mediates ROS homeostasis to confer drought resistance in Salvia miltiorrhiza through an ABA-independent pathway Authors : Tong Wang , Qing Yang , Shiyu Yao , Jing Yang , Jingying Liu , and Pengda Ma [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173607103.32777612/v1 215 views 143 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Drought stress is a primary environmental element restricting global crop productivity, quality and geographical distribution. Jasmonic acid (JA) plays a significant part in plant adversity defense, but many of its functions in drought response are unknown. Here, we report that SmMYC2 involves an ABA-independent drought stress response pathway and promotes drought resistance in Arabidopsis thaliana and Salvia miltiorrhiza . DNA affinity purification sequencing (DAP-seq) analysis uncovers the direct target genes of SmMYC2 related to adversity-responsive and hormone-signaling. Further studies revealed that SmMYC2 synergistically regulates three processes in drought response: (1) SmMYC2 directly bound to SmCAT ( catalase ) and SmPOD40 ( peroxidase 40 ) promoters and activated their transcription, thus triggering the elimination of reactive oxygen species (ROS) and raising the drought resistance of S. miltiorrhiza , (2) SmMYC2 directly activated SmDREB2D , a DREB (dehydration response element binding) transcription factor gene, which promoted drought adaptation of S. miltiorrhiza through further signal transduction, (3) In the feedback section, SmMYC2 upregulated the expression of JA synthesis and repressor genes, which could precisely control the magnitude, duration, and opportune moment of the defense-related reactions. Collectively, the study sheds light on the mechanism by which SmMYC2 advances drought tolerance at the transcriptional level via cascade regulation. SmMYC2 mediates ROS homeostasis to confer drought resistance in Salvia miltiorrhiza through an ABA-independent pathway Tong Wang 1, # , Qing Yang 1, # , Shiyu Yao 1 , Jing Yang 1 , Jingying Liu 1, * , Pengda Ma 1, * 1 College of Life Sciences, Northwest A&F University, Yangling, 712100, China # These authors contributed equally to this work * Correspondence: Jingying Liu (e-mail: [email protected] , Tel: +86 29 87082592) Pengda Ma (e-mail: [email protected] , Tel: +86 29 87082592); ABSTRACT Drought stress is a primary environmental element restricting global crop productivity, quality and geographical distribution. Jasmonic acid (JA) plays a significant part in plant adversity defense, but many of its functions in drought response are unknown. Here, we report that SmMYC2 involves an ABA-independent drought stress response pathway and promotes drought resistance in Arabidopsis thaliana and Salvia miltiorrhiza . DNA affinity purification sequencing (DAP-seq) analysis uncovers the direct target genes of SmMYC2 related to adversity-responsive and hormone-signaling. Further studies revealed that SmMYC2 synergistically regulates three processes in drought response: (1) SmMYC2 directly bound to SmCAT ( catalase ) and SmPOD40 ( peroxidase 40 ) promoters and activated their transcription, thus triggering the elimination of reactive oxygen species (ROS) and raising the drought resistance of S. miltiorrhiza , (2) SmMYC2 directly activated SmDREB2D , a DREB (dehydration response element binding) transcription factor gene, which promoted drought adaptation of S. miltiorrhiza through further signal transduction, (3) In the feedback section, SmMYC2 upregulated the expression of JA synthesis and repressor genes, which could precisely control the magnitude, duration, and opportune moment of the defense-related reactions. Collectively, the study sheds light on the mechanism by which SmMYC2 advances drought tolerance at the transcriptional level via cascade regulation. Keywords Drought stress, ROS, SmMYC2 , Salvia miltiorrhiza Introduction Drought stress is a pivotal environmental element confining plant development, usually leading to an imbalance in cellular osmotic pressure, which severely affects plant development and growth and reduces yields of most major crops (Fujita et al. 2006; Zhu, 2016). To sense and respond to drought stress, plants have developed various intricate defense systems by controlling physiological and biochemical processes (Pereira, 2016). Plant hormones are small-molecule signaling compounds, comprising jasmonic acid (JA), abscisic acid (ABA), ethylene (Eth) and auxin (IAA), which confer drought tolerance on plants, either independently or in a coordinated manner (Sharma et al. 2019; Zhao et al. 2021). JA is a well-known stress-resistant phytohormone that uses linolenic acid as a substrate and is synthesized through a battery of enzymes including lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid reductase (OPR) (Schaller and Stintzi 2009). The JA signaling pathway consists of different functional sections or modules that enable plants to respond physiologically to various internal and external (biotic and abiotic) stimuli (Chini et al., 2016). For example, AtLOX6 is necessary for the stress-induced cumulation of JA in the root system, and loss-of-function mutants of LOX6 are more vulnerable to being attacked by detritivorous crustaceans and more susceptible to drought (Grebner et al. 2013). ZmEREB57 activates JA signaling by directly binding to ZmAOC2 and improves salt tolerance in maize (Zhu et al. 2023). Nevertheless, the intrinsic modulatory mechanism of JA-mediated abiotic stress resistance in S. miltiorrhiza is unknown. Within the JA signaling pathway, MYC2 is a master transcription factor (TF). It is recognized as a positive modulator of downstream defense-associated genes (Zheng et al. 2023) that enhance plant tolerance to insect bites (Chen et al. 2024) and pathogenic agent infections (Du et al. 2017). MYC2 is highly flexible in regulating JA signaling: on the one hand, MYC2 can amplify the transcriptional output of JA signals through a positive feedback loop. For example, OsMYC2 controls diurnal flower-opening time in rice by directly activating the transcription of genes related to JA biosynthesis to enhance JA contents in lodicule cells (Zhu et al. 2024). CsMYC2 increases tea plant ( Camellia sinensis ) tolerance to osmotic stress by binding to CsLOX7 and CsAOS2 to activate their transcription and increase JA levels (Zhu et al. 2024). On the other hand, although JA pathway regulator MYC2 is a central node in the plant signaling network, sustained activation of MYC2 activity is potentially lethal. Therefore, MYC2 needs to be tightly controlled to optimize plant adaptation (Chico et al. 2020). The negative feedback loops that regulate JA-signaling are generated by the JA-induced repressor genes such as jasmonate zim-domain ( JAZ ) and JA-associated MYC2-like ( JAM ) (Liu et al. 2019; Sasaki-Sekimoto et al. 2013; Zhang et al. 2020). In particular, stable JAZs and some JAZ variants devoid of the jas motif are known to play a role in the negative feedback modulation of JA signaling (Moreno et al. 2013; Shyu et al. 2012). Our recent research demonstrates that the SmJAZ-SmMYC2-SmMYB36 module dynamically regulates the JA-mediated accumulation of tanshinones (Cao et al. 2024). Nonetheless, the biological processes and regulatory systems of MYC2 in drought stress response in S. miltiorrhiza are not clear. ROS, like superoxide anion (O 2 - ), hydrogen peroxide (H 2 O 2 ) and singlet oxygen ( 1 O 2 ), are constantly created in plants as byproducts of different metabolic activities. The antioxidant defense system of the plants is responsible for reducing/removing excess ROS to keep it at a stable level (Wang et al. 2024). The dynamic equilibrium between ROS synthesis and elimination in plants is upset by drought stress, which causes an excessive build-up of endocellular ROS and oxidative damage to cytoarchitectures (Hunyadi 2019). The enzymatic antioxidant system, which includes a set of ROS scavengers such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), helps plants combat ROS toxicity (Suzuki et al. 2012). Increased antioxidant enzyme activity has been demonstrated in numerous studies to help decrease the negative impacts of drought stress on multifarious plants, including Arabidopsis ( Arabidopsis thaliana ) (Cha et al. 2014), rice ( Oryza sativa ) (Ouyang et al. 2020), tobacco ( Nicotiana tabacum ) (Liang et al. 2022) and wheat ( Triticum aestivum ) (Yu et al. 2023). Overexpression of antioxidant enzyme-encoding genes can significantly increase antioxidant enzyme activity, thereby enhancing abiotic adversity resistance in plants. For example, CAT and POD-encoding genes have positive effects on abiotic stress response in distinct plant species (Xu et al. 2023; Zhai et al. 2024; Zhao et al. 2020). Dehydration response element binding (DREB) protein belongs to the AP2/ERF superfamily (Han et al. 2022). They specifically bind to dehydration-responsive elements (DREs) in the promoters of downstream genes and are vital contributors for the induction of the ABA-independent drought stress response (Rehman and Mahmood 2015). DREBs can be classified into DREB1-type or DREB2-type, and a great deal of research have reported their roles in positive responses to stress (Hu et al. 2023; Mei et al. 2022). However, it is unclear what mechanism triggers their expression during a drought in S. miltiorrhiza . S. miltiorrhiza is an extensively studied medicinal plant that is frequently utilized in the therapy of cardiovascular disorders (Wang et al. 2007; Zhou et al. 2005). Increasingly harsh environmental conditions, particularly global warming, have caused a gradual increase in droughts, resulting in a large drop in the area planted with S. miltiorrhiza and in its production (Liu et al. 2011; Wu et al. 2014). Hence, it is critical to boost the drought tolerance of S. miltiorrhiza . To date, we have found no systematic studies on the drought response mechanisms of S. miltiorrhiza . The present study confirmed that SmMYC2 played a proactive part in drought resilience in Arabidopsis and S. miltiorrhiza via an ABA-independent form. A set of genes associated with stress response and hormone signaling were ascertained by DNA affinity purification sequencing (DAP-seq) analysis, and the SmMYC2-mediated JA feedback loop, which includes JA synthesis and signaling repressor genes, was briefly depicted. Functional analyses of the antioxidant enzyme-encoding gene SmCAT and DREB2 -type gene SmDREB2D validated our working hypothesis. Overall, our study provided an important mechanistic cognition for unraveling the drought tolerance conferred by SmMYC2 to S. miltiorrhiza at the level of transcriptional regulation and provided a comprehensive understanding of the interaction of JA with other hormones. 2. Materials and Methods 2.1 Plant materials and stress treatments Plant materials utilized in the research included S. miltiorrhiza seedlings (provided by Tensil Pharmaceuticals Shangluo Branch), S. miltiorrhiza hairy root and A. thaliana seedlings (”Col-0”). S. miltiorrhiza and A. thaliana seedlings grew at 25°C under LD (16 h light/8 h dark) circumstances. The hairy roots were cultivated in 6,7-V liquid mediums and were incubated in an orbital shaker protected from light at 25°C. For gene expression analysis, 7-week-old S. miltiorrhiza plants were separately disposed with 100 μM MeJA, 100 μM ABA and 20% (W/V) PEG-6000 for 24 h. Leaves were collected at the designated times and quickly refrigerated at -80℃ for additional research. 2.2 RNA extraction and quantitative reverse transcriptional PCR (qRT-PCR) Total RNA from S. miltiorrhiza tissues was extracted with RNAprep Pure Plant Kit (TIANGEN, Beijing, China) ̵‌in line with manufacturer’s instruction. Extracted total RNA was utilized to prepare cDNA with PrimeScript RT reagent kit (TaKaRa, Dalian, Chain). qRT-PCR was carried out with a real-time PCR system (Bio-RAD CFX96, CA, USA) and Taq SYBR®Green qPCR premix kit (iScience, China). Table S1 lists the primers used for qRT-PCR. 2.3 Subcellular localization assay The fusion vector ( 35S:SmCAT-GFP ) and the control vector ( 35S:GFP ) were transformed into Agrobacterium tumefaciens strain GV3101, respectively, and transiently co-expressed in 5-week-old Nicotiana benthamiana leaves with peroxisome marker. After three days of infiltration, confocal imaging was observed by a Leica TCS SP8 laser scanning microscope. 2.4 Plasmid construction and generation of transgenic materials The full-length coding sequences (CDSs) of SmMYC2 , SmCAT and SmDREB2D were cloned and separately fused into the pCambia1300-Myc to construct the overexpression (OX) vectors. To silence SmMYC2 , SmCAT and SmDREB2D , a 300 bp fragment was separately cloned against the non-conserved domain of SmMYC2 , SmCAT and SmDREB2D and then recombined into the pCambia1300-pHANNBIAL, respectively, to generate RNAi vectors. Agrobacterium rhizogenes (ATCC15834)-mediated methods were utilized to obtain transgenic hairy roots of S. miltiorrhiza as depicted in our previous study (Pei et al. 2018). Since the acquisition of transgenic seedlings mediated by A. tumefaciens is time-consuming and laborious and highly susceptible to chimeras, we developed for the first time a new effective and feasible genetic transformation system of seedlings based on the well-established and rapid genetic transformation system of hairy roots in this research. Briefly, we first utilized A. rhizogenes (ATCC15834) to produce transgenic hairy roots and then transferred them to MS medium comprising 0.1 mg/L NAA (1-naphthlcetic acid) and 1 mg/L 6-BA (6-benzylaminopurine) at 25°C under LD (16 h light/8 h dark) circumstances. After about two weeks, the hairy roots could regenerate into plants. Transgenic Arabidopsis lines overexpressing SmMYC2 ( SmMYC2 -OE) used in this study have been generated in our previous research (Deng et al. 2023). 2.5 Phenotype analyses for drought and ABA sensitivity For Arabidopsis seed germination experiments, surface-sterilized seeds were spread on the following media: 1/2MS medium supplemented with different mannitol and ABA concentrations. Phenotypes were observed every day for a total of 8 days and germination rate measurements were recorded. For root length experiments in Arabidopsis seedlings, 4-day-old seedlings were incubated in 1/2 MS medium and they were then shifted to treatment media: 1/2 MS medium with varying mannitol and ABA concentrations added. Following that, they were placed vertically in the culture for 5 days and finally the length of the primary roots was recorded. For drought treatment of Arabidopsis plants, 5-week-old seedlings were placed in a 13-day anhydrous environment followed by 2 days of rehydration, and pre-treatment seedlings were used as controls and physiological parameters were measured. For drought treatment of S. miltiorrhiza plants, 8-week-old seedlings with analogous growth states were inflicted with 14 days of water deficit and 4 days of rehydration. The wild-type (WT) plants and seedlings transformed with the empty vectors (EV) were utilized as controls. Three biological replicates were executed for every experiment, and each replicate contained at least 20 plants of each genotype. After 10 days of drought, samples were taken for physiological analyses. For the treatment of S. miltiorrhiza hairy roots, the hairy roots induced by ATCC15834 without the plasmids (WT) and transformed with the empty vectors (EV) were utilized as controls. Three independent replicates of 10 hairy roots of each line were performed for every experiment. WT, EV and positive hairy roots were placed in Erlenmeyer flasks containing 6,7-V liquid medium for 28 days. Hairy roots with similar growth statuses were selected and then placed in a 6,7-V liquid medium containing 0 or 10% PEG-6000 for one week. 2.6 Determination of physiological parameters and histochemical staining H 2 O 2 and O 2 - content, and SOD, POD and CAT enzyme activities were determined using assay kits (Macklin, Shanghai, China). Changes in malondialdehyde (MDA), proline and chlorophyll content were detected as previously described (Pan et al. 2012). We use diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining to observe the in-situ accumulation of H 2 O 2 and O 2 - (Huang et al. 2013). 2.7 Quantification of JA and ABA contents JA and ABA were measured using ELISA detection kits (MLBIO, Shanghai, China). Briefly, 100 mg tissues were uniformized in 5 ml PBS (pH 7.4) and 500 μl methanol. A microplate reader (Tecan, Austria) was used to record the absorbance at 450 nm (OD450). 2.8 Determination of chlorophyll fluorescence parameters We used a fluorometer JUNIOR-PAM (Walz, Germany) to determine the leaf chlorophyll fluorescence parameters. Three plants were stochastically chosen from every treatment, and following 30 min of dark adjustment, the completely expanding leaf was snipped, unfolded and immobilized on the detecting set of the fluorometer. The test parameters were established in conformity to the previous research (Navarro et al. 2021). 2.9 DAP-seq analysis To explore the SmMYC2-binding profile in the promoter and downstream of genes, peak regions within 2000 bp of upstream and downstream of genes were calculated. Bowtie 2 was utilized to align the DAP-seq reads to the S. miltiorrhiza genome. The default parameters of Bowtie 2.2.3 were used, and only unique alignment results were reported. MACS2 was used to detect DAP-seq peaks (Zhang et al. 2008). 2.10 Yeast one-hybrid (Y1H) assay For Y1H assay, the open reading frame (ORF) of SmMYC2 was amplified and then fused to pB42AD vector to generate prey vectors. Specific promoter segments of SmCAT , SmPOD40 and SmDREB2D were inserted into the pLacZi vector to produce bait vectors. The Y1H assays were performed based on the product manuals (Clontech, Mountain View, CA, USA). Transformants grew on SD/-Trp/-Ura plates, and positive clones could grow and turn blue on SD/-Trp/-Ura plates containing X-gal. 2.11 Dual-luciferase (dual-LUC) assay The promoters of SmCAT , SmPOD40 and SmDREB2D were isolated and separately fused to the pGreenII 0800-LUC vector for reporter plasmids. The pCsGFPBT-SmMYC2 was utilized as the effector plasmid. These plasmids were transiently expressed in tobacco leaves with different combinations by A. tumefaciens . The areas around the injection site were gathered and tested for luminescence using a detection kit (TransGen, Beijing, China). Photographs of the LUC fluorescence were taken with a Lumazone Pylon 2048B imaging system. 2.12 Electrophoretic mobility shift assay (EMSA) A biotin-labeled SmCAT , SmPOD40 and SmDREB2D probe was synthesized by Sangon Biotech (https://www.sangon.com/). EMSA was carried out by a chemiluminescent EMSA kit (Beyotime, Beijing, China) based on the manufacturer’s instructions. MBP-SmMYC2 or MBP protein and biotin-labeled probes were blended in the binding buffer for 15 min. Protein-probe separation was performed by polyacrylamide gel electrophoresis. 2.13 Statistical analysis Data analysis was carried out by SPSS22.0 software (SPSS Inc.). The significance of differences among means was ascertained by one-way ANOVA or Student’s t -test (* p < 0.05, ** p < 0.01). 3. Results 3.1 Drought activates the antioxidant system and promotes the amassing of endogenous JA and ABA in S. miltiorrhiza To research the influences of drought stress on S. miltiorrhiza , WT seedlings were treated with drought and their physiological and biochemical indices were determined. Compared with normal conditions, the stress-treated plants accumulated more H 2 O 2 and O 2 - (Figure 1f,g), and induced up-regulation of POD and CAT activities (Figure 1h,i). To investigate the changes of endogenous JA in S. miltiorrhiza under drought stress, the transcript levels of key genes for JA synthesis were measured by qRT-PCR, and the expression of JA synthesis genes was up-regulated after drought treatment (Figure 1b-e). In addition, endogenous JA (Figure 1a) and ABA (Figure S1) contents were also examined, and it was found that the contents of the two hormones in the stress-treated had noticeably higher levels than in the control plants. In conclusion, it was shown that drought promoted the burst of ROS, the increase of antioxidant enzyme activities and the cumulation of modulatory hormones (ABA and JA) in S. miltiorrhiza . 3.2 Expression pattern analysis of SmMYC2 Given that the MYC2 TF is central to the JA pathway-mediated defense response, we focused on SmMYC2, which we previously identified, and it acts as a typical TF with transcriptional activation activity (Cao et al. 2024). To explore the response of SmMYC2 to various signals, we first analyzed its promoter sequence characteristics and identified various phytohormone and abiotic stress response-related elements in the 2000 bp promoter region of SmMYC2 (Figure 1j), such as MeJA-responsive elements (CGTCA and TGACG motifs), GA-responsive elements (TCTGTTG motif), auxin-responsive elements (TGA motif) and drought-responsive elements (DRE motif). In its region, we did not find an ABA-responsive element (ABRE; ACGTGG/TC). The publicly available transcriptome data showed that SmMYC2 was able to be significantly triggered by MeJA and drought but not in response to ABA treatment (Figure S2). qRT‐PCR further validated its expression patterns under multiple treatments. As shown in Figure 1k, SmMYC2 transcript levels were considerably higher than that of the control from 1-12 h after MeJA treatment and peaked at 1 h with a 7.5-fold increase. However, the expression level of SmMYC2 was almost unchanged compared to the control after 24 h of ABA treatment (Figure 1m). We then explored the expression pattern of SmMYC2 under drought treatment (induced by PEG). SmMYC2 was rapidly triggered after 1 h of treatment and peaked at 1 h which was about 5.8-fold of that of the control (Figure 1l). These findings demonstrated that SmMYC2 is involved in sensing drought signaling independently of ABA. 3.3 Analysis of the function of SmMYC2 in the drought and ABA responses in Arabidopsis To determine the effect of SmMYC2 in drought response, we first treated transgenic Arabidopsis with mannitol. In 1/2 MS medium, there was no obvious variation in seed germination and root length growth between WT and transgenic lines. Under mannitol treatment, the seed germination rate and root length of the transgenic lines were noteworthily greater than those of WT (Figure S3a,b), indicating that the expression of SmMYC2 reduced mannitol-induced harm and growth inhibition in Arabidopsis . To examine the role of SmMYC2 in drought resistance, 4-week-old adult Arabidopsis plants cultured in soil were subjected to drought treatment. Under normal circumstances, there were no obvious phenotypic differences between the lines. After 13 days of drought stress, leaf yellowing was less severe in the SmMYC2 -OE lines than in the WT, and after 2 days of rewatering, the transgenic lines displayed a tolerant phenotype compared to the WT (Figure 2a). After drought treatment, the proline and chlorophyll contents in the transgenic lines were clearly more than in the WT (Figure 2b,d). The WT plants had markedly higher MDA levels than the transgenic lines under drought conditions (Figure 2c), suggesting that WT experienced more serious membrane damage. To acquire deeper about the impact of drought stress on plant photosynthesis, we further analyzed some chlorophyll fluorescence parameters and drought reduced Fv/Fm , Y(II) and qP in both WT and OE lines, but the WT showed a higher decrease than the transgenic lines (Figure S3c-e). For NPQ, both WT and transgenic lines increased after treatment contrasted to normal conditions, but the increase was greater in WT (Figure S3f). DAB and NBT staining was executed to assess H 2 O 2 and O 2 - levels. There were no obvious distinctions in staining intensity between WT and transgenic plants under normal situations. Under drought treatment, brown and blue spots indicative of H 2 O 2 and O 2 - were more numerous and darker in WT than in the transgenic lines (Figure 2e). Furthermore, SOD, POD and CAT activities were signally stronger in the transgenic lines than in WT (Figure 2f-h). These consequences imply that SmMYC2 actively modulates the drought response in Arabidopsis . Considering that ABA treatment had no effect on SmMYC2 transcript levels, we hypothesized that SmMYC2 may function independently of ABA for drought stress resistance. To verify this assumption, we compared ABA sensitivity in WT and SmMYC2 -OE lines. As expected, the extent of inhibitory effect in seed germination (Figure S4a) and seedling root development (Figure S4b) by ABA was equal in all genotypes. To better clarify the molecular mechanisms of SmMYC2-mediated drought stress resistance, we detected the relative expression levels of three representative ABA-independent drought response genes in WT and SmMYC2 -OE plants under control conditions or after drought treatment. The expression of AtCOR15A , AtDREB2A and AtCBF4 was clearly higher in the SmMYC2 -OE lines than in WT under drought treatment (Figure 2i-k). 3.4 SmMYC2 confers drought stress resistance in S. miltiorrhiza through an ABA-independent pathway To investigate the function of SmMYC2 in S. miltiorrhiza , SmMYC2 overexpression ( SmMYC2 -OX) and RNA interference ( SmMYC2 -Ri) hairy roots were produced. We selected three lines (OX-1, OX-4 and OX-11) that exhibited high SmMYC2 expression levels and three lines (Ri-2, Ri-6 and Ri-9) with low SmMYC2 expression levels for further experiments (Figure S5). We first carried out a hairy root drought stress (induced by PEG) experiment. Hairy roots with similar status after synchronization were treated with 10% PEG-6000. After one week of treatment, the growth and fresh weight of SmMYC2 -OX hairy roots under PEG treatment were superior to those of the control, and the browning of hairy root tissues was significantly less, and the reverse phenotype was exhibited in the SmMYC2 -Ri lines (Figure S6a,b). Compared with SmMYC2 -OX, control lines showed higher ROS and MDA accumulation and lower SOD, POD, and CAT activities (Figure S6c-h), suggesting that SmMYC2 can alleviate drought-triggered oxidative stress damage by fostering the clearance of excess ROS. To further validate that SmMYC2 may operate independently from ABA in drought stress resistance, we utilized qRT-PCR to detect the transcript levels of SmNCED3 in different lines after PEG treatment, which has been functionally characterized as the rate-limiting enzyme of the ABA synthesis pathway in S. miltiorrhiza (Jia et al. 2018). The result discovered that the expression levels were comparable among the different lines after stress treatment. Importantly, we did not find differences in PEG-induced ABA accumulation (Figure S8a). For further attesting the function of SmMYC2 in S. miltiorrhiza , we produced SmMYC2 -overexpressing transgenic S. miltiorrhiza seedlings by Agrobacterium -mediated transformation. Two transgenic lines (OX-4 and OX-8) with high SmMYC2 expression were utilized for drought tolerance experiments (Figure S7). After 14 days of drought and 4 days of rewatering, the morphological analysis showed that the SmMYC2 -OX plants had less susceptibility to drought stress with delayed leaf wilting than control plants (Figure 3a). The MDA content of control plants was much higher than that of SmMYC2 -OX plants (Figure 3b), compliance with the morphological observations. The transgenic lines had distinctly lower H 2 O 2 and O 2 - levels (Figure 3d,e), as well as less DAB and NBT staining (Figure 3c). Control plants also displayed weaker POD and CAT activities than transgenic plants (Figure 3f,g). Furthermore, there were no clear differences in the transcript levels of SmNCED3 and the accumulation of endogenous ABA in SmMYC2 -OX and control plants under drought treatment (Figure S8b). Based on these findings in Arabidopsis and S. miltiorrhiza , we prove that SmMYC2 is a positive modulator of drought defense in S. miltiorrhiza and that its mode of action is to modulate the expression of drought-responsive genes in an ABA-independent manner. 3.5 Genome-wide identification of SmMYC2 binding sites To study the molecular mechanism of SmMYC2-mediated modulation of drought resistance, DAP-seq was used to detect SmMYC2 binding sites on a genomic scale. We consistently identified a total of 20754 enriched peaks in both biological replicates, corresponding to 12747 genes (Figure 4a), which were therefore regarded as high-confidence SmMYC2 binding regions. All reads within 2000 bp distribution upstream of transcription start site (TSS) and 2000 bp distribution downstream of inverse transcription termination site (TES) were counted using DeepTools software, and most of the binding sites were concentrated on the TSS (Figure 4b). An exhaustive survey of the binding profiles disclosed that not only were 18.86% of the binding sites for SmMYC2 situated within the promoter regions (up to 2 kb upstream from TSS), but also consisted of many binding sites in the 5’ UTRs (0.93%), 3’UTRs (1.3%), exons (3.82%), introns (13.53%), and intergenic regions (52.65%) (Figure 4c, Table S2). This suggests that SmMYC2 is a typical TF that binds to DNA promoters and regulates gene activity. To acquire additional information about the DNA-binding properties of SmMYC2, we used the MEME-chip in the Multiple Em for Motif Elicitation (MEME) program Suite to identify motifs that were notably enriched in peak gene sequences. The findings revealed that the most statistically overrepresented motif sequence was WTTWRRCACGTG (E value=5.7e-366), accounting for 86.18% of peak-related genes. Particularly, the nucleotide sequence of this motif originated from the essential sequence CA[C/T][G/A]TG, a recently reported MYC2 identification motif with A/T-rich flanking motifs centered at ±5 capable of increasing the specificity and/or stability of MYC2 binding (López-Vidriero et al. 2021), validating accuracy of our DAP-seq data. We identified two other motifs, DTGACAGS (E value=1.3×e-013) and WWTTAATTA (E value=2.7×e-007), which together accounted for 17.00% of the peak-associated genes (Figure 4d). Gene ontology (GO) enrichment analysis of the genes that possessed SmMYC2 binding sites disclosed enrichment for processes such as ”response to hormone” and ”response to stimulus” (Figure S9a, Table S3). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was then performed and we found that the target genes of SmMYC2 were related to a variety of metabolic pathways, including ”ubiquinone and other terpenoid-quinone biosynthesis”, ”plant hormone signal transduction” and ”peroxisome” (Figure S9b, Table S4). Among them, ”plant hormone signal transduction”, ”response to stimulus” and ”peroxisome” were closely related to plant responses to adversity. These results imply that SmMYC2 may regulate multiple biological pathways associated with stress through hormone-mediated signaling pathways. 3.6 SmMYC2 mediates the regulation of metabolic and stress pathways Based on the SmMYC2 target genes related to the ”response to stimulus” pathway being markedly enriched in GO enrichment analysis, we next focused on those target genes whose binding sites were located in promoter regions. Some of them had known stress-related functions (Figure S10a, Table S5), including universal stress protein (USP) gene, oxidative stress (OXS) gene, early dehydration-inducible (ERD) gene, dehydrin (DHN) gene, aquaporin (AQP) gene and class I trehalose-6-phosphate synthase (TPS) gene. Among them, DHN (Chiappetta et al. 2015; Falavigna et al. 2019; Verma et al. 2017) and class I TPS (Almeida et al. 2005; Han et al. 2016; Li et al. 2011) can resist drought stress by promoting the synthesis of osmoprotective substances in various species, and it has been demonstrated that SmDHN1 can actively participate in the response of S. miltiorrhiza to drought (Chen et al. 2021). Furthermore, the relative expression levels of SmDHN1 and SmTPS11 were remarkably up-regulated in SmMYC2 -OX plants (Figure S10b). Considering the SmMYC2 target genes connected with the ”peroxisome” pathway were conspicuously enriched in KEGG enrichment analysis, we next identified many genes whose binding sites were in promoter regions (Table S6). It was worth noting that genes encoding peroxisomal membrane protein (PMP), involved in peroxisomal biogenesis (PEX) and fatty acid oxidation (ACOX), as well as responsible for the construction of plant antioxidant network (CAT and POD) were all obviously up-regulated in SmMYC2 -OX seedlings by qRT-PCR assays (Figure 5). These upregulated target genes accounted for the vast majority of SmMYC2-targeted peroxisome pathway genes (eight tenths). It suggests that SmMYC2 can mediate the response of peroxisomes to environmental and cellular stimuli by altering their dimension, amount and proteome content. Of note, elevated antioxidant enzyme activity is a conserved reaction to drought and dehydration (Wang et al. 2024). Given that SmMYC2 target genes associated with the ”response to hormone” pathway were remarkably enriched both in GO and KEGG enrichment analyses, we next analyzed the genes whose binding sites were in promoter regions (Table S7). These typical genes participated in miscellaneous plant hormone synthesis and signaling pathways, including JA, ABA, IAA and Eth, and then we constructed simplified molecular networks (Figure S11). Interestingly, we identified many AP2/ERF superfamily members in the Eth pathway. Notably, these included two DREB family members that were upregulated by SmMYC2 (Figure S12), separately belonging to the DREB1 -type and DREB2 -type, which were able to participate in ABA-independent signal transduction pathways to improve plant drought survivability (Agarwal et al. 2017; Sakuma et al. 2006). Moreover, we discovered a total of four representative JA pathway-associated SmMYC2 target genes through an exhaustive exploration of our data and then investigated SmMYC2-mediated transcriptional modulation of them (Table S7). We identified a key gene for JA synthesis ( SmOPR3-5 ) (Xue et al. 2022), and its transcript was noticeably up-regulated in SmMYC2 -OX seedlings (Figure S13a,b). This manifests that SmMYC2 can positively feedback regulate the JA signaling in S. miltiorrhiza under drought stress. Meanwhile, a jasmonate resistant 6 (JAR6) gene, which encodes the enzyme responsible for generating the bioactive form of JA from the coupling of JA with isoleucine, was also characterized as the target of SmMYC, but no statistical significance of its expression level was observed between WT and SmMYC2 -OX plants (Figure S13c,d). This indicates that the foundational JAR level may be adequate for the transformation of extra JA into bioactive JA-Ile. In A. thaliana , JAZ proteins act as typical repressors of MYC2 (Chini et al. 2007). Interestingly, we recognized two JAZ genes ( SmJAZ1 and SmJAZ2 ) that were SmMYC2 directly up-regulated (Figure S13e-f), which supported the existence of negative feedback mechanisms of SmMYC2 in JA signaling. It is noteworthy that our recent study has found that both SmJAZ1 and SmJAZ2 can interact with SmMYC2 and inhibit its activity, thus weakening the JA signaling (Ma et al. 2022) These results suggest that SmMYC2 can form a feedback loop for fine regulation of the JA pathway by directly positively regulating the expression of SmOPR3-5 and SmJAZ1/2 . In summary, SmMYC2 can directly target and noteworthily activate a mass of genes related to metabolic pathways and stress responses. 3.7 Dynamic changes in JA accumulation and signaling in S. miltiorrhiza during drought Considering that JA-modulated gene transcription under both the wounding and pathogen infection is highly chronological (Du et al. 2017). We contrasted the temporal expression profiles of MYC2 , OPR3-5 and JAZ1/2 in response to drought (PEG-induced). qRT-PCR tests disclosed that although drought induced the expression of these genes in WT plants of S. miltiorrhiza , the peak time of their induction was distinct: the expression of MYC2 peaked at 1 h, while the expression of OPR3-5 peaked at 3 h and the expression of JAZ1/2 peaked at 6 h after drought (Figure S14a). The drought-triggered expression dynamics of these genes were basically conservative for their Arabidopsis homologs in response to MeJA (Hickman et al. 2017). We next measured the content of endogenous JA in the leaves at different time points after drought stress. The consequences implied endogenous JA had accumulated as early as at 1 h, then sharply increased at 3 h and achieved its maximum at 7 h. This was followed by a sharp decline at 7 h and until returned to normal levels (Figure S14b). The accumulation pattern was highly coincident with the expression profiles of the JA biosynthesis and signaling genes. In short, drought-induced JA-modulated genes in S. miltiorrhiza may be highly temporally ordered and SmMYC2 may function at a high hierarchical level. 3.8 SmMYC2 directly regulates core genes involved in ROS scavenging To explore the mechanism by which SmMYC2 exerted drought tolerance, based on the fact that SmMYC2 could directly upmodulate peroxisomal metabolism, we selected two key genes that may participate in drought defense through the antioxidant system for validation: SmCAT (SMil_00016105) and SmPOD40 (SMil_00005644). To verify that SmMYC2 directly regulates ROS homeostasis through these genes, we isolated the coding sequence of SmMYC2 and particular promoter fragments (500 bp) of these target genes and separately fused them to pB42AD and pLacZi vectors, which were then pairwise transformed into yeast cells for yeast one-hybrid (Y1H) assay. As shown in Figure 6a, only prey (pB42AD-SmMYC2) and bait (pLacZi- proSmCAT and proSmPOD40 ) co-transformed cells turned blue in chromogenic mediums containing X-Gal. After mutating the binding site in the promoter fragments of these target genes to AAAAAA, prey- and bait-transformed cells no longer turned blue in the chromogenic medium containing X-Gal (Figure S15a). These results confirmed the interaction of SmMYC2 with the promoters of SmCAT and SmPOD40 in yeast. We next implemented a dual-LUC assay in which we generated the recombinant 35S:: SmMYC2 effector and three reporters proSmCAT ::LUC and proSmPOD40 ::LUC. When both recombinant reporter and effector were existent, the fluorescence signal was brighter than the control, and the LUC activity measurements corresponded to the LUC imaging results (Figure 6b,c). After mutating the binding site in the promoter fragment of the target gene, the brightness of the fluorescence signal was almost the same regardless of whether the recombinant reporter and effector were present at the same time, and LUC activity measurements showed the same results as LUC imaging (Figure S15b). Moreover, we executed electrophoretic mobility shift assays (EMSAs) to verify that SmMYC2 could specifically bind to the SmCAT and SmPOD40 promoters. We noticed a shifted band for each promoter fragment when purified SmMYC2 was blended with the biotin-labeled DNA probe containing the binding motifs we determined in the DAP-seq analysis but not by the probe with the mutant binding motifs (Figure 6d). These findings revealed that SmMYC2 could bind to the promoters of SmCAT and SmPOD40 both in vitro and in vivo and activate their transcription. In short, our consequences suggest that SmMYC2 is a key positive transcriptional regulator mediating ROS homeostasis in S. miltiorrhiza and validate the dependability of DAP-seq for determining SmMYC2-binding genes. 3.9 SmCAT positively regulates drought tolerance in S. miltiorrhiza As it was validated that SmCAT is a target of SmMYC2, we cloned the full-length CDS of SmCAT from S. miltiorrhiza . Conserved structural domain analysis showed that SmCAT contained a catalase core domain (Catalase, Pfam: PF00199) and a catalase-relative immune response domain (Catalase-rel, Pfam: PF06628) (Figure S16a), which were essential for its function. Genome-wide analysis showed that the CAT family in S. miltiorrhiza contained only this one member, and bi-directional blast showed that SmCAT and AtCAT2 (responsible for most of the catalase activity) (Chen et al. 2020) were orthologous genes (Figure S16b, Table S8), which suggested that S. miltiorrhiza had retained only one of the most active family members to perform its function during the evolutionary process. Protein sequence alignment showed that SmCAT protein had a PTS1 motif (QKL) at its C-terminal like other peroxisomal CAT proteins (Figure S16c). This conserved motif is reported to ensure catalases enter the peroxisomes (Wang et al. 2017). To investigate the subcellular localization of SmCAT, SmCAT-GFP was transiently expressed together with a peroxisome-localized marker in N. benthamiana leaves. The result showed that SmCAT-GFP fluorescence signals overlapped with the peroxisomal marker mCherry-PTS1 (Figure S16d), indicating that SmCAT is localized to the peroxisome. The peroxisomal localization of SmCAT protein indicates that SmCAT may play a vital function in peroxisomal metabolism. To determine whether SmCAT was involved in drought stress response, we generated SmCAT -overexpressing plants and RNA interference plants (Figure S17). We selected two lines (OX-4 and OX-9) that exhibited high SmCAT transcript levels and two lines (Ri-1 and Ri-3) with low SmCAT expression levels for further study. Morphological analysis exhibited that the SmCAT -OX lines evinced traits indicating considerable tolerance to drought-induced damage and yellowing, whereas the SmCAT -Ri lines showed more severe leaf wilting (Figure 7a). Compared with control plants, SmCAT -OX lines had higher POD and CAT activities and cumulated less MDA, H 2 O 2 and O 2 - under drought treatment, while the opposite tendency was noticed in SmCAT -Ri plants (Figure 7b,d-g). Consistent with measurements, brown and blue spots indicative of H 2 O 2 and O 2 - were lighter in the SmCAT -OX lines than in the WT, while color was darker in SmCAT -Ri lines (Figure 7c). Altogether, these results determine that SmCAT improves drought resistance by maintaining ROS homeostasis. 3.10 SmDREB2D positively modulates drought resistance in S. miltiorrhiza Numerous studies have reported that bHLH TFs in plants can directly modulate the expression of DREB2 -type genes in response to drought treatment (Mao et al. 2024; Wei et al. 2021). Since SmDREB2D behaves as a potential direct target of SmMYC2 (Figure S12) and the expression profile of SmDREB2D under drought and ABA treatments was analogous to that of SmMYC2 (Figure S18a), we cloned the full-length CDS of SmDREB2D from S. miltiorrhiza . Multiple sequence alignment of SmDREB2D with other DREB proteins showed the existence of valine (V) at position 14 and glutamic acid (E) at position 19 at the N-terminal end of the SmDREB2D sequence (Figure S18b, Table S9). The structural domain analysis indicated that SmDREB2D had only one AP2 structural domain (Figure S18c). In summary, SmDREB2D belongs to the DREB2 -type. To ascertain the binding of SmMYC2 on the promoter of SmDREB2D , pLacZi- proSmDREB2D and mutant pLacZi- proSmDREB2D fusion vectors were gained for Y1H assay. The consequences displayed that only co-transformation of pB42AD-SmMYC2 and pLacZi- proSmDREB2D into yeast cells led to them turning blue on the chromogenic medium (Figure S19a,b). An EMSA was implemented to further attest whether SmMYC2 is specifically bound to the promoter of SmDREB2D . The shifted band was observed after adding a probe containing a G-box of the SmDREB2D promoter (Figure S19c). To verify the transcriptional modulation of SmDREB2D by SmMYC2, we conducted dual-LUC assays. The findings exhibited that SmMYC2 facilitated the transcriptional activity of SmDREB2D promoter (Figure S19d-f). To determine the effect of SmDREB2D in S. miltiorrhiza drought response, transgenic hairy roots overexpressing SmDREB2D or with SmDREB2D expression inhibited were acquired (Figure S20). We chose three independent lines (specified as OX-5, OX-7 and OX-10) that manifested high SmDREB2D expression levels and three independent lines (specified as Ri-2, Ri-12 and Ri-14) that manifested low SmDREB2D expression levels for the following analysis. To explore whether SmDREB2D also improves drought tolerance by affecting ROS homeostasis, we carried out a hairy root drought stress (induced by PEG) experiment. Hairy roots with similar status after synchronization were treated with 10% PEG-6000. After one week of treatment, the growth and fresh weight of SmDREB2D -OX hairy roots under PEG treatment were superior to those of the control, and the browning of hairy root tissues was significantly less, and the reverse phenotype was exhibited in the SmDREB2D -Ri lines (Figure S21a,b). Compared with control lines, SmDREB2D -OX lines showed less ROS accumulation and higher POD and CAT activities after PEG treatment, whereas the contrary trend was noticed in SmDREB2D -Ri lines. There was no significant difference in the SOD activity between WT and transgenic lines under normal and drought situations (Figure S21c-g). These findings manifest that SmDREB2D positively modulates drought resistance of S. miltiorrhiza . 4. Discussion Drought stress greatly limits crop geographical distribution (Kim et al. 2017; Kumar et al. 2019). Research on plenty of plant species has demonstrated the critical role MYC2 TF plays in modulating responses to drought (Xia et al. 2024; Zhao et al. 2023), yet the fundamental mechanisms are still mostly unclear. S. miltiorrhiza is a momentous herbal plant, and its dried root is commonly used clinically in traditional Chinese medicine, mainly for the cure of cardiovascular and cerebrovascular illnesses and other inflammatory diseases (Dong et al., 2011; Wang and Wu 2010; Xu et al. 2010). Due to its comparatively small genome (about 600 Mb), brief life span, low need for growth and high pharmacological worth (Li and Lu 2014), S. miltiorrhiza is becoming a model plant for traditional Chinese medicine. Although much has been reported about this species, research on the molecular mechanism of its response to drought is still lacking. Here, we identified a bHLH TF, SmMYC2, which positively modulates drought adaption in S. miltiorrhiza. It enhances drought resistance of S. miltiorrhiza in an ABA-independent manner by directly advancing the expression of SmCAT , SmPOD40 and SmDREB2D , etc. Last but not least, we found solid evidence for the role of SmMYC2 in the feedback region controlling JA signaling. Our findings offer a reference for future studies on drought-triggered JA signaling and MYC2 TF-mediated plant resistance mechanisms. In plants, the interaction of JA and ABA synergistically increases plant resistance to drought stress by increasing intracellular antioxidant enzyme content, further safeguarding the plant from ROS bursts (Abdelgawad et al. 2014; Devireddy et al. 2021; Ding et al. 2010). Similarly, S. miltiorrhiza was subjected to drought stress with increased ROS content and enhanced POD and CAT activities, conforming to the cumulation of JA and ABA (Figure 1, Figure S1). In JA-mediated drought response, MYC2 TF acts as a hub for JA signal transduction and responds to drought stress by activating downstream defense-related genes (Kazan and Manners 2013). The expression of drought stress-responsive genes is triggered via ABA-dependent and ABA-independent pathways, modulated by ABRE-binding and DRE-binding TF, respectively. In this research, we discovered that the promoter of SmMYC2 did not contain an ABRE motif but contained a DRE motif (Figure 1j), and overexpression of SmMYC2 reduced the accumulation of ROS, which significantly increased plant tolerance to drought stress, while silencing of SmMYC2 increased the sensitivity of S. miltiorrhiza to drought stress (Figures 2-3, Figures S3,6). Interestingly, SmMYC2 transcript levels were conspicuously induced after MeJA and PEG treatments, but no discernible changes were observed under ABA treatment (Figure 1k-m, Figure S2), suggesting that SmMYC2 may confer drought tolerance by an ABA-independent pathway. In support of the idea that SmMYC2 acts independently of ABA, we found that SmMYC2 transgenic Arabidopsis did not differ from WT in regulating ABA responses as measured by seed germination and root growth after ABA treatment (Figure S4). Most importantly, the studies from both overexpression and silencing of SmMYC2 in hairy roots and overexpression of SmMYC2 in seedlings showed that when the plants were placed under drought stress, the transcriptional levels of SmNCED3 as well as the accumulation of ABA were at the same level as that of the control plants (Figure S8). Overall, these findings unquestionably illustrate that SmMYC2 is a momentous positive regulator in the ABA-independent drought stress response pathway. Plant TFs are considered middle target genes in signal transduction pathways to modulate plant stress tolerance and are one of the most critical molecular mechanisms by which plants confront adversity (Li et al. 2023). In view of the positive affect of SmMYC2 in drought resistance, in order to uncover the modulatory mechanism of SmMYC2-mediated stress response, we used DAP-seq to recognize many genes that could participate in stress response and phytohormone synthesis and signal transduction (Tables S5-7). In the many stress-responsive genes, we found two conserved genes capable of improving drought tolerance ( SmDHN1 and SmTPS11 ). Up to now, most of the overexpression of DHN or TPS genes in plants has displayed better tolerance to drought stress by increasing dehydrin or trehalose levels (Garg et al. 2002; Jang et al. 2003; Karim et al. 2007). In natural environments, high temperatures and drought often occur simultaneously, which may be the main reason for the presence of multiple HSFs involved in stress regulation. Meanwhile, a cold-regulated (COR) gene participated in plant response to cold stress and three Na + /H + antiporter (NHX) genes responsible for regulating ionic homeostatic balance in plants had also been identified. Interestingly, we also discovered multiple genes involved in biotic stress response, including pathogenesis-related (PR) gene, resistance to Pseudomonas syringae pv. maculicola (RPM) gene, RPM interacting protein (RIN) gene, and non-race-specific disease resistance (NDR) gene. Moreover, several genes in the calcium signaling pathway and MAPK signaling pathway were also successfully recognized (Table S5). Collectively, our study supplies strong evidence that SmMYC2 can widely respond to biotic stresses and various kinds of abiotic stresses comprising drought, high temperature, cold and salinity. Plant hormones are intrinsic to coordinated adversity defense, and we found that SmMYC2 can target key genes that regulate multiple hormone metabolism, including JA, ABA, IAA and Eth (Figure S11). It has been manifested that MYC2 acts as a master switch in JA metabolism, including autoregulatory feedback loops (Liu et al. 2019; Wasternack and Song 2017). In the present research, we found that SmMYC2 was able to directly upregulate the OPR and JAZ genes in the JA pathway (Figure S13), suggesting that SmMYC2 can fine-tune the activation and inhibition of the cooperative and antergic activities of each component, resulting in the induction and/or termination of JA signaling at distinct levels. Interestingly, the drought-triggered induction of OPR and JAZ genes was obviously deferred relative to that of MYC2 (Figure S14a). The uncontrolled activation of MYC2 as a convergence node of numerous pathways can be extremely damaging or even fatal to the cell, and thus needs to be tightly regulated both spatially and temporally (Chico et al., 2020). Given that the de novo synthesis of stabilized JAZ repressors exhibits a characteristic delay relative to the induction of MYC2 genes (Chuang et al. 2010; Chuang and Howe 2009; Yan et al. 2007). Integrated JA accumulation mode (Figure S14b), we hypothesized that in the early stages of drought, SmMYC2 amplified JA signaling through the targeted activation of SmOPR3-5 , exacerbating the induction of downstream response genes by SmMYC2. In the late stage of drought, plants no longer required a defense response, at which time SmMYC2 repressed or terminated JA signaling through transcriptional activation of SmJAZ1/2 . This type of regulation will ensure the thorough enforcement of an already launched JA response. Interestingly, the ABA signaling pathway also had multiple target genes that were directly bound by SmMYC2 (Table S7), while we did not find any common genes (such as zeaxanthin epoxidase , NCED and aldehyde oxidase ) connected with the ABA synthesis pathway (Table S2). Since SmMYC2 was unable to sense ABA and might not involved in ABA biosynthesis but might directly bind the genes associated with the ABA signaling pathway, we supposed that SmMYC2 was an upstream modulator of ABA signal transduction and a vital node in crosstalk with the ABA pathway. Moreover, SmMYC2 had potential direct bonding relationships with plentiful key genes in the Eth synthesis and signal transduction (Table S7), suggesting that there are complex interactions between SmMYC2 and the Eth pathway. These results indicate that SmMYC2-mediated drought resistance is closely related to multiple signaling pathways, and its defense response to adversity stress encompasses multiple metabolic changes in a multitude of pathways, a complex network involving the interactions of many genes, which needs to be further investigated. As is known to all, drought stress creates an excessive buildup of ROS and causes harm to membrane lipids (Fedoroff et al., 2010; Schieber and Chandel, 2014). CAT and POD, as an important H 2 O 2 scavenging enzyme, exert a crucial influence on plant response to adversity (Bueso et al. 2007; Du et al. 2008). In this research, we exhibited that SmMYC2 could specifically bind to the promoters of SmCAT and SmPOD40 and transcriptionally activate their expression (Figure 6). Further functional verification identified the positive role of SmCAT in regulating drought resistance in S. miltiorrhiza (Figure 7). Collectively, these results indicate that the SmMYC2- SmCAT / SmPOD40 module can confer drought resistance to S. miltiorrhiza by directly promoting ROS scavenging. Moreover, many other peroxisome pathway genes also had been identified as potential targets of SmMYC2 and were remarkably upregulated by SmMYC2 (Figure 5, Table S6). It is now widely accepted that the peroxisome is the core hub of the cell to complete secondary metabolism, mainly responsible for the fatty acid oxidation and the removal of ROS, thus preserving the cell from oxidative stress damage. (Reumann and Bartel 2016). Activation of core genes involved in peroxisome biogenesis and fatty acid β-oxidation has been shown to be also a conserved response to drought and dehydration (Ebeed et al. 2018). This implies that peroxisomes play a significant and ancient evolutionary role in the perception and reaction processes to stress. It is therefore reasonable to conjecture that SmMYC2 contributes to the control of peroxisome dynamic equilibrium under drought stress. In conclusion, our research provides fresh perspectives on the effect of peroxisomes and peroxisome-related procedures in drought response and suggests that the role of the JA-peroxisome module in sensing and responding to drought stress warrants further study. To cope with drought stress, the expression of a particular class of genes must be quickly triggered in response to sign of water deficiency (Zhang et al. 2022). In this course, DREBs play crucial roles in the transcriptional activation of the ABA-independent drought stress response, especially DREB2-type members (Agarwal et al. 2017). In our research, SmMYC2 bound directly to the G-box in the SmDREB2D promoter and activated its expression (Figure S19), and the positive function of SmDREB2D in regulating S. miltiorrhiza drought adaption through ROS scavenging was confirmed (Figure S21). Sufficient evidence demonstrates that DREB1 -type genes are also positive modulators of drought resistance (Azzeme et al. 2017; Wang et al. 2022; Zhou et al. 2020). Of note, our omics data analysis showed SmDREB1E is most likely also a target of SmMYC2 (Figure S12). In a word, our findings suggest that the SmMYC2- SmDREBs module possibly contributes to SmMYC2-mediated ROS removal via further signaling. Additional research is needed to elaborate this assumption. 5. Conclusion Taken together, JA signaling is activated and promotes SmJAZ1/2 degradation by the 26S proteasome under drought stress. SmMYC2 is relieved from JAZ-mediated repression, allowing it to directly bind SmCAT , SmPOD40 and SmDREB2D promoters to enhance their expression, thereby restraining excess ROS amassing and improving the drought resistance of plants. In the feedback section, SmMYC2 flexibly controls JA signaling output by regulating JA synthesis and repressing gene expression (Figure 8). The molecular mechanism by which SmMYC2 modulates drought tolerance in S. miltiorrhiza is greatly important for acclimatization to climatic variation and S. miltiorrhiza production under drought. Acknowledgements This work was funded by the National Natural Science Foundation of China (No.32270278). Author Contributions JYL, PDM conceived and designed the research. TW, QY performed the experiments. SYY, JY analyzed the data. Conflict of Interest The authors declare no conflict of interest. Data availability The datasets supporting the results of this article are included within the article and its Supporting Information. References Abdelgawad, Z. A., Khalafaallah, A. A., & Abdallah, M. M. (2014) Impact of methyl jasmonate on antioxidant activity and some biochemical aspects of maize plant grown under water stress condition. Agricultural Sciences , 5(12), 1077-1088. Agarwal, P. K., Gupta, K., Lopato, S., & Agarwal, P. 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Keywords salvia miltiorrhiza smmyc2 drought stress hormones ros signaling Authors Affiliations Tong Wang Northwest A&F University College of Life Sciences View all articles by this author Qing Yang Northwest A&F University College of Life Sciences View all articles by this author Shiyu Yao Northwest A&F University College of Life Sciences View all articles by this author Jing Yang Northwest A&F University College of Life Sciences View all articles by this author Jingying Liu Northwest A&F University College of Life Sciences View all articles by this author Pengda Ma [email protected] Northwest A&F University College of Life Sciences View all articles by this author Metrics & Citations Metrics Article Usage 215 views 143 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Tong Wang, Qing Yang, Shiyu Yao, et al. SmMYC2 mediates ROS homeostasis to confer drought resistance in Salvia miltiorrhiza through an ABA-independent pathway. Authorea . 05 January 2025. 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