Physiological and Transcriptomic Cooperative Regulatory Mechanisms of Cotinus coggygria in Response to Drought and Rewatering Processes

Physiological and Transcriptomic Cooperative Regulatory Mechanisms of Cotinus coggygria in Response to Drought and Rewatering Processes | 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 Physiological and Transcriptomic Cooperative Regulatory Mechanisms of Cotinus coggygria in Response to Drought and Rewatering Processes Shiya Mao, Xinchun Liang, Yumeng Feng, Lulu Yang, Yiqian Xiao, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7457742/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Jan, 2026 Read the published version in BMC Plant Biology → Version 1 posted 12 You are reading this latest preprint version Abstract Background Global drought represents a pressing environmental challenge, necessitating a deeper comprehension of how plant species at various stages of drought response adapt to such stress. Cotinus coggygria , a deciduous tree species known for its autumn color transformation, holds significance for arid and semi-arid ecological contexts. Research investigating the detailed physiological and transcriptomic responses of C. coggygria to drought and subsequent rewatering is currently lacking. Results Seedlings of C. coggygria were subjected to five distinct drought durations (30, 50, 70, 90, and 110 days) followed by a 20-day rewatering period. Increasing drought severity led to reductions in seedling height, ground diameter, leaf water potential, and nitrogen and phosphorus contents across plant organs, while showing notable increases in stomatal traits, chlorophyll and carotenoid levels, as well as soluble protein and proline contents, ultimately bolstering the plant's ability to retain water. Towards the later stages of stress, heightened levels of hydrogen peroxide and malondialdehyde were observed, accompanied by diminished hydroxyl radical content, and augmented activities of peroxidase, catalase, and glutathione, indicative of antioxidant system modulation. Following short-term rewatering, most physiological parameters of C. coggygria did not fully recover to control levels. Transcriptomic analysis revealed 3443 up-regulated and 3891 down-regulated differentially expressed genes (DEGs) under 110 days of stress, and 1923 up-regulated and 1541 down-regulated DEGs following 20 days of rewatering, highlighting genes modulating phytohormone signaling pathways, metabolic pathways associated with key physiological indicators, and differentially expressed transcription factors. Conclusions The research revealed that C. coggygria demonstrated synchronized physiological and transcriptomic reactions to both drought stress and subsequent rehydration. These reactions encompassed alterations in growth metrics, nutrient levels, physiological characteristics, antioxidant system functionality, and gene expression profiles. The results offer significant understanding into the adaptive mechanisms of C. coggygria under drought stress conditions and may have implications for comprehending and mitigating drought effects on plant species in arid and semi-arid regions. Cotinus coggygria drought rewatering physiological response transcriptional regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background Drought is a significant abiotic stress factor that severely impacts plant growth and crop yields, affecting various physiological processes including growth, development, metabolism, and morphology [ 1 – 3 ]. Drought stress and subsequent rewatering are essential stages in the plant growth cycle [ 4 ], with rewatering serving as a recovery mechanism post-drought to restore growth and enable rapid plant development [ 5 ]. The investigation of physiological changes in plants during drought and rewatering conditions is crucial for understanding plant drought resistance mechanisms under varying water availability, thereby enhancing plant productivity and ecological adaptability. Drought stress exerts key effects on plants, altering pigment synthesis, osmoregulation, secondary metabolism, antioxidant systems, and gene expression [ 6 ]. The visible impact of drought on plants is the inhibition of morphological growth [ 7 ], with leaf morphology serving as a prominent indicator in drought studies [ 8 ]. Stomatal regulation, a common adaptive response to drought, helps plants maintain leaf water potential (LWP) stability and reduce gas exchange [ 9 ]. Chlorophylls (Chl) and carotenoids (Car) are pivotal for photosynthesis [ 7 ], with drought stress often leading to a reduction in chlorophyll contents [ 10 ]. Ecological stoichiometry, focusing on the balance of chemical elements in ecosystems [ 11 ], highlights the roles of carbon (C), nitrogen (N), and phosphorus (P) in plant growth and physiological processes [ 12 ]. C is an essential substrate and energy source for plant growth, while N and P are crucial nutrients and key elements for plant cell composition and metabolism [ 13 ]. Therefore, investigating the stoichiometric properties of C, N, and P in plants is valuable for understanding nutrient dynamics and utilization in plants. Plants commonly amass significant levels of reactive oxygen species (ROS) under drought conditions, leading to potential toxicity due to their excessive accumulation within plant tissues [ 14 ]. The antioxidant defense system plays a critical role in scavenging excess ROS to prevent cellular damage and maintain ROS homeostasis [ 15 , 16 ]. Malondialdehyde (MDA) levels, a product of membrane lipid peroxidation, reflect the extent of cell membrane damage under stress conditions. Osmoregulation plays a crucial role in the drought tolerance of plants, as it enables the maintenance of cell expansion even under drought stress conditions, thereby promoting plant growth [ 17 ]. Key osmoregulatory substances involved in this process are proline (Pro) and soluble protein (SP) [ 7 ]. The research by Wang et al [ 18 ] has shown that SP and Pro can serve as significant indicators for the identification of drought-resistant plant varieties. Transcriptomic methodologies have been extensively utilized to pinpoint genes orchestrating plant growth, development, and those exhibiting differential expression patterns under abiotic stress conditions [ 19 ]. Transcriptome sequencing has been pivotal in elucidating the molecular mechanisms governing plant responses to drought stress [ 20 ]. In the context of water deficit, phytohormones exhibit synergetic actions, with abscisic acid (ABA), salicylic acid (SA), cytokinin (CTK), ethylene (ETH), indole-3-acetic acid (IAA), jasmonic acid (JA), gibberellic acid (GA), and brassinosteroids (BR) playing crucial roles in aiding higher plants to surmount challenges posed by drought stress [ 21 ]. Elevated ABA levels have been observed to trigger the upregulation of numerous transcription factors (TFs) and genes, thereby activating downstream metabolic pathways [ 21 ]. Utilization of the weighted gene co-expression network analysis approach on drought-exposed Artemisia iliensis seedlings has revealed the pivotal involvement of various transcription factor families including WRKY, bHLH, NAC, AP2/ERF, MYB, GRAS, C2H2, MADS, and bZIP in mediating drought responses [ 22 ]. Numerous studies have investigated the physiological and biochemical characteristics of plants, yet there is a dearth of research on the physiological, biochemical, and gene expression alterations that occur during drought stress and subsequent recovery phases. Cotinus coggygria , a small deciduous tree belonging to the Anacardiaceae family and Cotinus genus, possesses notable attributes for soil and water conservation, landscape enhancement, and holds substantial medicinal, economic, and ornamental value. Current research efforts on C. coggygria primarily concentrate on breeding and afforestation [ 23 ], revealing significant research gaps in understanding its drought resistance mechanisms. Therefore, to study the physiological changes of drought stress as well as rewatering on leaf water potential, morphological growth and physiological structure, antioxidant system, osmotic substances of C. coggygria , as well as drought-resistant phytohormone signaling, differential expression patterns of genes related to physiological indicators, and the potential regulatory relationships among genes of the TFs family, can help C. coggygria drought tolerance indicators screening, for mining drought-resistant key genes and further elucidating the molecular regulatory mechanism to establish a framework. C. coggygria , as an autumn color-changing tree species, is of great significance to the application of tree species in arid and semi-arid landscapes. Methods Overview of the study area The research site is situated at the Forestry Station of Shanxi Agricultural University in Taigu District, Jinzhong City, Shanxi Province (112°57′54″ E, 37°42′78″ N). It experiences a temperate continental monsoon climate at an altitude of 1098 meters. The average annual temperature ranges from 5 to 10°C, with an annual precipitation of around 458 mm. The period from June to August receives the highest precipitation, constituting 70% of the annual total, whereas the lowest precipitation occurs from December to February. Experimental materials Transplant 3-year-old healthy seedlings of C. coggygria exhibiting consistent and vigorous growth into containers measuring 29.5 cm in width and 23.5 cm in height. The soil used in the experiment is taken from the garden soil of the Forestry Station. The pH value of the soil is 8.30, the total nitrogen content is 0.84 g·kg − 1 , the total phosphorus content is 0.48 g·kg − 1 , the available nitrogen content is 64.3 mg·kg − 1 , the available potassium content is 142.06 mg·kg − 1 , and the organic matter content is 12.06 g·kg − 1 . The soil's field capacity for water retention, as assessed by the ring knife method, is measured at 26.11%. The flowerpots are placed under a rain shelter. On April 30th, drought stress experiments were conducted with varying severity levels following the methodology outlined by Wang Kai et al [ 24 ]. Four soil moisture gradients were established: the control group (CK), mild stress (W1), moderate stress (W2), and severe stress (W3), corresponding to 80% ± 5%, 60% ± 5%, 40% ± 5%, and 20% ± 5% of the soil's field water holding capacity, respectively. Each group consisted of 40 pots, totaling 160 pots. Pots were placed under a rain shelter, and soil moisture content was measured using the gravimetric method [ 25 ]. Sampling was carried out after 30 d of drought stress treatment, followed by sampling every 20 d. A total of 5 samplings were carried out from the end of May to the middle of August. Subsequent to the drought stress period, a 20-day rehydration treatment was administered, with sampling conducted once. Determination of morphological growth and physiological indices The LWP at 6:00 AM and 12:00 PM was determined using a dew point potentiometer. The ground diameter (GD) of C.coggygria was measured with a vernier caliper (0.01 mm), and the seedling height (SH) was measured with a steel tape measure (accuracy of 0.1 cm). Leaf thickness at the upper, middle, and basal regions was measured with a thousandth caliper (0.001 mm), and the mean value was calculated. Stomatal number (SN) was quantified using Image J software, subsequently used to determine stomatal density (SD = SN per unit area) [ 26 ]. The contents of Chl and Car in leaves were quantified through ethanol extraction method [ 27 ]. The content of anthocyanin (Ant) was determined using the hydrochloric acid soaking method [ 28 ]. C, N, and P in leaves, stems, and roots were determined using the dry combustion method, Kjeldahl nitrogen analyzer method [ 29 ], and vanado-molybdate yellow colorimetry [ 30 ], respectively. Hydrogen peroxide (H₂O₂), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione (GSH), MDA, and Pro were all determined using the methods described in the Solarbio kit. The content of hydroxyl radical (·OH) was determined according to the method of the kit produced by Nanjing Jiancheng Bioengineering Institute. The content of SP was determined by the Coomassie Brilliant Blue G-250 staining method [ 27 ]. Transcriptome sequencing Fresh leaves from C. coggygria plants subjected to 110 days of drought treatment and 20 days post-rewatering in the CK and W3 treatments were harvested and rapidly frozen in liquid nitrogen. Each treatment comprised three independent replicates, resulting in a total of nine samples. Total RNA extraction was performed on these samples at Majorbio. Subsequently, a cDNA library was constructed and subjected to quality assessment before sequencing on the NovaSeq X Plus platform. De novo assembly of the clean data was conducted using Trinity software. The resulting transcriptome sequences were filtered, optimized, and assembled into Unigenes, which were then compared with the relevant database. DEGs were identified using the DESeq2 based on specified criteria (|log2FC| ≥ 1, FDR < 0.05). Data analysis Perform univariate and multivariate analysis of variance (ANOVA) on the dataset utilizing SPSS 26.0 software. Subsequently, conduct multiple comparisons using Duncan's test method to assess significant differences between groups. Results Effects of drought stress and subsequent rewatering on LWP and morphological growth of C. coggygria The prolonged drought and increased severity of drought led to a declining trend in both early morning leaf water potential (EMLWP) and midday leaf water potential (MDLWP) (Fig. 1 A, B). SH and GD tended to increase between treatment groups during the growth period of C. coggygria under sustained drought, and the intensification of the degree of stress led to the inhibition of growth of SH (except W1) and GD of C. coggygria between treatment groups (Fig. 1 C, D). W3 treatment leaves lost water and became thinner, and leaf thickness (LT) was lower than that of CK (Fig. 1 E). After 20 d of rewatering, the early morning LWP and midday LWP of W1, W2 and W3 treatment groups were still significantly lower than that of CK, and the W2 and W3 treatments SH, GD and LT were not restored to the CK level for the moment due to the short rewatering time (Fig. 1 ). Effect of drought stress and subsequent rewatering on stomatal traits of C. coggygria The SN and SD exhibited fluctuations among treatment groups under prolonged stress conditions (Fig. 2 ). With the intensification of drought stress, SN and SD gradually increased in all treatment groups (except CK) at 30–70 d of stress. At 110 d of stress, SD was higher in the W2 treatment group and lower in the W3 treatment group compared with CK. After rewatering, SN and SD were significantly higher in all treatment groups than in CK. Effects of drought stress and subsequent rewatering on C, N and P content of C. coggygria During prolonged drought stress, variations in carbon (C) content were observed in different plant organs, with a decrease followed by an increase in leaf C content, a decrease in stem C content (except W2), and a decreasing-then-increasing trend in root C content. Nitrogen (N) content showed a consistent decreasing trend across all organs in response to the different treatment groups. Phosphorus (P) content exhibited varied trends, characterized by a decline in leaf P content and a pattern of decrease followed by an increase in stem P content across different treatment conditions. In contrast, root P content exhibited a decreasing-then-increasing trend in CK and W1 treatments, diverging from the trends observed in W2 treatments. Under intensified drought stress, leaf C content was highest in the W3 treatment, followed by CK and W1 treatments at 30–70 days of stress, and W1 and W2 treatments surpassing CK treatment at 90–110 days of stress. Stem C content did not significantly differ between W3 and CK treatments at 30d-110 days of stress, with both exceeding W2 treatment. Root C content was highest in the W3 treatment at 30–70 days of stress, but CK surpassed W1 treatment at 110 days of stress (Fig. 3 A). Regarding N content, leaf N content was higher in W1 and W3 treatments compared to CK and W2 treatments during 30–50 days of stress, with no significant difference at 70 days, and W2 and W3 treatments surpassing W1 and CK treatments at 110 days of stress. During the 90 days stress period, stem N content was higher in the W3 treatment than in the other treatments. Root N content, in descending order, followed the sequence of W3 > CK > W1 > W2 (Fig. 3 B). Leaf P content was highest in the CK treatment at 30–90 days of stress, followed by a sequential decrease in P content across all treatment groups at 110 days of stress. Stem P content was greater in the CK treatment compared to the W3 treatment at 50–70 days of stress, and highest in the W3 treatment at 90–110 days of stress. Root P content at 30d-70d of stress showed that W1, W2 surpassed CK treatment, while at 90–110 days CK and W1 treatments surpassed W2 and W3 treatments (Fig. 3 C). After 20 days of rewatering, the C content in the W3-treated leaves and W2-treated stems/roots decreased respectively compared with CK, while the N content increased in each organ across all treatment groups, and the P content in the leaves/stems of all treatments increased whereas that in the W2/W3-treated roots decreased (Fig. 3 ). Effect of drought stress and subsequent rewatering on pigment content and leaf color parameters of C. coggygria Chlorophyll a, chlorophyll b, and total chlorophyll in leaf tissues exhibited an initial decrease followed by an increase over the course of drought stress, reaching their lowest levels on the 90th day (Fig. 4 A-C). Conversely, Car and Ant levels showed a consistent decline with prolonged drought exposure (Fig. 4 D-E). Under heightened drought stress, chlorophyll a, chlorophyll b, total chlorophyll, and Car contents displayed an increasing trend in all treatment groups. Ant levels in all treatment groups, except W3, exhibited a rise from 50 to 70 days of stress, whereas in the W3 group, Ant levels were suppressed during this period and decreased compared to CK by 90 to 110 days of stress. On the 20th day of rewatering, the levels of Chl a, Chl b, and Chl exhibited a progressive decline in all treatment groups (Fig. 4 A-C). The Car concentration did not vary significantly between groups W1 and W2 relative to the CK group, whereas there was a notable 57.22% rise in Car levels in group W3 compared to the CK group. Concurrently, the Ant concentrations in each treatment group exhibited increments of 46.02%, 73.03%, and 71.02%, respectively, in comparison to the CK group (Fig. 4 E). Effects of drought stress and subsequent rewatering on ROS levels and antioxidant mechanisms of C. coggygria The concentration of H 2 O 2 exhibited a pattern characterized by a decline, followed by an increase, and then a subsequent decline with the duration of stress in each treatment group, while the ·OH content displayed a pattern of decreasing, then elevating, followed by decreasing, and then increasing trends over time. SOD activity showed a relatively stable behavior. POD activity demonstrated divergent patterns among treatment groups, with consistent trends in CK, fluctuating trends in W1 and W2 groups, and increasing trends in W3 group. CAT activity demonstrated a pattern of initial increase followed by decrease with the duration of stress in the CK, and an overall trend of decrease followed by increase in the W1, W2, and W3 treatment groups. MDA content decreased after peaking at 70 days of drought treatment in all groups. GSH content exhibited an initial increase followed by a decrease in response to drought duration (Fig. 5 ). Upon reaching 70 days of drought stress, the ·OH content peaked in all treatment groups except W1 (Fig. 5 A). The H 2 O 2 content was significantly lower in all groups compared to CK at 30 days, while it was significantly higher at 50–90 days (Fig. 5 B). Among them, at 30d-90d of drought treatment, the MDA content gradually decreased with the decrease of soil moisture content, which was significantly lower than that of CK in all treatment groups. At 110th d of drought, MDA content increased sequentially with increasing drought (Fig. 5 C). SOD enzyme activity was significantly higher in W2 at 30 days, and lower in W3 at later stages of stress compared to CK (Fig. 5 D). POD activities were higher in W1 and W2 compared to CK at different stress durations (Fig. 5 E), while CAT activities were significantly higher in W1 and W2 at specific time points (Fig. 5 F). GSH content showed a gradual increase with prolonged drought (Fig. 5 G). Following rewatering, the H 2 O 2 content increased significantly in W1 and W2 compared to CK, with W2 showing the highest levels. The ·OH content in each treatment group did not exhibit significant variation and was found to be elevated compared to that of the CK. SOD enzyme activity was not fully recovered compared with 110d of stress, W2 treatment group was significantly higher than that of CK, and W3 treatment group had a stronger degree of stress, and SOD enzyme activity was still inhibited after rewatering. POD enzyme activities were significantly increased in the W1 and W2 treatment groups compared to CK, while POD enzyme activities remained lower in the W3 treatment group than in CK. CAT enzyme activity did not change significantly in the W2 and W3 treatment groups compared to 110 d of stress. The MDA content was significantly lower than that of CK, and the MDA content of the W3 treatment group was elevated compared with that of 110 d of stress, by 65.49%. The GSH content reached the maximum and was significantly higher than that of CK among the treatment groups, being 1.22, 1.28 and 1.12 times higher than that of CK, respectively (Fig. 5 ). Effect of drought stress and subsequent rewatering on osmoregulatory substances in C. coggygria At all periods of drought stress, the SP content of each treatment group, and the Pro content of CK, W1 and W2 treatment groups showed an increase followed by a decrease and then an increase, and the Pro content of the W3 treatment showed a smaller change (Fig. 6 ). There was no significant difference in SP content between CK treatment and other treatments in the pre-stress period, and the SP content of W2 and W3 treatment groups under 70d of stress was significantly higher than that of CK, with an increase of 3.54% and 4.63%, respectively; the SP content of W1 and W2 treatment groups under 90d of stress was significantly higher compared with that of CK, with an increase of 5.12% and 13.58%, and that of W3 treatment group under 110d of stress was also significantly higher compared with that of CK, with an increase of 6.80% (Fig. 6 A). The degree of drought significantly increased the Pro content, which was sequentially higher and significantly higher than CK among the treatment groups, with the greatest decrease in the 70th d of the drought treatment, with CK, W1 and W2 decreasing by 44.63, 78.04 and 80.92% compared to the 50th d of drought (Fig. 6 B). After rewatering, there was no significant difference in the SP content of each treatment group, and the Pro content was significantly higher than that of CK, which was 1.41, 1.60 and 2.81 times higher than that of CK, respectively (Fig. 6 ). Transcriptomic investigation of C. coggygria under drought stress and subsequent rewatering conditions To assess the gene expression pattern of C. coggygria , drought CK, Wd_3 and rewatering Wr_3 treatments were selected for high-throughput RNA sequencing. A total of 40901478 ~ 44714144 raw reads were obtained from the nine cDNA libraries, and 40627744 ~ 44397794 clean reads were obtained after eliminating the low-quality reads, and the percentage of localized reads for each sample was very high, ranging from 89.18–89.84% (Table 1). The clean reads library produced a percentage of Q30 bases above 95.29% and a percentage of Q20 bases above 98.55%, both with a GC content greater than 43.45%, and a comparison efficiency of 89.18%, indicating a good overall quality of the data. Gene expression abundance was less than normal water supply after drought treatment, and gene expression was largely restored after rewatering (Fig. 7 A). There were more DEGs for drought than for rewatering, and 1,785 genes were shared between drought and rewatering (Fig. 7 B), suggesting that these genes are most likely to respond to drought rewatering in C. coggygria . Analysis using DESeq2 software revealed that under drought conditions, 2684 genes were up-regulated and 4017 genes were down-regulated, whereas after rewatering, 1923 genes were up-regulated and 1541 genes were down-regulated in comparison to control (CK) (Fig. 7 C, D). Functional classification of DEGs was carried out by KEGG pathway analysis The KEGG enrichment analysis of the DEGs under drought conditions was enriched in 133 pathways, involving a total of 1,583 DEGs. Two pathways were significantly enriched, namely Metabolism and Environmental Information Processing. Among the two major categories of pathways, the pathways with the largest number of annotated genes are Plant hormone signal transduction, Starch and sucrose metabolism, Phenylpropanoid biosynthesis; MAPK signaling pathway - plant MAPK; and Biosynthesis of various plant secondary metabolites (Fig. 7 E). After rewatering, 711 DEGs in the leaves were annotated to 118 metabolic pathways. These pathways were significantly enriched in pathways such as Plant-pathogen interaction, ABC transporters, Phenylpropanoid biosynthesis, Biosynthesis of various plant secondary metabolites, and Flavonoid biosynthesis (Fig. 7 F). Compared with CK treatment, genes related to Metabolic, Biological and Environmental Information Processing were significantly expressed and the number of up-regulated expressed genes was much lower than the number of down-regulated expressed genes after Wd3 treatment, suggesting that drought stress significantly affected the metabolism and biosynthesis of C. coggygria . Compared with CK treatment, Wr3 treatment up-regulated 336 genes and down-regulated 375 genes, and the number of up-regulated expressed genes was lower than the number of down-regulated expressed genes. The results indicated that C. coggygria leaves used different mechanisms to resist drought stress. Effects of drought stress and subsequent rewatering on DEGs of phytohormone signaling in C. coggygria The KEGG term “phytohormone signaling” was significantly enriched during drought, and a total of 27 DEGs families were identified for key gene modules of phytohormone signaling, including IAA, CTK, GA, ABA, ETH, BR, SA, and JA, after drought and rewatering (Fig. 8 ). Twenty-six DEGs were enriched in the IAA signaling pathway during drought stress, and most of them were clustered in two gene modules, SAUR , AUX/IAA , and only the TRINITY_DN14656_c0_g1 was significantly up-regulated in the SAUR gene module. Five genes within the AUX/IAA gene module show upregulation, while two genes exhibit downregulation (Fig. 8 B). There are five genes in the CTK signaling pathway, AHP (TRINITY_DN17133_c0_g1) and ARR-B gene expression was up-regulated (Fig. 8 C). There were two DEGs involved in the GA signaling pathway, including one up-regulated GID1 gene and one down-regulated DELLA gene; the EBF1_2 gene of the ETH signaling pathway was up-regulated under drought stress (Fig. 8 D). Compared with CK, the expression of ABA signaling pathways PP2C and ABF genes was up-regulated in drought treatment, whereas the expression of most SNRK2 and PYL genes was down-regulated (Fig. 8 E). The results of ABA signaling indicated that drought stress induced PP2C and ABF genes, but repressed SNRK2 and PYL genes. There were eight DEGs in the BR signaling pathway under drought stress, including one in the up-regulated BAK1 gene, and the remaining seven genes were down-regulated (Fig. 8 F). The JA signaling pathway had a total of six DEGs, with MYC2 and most of the JAZ genes down-regulated (Fig. 8 G). There were five DEGs in the SA signaling pathway, with PR-1 (TRINITY_DN2251_c0_g2) and NPR1 genes down-regulated and the remaining genes up-regulated (Fig. 8 H). In total, 18 DEGs were identified as enriched during the rewatering process. Among these, 9 DEGs showed downregulation in their expression levels, encompassing 1 gene each from the AUX/IAA, PP2C, PR-1, PYL, and SAUR gene modules, as well as 2 genes each from the CYCD3 and TCH4 gene modules. The DEGs associated with various physiological parameters under drought and rewatering conditions Compared with CK, in Wd_3, the expression levels of six gene modules involved in Car synthesis were down-regulated, including CYP707A, NCED, LUT1, CYP97C1 and CCD8, while the expression levels of three gene modules were up-regulated, including VDE, NPQ1, CCD7 and crtZ. During the synthesis of Ant, the expression of one gene module was down-regulated. After rewatering, the expression of CYP707A and NCED involved in the Car synthesis process was down-regulated, and the expression of one gene module involved in Ant synthesis was up-regulated (Fig. 9 B). In the MAPK signaling metabolism, after rewatering, the TRIITY_DN41_c0_g1 related to H₂O₂ synthesis continued to be down-regulated (Fig. 9 C). Under stress, there were 5 DEGs related to proline in the metabolism of proline and arginine. The expression levels of 4 genes were up-regulated, and the expression of PRODH, fadM and putB genes was down-regulated. After rewatering, the genes related to proline synthesis basically returned to the level of CK (Fig. 9 D). There were 14 DEGs of POD, with 7 up-regulated genes and 7 down-regulated genes. Among them, the expression of ACSL and fadD was down-regulated under drought stress and continued to be down-regulated after rewatering (Fig. 9 E). Identification and analysis of differentially expressed TFs There were 28 different TFs families with a total of 241 genes in the DEGs of CK and Wd_3 under drought stress, including 28 AP2/ERF (11.6%), 25 bHLH (10.3%), 24 NAC (9.9%), 51 MYB (21.1%), 18 WRKY (7.4%), and 18 C2C2 (7.4%) family genes (Fig. 11A). There were 21 families of TFs and 241 gene numbers in the DEGs of Wr_3 and CK, and the most numerically significant genes were the AP2/ERF (20, 19%), bHLH (12, 11.4%), NAC (12, 11.4%), MYB (19, 18%), and WRKY (7, 6.6%) families (Fig. 10 B). Thus, the six most common and critical TFs of C. coggygria are MYB, AP2/ERF, bHLH, NAC, WRKY, and C2C2. The TFs families were categorized into at least two groups, one of which was positively and the other negatively correlated with drought stress, suggesting that these families could up- or down-regulate the expression profiles of enzyme-encoding genes. Through expression pattern analysis, TRINITY_DN40545_c0_g1 and TRINITY_DN6699_c0_g1 were found to be the AP2/ARFs most likely to be positively and negatively involved in the drought response of C. coggygria leaves, respectively (Fig. 10 C). TRINITY_DN806_c0_g1 and TRINITY_DN8552_c0_g1, TRINITY_DN7437_c0_g1 and TRINITY_DN563_c0_g1, TRINITY_DN163_c0_g1 and TRINITY_DN5469_c0_g2, TRINITY _DN15772_c0_g1 and TRINITY_DN2815_c0_g1 were the most likely genes among the MYB, NAC, WRKY, and C2C2 genes to be positively and negatively involved in the drought response, respectively (Figs. 10 -F, 10 H). For the bHLH family, TRINITY_DN4346_c0_g1 is most likely the gene negatively involved in drought response (Fig. 10 G). Discussion Drought stress induces notable alterations in both the external morphology and internal structure of plants [ 31 ]. This stress condition results in substantial water depletion from leaves [ 32 ]. Numerous research studies have demonstrated that drought stress has the potential to diminish the LWP of plants, thereby exerting an influence on their growth [ 33 , 34 ]. The reduction in LWP has the capacity to affect the solubility of biological compounds and disrupt ions crucial for cellular functions, consequently causing plant malfunction and the production of ROS [ 34 ]. Insufficient water availability leads to a deceleration in plant growth, prominently manifesting as diminished SH [ 35 ], reduced leaf area [ 36 ] and heightened LT [ 35 ]. The degree of recovery in compensatory plant responses subsequent to rehydration may be impacted by the severity and duration of preceding drought stress [ 37 ]. The decline in LWP observed in C. coggygria under stress aligns with similar findings in Triticum aestivum and Oryza sativa [ 38 , 39 ]. The intensification of stress levels resulted in the inhibition of SH (except W1) and GD growth. Research has indicated that mild drought stress exerts minimal effects on plant development; however, under moderate and severe stress conditions, there is a marked reduction in plant height [ 40 ]. As drought stress severity escalates, the W3 treatment demonstrated a thinning of leaves due to excessive water loss, in line with the observations of Lei et al study [ 41 ]. By day 110 of stress exposure, the W2 treatment exhibited an increase in LT, consistent with Park et al 's findings attributing leaf thickness to a thick cuticle layer and deeply depressed stomata, which help diminish water loss [ 42 ]. Other durations of drought stress did not significantly impact LT, potentially due to the brief stress period. Following rehydration, the LWP, SH, GD, and LT of C. coggygria failed to fully recover to the levels observed in the control group. The decline in plant biomass under drought stress is associated with both stomatal and non-stomatal factors, such as stomatal closure, leaf senescence, and chlorophyll degradation [ 33 ]. Stomatal regulation is a critical factor under abiotic stress conditions, influencing the equilibrium between plant transpiration and photosynthesis [ 43 ]. In the study, SN and SD gradually increased in the W1, W2 and W3 treatment groups during the pre-stress period, and the higher SD was due to the tight stacking of epidermal cells and the reduction in epidermal cell expansion [ 44 ]. Following rehydration, SN and SD in all treatment groups surpassed those in the CK, indicating that the stomatal traits of C. coggygria had not completely recovered post-rehydration. Drought stress induces ROS production, which causes severe oxidative damage to membrane lipids and is required to limit plant growth and development by reducing photosynthesis and nutrients [ 45 , 46 ], and elemental deficiencies lead to increased H 2 O 2 [ 47 ]. Nutrients play an important role in regulating plant stress tolerance [ 48 ], and a decrease in soil water content affects the release and mobility of elements [ 49 ]. C, N, and P content are the major elements for plant growth and development [ 50 ], N deficiency reduces the photosynthetic activity and longevity of leaves [ 47 ]. In a study by Gargallo-Garriga et al it was shown that under sustained natural drought, the content of N and P concentrations varied throughout the season [ 51 ]. The different trends of C, N and P contents of each organ with the prolongation of stress time in the experiment were due to the fact that the plant body, in order to maintain the stability of its own chemical composition, made corresponding feedbacks to the changes in the external environment [ 52 ]. In the leaves of C. coggygria , in the W1 treatment under short-term drought stress, the C metabolism activities suffered interference and the photosynthetic rate was reduced, resulting in less C uptake. But in the W1 and W2 treatments under long-term drought stress, needed to absorb more water to alleviate drought and resist drought, thus leading to an increase in the C content per unit area of the leaf [ 53 ]. W3 foliar treatment C concentration was not affected by drought treatment, which was in agreement with Zhang et al related study [ 49 ]. The higher root C content of the W3 treatment contributes to C uptake, which is an important strategy for nutrient retention under stress conditions [ 54 ]. As the degree of drought stress intensified, leaf N content gradually increased, indicating that N solubilization and transformation require the involvement of water, and lower soil water content makes N difficult to solubilize and less available [ 55 ]. The stem N content was higher in the W3 treatment than in the other treatments, probably due to the fact that the W3 treatment regulated growth through the stems, resulting in a change in the distribution of N in the plant, leading to an increase in N content. Root N content increased in W3-treated roots at 90 d of stress probably due to excessive water deficit leading to N recycling to sustain life activities. Changes in leaf, stem, and root P content across treatment groups suggest that drought limits the movement of soil nutrients [ 56 ]. After 20 d of rewatering, the leaves and roots of the W3 treatment group did not allow the plant to recover quickly due to irreversible damage, resulting in lower C content, in agreement with An et al study [ 57 ]. In the organs of C. coggygria under the W2 and W3 treatments, the N content will temporarily increase the assimilation, absorption, and utilization of N after rewatering to supplement the growth requirements, so as to promote the growth and repair of the plants. The P content in the leaves and roots of each treatment group increased, while the P content in the stems decreased. In the plant body, the stem mainly plays the roles of support, transportation and storage. When drought limits the adsorption and solubilization of inorganic phosphorus, the available phosphorus tends to be transported to other organs such as roots and leaves [ 58 ]. Three broad categories of plant reactions to drought include alterations in pigmentation, both qualitatively and quantitatively [ 59 ]. Chl is a key factor in plant photosynthesis and is responsible for the absorption, transfer and conversion of light energy [ 60 ]. Car can also play a role in scavenging ROS as a non-enzymatic antioxidant in addition to its role in light trapping during photosynthesis [ 59 , 61 ]. Increased drought stress in the study led to higher Chla, Chlb, and Chl contents in all treatment groups of C. coggygria leaves, suggesting that C. coggygria can increase light energy utilization by increasing Chl content to adapt to water deficit [ 61 ], which is in agreement with the studies of Periploca sepium [ 62 ] and Hordeum vulgare [ 63 ]. C. coggygria under drought stress is also able to reduce water evaporation by increasing Car content, releasing excess heat, and lowering leaf temperature [ 64 ]. Ant content showed an increasing trend with the intensification of stress, which is consistent with the study of Hodaei et al [ 65 ] that Ant shows an increase in content with water deficit, which promotes the antioxidant capacity of plants. The value of the color parameter b* gradually decreased in the later stages of stress in all treatment groups, and the leaf coloration was skewed towards a yellowish-blue tone. The decrease in C. coggygria Chla, Chlb, and Chl levels after short-term rewatering is due to plant nutrient deficiencies, which reduce the synthesis of Chl by dedicating more resources to growth and restoration. Drought-induced stress in plants typically results in physical and chemical harm, leading to compromised nutrient absorption, disrupted cellular processes, and the accumulation of ROS [ 66 ]. Among ROS, H 2 O 2 is known for its stability [ 67 ], while the ·OH exhibits high reactivity [ 68 ]. MDA is commonly used as a biomarker to evaluate the integrity of plant cell membranes [ 69 ]. ROS levels rise, triggering the activation of both enzymatic and non-enzymatic defense mechanisms [ 70 ]. SOD operates in chloroplasts, peroxisomes, and mitochondria [ 71 ], playing a key role in converting ·OH into H₂O₂. Subsequently, POD and CAT catalyze the decomposition of H₂O₂ into H₂O and O₂ [ 71 ]. GSH, a vital component of the non-enzymatic defense system, acts as a dithiol reducing agent that safeguards enzyme sulfhydryl groups, regenerates ASA, and reacts with singlet oxygen and ·OH [ 36 ]. With the decrease of soil water content in the experiment, the antioxidant activity of C. coggygria increased in the late stage of stress, which mitigated the damage of ·OH to the cells, promoted the decomposition of H₂O₂, and helped to maintain the balance of intracellular signaling. The changes in C. coggygria SOD enzyme activity among treatment groups were consistent with the study of Huang et al [ 72 ], which may be due to the temporary increase in SOD enzyme activity caused by water deficit. Due to the higher degree of stress, the SOD activities of W3 treatment were lower than those of CK, which was consistent with the study of Li et al [ 73 ], where the persistent drought and the increased degree of stress led to the damage of the plant cell membrane system, the inhibition of antioxidant enzyme synthesis, and the accumulation of ROS exceeded the scavenging capacity of the plant. During the pre-stress period, MDA content gradually decreased in all treatment groups, indicating that C. coggygria was able to acclimatize to drought for a sufficient period of time [ 72 ]. In the late stage of stress, the MDA content increased sequentially with the intensification of drought, which corresponded to the study of Zhao et al [ 74 ], and the W3 treatment was not significantly different from CK, indicating a limited capacity of the antioxidant system to regulate the antioxidant system [ 72 ]. At the same point in time, GSH content gradually increased with increasing drought. GSH gradually increased with the intensification of drought. The increase in GSH helps maintain normal immune system function and has antioxidant and detoxification effects [ 75 ]. After rewatering, the activities of ·OH and H₂O₂ in the W1 treatment group and W2 treatment group were both higher than those in CK. This is because rewatering still cannot effectively scavenge the ROS accumulated during drought stress. The antioxidant enzyme activities were lower in the W3 treatment group than in CK, indicating that the antioxidant system of C. coggygria was irreversibly damaged by severe drought. The significant increase in GSH content after rewatering was due to the fact that the processes of photosynthesis and respiration of the plant were enhanced, providing more energy and substrate for GSH synthesis, indicating that rewatering is favorable for C. coggygria to carry out self-repair [ 76 ]. Osmoregulation is recognized as an important physiological adaptive property associated with abiotic stresses [ 77 ]. Plants respond to stress by eliminating ROS through osmotic pressure accumulated in the cytoplasm and chloroplasts [ 78 ]. Pro is an osmoregulatory substance that improves osmoregulation in plants and also acts as an antioxidant in plants, scavenging ·OH and stabilizing cell membranes [ 2 , 79 ]. SP is also an osmoregulatory substance, and some studies have shown a sharp decrease in SP under drought stress [ 2 ], but others have shown a significant increase in SP content under drought treatment [ 80 , 81 ]. Trends in C. coggygria Pro and SP content varied throughout the period under study. Enhanced aridity leads to an increase in Pro content, which helps to stimulate the production of antioxidant enzyme activities [ 82 ]. The SP content of each treatment group gradually accumulated in the late stage of stress, which improved the osmoregulation ability of C. coggygria [ 83 ]. After rewatering, Pro content gradually decreased in each treatment group, while SP content basically returned to the normal level. Phytohormones are signaling compounds that regulate key aspects of growth, development, and environmental stress responses [ 84 ]. As a key stress hormone in plants, ABA plays the role of a central integrator in drought stress response, forming a complex regulatory network by activating adaptive signals and coordinating the interactions of multiple hormones [ 84 , 85 ]. When plants respond to drought stress, ABA binds to upstream PYR/PYLs receptors and inhibits PP2Cs negative regulators, and SnRK2s type protein kinase promotes ABA responses by activating ABF transcription factors through dephosphorylation of downstream targets [ 86 ]. ARF may bind to the transcriptional repressor Aux/IAA to inhibit IAA synthesis, thereby suppressing biomass accumulation to alleviate water deficit [ 87 ]. JAZ proteins negatively regulate JA-responsive genes [ 88 ], and under drought stress, the expression of JAR1 , a JA signaling gene, was significantly decreased, and the expression of COI1 , which is involved in stomatal movement, was increased [ 83 ]. BR signaling regulatory positive and negative factors activate SnRK2s to control the initiation and amplification of ABA signaling [ 89 ]. GA signaling regulates resistance by controlling cellular redox homeostasis, and increased DELLA activity interferes with ABA signaling [ 90 ]. Studies in Arabidopsis thaliana under drought stress have shown that by regulating the redundant negative regulators AHP2 , AHP3 and AHP5 in the CTK signaling pathway, AHP controls the drought response in both an ABA-dependent and an ABA-independent manner [ 91 ]. In addition, SA enhances plant drought tolerance by strengthening antioxidant defense, promoting osmotic fluid accumulation, increasing water use efficiency, and enhancing photosynthesis [ 92 ]. During the dual process of drought and rewatering in this study, it was shown that ABA and other hormone signaling pathways are involved in the adaptive response of C. coggygria to drought stress. Involvement in phytohormone signaling including 27 gene families such as PYR/PYLs , SnRK2 , IAAs and ARFs was observed in drought stress and rewatering, suggesting that the phytohormone signaling pathway may be related to drought and rewatering responses in C. coggygria . The study reported a down-regulation in the expression of 10 SAUR and 2 AUX/IAA genes within the IAA signaling pathway, resulting in the suppression of IAA synthesis and ultimately enhancing the drought tolerance of plants [ 20 ]. Drought stress induces phytohormone-related genes in C. coggygria to express and interact with each other, activating a comprehensive and complex phytohormone regulatory network to improve its adaptation and survival under water deficit. In cotton, the NCED gene is a key gene in the ABA synthesis pathway [ 93 ]. Under drought conditions, the biosynthesis of anthocyanins is regulated by MYB and bHLH transcription factors [ 94 ]. In potatoes, the upregulation of genes related to osmotic regulation corresponds to the osmotic response [ 95 ]. The class III peroxidase gene family in tea plants plays an important role in abiotic stress [ 96 ]. In the study, after drought treatment, the expression levels of most genes related to Car synthesis and Ant synthesis genes were downregulated, which was contrary to the accumulation of Car and Ant contents. It is likely that although the transcriptional levels of Car and Ant synthesis genes decreased, the translation efficiency of their mRNAs increased and their catalytic activities were enhanced, which is consistent with the research on Scutellaria baicalensis [ 97 ]. Under drought conditions, there were changes in the DEGs related to Pro content, H₂O₂, and POD, which were consistent with the changes in Pro content, H₂O₂ content, and POD enzyme activity. TFs play a variety of roles in the control of gene expression in plants and are required for the regulation of biological processes such as development and environmental stress responses [ 98 ]. Several families of transcription factors such as MYB, WRKY, AP2/ERF, NAC, bHLH, and C2C2 have been characterized and proved to be useful tools for enhancing drought tolerance in plants [ 20 , 99 , 100 ]. In addition, MYB transcription factors are critical in the biosynthesis of plant secondary metabolites, including anthocyanins, flavonoids, lignin and cuticle synthesis, and rely on ABA signaling to participate in the regulation of stomatal movement in plants under arid environments [ 101 ]. GmWRKY54 directly binds to the promoters of PYL8 , SRK2A , CIPK11 , and CPK3 and activates their expression, thereby improving drought tolerance in Glycine max [ 102 ]. In poplar, the bHLH family gene PebHLH35 responds to drought stress by positively regulating stomatal density, stomatal aperture and photosynthesis [ 103 ]. Similarly, overexpression of the stress-responsive NAC1 in rice conferred tolerance to severe drought stress without phenotypic or yield changes, whereas overexpression of OsNAC6 in rice led to improved water retention by controlling stomatal closure under dehydration stress [ 104 ]. Correlation network prediction revealed that AP2/ERF, WRKY, MYB, bHLH, and NAC affect plant drought tolerance by regulating the expression of downstream genes such as PP2C , JAZ , and SnRK2 [ 20 ]. The study identified the six predominant TF families in leaves as AP2/ERF, bHLH, NAC, MYB, WRKY, and C2C2. Drought stress was found to elevate the expression of these TFs, with their regulation being either up-regulated or down-regulated in reaction to stress and subsequent rewatering. This implies that they function as positive or negative regulators of drought stress in C. coggygria , respectively. Conclusions Prolonged exposure to drought stress triggers varying dynamic trends in the morphological structure and physiological parameters during the growth stages of C.coggygria . Elevated stress levels correspond to decreased N and P content in various organs of C. coggygria , resulting in diminished LWP and notable accumulations of SD, SN, photosynthetic pigments and osmolytes. This accumulation hinders growth while enhancing the water-holding capacity of C.coggygria . The antioxidant compounds in C.coggygria synergistically regulate the equilibrium between ROS production and scavenging within the plant. Nevertheless, studies highlight the limited regulatory capacity of C.coggygria in modulating its antioxidant defense system. Short-term rehydration fails to fully restore most physiological parameters of C.coggygria to control levels. Furthermore, analyses have unveiled the gene expression dynamics associated with hormone signaling pathways and alterations in drought-resilient physiological markers in C.coggygria . Transcription factors such as WRKYs, MYBs, bHLHs, AP2/ERFs, NACs, and C2C2s have been identified as potential key regulators, shedding light on crucial candidate genes for drought tolerance in C.coggygria . Abbreviations ROS Reactive oxygen species LWP Leaf water potential EMLWP Early morning leaf water potential MDLWP Midday leaf water potential SH Seeding height GD Ground diameter LT Leaf thickness SN Stomatal number SD Stomatal density C Carbon N Nitrogen P Phosphorus Chla Chlorophyll a Chlb Chlorophyll b Chl Chlorophyll Car Carotenoids Ant Anthocyanin H 2 O 2 Hydrogen peroxide ·OH Hydroxyl radical MAD Malondialdehyde SOD Superoxide dismutase POD Peroxidase CAT Catalase GSH Glutathione SP Soluble protein Pro Proline ABA Abscisic acid SA Salicylic acid CTK Cytokinin ETH Ethylene IAA Indole-3-acetic acid JA Jasmonic acid GA Gibberellic acid BR Brassinosteroids TFs Transcription factors. Declarations Author Contributions The thesis was written by S.M. and X.L.; the methodology was collected and organized by Y.F. and L.Y.; data collection was carried out by Y.X. and J.S.; data analysis and organization were done by X.C.; seedling maintenance was undertaken by J.B., X.W. and Y.Z.; and the experimental design, thesis conception, and experimental guidance were provided by K.Z. and X.Y.. Funding This research was funded by the Natural Science Foundation of Shanxi Province (202103021224144), the Biobreeding Project of Shanxi Agricultural University (YZGC138), the Special Project for Forest and Grass Germplasm Resources Investigation of Shanxi Forestry and Grassland Bureau (QT2024007), the Postgraduate Research Innovation Project (2023KY346), the Key Scientific Research Project of Shanxi Road & Bridge Group (SXLQ-XY-3-002-2023), the Transportation Construction Technology Research Project of Zhongzi Huake (2024-GSGL-01). Availability of data and materials The datasets generated and analysed during the current study are available in the NCBI SRA with the accession number PRJNA1315001. Ethics approval and consent to participate The plant materials used in this study were 3-year-old healthy seedlings of C. coggygria growing in the Forestry Station of Shanxi Agricultural University, Jinzhong, China. And no permits are required for the collection of plant samples. This study did not require ethical approval or consent, as it did not involve any endangered or protected species. Consent for publication Not applicable. Competing Interest The authors declare that they have no competing interests. References Cohen I, Zandalinas SI, Huck C, Fritschi FB, Mittler R. Meta-analysis of drought and heat stress combination impact on crop yield and yield components. Physiol Plant. 2021;171(1):66–76. Tan W, Li W, Li J, Liu D, Xing W. 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Overexpression of CgbHLH001 , a Positive Regulator to Adversity, Enhances the Photosynthetic Capacity of Maize Seedlings under Drought Stress. Agronomy-Basel. 2022;12(5):1149. Nakashima K, Tran L-SP, Van Nguyen D, Fujita M, Maruyama K, Todaka D, Ito Y, Hayashi N, Shinozaki K, Yamaguchi-Shinozaki K. Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant journal: cell Mol biology. 2007;51(4):617–30. Tables Table 1 Raw sequencing data and quality control checks of nine C. coggygria leaf cDNA libraries. Sample Raw reads Clean reads Q20 (%) Q30 (%) GC content (%) Mapped ratio CK_1 42715214 42414146 98.59 95.41 43.70 89.33% CK_2 41996726 41694568 98.65 95.59 43.62 88.97% CK_3 43477334 43160840 98.61 95.47 43.65 89.40% Wd3_1 43536722 43243182 98.67 95.67 43.53 89.51% Wd3_2 41364718 41069882 98.55 95.29 43.45 89.18% Wd3_3 43572584 43270142 98.69 95.72 43.63 89.84% Wr3_1 40901478 40627744 98.63 95.53 43.55 89.51% Wr3_2 43547294 43251580 98.63 95.52 43.63 89.83% Wr3_3 44714144 44397794 98.70 95.75 43.54 89.40% Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 26 Jan, 2026 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 17 Oct, 2025 Reviews received at journal 25 Sep, 2025 Reviews received at journal 13 Sep, 2025 Reviewers agreed at journal 13 Sep, 2025 Reviews received at journal 12 Sep, 2025 Reviewers agreed at journal 12 Sep, 2025 Reviewers agreed at journal 11 Sep, 2025 Reviewers invited by journal 11 Sep, 2025 Editor assigned by journal 11 Sep, 2025 Editor invited by journal 10 Sep, 2025 Submission checks completed at journal 09 Sep, 2025 First submitted to journal 09 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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02:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7457742/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7457742/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-026-08154-0","type":"published","date":"2026-01-26T15:58:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91641408,"identity":"2329774f-f8f0-4adf-874b-cab39b0622f4","added_by":"auto","created_at":"2025-09-18 14:54:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":575991,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of drought stress and subsequent rewatering on leaf water potential and growth parameters of \u003cem\u003eC. coggygria.\u003c/em\u003e (A) EMLWP, early morning leaf water potential; (B) MDLWP, midday leaf water potential; (C) SH, seeding height; (D) GD, ground diamete; (E) LT, leaf thickness. Significant differences within groups were tested using univariate ANOVA, and significant differences between groups were tested using multivariate ANOVA. Statistical significance was determined by a p-value \u0026lt; 0.05. Uppercase letters indicate within-group differences, while lowercase letters indicate between-group differences. Abbreviations: D30d denotes 30 days of stress; R20d denotes 20 days after rehydration; CK denotes the control group; W1 denotes mild stress; W2 denotes moderate stress; W3 denotes severe stress.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457742/v1/392393a79310fb7c61105d71.jpg"},{"id":91641407,"identity":"7d9a4ea9-7986-4da2-b90c-257d95de4b83","added_by":"auto","created_at":"2025-09-18 14:54:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":234434,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of drought stress and subsequent rewatering on stomatal traits of \u003cem\u003eC. coggygria.\u003c/em\u003e (A): SN, stomatal number; (B) SD, stomatal density. Significant differences within groups were tested using univariate ANOVA, and significant differences between groups were tested using multivariate ANOVA. Statistical significance was determined by a p-value \u0026lt; 0.05. Uppercase letters (A, B, C, etc.) indicate within-group differences, while lowercase letters (a, b, c, etc.) indicate between-group differences. Abbreviations: D30d denotes 30 days of stress; R20d denotes 20 days after rehydration; CK denotes the control group; W1 denotes mild stress; W2 denotes moderate stress; W3 denotes severe stress.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457742/v1/45130f09f260a50821908629.jpg"},{"id":91641409,"identity":"ad540d2a-8fc2-482b-92ae-e4c62e2e9dab","added_by":"auto","created_at":"2025-09-18 14:54:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":497091,"visible":true,"origin":"","legend":"\u003cp\u003eStoichiometric alterations in \u003cem\u003eC. coggygria\u003c/em\u003e induced by drought stress and subsequent rewatering. (A) C, carbon content of organs; (B) N, nitrogen content of organs; (C) P, phosphorus content of organs. Significant differences within groups were tested using univariate ANOVA, and significant differences between groups were tested using multivariate ANOVA. Statistical significance was determined by a p-value \u0026lt; 0.05. Uppercase letters (A, B, C, etc.) indicate within-group differences, while lowercase letters (a, b, c, etc.) indicate between-group differences. Abbreviations: D30d denotes 30 days of stress; R20d denotes 20 days after rehydration; CK denotes the control group; W1 denotes mild stress; W2 denotes moderate stress; W3 denotes severe stress.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457742/v1/d091bdaf5943d2e73618e575.jpg"},{"id":91641411,"identity":"5689d339-44de-4157-bc34-4656fe59aac6","added_by":"auto","created_at":"2025-09-18 14:54:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":560072,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of drought stress and subsequent rewatering on pigment content of \u003cem\u003eC. coggygria. \u003c/em\u003e(A) Chla, chlorophyll a; (B) Chlb, chlorophyll b; (C) Chl, chlorophyl; (D) Car, carotenoids; (E) Ant, anthocyanidin. Significant differences within groups were tested using univariate ANOVA, and significant differences between groups were tested using multivariate ANOVA. Statistical significance was determined by a p-value \u0026lt; 0.05. Uppercase letters (A, B, C, etc.) indicate within-group differences, while lowercase letters (a, b, c, etc.) indicate between-group differences. Abbreviations: D30d denotes 30 days of stress; R20d denotes 20 days after rehydration; CK denotes the control group; W1 denotes mild stress; W2 denotes moderate stress; W3 denotes severe stress.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457742/v1/96a3c6ed3c85b418647a0060.jpg"},{"id":91641840,"identity":"c3625e93-2f43-4468-985c-07d1a7145c39","added_by":"auto","created_at":"2025-09-18 15:02:44","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":481487,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of drought stress and subsequent rewatering on ROS levels and antioxidant systems.\u003cem\u003e \u003c/em\u003e(A) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, hydrogen peroxide; (B) ·OH, hydroxyl radical; (C) MAD, malondialdehyde; (D) SOD, superoxide dismutase; (E) POD, peroxidase; (F) CAT, catalase; (G) GSH, glutathione. Significant differences within groups were tested using univariate ANOVA, and significant differences between groups were tested using multivariate ANOVA. Statistical significance was determined by a p-value \u0026lt; 0.05. Uppercase letters (A, B, C, etc.) indicate within-group differences, while lowercase letters (a, b, c, etc.) indicate between-group differences. Abbreviations: D30d denotes 30 days of stress; R20d denotes 20 days after rehydration; CK denotes the control group; W1 denotes mild stress; W2 denotes moderate stress; W3 denotes severe stress.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457742/v1/c4c52cc460dde4d8e62537e7.jpg"},{"id":91641414,"identity":"18dae13a-aa0b-4efe-9523-ce1496358ab3","added_by":"auto","created_at":"2025-09-18 14:54:44","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":227998,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of drought stress and subsequent rewatering on osmoregulatory substances. (A) SP, soluble protein; (B)Pro, proline. Significant differences within groups were tested using univariate ANOVA, and significant differences between groups were tested using multivariate ANOVA. Statistical significance was determined by a p-value \u0026lt; 0.05. Uppercase letters (A, B, C, etc.) indicate within-group differences, while lowercase letters (a, b, c, etc.) indicate between-group differences. Abbreviations: D30d denotes 30 days of stress; R20d denotes 20 days after rehydration; CK denotes the control group; W1 denotes mild stress; W2 denotes moderate stress; W3 denotes severe stress.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457742/v1/74f70ad8b40c5ed6a7a17520.jpg"},{"id":91641417,"identity":"27cde84e-ce88-460b-a60a-4bb295474f8e","added_by":"auto","created_at":"2025-09-18 14:54:44","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":501066,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of differentially expressed genes in \u003cem\u003eC. coggygria\u003c/em\u003e for drought stress and subsequent rewatering. (A) Overall plot of the distribution of expression of genes expressed in each treatment. (B) Venn diagram for gene set analysis between treatments. (C-D) Volcano maps of drought and rewatering differentially expressed genes using DESq2 to identify DEGs with a setting of |log2FC| ≥ 1 and an error rate (FDR) \u0026lt; 0.05. (E-F) KEGG enrichment analysis of differentially expressed genes.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457742/v1/234ad9902047c82fd77f24e9.jpg"},{"id":91641423,"identity":"7724a2c9-aa8a-4e71-8d73-fb538c56ca61","added_by":"auto","created_at":"2025-09-18 14:54:44","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":574206,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of differentially expressed genes related to plant hormone signaling pathways. (A) Schematic diagram of the plant hormone signaling pathway, with the key coding genes involved shaded. (B-H) Heatmap of differentially expressed genes for indole-3-acetic acid (IAA), cytokinin (CTK), gibberellin acid (GA) and ethylene (ETH), abscisic acid (ABA), brassinosteroids (BR), jasmonic acid (JA), salicylic acid (SA).\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457742/v1/0be7617f9513a353f06fc00b.jpg"},{"id":91641845,"identity":"669e19ee-8439-4283-b410-d23a7802af68","added_by":"auto","created_at":"2025-09-18 15:02:44","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":612372,"visible":true,"origin":"","legend":"\u003cp\u003eExamination of genes with varying expression patterns associated with physiological indicators during drought stress and subsequent rewatering conditions. (A) Illustration depicting the pathway of DEGs influencing physiological indicators in response to drought stress and subsequent rewatering, highlighting key coding genes. (B-E) Heatmaps of DEGs for carotenoids (Car), anthocyanins (Ant), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), proline (Pro), and peroxidase (POD).\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457742/v1/7026045085016e76531e69ac.jpg"},{"id":91641424,"identity":"6fe560df-44f2-470b-9ca4-a45f0c26bc9f","added_by":"auto","created_at":"2025-09-18 14:54:44","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":904916,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification and analysis of DEGs of transcription factor families under drought stress and subsequent rewatering conditions. (A-B): Categorization of transcription factor families in response to drought stress and subsequent rewatering. (C-H) Heatmapsillustrating the differential expression of genes encoding AP2/ERF, MYB, NAC, WRKY, bHLH, and C2C2 transcription factors.\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457742/v1/fe469103d24c37b164624961.jpg"},{"id":101690682,"identity":"bde9f829-112d-4b17-a4f1-acca9326e7d5","added_by":"auto","created_at":"2026-02-02 16:07:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6537605,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7457742/v1/0590e7a3-c5f9-44fa-9fea-dcec65db6b2d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Physiological and Transcriptomic Cooperative Regulatory Mechanisms of Cotinus coggygria in Response to Drought and Rewatering Processes","fulltext":[{"header":"Background","content":"\u003cp\u003eDrought is a significant abiotic stress factor that severely impacts plant growth and crop yields, affecting various physiological processes including growth, development, metabolism, and morphology [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Drought stress and subsequent rewatering are essential stages in the plant growth cycle [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], with rewatering serving as a recovery mechanism post-drought to restore growth and enable rapid plant development [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The investigation of physiological changes in plants during drought and rewatering conditions is crucial for understanding plant drought resistance mechanisms under varying water availability, thereby enhancing plant productivity and ecological adaptability.\u003c/p\u003e\u003cp\u003eDrought stress exerts key effects on plants, altering pigment synthesis, osmoregulation, secondary metabolism, antioxidant systems, and gene expression [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The visible impact of drought on plants is the inhibition of morphological growth [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], with leaf morphology serving as a prominent indicator in drought studies [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Stomatal regulation, a common adaptive response to drought, helps plants maintain leaf water potential (LWP) stability and reduce gas exchange [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Chlorophylls (Chl) and carotenoids (Car) are pivotal for photosynthesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], with drought stress often leading to a reduction in chlorophyll contents [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Ecological stoichiometry, focusing on the balance of chemical elements in ecosystems [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], highlights the roles of carbon (C), nitrogen (N), and phosphorus (P) in plant growth and physiological processes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. C is an essential substrate and energy source for plant growth, while N and P are crucial nutrients and key elements for plant cell composition and metabolism [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, investigating the stoichiometric properties of C, N, and P in plants is valuable for understanding nutrient dynamics and utilization in plants.\u003c/p\u003e\u003cp\u003ePlants commonly amass significant levels of reactive oxygen species (ROS) under drought conditions, leading to potential toxicity due to their excessive accumulation within plant tissues [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The antioxidant defense system plays a critical role in scavenging excess ROS to prevent cellular damage and maintain ROS homeostasis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Malondialdehyde (MDA) levels, a product of membrane lipid peroxidation, reflect the extent of cell membrane damage under stress conditions. Osmoregulation plays a crucial role in the drought tolerance of plants, as it enables the maintenance of cell expansion even under drought stress conditions, thereby promoting plant growth [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Key osmoregulatory substances involved in this process are proline (Pro) and soluble protein (SP) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The research by Wang \u003cem\u003eet al\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] has shown that SP and Pro can serve as significant indicators for the identification of drought-resistant plant varieties.\u003c/p\u003e\u003cp\u003eTranscriptomic methodologies have been extensively utilized to pinpoint genes orchestrating plant growth, development, and those exhibiting differential expression patterns under abiotic stress conditions [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Transcriptome sequencing has been pivotal in elucidating the molecular mechanisms governing plant responses to drought stress [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the context of water deficit, phytohormones exhibit synergetic actions, with abscisic acid (ABA), salicylic acid (SA), cytokinin (CTK), ethylene (ETH), indole-3-acetic acid (IAA), jasmonic acid (JA), gibberellic acid (GA), and brassinosteroids (BR) playing crucial roles in aiding higher plants to surmount challenges posed by drought stress [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Elevated ABA levels have been observed to trigger the upregulation of numerous transcription factors (TFs) and genes, thereby activating downstream metabolic pathways [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Utilization of the weighted gene co-expression network analysis approach on drought-exposed \u003cem\u003eArtemisia iliensis\u003c/em\u003e seedlings has revealed the pivotal involvement of various transcription factor families including WRKY, bHLH, NAC, AP2/ERF, MYB, GRAS, C2H2, MADS, and bZIP in mediating drought responses [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNumerous studies have investigated the physiological and biochemical characteristics of plants, yet there is a dearth of research on the physiological, biochemical, and gene expression alterations that occur during drought stress and subsequent recovery phases. \u003cem\u003eCotinus coggygria\u003c/em\u003e, a small deciduous tree belonging to the Anacardiaceae family and \u003cem\u003eCotinus\u003c/em\u003e genus, possesses notable attributes for soil and water conservation, landscape enhancement, and holds substantial medicinal, economic, and ornamental value. Current research efforts on \u003cem\u003eC. coggygria\u003c/em\u003e primarily concentrate on breeding and afforestation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], revealing significant research gaps in understanding its drought resistance mechanisms. Therefore, to study the physiological changes of drought stress as well as rewatering on leaf water potential, morphological growth and physiological structure, antioxidant system, osmotic substances of \u003cem\u003eC. coggygria\u003c/em\u003e, as well as drought-resistant phytohormone signaling, differential expression patterns of genes related to physiological indicators, and the potential regulatory relationships among genes of the TFs family, can help \u003cem\u003eC. coggygria\u003c/em\u003e drought tolerance indicators screening, for mining drought-resistant key genes and further elucidating the molecular regulatory mechanism to establish a framework. \u003cem\u003eC. coggygria\u003c/em\u003e, as an autumn color-changing tree species, is of great significance to the application of tree species in arid and semi-arid landscapes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eOverview of the study area\u003c/h2\u003e\u003cp\u003eThe research site is situated at the Forestry Station of Shanxi Agricultural University in Taigu District, Jinzhong City, Shanxi Province (112\u0026deg;57\u0026prime;54\u0026Prime; E, 37\u0026deg;42\u0026prime;78\u0026Prime; N). It experiences a temperate continental monsoon climate at an altitude of 1098 meters. The average annual temperature ranges from 5 to 10\u0026deg;C, with an annual precipitation of around 458 mm. The period from June to August receives the highest precipitation, constituting 70% of the annual total, whereas the lowest precipitation occurs from December to February.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExperimental materials\u003c/h3\u003e\n\u003cp\u003eTransplant 3-year-old healthy seedlings of \u003cem\u003eC. coggygria\u003c/em\u003e exhibiting consistent and vigorous growth into containers measuring 29.5 cm in width and 23.5 cm in height. The soil used in the experiment is taken from the garden soil of the Forestry Station. The pH value of the soil is 8.30, the total nitrogen content is 0.84 g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the total phosphorus content is 0.48 g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the available nitrogen content is 64.3 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the available potassium content is 142.06 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the organic matter content is 12.06 g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The soil's field capacity for water retention, as assessed by the ring knife method, is measured at 26.11%. The flowerpots are placed under a rain shelter.\u003c/p\u003e\u003cp\u003eOn April 30th, drought stress experiments were conducted with varying severity levels following the methodology outlined by Wang Kai et al [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Four soil moisture gradients were established: the control group (CK), mild stress (W1), moderate stress (W2), and severe stress (W3), corresponding to 80% \u0026plusmn; 5%, 60% \u0026plusmn; 5%, 40% \u0026plusmn; 5%, and 20% \u0026plusmn; 5% of the soil's field water holding capacity, respectively. Each group consisted of 40 pots, totaling 160 pots. Pots were placed under a rain shelter, and soil moisture content was measured using the gravimetric method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Sampling was carried out after 30 d of drought stress treatment, followed by sampling every 20 d. A total of 5 samplings were carried out from the end of May to the middle of August. Subsequent to the drought stress period, a 20-day rehydration treatment was administered, with sampling conducted once.\u003c/p\u003e\n\u003ch3\u003eDetermination of morphological growth and physiological indices\u003c/h3\u003e\n\u003cp\u003eThe LWP at 6:00 AM and 12:00 PM was determined using a dew point potentiometer. The ground diameter (GD) of \u003cem\u003eC.coggygria\u003c/em\u003e was measured with a vernier caliper (0.01 mm), and the seedling height (SH) was measured with a steel tape measure (accuracy of 0.1 cm). Leaf thickness at the upper, middle, and basal regions was measured with a thousandth caliper (0.001 mm), and the mean value was calculated. Stomatal number (SN) was quantified using Image J software, subsequently used to determine stomatal density (SD\u0026thinsp;=\u0026thinsp;SN per unit area) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The contents of Chl and Car in leaves were quantified through ethanol extraction method [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The content of anthocyanin (Ant) was determined using the hydrochloric acid soaking method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. C, N, and P in leaves, stems, and roots were determined using the dry combustion method, Kjeldahl nitrogen analyzer method [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and vanado-molybdate yellow colorimetry [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], respectively.\u003c/p\u003e\u003cp\u003eHydrogen peroxide (H₂O₂), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione (GSH), MDA, and Pro were all determined using the methods described in the Solarbio kit. The content of hydroxyl radical (\u0026middot;OH) was determined according to the method of the kit produced by Nanjing Jiancheng Bioengineering Institute. The content of SP was determined by the Coomassie Brilliant Blue G-250 staining method [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eTranscriptome sequencing\u003c/h3\u003e\n\u003cp\u003eFresh leaves from \u003cem\u003eC. coggygria\u003c/em\u003e plants subjected to 110 days of drought treatment and 20 days post-rewatering in the CK and W3 treatments were harvested and rapidly frozen in liquid nitrogen. Each treatment comprised three independent replicates, resulting in a total of nine samples. Total RNA extraction was performed on these samples at Majorbio. Subsequently, a cDNA library was constructed and subjected to quality assessment before sequencing on the NovaSeq X Plus platform. De novo assembly of the clean data was conducted using Trinity software. The resulting transcriptome sequences were filtered, optimized, and assembled into Unigenes, which were then compared with the relevant database. DEGs were identified using the DESeq2 based on specified criteria (|log2FC| \u0026ge; 1, FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003ePerform univariate and multivariate analysis of variance (ANOVA) on the dataset utilizing SPSS 26.0 software. Subsequently, conduct multiple comparisons using Duncan's test method to assess significant differences between groups.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eEffects of drought stress and subsequent rewatering on LWP and morphological growth of\u003c/b\u003e \u003cb\u003eC. coggygria\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe prolonged drought and increased severity of drought led to a declining trend in both early morning leaf water potential (EMLWP) and midday leaf water potential (MDLWP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). SH and GD tended to increase between treatment groups during the growth period of \u003cem\u003eC. coggygria\u003c/em\u003e under sustained drought, and the intensification of the degree of stress led to the inhibition of growth of SH (except W1) and GD of \u003cem\u003eC. coggygria\u003c/em\u003e between treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). W3 treatment leaves lost water and became thinner, and leaf thickness (LT) was lower than that of CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter 20 d of rewatering, the early morning LWP and midday LWP of W1, W2 and W3 treatment groups were still significantly lower than that of CK, and the W2 and W3 treatments SH, GD and LT were not restored to the CK level for the moment due to the short rewatering time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of drought stress and subsequent rewatering on stomatal traits of\u003c/b\u003e \u003cb\u003eC. coggygria\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe SN and SD exhibited fluctuations among treatment groups under prolonged stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). With the intensification of drought stress, SN and SD gradually increased in all treatment groups (except CK) at 30\u0026ndash;70 d of stress. At 110 d of stress, SD was higher in the W2 treatment group and lower in the W3 treatment group compared with CK. After rewatering, SN and SD were significantly higher in all treatment groups than in CK.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of drought stress and subsequent rewatering on C, N and P content of\u003c/b\u003e \u003cb\u003eC. coggygria\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDuring prolonged drought stress, variations in carbon (C) content were observed in different plant organs, with a decrease followed by an increase in leaf C content, a decrease in stem C content (except W2), and a decreasing-then-increasing trend in root C content. Nitrogen (N) content showed a consistent decreasing trend across all organs in response to the different treatment groups. Phosphorus (P) content exhibited varied trends, characterized by a decline in leaf P content and a pattern of decrease followed by an increase in stem P content across different treatment conditions. In contrast, root P content exhibited a decreasing-then-increasing trend in CK and W1 treatments, diverging from the trends observed in W2 treatments. Under intensified drought stress, leaf C content was highest in the W3 treatment, followed by CK and W1 treatments at 30\u0026ndash;70 days of stress, and W1 and W2 treatments surpassing CK treatment at 90\u0026ndash;110 days of stress. Stem C content did not significantly differ between W3 and CK treatments at 30d-110 days of stress, with both exceeding W2 treatment. Root C content was highest in the W3 treatment at 30\u0026ndash;70 days of stress, but CK surpassed W1 treatment at 110 days of stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Regarding N content, leaf N content was higher in W1 and W3 treatments compared to CK and W2 treatments during 30\u0026ndash;50 days of stress, with no significant difference at 70 days, and W2 and W3 treatments surpassing W1 and CK treatments at 110 days of stress. During the 90 days stress period, stem N content was higher in the W3 treatment than in the other treatments. Root N content, in descending order, followed the sequence of W3\u0026thinsp;\u0026gt;\u0026thinsp;CK\u0026thinsp;\u0026gt;\u0026thinsp;W1\u0026thinsp;\u0026gt;\u0026thinsp;W2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Leaf P content was highest in the CK treatment at 30\u0026ndash;90 days of stress, followed by a sequential decrease in P content across all treatment groups at 110 days of stress. Stem P content was greater in the CK treatment compared to the W3 treatment at 50\u0026ndash;70 days of stress, and highest in the W3 treatment at 90\u0026ndash;110 days of stress. Root P content at 30d-70d of stress showed that W1, W2 surpassed CK treatment, while at 90\u0026ndash;110 days CK and W1 treatments surpassed W2 and W3 treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter 20 days of rewatering, the C content in the W3-treated leaves and W2-treated stems/roots decreased respectively compared with CK, while the N content increased in each organ across all treatment groups, and the P content in the leaves/stems of all treatments increased whereas that in the W2/W3-treated roots decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of drought stress and subsequent rewatering on pigment content and leaf color parameters of\u003c/b\u003e \u003cb\u003eC. coggygria\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChlorophyll a, chlorophyll b, and total chlorophyll in leaf tissues exhibited an initial decrease followed by an increase over the course of drought stress, reaching their lowest levels on the 90th day (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Conversely, Car and Ant levels showed a consistent decline with prolonged drought exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E). Under heightened drought stress, chlorophyll a, chlorophyll b, total chlorophyll, and Car contents displayed an increasing trend in all treatment groups. Ant levels in all treatment groups, except W3, exhibited a rise from 50 to 70 days of stress, whereas in the W3 group, Ant levels were suppressed during this period and decreased compared to CK by 90 to 110 days of stress.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOn the 20th day of rewatering, the levels of Chl a, Chl b, and Chl exhibited a progressive decline in all treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). The Car concentration did not vary significantly between groups W1 and W2 relative to the CK group, whereas there was a notable 57.22% rise in Car levels in group W3 compared to the CK group. Concurrently, the Ant concentrations in each treatment group exhibited increments of 46.02%, 73.03%, and 71.02%, respectively, in comparison to the CK group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of drought stress and subsequent rewatering on ROS levels and antioxidant mechanisms of\u003c/b\u003e \u003cb\u003eC. coggygria\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exhibited a pattern characterized by a decline, followed by an increase, and then a subsequent decline with the duration of stress in each treatment group, while the \u0026middot;OH content displayed a pattern of decreasing, then elevating, followed by decreasing, and then increasing trends over time. SOD activity showed a relatively stable behavior. POD activity demonstrated divergent patterns among treatment groups, with consistent trends in CK, fluctuating trends in W1 and W2 groups, and increasing trends in W3 group. CAT activity demonstrated a pattern of initial increase followed by decrease with the duration of stress in the CK, and an overall trend of decrease followed by increase in the W1, W2, and W3 treatment groups. MDA content decreased after peaking at 70 days of drought treatment in all groups. GSH content exhibited an initial increase followed by a decrease in response to drought duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUpon reaching 70 days of drought stress, the \u0026middot;OH content peaked in all treatment groups except W1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content was significantly lower in all groups compared to CK at 30 days, while it was significantly higher at 50\u0026ndash;90 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Among them, at 30d-90d of drought treatment, the MDA content gradually decreased with the decrease of soil moisture content, which was significantly lower than that of CK in all treatment groups. At 110th d of drought, MDA content increased sequentially with increasing drought (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). SOD enzyme activity was significantly higher in W2 at 30 days, and lower in W3 at later stages of stress compared to CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). POD activities were higher in W1 and W2 compared to CK at different stress durations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), while CAT activities were significantly higher in W1 and W2 at specific time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). GSH content showed a gradual increase with prolonged drought (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003eFollowing rewatering, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content increased significantly in W1 and W2 compared to CK, with W2 showing the highest levels. The \u0026middot;OH content in each treatment group did not exhibit significant variation and was found to be elevated compared to that of the CK. SOD enzyme activity was not fully recovered compared with 110d of stress, W2 treatment group was significantly higher than that of CK, and W3 treatment group had a stronger degree of stress, and SOD enzyme activity was still inhibited after rewatering. POD enzyme activities were significantly increased in the W1 and W2 treatment groups compared to CK, while POD enzyme activities remained lower in the W3 treatment group than in CK. CAT enzyme activity did not change significantly in the W2 and W3 treatment groups compared to 110 d of stress. The MDA content was significantly lower than that of CK, and the MDA content of the W3 treatment group was elevated compared with that of 110 d of stress, by 65.49%. The GSH content reached the maximum and was significantly higher than that of CK among the treatment groups, being 1.22, 1.28 and 1.12 times higher than that of CK, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of drought stress and subsequent rewatering on osmoregulatory substances in\u003c/b\u003e \u003cb\u003eC. coggygria\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt all periods of drought stress, the SP content of each treatment group, and the Pro content of CK, W1 and W2 treatment groups showed an increase followed by a decrease and then an increase, and the Pro content of the W3 treatment showed a smaller change (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). There was no significant difference in SP content between CK treatment and other treatments in the pre-stress period, and the SP content of W2 and W3 treatment groups under 70d of stress was significantly higher than that of CK, with an increase of 3.54% and 4.63%, respectively; the SP content of W1 and W2 treatment groups under 90d of stress was significantly higher compared with that of CK, with an increase of 5.12% and 13.58%, and that of W3 treatment group under 110d of stress was also significantly higher compared with that of CK, with an increase of 6.80% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The degree of drought significantly increased the Pro content, which was sequentially higher and significantly higher than CK among the treatment groups, with the greatest decrease in the 70th d of the drought treatment, with CK, W1 and W2 decreasing by 44.63, 78.04 and 80.92% compared to the 50th d of drought (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter rewatering, there was no significant difference in the SP content of each treatment group, and the Pro content was significantly higher than that of CK, which was 1.41, 1.60 and 2.81 times higher than that of CK, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptomic investigation of\u003c/b\u003e \u003cb\u003eC. coggygria\u003c/b\u003e \u003cb\u003eunder drought stress and subsequent rewatering conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the gene expression pattern of \u003cem\u003eC. coggygria\u003c/em\u003e, drought CK, Wd_3 and rewatering Wr_3 treatments were selected for high-throughput RNA sequencing. A total of 40901478\u0026thinsp;~\u0026thinsp;44714144 raw reads were obtained from the nine cDNA libraries, and 40627744\u0026thinsp;~\u0026thinsp;44397794 clean reads were obtained after eliminating the low-quality reads, and the percentage of localized reads for each sample was very high, ranging from 89.18\u0026ndash;89.84% (Table\u0026nbsp;1). The clean reads library produced a percentage of Q30 bases above 95.29% and a percentage of Q20 bases above 98.55%, both with a GC content greater than 43.45%, and a comparison efficiency of 89.18%, indicating a good overall quality of the data. Gene expression abundance was less than normal water supply after drought treatment, and gene expression was largely restored after rewatering (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). There were more DEGs for drought than for rewatering, and 1,785 genes were shared between drought and rewatering (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), suggesting that these genes are most likely to respond to drought rewatering in \u003cem\u003eC. coggygria\u003c/em\u003e. Analysis using DESeq2 software revealed that under drought conditions, 2684 genes were up-regulated and 4017 genes were down-regulated, whereas after rewatering, 1923 genes were up-regulated and 1541 genes were down-regulated in comparison to control (CK) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eFunctional classification of DEGs was carried out by KEGG pathway analysis\u003c/h3\u003e\n\u003cp\u003eThe KEGG enrichment analysis of the DEGs under drought conditions was enriched in 133 pathways, involving a total of 1,583 DEGs. Two pathways were significantly enriched, namely Metabolism and Environmental Information Processing. Among the two major categories of pathways, the pathways with the largest number of annotated genes are Plant hormone signal transduction, Starch and sucrose metabolism, Phenylpropanoid biosynthesis; MAPK signaling pathway - plant MAPK; and Biosynthesis of various plant secondary metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). After rewatering, 711 DEGs in the leaves were annotated to 118 metabolic pathways. These pathways were significantly enriched in pathways such as Plant-pathogen interaction, ABC transporters, Phenylpropanoid biosynthesis, Biosynthesis of various plant secondary metabolites, and Flavonoid biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Compared with CK treatment, genes related to Metabolic, Biological and Environmental Information Processing were significantly expressed and the number of up-regulated expressed genes was much lower than the number of down-regulated expressed genes after Wd3 treatment, suggesting that drought stress significantly affected the metabolism and biosynthesis of \u003cem\u003eC. coggygria\u003c/em\u003e. Compared with CK treatment, Wr3 treatment up-regulated 336 genes and down-regulated 375 genes, and the number of up-regulated expressed genes was lower than the number of down-regulated expressed genes. The results indicated that \u003cem\u003eC. coggygria\u003c/em\u003e leaves used different mechanisms to resist drought stress.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of drought stress and subsequent rewatering on DEGs of phytohormone signaling in\u003c/b\u003e \u003cb\u003eC. coggygria\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe KEGG term \u0026ldquo;phytohormone signaling\u0026rdquo; was significantly enriched during drought, and a total of 27 DEGs families were identified for key gene modules of phytohormone signaling, including IAA, CTK, GA, ABA, ETH, BR, SA, and JA, after drought and rewatering (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Twenty-six DEGs were enriched in the IAA signaling pathway during drought stress, and most of them were clustered in two gene modules, \u003cem\u003eSAUR\u003c/em\u003e, \u003cem\u003eAUX/IAA\u003c/em\u003e, and only the TRINITY_DN14656_c0_g1 was significantly up-regulated in the \u003cem\u003eSAUR\u003c/em\u003e gene module. Five genes within the AUX/IAA gene module show upregulation, while two genes exhibit downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). There are five genes in the CTK signaling pathway, \u003cem\u003eAHP\u003c/em\u003e (TRINITY_DN17133_c0_g1) and \u003cem\u003eARR-B\u003c/em\u003e gene expression was up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). There were two DEGs involved in the GA signaling pathway, including one up-regulated \u003cem\u003eGID1\u003c/em\u003e gene and one down-regulated \u003cem\u003eDELLA\u003c/em\u003e gene; the \u003cem\u003eEBF1_2\u003c/em\u003e gene of the ETH signaling pathway was up-regulated under drought stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Compared with CK, the expression of ABA signaling pathways \u003cem\u003ePP2C\u003c/em\u003e and \u003cem\u003eABF\u003c/em\u003e genes was up-regulated in drought treatment, whereas the expression of most \u003cem\u003eSNRK2\u003c/em\u003e and \u003cem\u003ePYL\u003c/em\u003e genes was down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). The results of ABA signaling indicated that drought stress induced \u003cem\u003ePP2C\u003c/em\u003e and \u003cem\u003eABF\u003c/em\u003e genes, but repressed \u003cem\u003eSNRK2\u003c/em\u003e and \u003cem\u003ePYL\u003c/em\u003e genes. There were eight DEGs in the BR signaling pathway under drought stress, including one in the up-regulated \u003cem\u003eBAK1\u003c/em\u003e gene, and the remaining seven genes were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). The JA signaling pathway had a total of six DEGs, with \u003cem\u003eMYC2\u003c/em\u003e and most of the \u003cem\u003eJAZ\u003c/em\u003e genes down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). There were five DEGs in the SA signaling pathway, with \u003cem\u003ePR-1\u003c/em\u003e (TRINITY_DN2251_c0_g2) and \u003cem\u003eNPR1\u003c/em\u003e genes down-regulated and the remaining genes up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH). In total, 18 DEGs were identified as enriched during the rewatering process. Among these, 9 DEGs showed downregulation in their expression levels, encompassing 1 gene each from the AUX/IAA, PP2C, PR-1, PYL, and SAUR gene modules, as well as 2 genes each from the CYCD3 and TCH4 gene modules.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eThe DEGs associated with various physiological parameters under drought and rewatering conditions\u003c/h3\u003e\n\u003cp\u003eCompared with CK, in Wd_3, the expression levels of six gene modules involved in Car synthesis were down-regulated, including CYP707A, NCED, LUT1, CYP97C1 and CCD8, while the expression levels of three gene modules were up-regulated, including VDE, NPQ1, CCD7 and crtZ. During the synthesis of Ant, the expression of one gene module was down-regulated. After rewatering, the expression of CYP707A and NCED involved in the Car synthesis process was down-regulated, and the expression of one gene module involved in Ant synthesis was up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). In the MAPK signaling metabolism, after rewatering, the TRIITY_DN41_c0_g1 related to H₂O₂ synthesis continued to be down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). Under stress, there were 5 DEGs related to proline in the metabolism of proline and arginine. The expression levels of 4 genes were up-regulated, and the expression of PRODH, fadM and putB genes was down-regulated. After rewatering, the genes related to proline synthesis basically returned to the level of CK (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). There were 14 DEGs of POD, with 7 up-regulated genes and 7 down-regulated genes. Among them, the expression of ACSL and fadD was down-regulated under drought stress and continued to be down-regulated after rewatering (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eIdentification and analysis of differentially expressed TFs\u003c/h2\u003e\u003cp\u003eThere were 28 different TFs families with a total of 241 genes in the DEGs of CK and Wd_3 under drought stress, including 28 AP2/ERF (11.6%), 25 bHLH (10.3%), 24 NAC (9.9%), 51 MYB (21.1%), 18 WRKY (7.4%), and 18 C2C2 (7.4%) family genes (Fig.\u0026nbsp;11A). There were 21 families of TFs and 241 gene numbers in the DEGs of Wr_3 and CK, and the most numerically significant genes were the AP2/ERF (20, 19%), bHLH (12, 11.4%), NAC (12, 11.4%), MYB (19, 18%), and WRKY (7, 6.6%) families (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB). Thus, the six most common and critical TFs of \u003cem\u003eC. coggygria\u003c/em\u003e are MYB, AP2/ERF, bHLH, NAC, WRKY, and C2C2. The TFs families were categorized into at least two groups, one of which was positively and the other negatively correlated with drought stress, suggesting that these families could up- or down-regulate the expression profiles of enzyme-encoding genes. Through expression pattern analysis, TRINITY_DN40545_c0_g1 and TRINITY_DN6699_c0_g1 were found to be the \u003cem\u003eAP2/ARFs\u003c/em\u003e most likely to be positively and negatively involved in the drought response of \u003cem\u003eC. coggygria\u003c/em\u003e leaves, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC). TRINITY_DN806_c0_g1 and TRINITY_DN8552_c0_g1, TRINITY_DN7437_c0_g1 and TRINITY_DN563_c0_g1, TRINITY_DN163_c0_g1 and TRINITY_DN5469_c0_g2, TRINITY _DN15772_c0_g1 and TRINITY_DN2815_c0_g1 were the most likely genes among the MYB, NAC, WRKY, and C2C2 genes to be positively and negatively involved in the drought response, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e-F, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eH). For the \u003cem\u003ebHLH\u003c/em\u003e family, TRINITY_DN4346_c0_g1 is most likely the gene negatively involved in drought response (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDrought stress induces notable alterations in both the external morphology and internal structure of plants [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This stress condition results in substantial water depletion from leaves [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Numerous research studies have demonstrated that drought stress has the potential to diminish the LWP of plants, thereby exerting an influence on their growth [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The reduction in LWP has the capacity to affect the solubility of biological compounds and disrupt ions crucial for cellular functions, consequently causing plant malfunction and the production of ROS [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Insufficient water availability leads to a deceleration in plant growth, prominently manifesting as diminished SH [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], reduced leaf area [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and heightened LT [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The degree of recovery in compensatory plant responses subsequent to rehydration may be impacted by the severity and duration of preceding drought stress [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe decline in LWP observed in \u003cem\u003eC. coggygria\u003c/em\u003e under stress aligns with similar findings in \u003cem\u003eTriticum aestivum\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The intensification of stress levels resulted in the inhibition of SH (except W1) and GD growth. Research has indicated that mild drought stress exerts minimal effects on plant development; however, under moderate and severe stress conditions, there is a marked reduction in plant height [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. As drought stress severity escalates, the W3 treatment demonstrated a thinning of leaves due to excessive water loss, in line with the observations of Lei \u003cem\u003eet al\u003c/em\u003e study [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. By day 110 of stress exposure, the W2 treatment exhibited an increase in LT, consistent with Park \u003cem\u003eet al\u003c/em\u003e 's findings attributing leaf thickness to a thick cuticle layer and deeply depressed stomata, which help diminish water loss [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Other durations of drought stress did not significantly impact LT, potentially due to the brief stress period. Following rehydration, the LWP, SH, GD, and LT of \u003cem\u003eC. coggygria\u003c/em\u003e failed to fully recover to the levels observed in the control group.\u003c/p\u003e\u003cp\u003eThe decline in plant biomass under drought stress is associated with both stomatal and non-stomatal factors, such as stomatal closure, leaf senescence, and chlorophyll degradation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Stomatal regulation is a critical factor under abiotic stress conditions, influencing the equilibrium between plant transpiration and photosynthesis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In the study, SN and SD gradually increased in the W1, W2 and W3 treatment groups during the pre-stress period, and the higher SD was due to the tight stacking of epidermal cells and the reduction in epidermal cell expansion [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Following rehydration, SN and SD in all treatment groups surpassed those in the CK, indicating that the stomatal traits of \u003cem\u003eC. coggygria\u003c/em\u003e had not completely recovered post-rehydration.\u003c/p\u003e\u003cp\u003eDrought stress induces ROS production, which causes severe oxidative damage to membrane lipids and is required to limit plant growth and development by reducing photosynthesis and nutrients [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and elemental deficiencies lead to increased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Nutrients play an important role in regulating plant stress tolerance [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], and a decrease in soil water content affects the release and mobility of elements [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. C, N, and P content are the major elements for plant growth and development [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], N deficiency reduces the photosynthetic activity and longevity of leaves [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In a study by Gargallo-Garriga \u003cem\u003eet al\u003c/em\u003e it was shown that under sustained natural drought, the content of N and P concentrations varied throughout the season [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe different trends of C, N and P contents of each organ with the prolongation of stress time in the experiment were due to the fact that the plant body, in order to maintain the stability of its own chemical composition, made corresponding feedbacks to the changes in the external environment [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In the leaves of \u003cem\u003eC. coggygria\u003c/em\u003e, in the W1 treatment under short-term drought stress, the C metabolism activities suffered interference and the photosynthetic rate was reduced, resulting in less C uptake. But in the W1 and W2 treatments under long-term drought stress, needed to absorb more water to alleviate drought and resist drought, thus leading to an increase in the C content per unit area of the leaf [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. W3 foliar treatment C concentration was not affected by drought treatment, which was in agreement with Zhang \u003cem\u003eet al\u003c/em\u003e related study [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The higher root C content of the W3 treatment contributes to C uptake, which is an important strategy for nutrient retention under stress conditions [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. As the degree of drought stress intensified, leaf N content gradually increased, indicating that N solubilization and transformation require the involvement of water, and lower soil water content makes N difficult to solubilize and less available [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The stem N content was higher in the W3 treatment than in the other treatments, probably due to the fact that the W3 treatment regulated growth through the stems, resulting in a change in the distribution of N in the plant, leading to an increase in N content. Root N content increased in W3-treated roots at 90 d of stress probably due to excessive water deficit leading to N recycling to sustain life activities. Changes in leaf, stem, and root P content across treatment groups suggest that drought limits the movement of soil nutrients [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAfter 20 d of rewatering, the leaves and roots of the W3 treatment group did not allow the plant to recover quickly due to irreversible damage, resulting in lower C content, in agreement with An \u003cem\u003eet al\u003c/em\u003e study [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In the organs of \u003cem\u003eC. coggygria\u003c/em\u003e under the W2 and W3 treatments, the N content will temporarily increase the assimilation, absorption, and utilization of N after rewatering to supplement the growth requirements, so as to promote the growth and repair of the plants. The P content in the leaves and roots of each treatment group increased, while the P content in the stems decreased. In the plant body, the stem mainly plays the roles of support, transportation and storage. When drought limits the adsorption and solubilization of inorganic phosphorus, the available phosphorus tends to be transported to other organs such as roots and leaves [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThree broad categories of plant reactions to drought include alterations in pigmentation, both qualitatively and quantitatively [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Chl is a key factor in plant photosynthesis and is responsible for the absorption, transfer and conversion of light energy [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Car can also play a role in scavenging ROS as a non-enzymatic antioxidant in addition to its role in light trapping during photosynthesis [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Increased drought stress in the study led to higher Chla, Chlb, and Chl contents in all treatment groups of \u003cem\u003eC. coggygria\u003c/em\u003e leaves, suggesting that \u003cem\u003eC. coggygria\u003c/em\u003e can increase light energy utilization by increasing Chl content to adapt to water deficit [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], which is in agreement with the studies of \u003cem\u003ePeriploca sepium\u003c/em\u003e [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] and \u003cem\u003eHordeum vulgare\u003c/em\u003e [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. \u003cem\u003eC. coggygria\u003c/em\u003e under drought stress is also able to reduce water evaporation by increasing Car content, releasing excess heat, and lowering leaf temperature [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Ant content showed an increasing trend with the intensification of stress, which is consistent with the study of Hodaei \u003cem\u003eet al\u003c/em\u003e [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] that Ant shows an increase in content with water deficit, which promotes the antioxidant capacity of plants. The value of the color parameter b* gradually decreased in the later stages of stress in all treatment groups, and the leaf coloration was skewed towards a yellowish-blue tone. The decrease in \u003cem\u003eC. coggygria\u003c/em\u003e Chla, Chlb, and Chl levels after short-term rewatering is due to plant nutrient deficiencies, which reduce the synthesis of Chl by dedicating more resources to growth and restoration.\u003c/p\u003e\u003cp\u003eDrought-induced stress in plants typically results in physical and chemical harm, leading to compromised nutrient absorption, disrupted cellular processes, and the accumulation of ROS [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Among ROS, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is known for its stability [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], while the \u0026middot;OH exhibits high reactivity [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. MDA is commonly used as a biomarker to evaluate the integrity of plant cell membranes [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. ROS levels rise, triggering the activation of both enzymatic and non-enzymatic defense mechanisms [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. SOD operates in chloroplasts, peroxisomes, and mitochondria [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e], playing a key role in converting \u0026middot;OH into H₂O₂. Subsequently, POD and CAT catalyze the decomposition of H₂O₂ into H₂O and O₂ [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. GSH, a vital component of the non-enzymatic defense system, acts as a dithiol reducing agent that safeguards enzyme sulfhydryl groups, regenerates ASA, and reacts with singlet oxygen and \u0026middot;OH [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWith the decrease of soil water content in the experiment, the antioxidant activity of \u003cem\u003eC. coggygria\u003c/em\u003e increased in the late stage of stress, which mitigated the damage of \u0026middot;OH to the cells, promoted the decomposition of H₂O₂, and helped to maintain the balance of intracellular signaling. The changes in \u003cem\u003eC. coggygria\u003c/em\u003e SOD enzyme activity among treatment groups were consistent with the study of Huang et al [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], which may be due to the temporary increase in SOD enzyme activity caused by water deficit. Due to the higher degree of stress, the SOD activities of W3 treatment were lower than those of CK, which was consistent with the study of Li \u003cem\u003eet al\u003c/em\u003e [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], where the persistent drought and the increased degree of stress led to the damage of the plant cell membrane system, the inhibition of antioxidant enzyme synthesis, and the accumulation of ROS exceeded the scavenging capacity of the plant. During the pre-stress period, MDA content gradually decreased in all treatment groups, indicating that \u003cem\u003eC. coggygria\u003c/em\u003e was able to acclimatize to drought for a sufficient period of time [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. In the late stage of stress, the MDA content increased sequentially with the intensification of drought, which corresponded to the study of Zhao \u003cem\u003eet al\u003c/em\u003e [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e], and the W3 treatment was not significantly different from CK, indicating a limited capacity of the antioxidant system to regulate the antioxidant system [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. At the same point in time, GSH content gradually increased with increasing drought. GSH gradually increased with the intensification of drought. The increase in GSH helps maintain normal immune system function and has antioxidant and detoxification effects [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAfter rewatering, the activities of \u0026middot;OH and H₂O₂ in the W1 treatment group and W2 treatment group were both higher than those in CK. This is because rewatering still cannot effectively scavenge the ROS accumulated during drought stress. The antioxidant enzyme activities were lower in the W3 treatment group than in CK, indicating that the antioxidant system of \u003cem\u003eC. coggygria\u003c/em\u003e was irreversibly damaged by severe drought. The significant increase in GSH content after rewatering was due to the fact that the processes of photosynthesis and respiration of the plant were enhanced, providing more energy and substrate for GSH synthesis, indicating that rewatering is favorable for \u003cem\u003eC. coggygria\u003c/em\u003e to carry out self-repair [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOsmoregulation is recognized as an important physiological adaptive property associated with abiotic stresses [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Plants respond to stress by eliminating ROS through osmotic pressure accumulated in the cytoplasm and chloroplasts [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Pro is an osmoregulatory substance that improves osmoregulation in plants and also acts as an antioxidant in plants, scavenging \u0026middot;OH and stabilizing cell membranes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. SP is also an osmoregulatory substance, and some studies have shown a sharp decrease in SP under drought stress [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], but others have shown a significant increase in SP content under drought treatment [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTrends in \u003cem\u003eC. coggygria\u003c/em\u003e Pro and SP content varied throughout the period under study. Enhanced aridity leads to an increase in Pro content, which helps to stimulate the production of antioxidant enzyme activities [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. The SP content of each treatment group gradually accumulated in the late stage of stress, which improved the osmoregulation ability of \u003cem\u003eC. coggygria\u003c/em\u003e [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. After rewatering, Pro content gradually decreased in each treatment group, while SP content basically returned to the normal level.\u003c/p\u003e\u003cp\u003ePhytohormones are signaling compounds that regulate key aspects of growth, development, and environmental stress responses [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. As a key stress hormone in plants, ABA plays the role of a central integrator in drought stress response, forming a complex regulatory network by activating adaptive signals and coordinating the interactions of multiple hormones [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. When plants respond to drought stress, ABA binds to upstream \u003cem\u003ePYR/PYLs\u003c/em\u003e receptors and inhibits \u003cem\u003ePP2Cs\u003c/em\u003e negative regulators, and \u003cem\u003eSnRK2s type\u003c/em\u003e protein kinase promotes ABA responses by activating \u003cem\u003eABF\u003c/em\u003e transcription factors through dephosphorylation of downstream targets [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. \u003cem\u003eARF\u003c/em\u003e may bind to the transcriptional repressor \u003cem\u003eAux/IAA\u003c/em\u003e to inhibit IAA synthesis, thereby suppressing biomass accumulation to alleviate water deficit [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. JAZ proteins negatively regulate JA-responsive genes [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e], and under drought stress, the expression of \u003cem\u003eJAR1\u003c/em\u003e, a JA signaling gene, was significantly decreased, and the expression of \u003cem\u003eCOI1\u003c/em\u003e, which is involved in stomatal movement, was increased [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. BR signaling regulatory positive and negative factors activate \u003cem\u003eSnRK2s\u003c/em\u003e to control the initiation and amplification of ABA signaling [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. GA signaling regulates resistance by controlling cellular redox homeostasis, and increased \u003cem\u003eDELLA\u003c/em\u003e activity interferes with ABA signaling [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. Studies in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e under drought stress have shown that by regulating the redundant negative regulators \u003cem\u003eAHP2\u003c/em\u003e, \u003cem\u003eAHP3\u003c/em\u003e and \u003cem\u003eAHP5\u003c/em\u003e in the CTK signaling pathway, \u003cem\u003eAHP\u003c/em\u003e controls the drought response in both an ABA-dependent and an ABA-independent manner [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. In addition, SA enhances plant drought tolerance by strengthening antioxidant defense, promoting osmotic fluid accumulation, increasing water use efficiency, and enhancing photosynthesis [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. During the dual process of drought and rewatering in this study, it was shown that ABA and other hormone signaling pathways are involved in the adaptive response of \u003cem\u003eC. coggygria\u003c/em\u003e to drought stress. Involvement in phytohormone signaling including 27 gene families such as \u003cem\u003ePYR/PYLs\u003c/em\u003e, \u003cem\u003eSnRK2\u003c/em\u003e, IAAs and \u003cem\u003eARFs\u003c/em\u003e was observed in drought stress and rewatering, suggesting that the phytohormone signaling pathway may be related to drought and rewatering responses in \u003cem\u003eC. coggygria\u003c/em\u003e. The study reported a down-regulation in the expression of 10 SAUR and 2 AUX/IAA genes within the IAA signaling pathway, resulting in the suppression of IAA synthesis and ultimately enhancing the drought tolerance of plants [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Drought stress induces phytohormone-related genes in \u003cem\u003eC. coggygria\u003c/em\u003e to express and interact with each other, activating a comprehensive and complex phytohormone regulatory network to improve its adaptation and survival under water deficit.\u003c/p\u003e\u003cp\u003eIn cotton, the \u003cem\u003eNCED\u003c/em\u003e gene is a key gene in the ABA synthesis pathway [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. Under drought conditions, the biosynthesis of anthocyanins is regulated by MYB and bHLH transcription factors [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. In potatoes, the upregulation of genes related to osmotic regulation corresponds to the osmotic response [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]. The class III peroxidase gene family in tea plants plays an important role in abiotic stress [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e]. In the study, after drought treatment, the expression levels of most genes related to Car synthesis and Ant synthesis genes were downregulated, which was contrary to the accumulation of Car and Ant contents. It is likely that although the transcriptional levels of Car and Ant synthesis genes decreased, the translation efficiency of their mRNAs increased and their catalytic activities were enhanced, which is consistent with the research on \u003cem\u003eScutellaria baicalensis\u003c/em\u003e [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e]. Under drought conditions, there were changes in the DEGs related to Pro content, H₂O₂, and POD, which were consistent with the changes in Pro content, H₂O₂ content, and POD enzyme activity.\u003c/p\u003e\u003cp\u003eTFs play a variety of roles in the control of gene expression in plants and are required for the regulation of biological processes such as development and environmental stress responses [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. Several families of transcription factors such as MYB, WRKY, AP2/ERF, NAC, bHLH, and C2C2 have been characterized and proved to be useful tools for enhancing drought tolerance in plants [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e, \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e]. In addition, MYB transcription factors are critical in the biosynthesis of plant secondary metabolites, including anthocyanins, flavonoids, lignin and cuticle synthesis, and rely on ABA signaling to participate in the regulation of stomatal movement in plants under arid environments [\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e]. \u003cem\u003eGmWRKY54\u003c/em\u003e directly binds to the promoters of \u003cem\u003ePYL8\u003c/em\u003e, \u003cem\u003eSRK2A\u003c/em\u003e, \u003cem\u003eCIPK11\u003c/em\u003e, and \u003cem\u003eCPK3\u003c/em\u003e and activates their expression, thereby improving drought tolerance in \u003cem\u003eGlycine max\u003c/em\u003e [\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e]. In poplar, the bHLH family gene \u003cem\u003ePebHLH35\u003c/em\u003e responds to drought stress by positively regulating stomatal density, stomatal aperture and photosynthesis [\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e]. Similarly, overexpression of the stress-responsive \u003cem\u003eNAC1\u003c/em\u003e in rice conferred tolerance to severe drought stress without phenotypic or yield changes, whereas overexpression of \u003cem\u003eOsNAC6\u003c/em\u003e in rice led to improved water retention by controlling stomatal closure under dehydration stress [\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e]. Correlation network prediction revealed that AP2/ERF, WRKY, MYB, bHLH, and NAC affect plant drought tolerance by regulating the expression of downstream genes such as \u003cem\u003ePP2C\u003c/em\u003e, \u003cem\u003eJAZ\u003c/em\u003e, and \u003cem\u003eSnRK2\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The study identified the six predominant TF families in leaves as AP2/ERF, bHLH, NAC, MYB, WRKY, and C2C2. Drought stress was found to elevate the expression of these TFs, with their regulation being either up-regulated or down-regulated in reaction to stress and subsequent rewatering. This implies that they function as positive or negative regulators of drought stress in \u003cem\u003eC. coggygria\u003c/em\u003e, respectively.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eProlonged exposure to drought stress triggers varying dynamic trends in the morphological structure and physiological parameters during the growth stages of \u003cem\u003eC.coggygria\u003c/em\u003e. Elevated stress levels correspond to decreased N and P content in various organs of \u003cem\u003eC. coggygria\u003c/em\u003e, resulting in diminished LWP and notable accumulations of SD, SN, photosynthetic pigments and osmolytes. This accumulation hinders growth while enhancing the water-holding capacity of \u003cem\u003eC.coggygria\u003c/em\u003e. The antioxidant compounds in \u003cem\u003eC.coggygria\u003c/em\u003e synergistically regulate the equilibrium between ROS production and scavenging within the plant. Nevertheless, studies highlight the limited regulatory capacity of \u003cem\u003eC.coggygria\u003c/em\u003e in modulating its antioxidant defense system. Short-term rehydration fails to fully restore most physiological parameters of \u003cem\u003eC.coggygria\u003c/em\u003e to control levels. Furthermore, analyses have unveiled the gene expression dynamics associated with hormone signaling pathways and alterations in drought-resilient physiological markers in \u003cem\u003eC.coggygria\u003c/em\u003e. Transcription factors such as WRKYs, MYBs, bHLHs, AP2/ERFs, NACs, and C2C2s have been identified as potential key regulators, shedding light on crucial candidate genes for drought tolerance in \u003cem\u003eC.coggygria\u003c/em\u003e.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eReactive oxygen species\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLWP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLeaf water potential\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEMLWP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEarly morning leaf water potential\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMDLWP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMidday leaf water potential\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSeeding height\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGround diameter\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLeaf thickness\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eStomatal number\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eStomatal density\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCarbon\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNitrogen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhosphorus\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eChla\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eChlorophyll a\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eChlb\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eChlorophyll b\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eChl\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eChlorophyll\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCar\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCarotenoids\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAnt\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAnthocyanin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHydrogen peroxide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u0026middot;OH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHydroxyl radical\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMAD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMalondialdehyde\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSOD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSuperoxide dismutase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePOD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePeroxidase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCAT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCatalase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGSH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlutathione\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSoluble protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePro\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eProline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eABA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAbscisic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSalicylic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCTK\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCytokinin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eETH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEthylene\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIAA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIndole-3-acetic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eJA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eJasmonic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGibberellic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBrassinosteroids\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTFs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTranscription factors.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe thesis was written by S.M. and X.L.; the methodology was collected and organized by Y.F. and L.Y.; data collection was carried out by Y.X. and J.S.; data analysis and organization were done by X.C.; seedling maintenance was undertaken by J.B., X.W. and Y.Z.; and the experimental design, thesis conception, and experimental guidance were provided by K.Z. and X.Y..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Natural Science Foundation of Shanxi Province (202103021224144), the Biobreeding Project of Shanxi Agricultural University (YZGC138), the Special Project for Forest and Grass Germplasm Resources Investigation of Shanxi Forestry and Grassland Bureau (QT2024007), the Postgraduate Research Innovation Project (2023KY346), the Key Scientific Research Project of Shanxi Road \u0026amp; Bridge Group (SXLQ-XY-3-002-2023), the Transportation Construction Technology Research Project of Zhongzi Huake (2024-GSGL-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available in the NCBI SRA with the accession number PRJNA1315001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plant materials used in this study were 3-year-old healthy seedlings of \u003cem\u003eC. coggygria\u0026nbsp;\u003c/em\u003egrowing in the Forestry Station of Shanxi Agricultural University, Jinzhong, China. And no permits are required for the collection of plant samples. This study did not require ethical approval or consent, as it did not involve any endangered or protected species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCohen I, Zandalinas SI, Huck C, Fritschi FB, Mittler R. Meta-analysis of drought and heat stress combination impact on crop yield and yield components. Physiol Plant. 2021;171(1):66\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTan W, Li W, Li J, Liu D, Xing W. Drought resistance evaluation of sugar beet germplasms by response of phenotypic indicators. 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Plant journal: cell Mol biology. 2007;51(4):617\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eRaw sequencing data and quality control checks of nine \u003cem\u003eC. coggygria\u003c/em\u003e leaf cDNA libraries.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"544\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 8.63971%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1765%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRaw reads\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.4412%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClean reads\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.3162%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eQ20 (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.5%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eQ30 (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.4853%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGC content (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.4412%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMapped ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 8.63971%;\"\u003e\n \u003cp\u003eCK_1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1765%;\"\u003e\n \u003cp\u003e42715214\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.4412%;\"\u003e\n \u003cp\u003e42414146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 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valign=\"top\" style=\"width: 19.4853%;\"\u003e\n \u003cp\u003e43.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.4412%;\"\u003e\n \u003cp\u003e89.83%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 8.63971%;\"\u003e\n \u003cp\u003eWr3_3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.1765%;\"\u003e\n \u003cp\u003e44714144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.4412%;\"\u003e\n \u003cp\u003e44397794\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.3162%;\"\u003e\n \u003cp\u003e98.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.5%;\"\u003e\n \u003cp\u003e95.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19.4853%;\"\u003e\n \u003cp\u003e43.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15.4412%;\"\u003e\n \u003cp\u003e89.40%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cotinus coggygria, drought, rewatering, physiological response, transcriptional regulation","lastPublishedDoi":"10.21203/rs.3.rs-7457742/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7457742/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eGlobal drought represents a pressing environmental challenge, necessitating a deeper comprehension of how plant species at various stages of drought response adapt to such stress. \u003cem\u003eCotinus coggygria\u003c/em\u003e, a deciduous tree species known for its autumn color transformation, holds significance for arid and semi-arid ecological contexts. Research investigating the detailed physiological and transcriptomic responses of \u003cem\u003eC. coggygria\u003c/em\u003e to drought and subsequent rewatering is currently lacking.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eSeedlings of \u003cem\u003eC. coggygria\u003c/em\u003e were subjected to five distinct drought durations (30, 50, 70, 90, and 110 days) followed by a 20-day rewatering period. Increasing drought severity led to reductions in seedling height, ground diameter, leaf water potential, and nitrogen and phosphorus contents across plant organs, while showing notable increases in stomatal traits, chlorophyll and carotenoid levels, as well as soluble protein and proline contents, ultimately bolstering the plant's ability to retain water. Towards the later stages of stress, heightened levels of hydrogen peroxide and malondialdehyde were observed, accompanied by diminished hydroxyl radical content, and augmented activities of peroxidase, catalase, and glutathione, indicative of antioxidant system modulation. Following short-term rewatering, most physiological parameters of \u003cem\u003eC. coggygria\u003c/em\u003e did not fully recover to control levels. Transcriptomic analysis revealed 3443 up-regulated and 3891 down-regulated differentially expressed genes (DEGs) under 110 days of stress, and 1923 up-regulated and 1541 down-regulated DEGs following 20 days of rewatering, highlighting genes modulating phytohormone signaling pathways, metabolic pathways associated with key physiological indicators, and differentially expressed transcription factors.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe research revealed that \u003cem\u003eC. coggygria\u003c/em\u003e demonstrated synchronized physiological and transcriptomic reactions to both drought stress and subsequent rehydration. These reactions encompassed alterations in growth metrics, nutrient levels, physiological characteristics, antioxidant system functionality, and gene expression profiles. The results offer significant understanding into the adaptive mechanisms of \u003cem\u003eC. coggygria\u003c/em\u003e under drought stress conditions and may have implications for comprehending and mitigating drought effects on plant species in arid and semi-arid regions.\u003c/p\u003e","manuscriptTitle":"Physiological and Transcriptomic Cooperative Regulatory Mechanisms of Cotinus coggygria in Response to Drought and Rewatering Processes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 14:54:39","doi":"10.21203/rs.3.rs-7457742/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-17T07:42:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-25T15:14:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-14T01:35:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54658555871898834550407257030654444782","date":"2025-09-14T01:12:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-12T08:42:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"278111970629425918094362789276184681627","date":"2025-09-12T08:19:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331782667591832782700813718384036462583","date":"2025-09-11T10:17:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-11T10:07:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-11T09:48:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-10T06:25:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-09T14:55:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-09-09T14:50:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"752fa1c9-d5c6-4bd3-9b15-48e84cb21c4e","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:03:41+00:00","versionOfRecord":{"articleIdentity":"rs-7457742","link":"https://doi.org/10.1186/s12870-026-08154-0","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2026-01-26 15:58:30","publishedOnDateReadable":"January 26th, 2026"},"versionCreatedAt":"2025-09-18 14:54:39","video":"","vorDoi":"10.1186/s12870-026-08154-0","vorDoiUrl":"https://doi.org/10.1186/s12870-026-08154-0","workflowStages":[]},"version":"v1","identity":"rs-7457742","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7457742","identity":"rs-7457742","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}