Physiological and transcriptomic cooperative regulatory mechanisms of Cotinus Coggygria in response to drought and rewatering processes

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. Presently, investigations into C. coggygria primarily concentrate on the effect of drought on its leaf color, with limited examination of the fundamental mechanisms involved. Through the merging of physiological and transcriptomic data analysis of C. coggygria exposed to drought conditions, crucial physiological markers and alterations in metabolic pathways under drought stress can be clarified, which holds significant importance for the utilization of ornamental tree species in arid and semi-arid areas.

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 growth parameters, leaf water potential, and nitrogen and phosphorus contents across plant organs, while showing notable increases in stomatal traits, pigment content and osmotic adjustment substances. Towards the later stages of stress, there was an accumulation of hydrogen peroxide content, alongside a reduction in hydroxyl radical content, coupled with elevations in levels of antioxidant enzymes and compounds. The stress-induced response amplifies water retention attributes at the cost of the growth of C. coggygria. Following short-term rewatering, most physiological parameters of C. coggygria did not fully recover to control levels. Transcriptomic analysis revealed 2684 up-regulated and 4017 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.

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]. Water management history, including reservoir construction in arid regions, underscores the broader environmental context in which plant drought studies are situated [4]. Drought stress and subsequent rewatering are essential stages in the plant growth cycle [5], with rewatering serving as a recovery mechanism post-drought to restore growth and enable rapid plant development [6]. Recent hydrological studies emphasize the role of soil water movement in optimizing irrigation strategies in arid farmlands, supporting the ecological relevance of assessing drought responses in woody plants [7].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. Comparative analyses of stress responses in woody species, such as mangroves under varying ecosystem conditions, also reveal how plant resilience strategies differ across ecological contexts [8]. C. 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 [9], revealing significant research gaps in understanding its drought resistance mechanisms.

Drought stress exerts key effects on plants, altering pigment synthesis, osmoregulation, secondary metabolism, antioxidant systems, and gene expression [10]. The visible impact of drought on plants is the inhibition of morphological growth [11], with leaf morphology serving as a prominent indicator in drought studies [12]. Stomatal regulation, a common adaptive response to drought, helps plants maintain leaf water potential (LWP) stability and reduce gas exchange [13]. Chlorophylls (Chl) and carotenoids (Car) are pivotal for photosynthesis [11], with drought stress often leading to a reduction in chlorophyll contents [14]. Ecological stoichiometry, focusing on the balance of chemical elements in ecosystems [15], highlights the roles of carbon (C), nitrogen (N), and phosphorus (P) in plant growth and physiological processes [16]. 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 [17]. 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 [18]. The antioxidant defense system plays a critical role in scavenging excess ROS to prevent cellular damage and maintain ROS homeostasis [19, 20]. 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 [21]. Key osmoregulatory substances involved in this process are proline (Pro) and soluble protein (SP) [11]. The research by Wang et al. [22] 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 [23]. Transcriptome sequencing has been pivotal in elucidating the molecular mechanisms governing plant responses to drought stress [24]. 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 [25]. Elevated ABA levels have been observed to trigger the upregulation of numerous transcription factors (TFs) and genes, thereby activating downstream metabolic pathways [25]. 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 [26]. Upon exposure to various stressors or defense cues, the upregulation of WRKY transcription factors in plants is promptly elicited. These WRKY proteins are established to participate in plant defense mechanisms, growth, metabolic activities, and modulation of hormone signaling pathways, among other functions [27]. bHLH transcription factors govern the transcriptional regulation of flavonoid biosynthesis in numerous woody species, including the anthocyanin biosynthesis pathway [28]. In rice, approximately 65% of MYB genes expressed in seedlings exhibit distinctive regulatory patterns under drought conditions [29].

The leaf functional traits of C. coggygria seedlings show distinct variations under different drought stress conditions across various provenances [30]. C. coggygria seedlings can adapt to short-term drought environments by adjusting their root morphology and exhibit greater sensitivity to drought conditions in full-light environments compared to shaded conditions [31, 32]. To date, there have been no joint studies on the physiology and transcriptome of C. coggygria under drought stress. Therefore, here we aim to: (1) characterize physiological responses of C. coggygria leaves to graded drought and rewatering; (2) identify differentially expressed genes (DEGs) under severe drought and after rehydration; and (3) link DEGs to key stress-related pathways. It is expected to provide support for the screening of drought resistance indicators, the mining of key drought-tolerant genes, and the further clarification of molecular regulatory mechanisms of C. coggygria.

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 around 800 m. 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%.

On April 30th, drought stress experiments were conducted with varying severity levels following the methodology outlined by Wang Kai et al. [33]. 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 [34]. 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. The control group remains CK.

Determination of morphological growth and physiological indices

Nine seedlings were chosen through random selection, with 9 replicates allocated to each group. 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.

Thirty fully expanded mature leaves were collected from 3 randomly selected seedlings in each group. These leaves were randomly mixed and used for the determination of various physiological parameters, with 3 biological replicates. From the three pots of seedlings in each group, 3 minor branches were randomly selected. A dew point potentiometer was used to measure their leaf water potential (LWP) at 6:00 a.m. and 12:00 p.m. respectively. The remaining leaves, stems, and roots were collected and dried in the laboratory for the determination of nitrogen (N), phosphorus (P), and potassium (K), with 3 biological replicates set. For stomatal measurement, 3 leaves were randomly selected, 3 replicates were set for each leaf, and the nail polish imprint method was used to measure the stomata. Stomatal number (SN) was quantified using Image J software, subsequently used to determine stomatal density (SD = SN per unit area) [35]. The contents of Chl and Car in leaves were quantified through ethanol extraction method [36]. The content of anthocyanin (Ant) was determined using the hydrochloric acid soaking method [37]. 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 [36]. C, N, and P in leaves, stems, and roots were determined using the dry combustion method, Kjeldahl nitrogen analyzer method [38], and vanado-molybdate yellow colorimetry [39], respectively.

Transcriptome sequencing

Mature and fully expanded 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 Bio-pharm Technology Co., Ltd. For a single library construction, the requirements are as follows: total RNA amount of 1 µg, concentration ≥ 30 ng/µL, RQN > 6.5, and OD_260/280 ratio ranging from 1.8 to 2.2. Subsequently, a cDNA library was constructed and subjected to quality assessment before sequencing, resulting in a total number of reads of 385,826,214. Trinity v2.8.5 software was used for de novo assembly of all clean data. The resulting transcriptome sequences were filtered, optimized, and assembled into Unigenes, which were then compared with the NR, Swiss-prot, Pfam, EggNOG, GO, and KEGG databases. DEGs were identified using the DESeq2 v1.42.0 based on specified criteria (|log₂FC| ≥ 1, FDR < 0.05).

Data analysis

Data were analyzed using SPSS 26.0. The determination of the same physiological index among different treatment groups was conducted using one-way analysis of variance (ANOVA), which belongs to between-group comparison. Since the measurements were performed on the same batch of seedlings, repeated-measures analysis of variance (ANOVA) was used for the determinations of the same treatment group at different time points, which belongs to within-group comparison. Subsequently, conduct multiple comparisons using Duncan’s test method to assess significant differences between groups. Graphs were plotted using Origin 2022 software.

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. 1A, B). While both SH and GD increased over time, the growth was more significantly suppressed under severe drought conditions (Fig. 1C, D). W3 treatment leaves lost water and became thinner, and leaf thickness (LT) was lower than that of CK (p < 0.05) (Fig. 1E).

Fig. 1.

Effects of drought stress and subsequent rewatering on leaf water potential and growth parameters of C. coggygria. A EMLWP, early morning leaf water potential; B MDLWP, midday leaf water potential; C SH, seeding height; D GD, ground diameter; E LT, leaf thickness. Significant differences within groups under the same treatment at different time points were analyzed using repeated measures ANOVA. Significant differences between groups at the same time point but under different treatments were analyzed using one-way ANOVA. Statistical significance was determined by a p-value < 0.05. Uppercase letters indicate within-group differences, while lowercase letters indicate between-group differences. 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

After 20 d of rewatering, the EMLWP and MDLWP of different treatment groups were still significantly lower than that of well-watered CK (p < 0.05), and the SH, GD and LT of W2 and W3 treatments 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, the SN and SD of the W1, W2, and W3 treatment groups increased gradually during the early and middle stages of stress. At 110 d of stress, SD was significantly higher in the W2 treatment group and significantly lower in the W3 treatment group compared with CK (p < 0.05). After rewatering, SN and SD were significantly higher in all treatment groups than in CK (p < 0.05).

Fig. 2.

Effects of drought stress and subsequent rewatering on stomatal traits of C. coggygria. A SN, stomatal number; B SD, stomatal density. Significant differences within groups under the same treatment at different time points were analyzed using repeated measures ANOVA. Significant differences between groups at the same time point but under different treatments were analyzed using one-way ANOVA. Statistical significance was determined by a p-value < 0.05. Uppercase letters (A, B, C, etc.) indicate within-group differences, while lowercase letters (a, b, c, etc.) indicate between-group differences. 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

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. In the late stage of stress, the leaf C content in the CK group decreased. From day 90 to day 110, the leaf C content in the W1 and W2 treatments was significantly higher than that in the CK treatment (p < 0.05). Throughout the entire period, the stem C content in the CK treatment showed an increasing trend, while that in the W2 treatment was significantly lower than in the CK treatment (p < 0.05). In the middle stage of stress, the root C content in the CK treatment decreased. On day 70, the root C content in the W3 treatment was significantly higher than that in the CK treatment (p < 0.05) (Fig. 3A). In the early and late stages of stress, the leaf nitrogen content in all treatment groups increased. On day 70, there was no significant difference among the treatment groups (p>0.05). Throughout the entire period, the stem N content in the CK treatment decreased, and the W3 treatment was significantly higher than the other treatments (p < 0.05). Meanwhile, the root N content in the CK treatment also showed a decreasing trend over the entire period; however, on day 90, the root N content in the W3 treatments was significantly higher than that in the CK treatment (p < 0.05) (Fig. 3B). The leaf P content and stem P content of the CK treatment increased in the middle stage of stress and decreased in the late stage of stress, while the change trend of root P content was opposite to that of leaf phosphorus content. On day 110, the leaf P content in the W1 and W2 treatments was significantly higher than that in the CK treatment (p < 0.05). For stem P content, the W3 treatment was significantly higher than the other treatments (p < 0.05). In terms of root P content, the W2 and W3 treatments were significantly lower than the CK treatment (p < 0.05) (Fig. 3C).

Fig. 3.

Stoichiometric alterations in C. coggygria 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 under the same treatment at different time points were analyzed using repeated measures ANOVA. Significant differences between groups at the same time point but under different treatments were analyzed using one-way ANOVA. Statistical significance was determined by a p-value < 0.05. Uppercase letters (A, B, C, etc.) indicate within-group differences, while lowercase letters (a, b, c, etc.) indicate between-group differences. 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

After 20 days of rewatering, the C content in the W3-treated leaves and W2-treated stems/roots significantly lower respectively compared with CK (p < 0.05). 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 significantly (p < 0.05) (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. 4A-C). Conversely, Car and Ant levels showed a consistent decline with prolonged drought exposure (Fig. 4D-E). Under heightened drought stress, chlorophyll a, chlorophyll b, total chlorophyll, and Car contents displayed an increasing trend in all treatment groups. The Ant content of all treatment groups increased gradually in the middle stage of stress and decreased in the late stage of stress. From day 50 to day 70, the Ant content in the W1 and W2 treatments was significantly higher than that in the CK treatment (p < 0.05), while the Ant content in the W3 treatment was inhibited.

Fig. 4.

Effect of drought stress and subsequent rewatering on pigment content of C. coggygria. A Chla, chlorophyll a; B Chlb, chlorophyll b; C Chl, chlorophyl; D Car, carotenoids; E Ant, anthocyanidin. Significant differences within groups under the same treatment at different time points were analyzed using repeated measures ANOVA. Significant differences between groups at the same time point but under different treatments were analyzed using one-way ANOVA. Statistical significance was determined by a p-value < 0.05. Uppercase letters (A, B, C, etc.) indicate within-group differences, while lowercase letters (a, b, c, etc.) indicate between-group differences. 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

On the 20th day of rewatering, the levels of Chl a, Chl b, and Chl exhibited a progressive decline in all treatment groups (Fig. 4A-C). The Car concentration did not vary significantly between groups W1 and W2 relative to the CK group (p>0.05). Whereas the Car content in the W3 treatment, as well as the Ant content in all treatment groups, were significantly higher than those in the CK (p < 0.05) (Fig. 4E).

Effects of drought stress and subsequent rewatering on ROS levels and antioxidant mechanisms of C. coggygria

The concentration of H₂O₂ 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).

Fig. 5.

Effect of drought stress and subsequent rewatering on ROS levels and antioxidant systems. A H₂O₂, hydrogen peroxide; B ·OH, hydroxyl radical; C MDA, malondialdehyde; D SOD, superoxide dismutase; E POD, peroxidase; F CAT, catalase; G GSH, glutathione. Significant differences within groups under the same treatment at different time points were analyzed using repeated measures ANOVA. Significant differences between groups at the same time point but under different treatments were analyzed using one-way ANOVA. Statistical significance was determined by a p-value < 0.05. Uppercase letters (A, B, C, etc.) indicate within-group differences, while lowercase letters (a, b, c, etc.) indicate between-group differences. 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

In the middle stage of stress, the ·OH content peaked in all treatment groups except W1 (Fig. 5A). The H₂O₂ content was significantly lower in all groups compared to CK at 30 days, while it was significantly higher at 50–90 days (p < 0.05) (Fig. 5B). Among them, in the early and middle stages of stress, the MDA content gradually decreased with the decrease of soil moisture content, which was significantly lower than that of CK in all treatment groups (p < 0.05). At the late stage of stress, MDA content increased sequentially with increasing drought (Fig. 5C). The activities of SOD, CAT, and POD showed inconsistent changes across different treatment groups with varying stress intensities and different periods. SOD enzyme activity was significantly higher in W2 at 30 days, and lower in W3 at later stages of stress compared to CK (p < 0.05) (Fig. 5D). POD activities were significantly higher in W1 and W2 compared to CK at different stress durations (Fig. 5E), while CAT activities were significantly higher in W1 and W2 at specific time points (p < 0.05) (Fig. 5F). GSH content showed a gradual increase with prolonged drought (Fig. 5G).

Following rewatering, the H₂O₂ content increased significantly in W1 and W2 compared to CK (p < 0.05), 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 (p>0.05). SOD enzyme activity was not fully recovered compared with 110d of stress, W2 treatment group was significantly higher than that of CK (p < 0.05), 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 significantly lower in the W3 treatment group than in CK (p < 0.05). CAT enzyme activity did not change significantly in the W2 and W3 treatment groups compared to 110 d of stress (p>0.05). The MDA content was significantly lower than that of CK (p < 0.05). The GSH content reached the maximum and was significantly higher than that of CK among the treatment groups (p < 0.05) (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. In the middle and late stages of stress, the SP content showed an increasing trend. The SP content of W2 and W3 treatment groups under 70d of stress was significantly higher than that of CK; the SP content of W1 and W2 treatment groups under 90d of stress was significantly higher compared with that of CK, and that of W3 treatment group under 110d of stress was also significantly higher compared with that of CK (p < 0.05) (Fig. 6A). The degree of drought significantly increased the Pro content, which was sequentially higher and significantly higher than CK among the treatment groups (p < 0.05), with the greatest decrease in the 70th d of the drought treatment (Fig. 6B).

Fig. 6.

Effect of drought stress and subsequent rewatering on osmoregulatory substances. A SP, soluble protein; B Pro, proline. Significant differences within groups under the same treatment at different time points were analyzed using repeated measures ANOVA. Significant differences between groups at the same time point but under different treatments were analyzed using one-way ANOVA. Statistical significance was determined by a p-value < 0.05. Uppercase letters (A, B, C, etc.) indicate within-group differences, while lowercase letters (a, b, c, etc.) indicate between-group differences. 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

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. (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 40,901,478 ~ 44,714,144 raw reads were obtained from the nine cDNA libraries, and 40,627,744 ~ 44,397,794 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% to 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. 7A). There were more DEGs for drought than for rewatering, and 1,785 genes were shared between drought and rewatering (Fig. 7B), 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. 7C, D).

Fig. 7.

Analysis of DEGs in C. coggygria for drought stress and subsequent rewatering. A Distribution of gene expression across various treatments. B Venn diagram for DEGs. C-D Volcano maps of DEGs. E-F KEGG enrichment analysis of DEGs

Table 1.

Raw sequencing data and quality assessment verifications of nine leaf cDNA libraries from C. coggygria

Sample	Raw reads	Clean reads	Q20 (%)	Q30 (%)	GC content (%)	Mapped ratio (%)
CK_1	42,715,214	42,414,146	98.59	95.41	43.70	89.33
CK_2	41,996,726	41,694,568	98.65	95.59	43.62	88.97
CK_3	43,477,334	43,160,840	98.61	95.47	43.65	89.40
Wd3_1	43,536,722	43,243,182	98.67	95.67	43.53	89.51
Wd3_2	41,364,718	41,069,882	98.55	95.29	43.45	89.18
Wd3_3	43,572,584	43,270,142	98.69	95.72	43.63	89.84
Wr3_1	40,901,478	40,627,744	98.63	95.53	43.55	89.51
Wr3_2	43,547,294	43,251,580	98.63	95.52	43.63	89.83
Wr3_3	44,714,144	44,397,794	98.70	95.75	43.54	89.40

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. 7E). 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. 7F). 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. 8A). 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. 8B). There are five genes in the CTK signaling pathway, AHP (TRINITY_DN17133_c0_g1) and ARR-B gene expression was up-regulated (Fig. 8C). 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. 8D). 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. 8E). 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. 8F). The JA signaling pathway had a total of six DEGs, with MYC2 and most of the JAZ genes down-regulated (Fig. 8G). 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. 8H). 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.

Fig. 8.

Analysis of DEGs 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 DEGs in different 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 (Fig. 9A). 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. 9B). In the MAPK signaling metabolism, after rewatering, the TRIITY_DN41_c0_g1 related to H₂O₂ synthesis continued to be down-regulated (Fig. 9C). 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. 9D). 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. 9E).

Fig. 9.

Examination of genes with varying expression patterns associated with physiological indicators during drought stress and subsequent rewatering conditions. A Diagram showing the cascade of DEGs affecting physiological parameters in reaction to drought stress and subsequent rehydration, with emphasis on pivotal coding genes. B-E Heatmaps of DEGs in different gene modules

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. 10A). There were 21 families of TFs and 105 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. 10B). 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. 10C). 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 (Fig. 10D-F, and H). For the bHLH family, TRINITY_DN4346_c0_g1 is most likely the gene negatively involved in drought response (Fig. 10G).

Fig. 10.

Identification and analysis of DEGs belonging to 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 Heatmaps illustrating the DEGs encoding AP2/ERF, MYB, NAC, WRKY, bHLH, and C2C2 transcription factors

Discussion

Drought stress induces notable alterations in both the external morphology and internal structure of plants [40]. This stress condition results in substantial water depletion from leaves [41]. Numerous research studies have demonstrated that drought stress has the potential to diminish the LWP of plants, thereby exerting an influence on their growth [42, 43]. 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 [43]. Insufficient water availability leads to a deceleration in plant growth, prominently manifesting as diminished SH [44], reduced leaf area [45], heightened LT [44], and closured stomata [42]. The degree of recovery in compensatory plant responses subsequent to rehydration may be impacted by the severity and duration of preceding drought stress [46].

The decline in LWP observed in C. coggygria under stress aligns with similar findings in Triticum aestivum and Oryza sativa [47, 48]. 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 [49]. 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 [50]. 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 [51]. Other durations of drought stress did not significantly impact LT, potentially due to the brief stress period. 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 [52]. Following rehydration, the LWP, SH, GD, LT and stomatal traits of C. coggygria failed to fully recover to the levels observed in the control group. Studies on vulnerable conifers like Juniperus thurifera demonstrate similar importance of dendrometric traits in ecological adaptation under drought [53].

Drought stress triggers the generation of ROS, essential for restricting plant growth and development through the inhibition of photosynthesis and nutrient absorption [54, 55]. Insufficient elemental levels result in elevated H₂O₂ production [56]. Nutrients play an important role in regulating plant stress tolerance [57], and a decrease in soil water content affects the release and mobility of elements [58]. C, N, and P content are the major elements for plant growth and development [59], N deficiency reduces the photosynthetic activity and longevity of leaves [56]. 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 [60].

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 [61]. The higher root C content of the W3 treatment contributes to C uptake, which is an important strategy for nutrient retention under stress conditions [62]. With the intensification of drought stress, the leaf N content gradually increases, which indicates that the dissolution and transformation of N require the participation of water [63]. Similarly, microbial inoculation with Bacillus subtilis has been shown to optimize nutrient uptake and water use efficiency in cotton under arid conditions [64]. 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 [65].

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 [66]. 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 [67].

Three broad categories of plant reactions to drought include alterations in pigmentation, both qualitatively and quantitatively [68]. Chl is a key factor in plant photosynthesis and is responsible for the absorption, transfer and conversion of light energy [69]. Car can also play a role in scavenging ROS as a non-enzymatic antioxidant in addition to its role in light trapping during photosynthesis [68, 70]. Unlike in herbaceous crops, C. coggygria showed a chlorophyll rebound after prolonged drought, suggesting species-specific acclimation. 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 [70], which is in agreement with the studies of Periploca sepium [71] and Hordeum vulgare [72]. C. coggygria under drought stress is also able to reduce water evaporation by increasing Car content, releasing excess heat, and lowering leaf temperature [73]. Ant content showed an increasing trend with the intensification of stress, which is consistent with the study of Hodaei et al. [74] indicating that Ant levels rise under water deficit conditions, thereby enhancing the plants’ antioxidant capabilities. 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 Chla, Chlb, and Chl levels in C. coggygria following brief rewatering is due to nutrient deficiencies, leading to decreased Chl synthesis as resources are prioritized for growth and recovery processes.

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. [75], 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. [76], 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 [75]. 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. [77]. Under severe drought (W3), increased POD/CAT activities and GSH content suggest an upregulated antioxidant defense, whereas in moderate stress these changes were milder, consistent with previous findings.

Even after rewatering, the plant remains ineffective in scavenging the ROS that have accumulated during the period of drought stress. 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 [78].

Osmoregulation is recognized as an important physiological adaptive property associated with abiotic stresses [79]. Variations in the Pro and SP content of C. coggygria exhibited diverse patterns over the course of the study period. Elevated aridity conditions result in heightened proline content, facilitating the enhancement of antioxidant enzyme activities [80]. The SP content of each treatment group gradually accumulated in the late stage of stress, which improved the osmoregulation ability of C. coggygria [81]. 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 [82]. 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 [82, 83]. 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 [84]. ARF may bind to the transcriptional repressor Aux/IAA to inhibit IAA synthesis, thereby suppressing biomass accumulation to alleviate water deficit [85]. JAZ proteins negatively regulate JA-responsive genes [86]. During periods of drought stress, the level of JAR1 gene expression, responsible for JA signaling, decreased notably, while the expression of COI1, which plays a role in regulating stomatal movement, increased [81]. BR signaling regulatory positive and negative factors activate SnRK2s to control the initiation and amplification of ABA signaling [87]. GA signaling regulates resistance by controlling cellular redox homeostasis, and increased DELLA activity interferes with ABA signaling [88]. Furthermore, SA boosts plant resilience to drought by bolstering antioxidant defenses, facilitating osmotic adjustment, improving water utilization efficiency, and enhancing photosynthetic activity [89]. 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 modules 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 [24]. Water scarcity triggers the expression of phytohormone-related genes in C. coggygria, leading to their interaction and activation of an intricate phytohormone regulatory network. This comprehensive network enhances the plant’s ability to adapt and survive in drought conditions. Subsequent to drought exposure, the majority of genes associated with Car and Ant synthesis exhibited reduced expression levels, in contrast to the observed accumulation of Car and Ant contents. This discrepancy suggests a potential scenario wherein while the transcriptional activity of Car and Ant synthesis genes decreased post-drought treatment, there was a probable increase in the translation efficiency of their mRNAs and enhancement of their catalytic activities. This phenomenon aligns with existing findings on Scutellaria baicalensis [90]. During drought stress, alterations were observed in DEGs linked to Pro content, H₂O₂ levels, and POD activity, which corresponded with fluctuations in Pro content, H₂O₂ levels, and POD enzyme activity. Similar regulatory interactions between PTEN/Akt/mTOR signaling and oxidative stress responses have been noted in mammalian systems, highlighting the conserved role of redox pathways across kingdoms [91].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 [92]. 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 [24, 93, 94]. This aligns with previous evidence where MYB transcription factors, such as PgMYB2 in Panax ginseng, positively regulated drought-associated metabolic pathways [95]. GmWRKY54 directly binds to the promoters of PYL8, SRK2A, CIPK11, and CPK3 and activates their expression, thereby improving drought tolerance in Glycine max [96]. In poplar, the bHLH family gene PebHLH35 responds to drought stress by positively regulating stomatal density, stomatal aperture and photosynthesis [97]. 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 [98]. Consistent with our findings, wheat HSP17.4 has been shown to enhance plant stress tolerance by interacting with TaHOP and stabilizing protein folding under stress [75]. 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 [24]. 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. Recent genome-wide analyses of WUSCHEL-related homeobox genes in ramie also suggest their role in root development and stress adaptation, consistent with TF-driven regulation observed here [99].

To our knowledge, this is the first integrative study of C. coggygria under prolonged drought and rehydration. We identified key phytohormone-related DEGs and TFs not previously reported in this species. Nevertheless, certain limitations exist: (1) Transcriptome data were solely obtained at the conclusion of the drought period and post-rehydration, thus failing to capture the dynamic gene expression changes throughout the stress progression; (2) Sole focus was placed on leaves, overlooking potential root responses; (3) The experimental setup utilizing potted plants under sheltered conditions may not fully replicate field settings. Furthermore, the DEGs highlighted in the transcriptome were not subjected to validation through qRT-PCR. Subsequent research endeavors should address the aforementioned shortcomings.

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. The stress responses in C. coggygria lead to an elevation in water retention characteristics, while inhibiting the growth of the plant. The antioxidant compounds in C. coggygria synergistically regulate the equilibrium between ROS production and scavenging within the plant. 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. This study provides the first comprehensive view of how C. coggygria coordinates physiological changes with transcriptomic adjustments under extreme drought and recovery. In particular, we identify candidate drought-responsive genes (e.g. specific TFs) for future functional studies.

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

H₂O₂

Hydrogen peroxide

·OH

Hydroxyl radical

MDA

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

Authors’ contributions

The manuscript was written by S.M. and X.L.; the review comments have been revised under guidance by P.Z.; 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).

Data availability

The datasets generated and analysed during the current study are available in the NCBI SRA with the accession number PRJNA1315001.

Declarations

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 interests

The authors declare no competing interests.