The study of physiological response mechanism and metabolomics on B. chinensis under drought

The study of physiological response mechanism and metabolomics on B. chinensis under drought | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The study of physiological response mechanism and metabolomics on B. chinensis under drought Yan Xiong, Mingjing Li, Xiaoyu Zhang, Xue Lei, Shuchang Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6961345/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Oct, 2025 Read the published version in BMC Plant Biology → Version 1 posted 10 You are reading this latest preprint version Abstract Background Drought has become an increasingly important environmental problem worldwide and is one of the major abiotic stress factors affecting crop growth and development. Therefore, elucidating the mechanism of drought resistance in plants is of great significance to improve the drought resistance of plants and to cultivate drought-resistant varieties. B. chinensis is an important medicinal plant and garden plant in China, and drought is one of the factors limiting the growth of B. chinensis , so it has become a research hotspot to investigate the physiological response mechanism of B. chinensis to drought. Results In this study, we investigated the physiological and biochemical response mechanisms of the root system of B. chinensis using B. chinensis seedlings as experimental materials, and conducted metabolomics analysis of the root system of B. chinensis using liquid chromatography-mass spectrometry (LC-MS) to verify the mechanisms of B. chinensis in response to drought stress after 0, 7, 14, 21, 28 d of drought and after re-watering.The results showed that: (1) with the prolongation of drought stress, the relative water content gradually decreased, the diameter of root, the diameter of the mid-column, the thickness of the cortex, and the diameter of the xylem all showed an upward trend and then a downward trend, the root activity gradually declined, the root structure was impaired, the cortex was not arranged and ruptured. (2) With the prolongation of drought stress, the cell membrane function was impaired, the relative conductivity and malondialdehyde content of roots tended to increase, the content of osmoregulatory substances such as soluble proteins and free proline also accumulated in large quantities in the roots, and the content of hydrogen peroxide and superoxide anion gradually increased. Meanwhile, the activities of peroxidase, catalase, glutathione reductase, ascorbate peroxidase and monodehydroascorbate reductase in the roots reached the maximum value after 28 d of drought, and all of them recovered to a different extent after re-watering. (3) A total of 656 differential metabolites were screened in the root system of B. chinensis under drought stress. In addition, KEGG enrichment analysis revealed that the metabolic pathways involved in the differential metabolites were mainly glycerophospholipid metabolism, sphingolipid metabolism, amino acid metabolism, tyrosine metabolism, arachidonic acid metabolism, flavonoid biosynthesis, steroid biosynthesis and tyrosine metabolism. Among them, the differences in flavonoid biosynthesis varied greatly, suggesting that flavonoids play an important role in the response to drought stress in B. chinensis . (4) The weighted gene co-expression network analysis WGCNA revealed high correlations between the three metabolite modules and root growth parameters and physiological and biochemical index parameters. Among them, the Meblue module was positively correlated with root activity, the Megreen module was positively correlated with catalase, and the Meturquoise module was positively correlated with malondialdehyde, catalase, soluble proteins, and relative conductivity. Conclusions This study reveals the defense response mechanism of B. chinensis under drought, and also analyzes the differential metabolites of B. chinensis under drought stress according to the changes in the quantity of related metabolites and explores the key metabolic pathways related to drought resistance, with a view to laying a theoretical foundation for physiological and metabolic studies revealing B. chinensis response to drought stress. B. chinensis Drought stress Physiology Response mechanism Metabolome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Introduction Plants growth and development are affected by various biotic and abiotic stresses. Among them, abiotic stresses have a particularly effect on plant growth. Accordingly, plants are vulnerable to the threat of abiotic stresses such as drought, flooding, high temperature, low temperature, salinity, light, chemical and mechanical stresses. Among them, drought is a serious and complex abiotic stress, which can have a negative impact on plant growth and crop yield [ 1 ]. In general, the root system is an important organ for plants to extract soil water and nutrients, and is also one of the key organs for material and energy exchange with the external environment [ 2 ]. When the plant root system is affected by drought stress, it will trigger a series of unfavorable effects such as impeded water uptake, cell structure and function damage, physiological and metabolic disorders, etc., which will ultimately lead to a decline in crop yields [ 3 , 4 ]. However, some response mechanism of plant to drought stress is not clear. Plants have developed a multifaceted network to withstand stressful environments [ 5 ]. Generally, when the root system senses the effects of environmental stress, chemical signals are generated and transported to the soil, which in turn cause a series of growth and physiological and biochemical responses of plants [ 6 ]. During drought stress, unsaturated fatty acids in the cell membrane are affected by free radicals leading to lipid peroxidation, while malondialdehyde content and relative conductivity also increase [ 7 , 8 ]. Drought also leads to loss of cell expansion pressure and dehydration, so the accumulation of osmolytes is crucial for osmoprotection and osmoregulation, and plants accumulate large amounts of osmoregulatory substances to maintain normal physiological metabolism in the cell, prevent cell dehydration, and alleviate the damage of drought to the plant to a certain extent [ 9 ]. In addition, when plants are subjected to drought stress, a large amount of reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ), superoxide anion radicals (O2 ·- ), and hydroxyl radicals (-OH), etc [ 10 ]. In general, excessive accumulation of ROS can cause oxidative damage to proteins, nucleic acids, and lipids, and ultimately lead to plant cell death [ 11 ]. At the same time, plants have sufficient antioxidant enzymes and non-enzymatic antioxidants to reduce excessive ROS accumulation in different organelles and reduce oxidative damage under drought conditions. In order to maintain a balance between ROS production and scavenging, plants have also evolved two different biological processes to cope with ROS, as well as to scavenge ROS through enzymatic and non-enzymatic processes [ 12 ]. Therefore, to cope drought stress, plants have evolved complex system. Plants response to adversity stress is a complex biological process, the most important of which is the alteration of metabolites. During drought stress, plants can maintain homeostasis by regulating a complex regulatory network consisting of genes-proteins-metabolites-phenotypes to achieve defense against drought stress, and thus metabolites can reflect the physiological state of plants during growth [ 13 ]. Under drought stress, the content of metabolites such as amino acids, flavonoids and phenolics increased significantly in plants [ 14 ]. Among them, metabolites such as flavonoids and phenolamines are widely involved in plant growth and development, physiological processes and plant stress response [ 15 , 16 ]. In addition, sugars such as mannitol, sorbitol, sucrose, fructans, and amino acid metabolites such as proline act as osmotic factors in plants to resist drought. There are also many small molecules such as anthocyanins, carotenoids and glutathione metabolites that can protect the plants from the damage caused by drought stress by scavenging excess ROS [ 17 ]. Therefore, metabolites are the basic substances that constitute the life activities and are the concentration of multiple responses. In recent years, metabolomics has been widely used to study drought tolerance in plants with different drought tolerance ability. Therefore, some metabolites under drought stress can be identified, screened and analyzed by metabolomics to reveal the mechanism of drought tolerance in plants [ 18 ]. Belamcanda chinensis L. is a perennial herb belonging to the genus Belamcanda in the family Iridaceae, and is a cash crop with high medicinal and ornamental value [ 19 ]. Its rhizomes have important roles in antioxidant, anti-inflammatory and anti-tumor properties [ 20 ]. In addition, its drought-resistant characteristics make it widely used in parks and landscapes. However, the current study only illustrates the drought resistance of B. chinensis under drought stress, but there is a lack of clarity about the mechanism of drought resistance. Therefore, it is important to understand the drought tolerance mechanism of B. chinensis to safeguard its yield and quality. In this study, we analyzed the growth morphology and physiological and biochemical indexes of B. chinensis under drought stress. At the same time, we also analyzed the differential metabolites of B. chinensis under drought stress using metabolomics to provied a theoretical foundation for the elucidation of physiological and metabolic mechanism of B. chinensis in response to drought stress. Materials and methods Plant materials and growing contitions The B. chinensis L. seedlings used in this study were provided by Institute of Natural Resources and Ecology, Heilongjiang Academy of Sciences (HAS). Two-year old B. chinensis L. seedlings with uniform growth were selected and planted in plastic nutrient pots (soil: perlite = 2:1). All the seedlings were divided into two groups including to the control and the treatment, and watered to reach 70%-80% of the soil water content before drought treatment. For the control group, the seedlings were fully irrigated every 7 d to saturate the soil and for the treatment group, the seedlings were continuously irrigated for 28 d, and re-watering was carried out on the 28 th day. The morphology was observed and photographed on the 0 d, 7 d, 14 d, 21 d, 28 d and after re-watering. Additionally, the seedlings roots were also selected in liquid nitrogen and stored in a refrigerator at -80℃ for further use. Determination of roots phenotype and related physiological indexes Determination of basic morphological parameters Relative water content was determined by drying method of Lang [21]. Root activity was determined by triphenyltetrazolium chloride (TTC) method of Ruf [22]. To observe root morphology, the roots were separated from the soil, the soil was carefully removed to avoid root damage and then the roots were scanned using a desktop scanner (Wanshen LA-S series plant image root analysis system software) . The observation of scanning electron microscopy was referred to the method of Shi [23]. Determination of the degree of damage to the membrane system and osmotic regulatory substances The conductivity was determined by the conductivity meter method of Rhoades [24]. Malondialdehyde (MDA) content was determined by TBA method of Mao [25]. Soluble proteins were determined by the G-250 method using the Thomas Blue method of Behnamnia [26]. Proline (Pro) was determined by the G-250 method using the Thomas Blue method of Behnamnia [26]. Determination of reactive oxygen (ROS) content Superoxide anion (O 2 ·- ) was determined by hydroxylamine oxidation Kono [27]. Hydrogen peroxide (H 2 O 2 ) was determined by the KI method of Wang [28]. Histochemical staining of O 2 ·- and H 2 O 2 was carried out by 3,3-diaminobenzidine (DAB) and nitinol Blue Tetrazolium (NBT), respectivly. Determination of Antioxidant enzyme activity The peroxidase (POD) is determined by the method of Madamanchi [29]. Determination of Catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) were used the method of Deng [30]. The glutathione reductase (GR) activity is analyzed using the method of Behnamnia [26]. Monodehydroascorbate reductase (MDHAR) was determined by using the G0213F kit (Suzhou Grace Biotechnology Co.). Determination of antioxidant content The root samples were thoroughly ground. Then 3 mL of 5% sulfosalicylic acid solution was added and centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was used for the determination of antioxidant content. Then, the contents of reduced glutathione (GSH) content and oxidized glutathione (GSSG) were determinated according to the method of Deng [30]. Metabolomics analysis The samples were sent to Shanghai Boyun Biotechnology Co. for the extraction of sample metabolites. The supernatant was analyzed by liquid chromatography-tandem time-of-flight mass spectrometry UPLC-Q-TOF/MS. In addition, quality control (QC) samples were prepared by mixing all core samples. During instrumental analysis, a QC sample was inserted next to each of the six test samples to check the reproducibility of the analytical process. The samples were sent to Shanghai Boyun Biotechnology Co., Ltd. for the metabolomics of B. chinensis roots, and the raw data were integrated, corrected, peak-aligned, and normalized by the metabolomics processing software progenesis QI (Waters Corporation, Milford, USA), and the corresponding metabolite content was expressed as the chromatographic peak-area integral to ultimately obtain a data matrix. Statistical analysis All data are shown using the mean ± standard deviation (SD) and were analyzed using SPSS 25 statistical software (Chicago, IL, USA). Differences among groups were compared using (One-way analysis of variance, ANOVA) followed by Duncan's new multiple range test. P -values < 0.05 were considered significant. GraphPad Prism 8.0.2 software were used for graphing figures. Results Effects of drought stress on phenotypic characterization Effects of drought stress on morphological parameters of B. chinensis Compared with the control, the number of lateral roots at 28 d of drought stress increased significantly and became thinner, the degree of lignification increased after drought treatment, and the color gradually deepened from white to yellow-brown, and the morphology and color of the root system recovered after re-watering. Meanwhile, the root water content showed a decreasing trend with the prolongation of the stress time, which was the lowest at 28 d of stress, 66.22%, and lower than that of the control group. The root water content recovered after re-watering (Fig. 1). Influence of drought stress on growth parameters of B. chinensis The root fresh weight and root dry weight of the control group gradually increased, the root fresh weight of the treatment group showed a trend of increasing and then slowly decreasing with the extension of drought stress, and the root fresh weight recovered after re-watering, but still lower than that of the control group (Table 1). The root dry weight of the treatment group showed a trend of increasing and then decreasing, and after re-watering, the root dry weight recovered, but did not recover to the level of the control group. Table 1 Changes of biomass in B. chinensis under drought Index Treatment days 0 7 14 21 28 35 Root fresh weight (g) Control 3.4±0.1de 4.2±0.2cd 4.8±0.3bc 5.2±0.4b 5.3±0.5b 6.1±0.5a Treatment 3.8±0.8d 3.6±0.1de 4.2±0.7cd 2.9±0.5ef 2.2±0.6f 2.7±0.7ef Root dry weight (g) Control 0.6±0.1g 0.8±0.1ef 0.9±0.1de 1.0±0.1c 1.2±0.1b 1.6±0.1a Treatment 0.6±0.1g 0.9±0.1def 0.9±0.1cd 0.8±0.1ef 0.8±0.1f 0.8±0.1f Data are Mean ± SE, different letters indicate significant differences ( P < 0.05). Effect of drought stress on root vigor of B. chinensis The trend of root activity of the control group from 7 to 35 d was decreasing-rising-decreasing (Fig. 2). Under drought stress, the root activity of the shotguns decreased significantly, and the longer the duration of the stress, the lower the root activity was, and the difference was significant compared with that of the control group at 28 d. The root activity increased after the re-watering, but it was still lower than that of the control group. Effects of drought stress on the structure of the root system of B. chinensis The epidermal cells of the control root system were intact , and the thin-walled cells of the cortex were regular in shape and small in size, and closely and neatly arranged (Fig. 3). In the treatment group, there was no significant difference in the anatomical structure of the root system compared with the control at 7 d and 14 d of drought stress, and both grew well. At 21 d, the root system showed slight drought damage, and the thin-walled cells of the root cortex were more tightly packed and irregularly arranged, with larger cellular gaps, and at 28 d, the damage to the root system structure was more serious, with cortical breakage and other traits. The number of root conduits was less affected by drought stress at 0 d-7 d, the number of conduits did not change significantly, and the shape of conduits was fuller, at 14 d the number of conduits in each group increased faster, the conduits became thinner and increased, but the shape remained more intact, and with the prolongation of the time of drought stress, the conduits continued to become thinner and increase in number, and the shape was distorted, and the mid-columns appeared to be broken and shriveled deformed, and the order of the mid-columns were also broken. After re-watering, there was some recovery of root cortex breaks and some recovery of conduit and mesocolon morphology. Diameter of root, diameter of the xylem, root cross-sectional area and diameter of the mid-column of the control group increased to a certain extent, the thickness of cortex was not significantly thickened, the mid-column structure was intact, and the morphology of the conduit was relatively full (Table 2). The diameter of root of the treatment group showed a trend of increasing and then decreasing, and showed a downward trend at 14-28 d, with a significant difference compared with that of the control. The diameter of the mid-column showed an upward and then downward trend, and the diameter of the mid-column was 2,921.40 μm at 28 d of drought stress. The diameter of the mid-column was 2921.40 μm at 28 d of drought stress, which was significantly different from the control, but still lower than the control, the thickness of the cortex showed a trend of increasing and then decreasing under drought stress, which was increasing at 0-21 d and rapidly increasing at 7 d, and then decreasing at 21-28 d, which was 3346.47 μm at 28 d, which was significantly different from the control. The diameter of the xylem showed an increasing and then decreasing trend, gradually decreasing at 7 d and reaching a minimum value of 484.84 μm at 28 d, root cross-sectional area and mid-column area showed an increasing and then decreasing trend with the extension of drought stress, and the root cross-sectional area and mid-column area were 70.38×10 6 μm 2 and 26.82×10 6 μm 2 , respectively, at the time of 28 d of drought stress, and the differences were significant when compared with that of the control. All anatomical parameters recovered after re-watering, but not to control levels. Table 2 Changes of structural parameters of stem root system under drought Index Treatment days 0 7 14 21 28 35 Diameter of Root (μm) Control 3918.0±25.6h 4110.0±49.9g 4634.8±45.2f 5140.3±18.6c 6275.3±22.9b 6576.1±26.9a Treatment 3958.9±30.4h 4138.2±14.0g 5030.2±29.6d 4813.2±29.6e 4666.6±49.9e 5013.3±79.9d Diameter of the mid-column(μm) Control 2119.2±9.8h 2351.3±13.9g 2938.9±24.4d 3011.9±25.9c 3237.2±30.0ab 3278.7±19.9a Treatment 2116.3±26.1 h 2356.5±17.3f 2882.2±47.9e 2990.5±24.9c 2921.4±21.5de 3209.9±27.5b Thickness of cortex (μm) Control 3138.5±13.4g 3147.9±29.5g 3625.8±24.8e 3960.9±28.9c 5138.4±14.9a 5197.9±34.1a Treatment 3168.5±33.9g 3196.5±33.4fg 3609.1±11.2d 3658.7±29.3d 3346.5±24.2ef 4338.4±30.5b Diameter of the xylem (μm) Control 604.9±14.4d 611.7±10.7cd 626.3±15.4bc 620.6±6.1cd 640.1±3.4ab 650.9±6.9a Treatment 605.8±3.2d 619.3±3.2cd 561.2±10.2e 549.9±7.3ef 484.8±10.9g 539.4±7.9ef Root cross-sectional area (10 6 μm 2 ) Control 48.1±0.1i 53.1±1.3h 67.5±1.3f 82.9±0.6c 123.4±0.7b 135.6±1.1a Treatment 52.2±1.5h 61.9±0.4g 79.5±0.9d 72.8±2.4e 70.4±3.9ef 78.9±2.5d Mid-column area (10 6 μm 2 ) Control 14.5±0.6h 16.9±0.9g 27.1±0.4cde 28.5±0.5c 32.3±1.7a 33.5±1.5a Treatment 13.8±0.6h 19.5±0.8f 26.1±0.9e 28.1±0.5cd 26.8±0.4de 30.6±0.5b Data are Mean ± SE, different letters indicate significant differences ( P < 0.05). Effects of drought stress on physiological and biochemical indexes in roots of B. chinensis Effect of drought stress on cell membrane permeability The relative conductivity of roots did not change significantly in the control group, and under drought stress, the relative conductivity of roots showed an increasing trend and was significantly higher than that of the control at 28 d of drought stress, and recovered after re-watering (Fig. 4). The changes of MDA content of root in the control group were basically stable, with the extension of drought stress time, the MDA content of root in the treatment group gradually increased, and the MDA content of root was significantly higher than that of the control group at 21 d. After re-watering, the MDA content of root decreased to the control level. Effects of drought stress on osmoregulators The trend of soluble protein (Sp) content in the root in the control group was first increasing and then decreasing (Fig. 5).With the prolongation of drought stress, soluble protein (Sp) content in the treatment group gradually increased and was significantly higher than that of the control at 28 d of stress, and after re-watering, soluble protein (Sp) content in recovered, but did not return to the control level. Proline content is altered in plants under drought stress. The Proline content of the control group did not change significantly, and the Proline content of roots in the treatment group was higher than that of the control group at 28 d. The Proline content of roots decreased after re-watering. Effect of drought stress on reactive oxygen species content There was no significant change in the H 2 O 2 content of roots under normal conditions, and the H 2 O 2 content of roots showed an increasing trend with the prolongation of drought stress, and it was significantly higher than that of the control group at 28 d (Fig. 6). At the same time, the H 2 O 2 content produced by the root system reached the maximum at 28 d of drought stress, and after re-watering, the H 2 O 2 content was restored to the level of the control. The O 2 ·- production rate of the root system did not change significantly under normal conditions. With the prolongation of drought stress, the O 2 ·- production rate of the root system showed an increasing trend, which was significantly higher than that of the control group at 28 d, and the production rate reached the maximum value, which could be recovered to a certain extent after re-watering. Effects of drought stress on reactive oxygen species staining profiles In order to detect the reactive oxygen content more intuitively, DAB and NBT staining were used to detect the changes of H 2 O 2 and O 2 ·- content under different days of drought stress in B. chinensis , respectively.The reaction between H 2 O 2 and DAB can produce brown precipitate, and the degree of staining can indicate the accumulation of hydrogen peroxide. The DAB staining of the roots in the treatment group did not change significantly at 0-14 d compared with the control, indicating that the H 2 O 2 content accumulated less (Fig. 7). the staining area of the roots increased and the color became darker at 21 d, and the staining area of the roots was large and the staining was aggravated at 28 d, which indicated that the highest amount of H 2 O 2 was produced at this time. The reaction of O 2 ·- with NBT can form a blue polymeric precipitate, which can indicate the accumulation of superoxide under stress. The NBT staining of the root system of the treatment group was light blue at 7 d, indicating that the O 2 ·- content accumulated less. the staining area of the root system increased and deepened from 14 d to 21 d, and then increased and deepened at 28 d. The NBT staining of the root system of the treatment group was light blue at 7 d, indicating that the O 2 ·- content accumulated less. The degree of DAB and NBT staining in the root system recovered after re-watering. Effect of drought stress on antioxidant enzymes The SOD activity of roots in the control group basically remained unchanged before 28 d, and the activity increased at 28-35 d (Fig. 8). The SOD activity of roots in the treatment group showed an increasing trend at 7-21 d of stress, and then showed a decreasing trend with the prolongation of the stress time, and the SOD activity reached the maximum value at 21 d of stress, which was significantly different from that of the control. In the control group, the POD activity of roots basically remained unchanged, in the treatment group, the POD activity of roots showed an increasing trend with the prolongation of drought stress, and reached the maximum value at 28 d, with a significant difference compared with the control. In the control group, the CAT activity of roots showed a decreasing-rising-decreasing trend, whereas in the treated group, the CAT activity of root basically remained unchanged from 0 to 14 d, and then increased with the prolongation of the stress time, and reached the maximum value at 28 d, with a significant difference compared with that of the control. The GR activity of roots in the control group basically remained unchanged, and the GR activity of roots under drought stress showed an increasing trend. The APX activity of roots in the control group showed a decreasing and then increasing trend, and the APX activity of roots in the treatment group gradually increased with the prolongation of the drought stress time, there was no significant change in the MDHAR activity of roots in the control group, and it gradually increased with the prolongation of the drought stress time. The enzyme activities at 28 d of drought stress were all significantly higher than the control ( P < 0.05), and the POD, CAT, GR , APX and MDHAR activities of roots increased 1.8, 2.4, 4.2, 2.0 and 6.4 times, respectively, compared with the control, and the POD, CAT, GR, APX and MDHAR activities of roots decreased significantly after re-watering. The POD, CAT and GR activities of roots recovered but not to the control level, and the SOD, APX and MDHAR activities of roots recovered to the control level. Effect of drought stress on antioxidants GR catalyzes the reduction of GSSG to GSH, thereby maintaining GSH levels and the GSH/GSSG ratio. The GSH, GSSG and GSH+GSSG content of roots in the control group remained basically unchanged, while the GSH, GSSG and GSH+GSSG content of roots in the treatment group showed an increasing trend, and the GSH, GSSG and GSH+GSSG content of roots in the treatment group reached the maximum at 28 d. GSH/GSSG in the root system of the control group showed an increasing and then decreasing trend from 0 to 21 d, and the change was not obvious from 21 to 35 d. The trend of GSH/GSSG in the root system of the treatment group basically remained unchanged, and the root system GSH/GSSG were lower than that of the control group. After re-watering, the GSH content of roots recovered to the control level, and both the GSSG content and GSH+GSSG recovered, but not to the control level, and the GSH/GSSG of the root system recovered to the control level (Fig. 9). Metabolomics analysis Principal component analysis of the sample The two principal components PC1 and PC2 explained 29.3% and 13.1% of the variability in the control group, and the two principal components PC1 and PC2 explained 21.9% and 13.7% of the variability in the treatment group, and the six biological replicates of each sample could be clustered well, indicating that the differences between core samples within the group were small, and the experiment was reproducible and reliable. In addition, there was a clear trend of separation of core samples between the drought treatments, indicating significant metabolic differences in the drought treatments (Fig. 10). Differential metabolite screening and identification The P -value of the t-test combined with the variable importance projection (VIP) value of the OPLS-DA model was adopted to screen for differential metabolites between the different groups, and the criteria for screening were VIP > 1.0 and significance at P < 0.05. Screening for differential metabolites With 58 significantly different metabolites between C7 and T7, 150 significantly different metabolites between C14 and T14, 238 significantly different metabolites between C21 and T21, 272 significantly different metabolites between C28 and T28, and 172 significantly different metabolites between C35 and T35 (Fig. 11). Thus, it can be found that the number of differential metabolites increased with the increase of drought stress time. In addition, the differential metabolites in five groups, C7-vs-T7, C14-vs-T14, C21-vs-T21, C28-vs-T28, and C35-vs-T35, were categorized into 13 classes, mainly consisting of 172 flavonoids, 48 fatty acyls, 39 steroids and steroid derivatives, 29 glycerophospholipids, and 21 Benzene and substituted derivatives, 23 Prenol lipids, 20 carboxylic acids and derivatives, 18 organoxygen compounds, 6 guanosine monophosphate, 6 coumarins and derivatives, 3 phenols, 2 phenylpropanoic acids and 274 other compounds. In order to determine the correlation between different metabolites and drought stress at different times, the analysis of the amount of change was carried out. Differential metabolites were clustered by means, and the results are shown in FigS 1. Differential metabolites were classified into 16 different clusters (C1-C16), in which the metabolites in the treatment groups C2, C3, C4, C9, C10, C12, C15, and C16 showed a decreasing and then increasing trend, and their contents recovered after re-watering, while the contents of the metabolites in C11 and C14 showed an increasing trend in drought stress for 21 d. The metabolites in C1 and C13 showed a continuous decreasing trend, while the contents of the metabolites in C1 and C13 showed a recovery after re-watering. The metabolites in C11 and C14 showed an increasing trend at 21 d of drought stress, while the metabolites in C1 and C13 showed a continuous decreasing trend and recovered after re-watering. Changes in differential metabolite content Changes of flavonoids content The contents of metabolites such as naringenin, lignocerosin 5-galactoside and rutin increased with the extension of drought stress and recovered after re-watering, whereas the contents of flavonoids such as apigenin, mangostin and naringenin 4-O-glucosidic acid showed a decreasing tendency under drought stress and still did not recover after re-watering. Another part of flavonoids such as apigenin 7-lactic acid, gallocatechin, naringenin 3-O-glucosiduronic acid, etc. showed significant increase in content at 14 and 21 d of drought, but showed a decreasing trend at 28 d of drought, and did not recover after re-watering. The contents of kaempferol, lignocerotoxin, hesperidin, quercetin, apigenin 7-xyloside, naringenin-5-O-glucosiduronic acid, lignocerotoxin 5-galactoside, and hesperidin 7-O-glucosiduronic acid did not change significantly at the early stage of the drought stress, and showed an increasing trend with the prolongation of the drought stress time, and then declined after re-watering (Fig. 12). Changes of content on amino acid compounds The contents of threonylvaline, glutamyltryptophan, isoleucine, phenylalanine and tryptophan increased at 28 d of drought stress and decreased after re-watering (Fig. 13). In addition, phenylalanine, as a precursor substance for the synthesis of phenolic compounds, was associated with changes in the content of its downstream compounds. The content of phenylalanine increased after drought stress and was always higher than that of the control, and the content of phenylalanine was lower than that of the control after re-watering. The contents of histidine, phenylalanyl-alanine, histidinol, alanine tryptophan, asparagine, aspartic acid and valine were the highest at 14 d of drought, and decreased with the increase of drought stress time. Changes of endogenous hormone content The ABA content of the control group did not change significantly from 0 to 35 d, and the ABA content of the control group was maintained at a low level, while the ABA content of the treatment group basically remained unchanged at the beginning of the drought stress, but with the prolongation of the drought stress time, especially at 28 d of the drought stress, the ABA content of the roots was significantly higher than that of the control, and the ABA content of the roots significantly declined after the re-watering (Fig. 15). The JA content of the control group remained basically unchanged from 0-28 d and showed an increasing trend at 35 d. In the treatment group, at 0-14 d of drought stress, the JA content of the root system was significantly not different from that of the control group, and with the extension of the stress time, the JA content in the root system was significantly higher than that of the control at 28 d and differed significantly from that of the control, and the JA content of the root system decreased after re-watering and was significantly lower than that of the control. The content of salicylic acid was higher than that of abscisic acid and jasmonic acid under drought stress. The SA content of control roots gradually increased from 0-35 d. The SA content of treated roots showed an increasing trend until 21 d, and was significantly higher than that of control at 21 d of stress, whereas the SA content decreased from 21 to 28 d of stress, and there was no significant difference between the SA content of roots and that of control at 28 d of stress, and the SA content of roots recovered after re-watering. Changes of other metabolite content Under drought stress, stearic acid, linolenic acid and arachidonic acid content were all in a decreasing trend and significantly lower than the control, arachidonic acid content content of the control group did not change significantly, with the extension of drought stress time, arachidonic acid content showed a trend of increasing and then decreasing, with the highest content in drought for 21 d, and the treatment groups were all higher than the control group, and the content of nutriacholic acid showed an increasing trend with the extension of drought stress time. The content of vitamin D 3 and phytol showed an increasing and then decreasing trend with the prolongation of drought stress, and the difference was significant at 21 d. The content of cinnamic acid showed a decreasing trend and was lower than that of the control group, while the content of adenine showed an increasing trend, and the difference of the content was more significant at 28 d, and it was higher than that of the control group (Fig. 16). Correlation analysis of metabolite content Rutin was positively correlated with the content of hesperidin with a correlation coefficient r of 0.68, and negatively correlated with the content of stearic acid with a correlation coefficient r of -0.53. PA (16:0/16:0) was negatively correlated with the content of Alpha-linolenic acid with a correlation coefficient r of -0.54. PG (12:0/15:0) was negatively correlated with PG (17:0/19:0) content, with a correlation coefficient r of -0.66, and positively correlated with arachidonic acid and stearic acid content, with correlation coefficients r of 0.53 and 0.59, respectively. Phenylalanine was positively correlated with valine content with a correlation coefficient r of 0.50. Arachidic acid was positively correlated with phytol content with a correlation coefficient of 0.77, and negatively correlated with Alpha-linolenic acid, arachidonic acid and stearic acid content with correlation coefficients r of -0.53, -0.58 and -0.59, respectively. Arachidonic acid content was negatively correlated with phytol content with a correlation coefficient of -0.72, and positively correlated with stearic acid content with a correlation coefficient r of 0.53. Quercetin content had no correlation with myristic acid content or with any of the other metabolites (Fig. 17). Metabolic pathway analysis of differential metabolites KEGG enrichment analysis In order to better explain the role of metabolites in metabolic pathways, the differential metabolites were analyzed by KEGG pathway enrichment. The metabolites were mainly involved in a total of 49 metabolic pathways, of which the key metabolic pathways were amino acid biosynthesis, arachidonic acid metabolism, flavonoid and flavonol biosynthesis, glycerophospholipid metabolism, steroid biosynthesis, tyrosine metabolism, zeatin biosynthesis, monoterpene biosynthesis, and fatty acid biosynthesis pathways. The KEGG metabolic pathway of C7-vs-T7 is dominated by the pathways of glycerophospholipid metabolism, fatty acid biosynthesis, unsaturated fatty acid biosynthesis, histidine metabolism, aminoglycan and nucleotide sugar metabolism, and biosynthesis of secondary metabolites (Fig. 18). The KEGG metabolic pathways of C14-vs-T14 are mainly cysteine and methionine metabolism, arachidonic acid metabolism, alanine, phytohormone signaling, aspartate and glutamate metabolism, amino acid metabolism, degradation of valine, leucine, and isoleucine, biosynthesis of valine, leucine, and isoleucine, pantothenic acid and coenzyme a biosynthesis, diterpene biosynthesis, and arginine biosynthesis synthesis and other pathways enriched. The KEGG metabolic pathway of C21-vs-T21 focuses on the pathways of zeatin biosynthesis, fatty acid biosynthesis, degradation of valine, leucine, and isoleucine, valine, leucine, and isoleucine biosynthesis, oleuropein steroid biosynthesis, and carotenoid biosynthesis. The KEGG metabolic pathway of C28-vs-T28 mainly focuses on the pathways of sesquiterpene and triterpene biosynthesis, fatty acid biosynthesis, flavonoid and flavonol biosynthesis, tryptophan metabolism, fatty acid degradation, sphingolipid metabolism, isoflavone biosynthesis, ether lipid metabolism, pyrimidine metabolism, terpenoid biosynthesis, and indole alkaloid biosynthesis.The KEGG pathway of C35-vs-T35 KEGG metabolic pathways were mainly focused on histidine metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, β-alanine metabolism, monoterpene biosynthesis, and carotenoid biosynthesis. Among them, KEGG metabolic pathways at 28 d of drought were more involved, mainly including more than ten metabolic pathways. Metabolic network analysis Phospho-6-glucose is converted to acetyl coenzyme A and phospho-6-lactose, respectively (Fig. 19). Acetyl coenzyme A enters fatty acid metabolism and the metabolites involved in its pathway are stearic acid, α-linolenic acid, linolenic acid, γ-linolenic acid, 17-hydroxylinolenic acid, arachidonic acid and jasmonic acid. Among them, the contents of α-linolenic acid, γ-linolenic acid and stearic acid decreased, and the contents of arachidonic acid and jasmonic acid increased significantly with the prolongation of drought stress. Phospho-6-lactose enters the phenylalanine pathway by generating pyruvate, and involved in this metabolic pathway are asparagine, aspartic acid, valine, isoleucine, proline, histidine, and serine. The isoleucine and proline contents increased with the duration of stress, and after re-watering, the contents returned to the control level, implying that proline plays a crucial role in coping with drought stress. Amino acids and their derivatives are major factors in plant response to drought stress, and specific amino acids can retard protein degradation under drought stress. The contents of asparagine, aspartic acid and valine were highest at 7 d of drought stress, and the content of histidine decreased with the duration of drought stress. The large accumulation of amino acids and derivatives regulates plant drought stress tolerance through osmotic homeostasis and maintenance of the stability of the cell membrane structure. Phospho-6-lactose is converted to phenylalanine, a precursor of many key secondary metabolic pathways that directly affect cellular osmoregulation and improve plant drought tolerance, and subsequently to flavonoid metabolism. Flavonoids are an important class of secondary metabolites in plants with protective roles in plant development and in response to biotic and abiotic stresses. We identified nine metabolites associated with flavonoid metabolism including: hesperidin, naringenin, apigenin, lignan, kaempferol, quercetin, populin, epicatechin and rutin. The contents of the nine metabolites did not change significantly under normal conditions, and at 28 d of drought stress, naringenin, apigenin, lignans, kaempferol, rutin and epicatechin contents increased, while the changes of hesperidin and populin were not significant, suggesting that flavonoids have a key role in coping with drought stress. Vitamin D, tyrosine, homovanillic acid and 3-methoxytyramine were also detected in this study. Vitamin D3, Homovanillic acid and 3-methoxytyramine content increased at 28 d under drought stress, while tyrosine and sphingosine content decreased. Weighted gene co- expression network analysis ， WGCNA Hierarchical clustering tree Weighted gene co-expression network analysis is a biological method for analyzing patterns of metabolite associations between different samples, allowing clustering of metabolites with similar expression patterns and analyzing associations between modules and specific traits and phenotypes. To further elucidate the complex regulatory network of B. chinensis in response to drought stress, total differential metabolites from the root system were used for weighted gene co-expression network analysis. A co-expression network was constructed using WGCNA to further explore the relationship between the 656 differential metabolites screened in the root system and root growth parameters and physiological and biochemical index parameters, and a total of nine metabolite modules were identified, with metabolites that did not belong to these modules indicated in gray (Fig. 20). Correlation analysis of modules with the matrix of physiological and biochemical traits To delve into the modules associated with drought stress, the metabolites characterizing the metabolite accumulation profiles contained in each module were calculated and, in addition, each co-expressed module was correlated with growth and physiological and biochemical parameters by means of Pearson's correlation coefficient. The Meblue (blue) module is the key module positively correlated with root activity, the Megreen (green) module is the module positively correlated with catalase, and the Meturquoise (turquoise) module is the key module positively correlated with malondialdehyde, catalase, soluble proteins, and relative conductivity (Fig. 21). Discussion Effect of drought stress on phenotypic parameters of the root system of B. chinensis The effects of drought stress on phytomass are mainly manifested in plant growth and development. Normally, under drought stress, the number of lateral roots in the root system of B. chinensis increased significantly and became thinner, and the color changed from white to yellow-brown, and the morphology recovered after re-watering, suggesting that the plasticity of the root system is a kind of adaptive mechanism for B. chinensis to cope with the environmental stresses. Relative water content is an important measure of the water status of all parts of the plant and a reflection of the plant's ability to hold water. In this study, with the prolongation of the drought stress time, the relative water content of roots of B. chinensis showed a decreasing trend, and after re-watering, it could be regulated by itself by different degrees of recovery, and this result was similar to the trend of the relative water content of the root system of subtropical conifers under drought stress [ 31 ]. Drought stress significantly affected the accumulation and distribution of plant biomass [ 32 ]. Shan [ 33 ] found significant changes in physiological traits and biomass allocation of Reaumuria soongorica root system under drought stress compared to control, similar results were obtained in this study. In this study, with the prolongation of drought stress, the root dry weight showed an increasing and then decreasing trend, while the dry weight of the aboveground part showed an overall increasing and then stabilizing trend. Under drought stress, the plant growth index's will also be changed accordingly, and the plant root system usually shows the corresponding morphological structure and physiological characteristics with the change of environment [ 34 ]. In this study the diameter of root, the diameter of the mid-column, the thickness of the cortex, and the diameter of the xylem all showed an upward trend and then a downward trend, the root structure was impaired, the cortex was not arranged and ruptured, which could be attributed to the fact that in the early stage of the drought, the B. chinensis enhanced the water uptake capacity by actively adjusting the root morphology (e.g., increasing the diameter of root and the cortex thickness), and that persistent drought resulted in the disorder of the osmotic regulation capacity, the cellular water loss increased, protoplasts contracted, and ultimately led to the damage of root system structure. Effects of drought stress on physiological and biochemical indexes of the root system of B. chinensis When plants are under drought stress, ROS production and removal will be out of balance. Drought leads to an increase in reactive oxygen radicals, subjecting plant cells to oxidative stress. When ROS exceed the capacity of the ROS scavenging system, it will cause the accumulation of MDA and oxidative damage, greater cell membrane permeability, and loss of intracellular substances, which will cause an increase in REC [ 35 ]. In this study, MDA content and REC in roots gradually increased under drought stress and recovered after re-watering, which was similar to the findings of Filippou [ 36 ] and Liu [ 37 ]. Plants subjected to drought stress reduce damage to themselves by increasing the levels of osmoregulators such as Sp and Pro. It was found that drought stress significantly increased the content of osmoregulatory substances in plants such as lily and Acorus calamus [ 38 ]. In this study, Sp and Pro contents in the root system of B. chinensis increased with the prolongation of drought stress, suggesting that the plant root system can protect itself from dehydration damage by accumulating large amounts of osmoregulators under drought stress. Usually, to reduce the damaging effects of ROS, plants can regulate their drought tolerance through enzymatic reactions of antioxidant enzymes and the content of antioxidants in the ascorbate-glutathione cycle (ASA-GSH). Among them, the antioxidant enzymes SOD, POD, and CAT scavenge ROS, and in general, increasing enzyme activities under drought conditions are indicative of strong drought tolerance [ 39 ]. In this study, it was found that SOD activity in the root system of B. chinensis showed an increasing and then decreasing trend with drought stress time, and both POD and CAT activities in the root system showed an increasing trend, which was similar to the performance of the results of the study by Iqbal [ 40 ], and both enzyme activities recovered after re-watering. Thus, the AsA-GSH cycle plays an important role in the antioxidant protection of plants under drought stress. In this study, the ASA-GSH cycle and the antioxidant enzyme activities that maintain the cycle homeostasis showed a gradual increase, and the results were similar to the findings of Shan [ 41 ] on drought tolerance in wheat. Effects of drought stress on metabolites in the root system of B. chinensis Under adversity stress, plants regulate the content of multiple metabolites through a complex metabolic network, thus alleviating the damage caused by adversity stress. Meanwhile, the changes of metabolites in plants can be analyzed by metabolomics as a way to understand the response mechanism of plants to adversity stress [ 42 ].In this study, a total of 656 differential metabolites were screened by LC-MS metabolomics, and the changes of metabolites under drought stress were characterized, as well as the important metabolic pathways that might be involved in the process of adapting to the arid environment. It has been shown that amino acid accumulation can regulate plant defense against drought stress through osmotic homeostasis and maintenance of cell membrane structural stability [ 43 ]. It was found that the amino acid content in the root system of wheat [ 44 ] and sorghum [ 45 ] tended to increase after environmental stresses, e.g., drought stress increased the content of several free amino acids in the root system of sorghum. In this study, isoleucine, phenylalanine and tryptophan contents within the root system of B. chinensis showed a significant increasing trend under drought stress, while histidine, valine, aspartic acid and asparagine contents all showed an increasing and then decreasing trend. This may be due to the disruption of cell membrane structure when the roots were subjected to severe drought stress, which led to an increase in amino acid content, suggesting that B. chinensis roots can maintain the osmotic balance required for root growth by increasing isoleucine, phenylalanine and tryptophan content. Flavonoids, as the most important secondary metabolites in plants, act as antioxidants in vivo and to some extent mitigate the damage caused by drought in plants [ 46 ]. In this study, flavonoids such as kaempferol, lignans, hesperidin, quercetin, lignans, naringenin, and rutin were significantly accumulated under drought stress.The results of the this study were similar to those of Rao [ 47 ] and Hodaei [ 48 ], which indicated that the antioxidant capacity of the root system of B. chinensis was increased, thereby balancing the excess free radicals produced by the drought stress. After re-watering, it recovered to the control level, showing the sensitivity of metabolites to drought stress, which reconfirmed the positive regulatory effect of flavonoids on drought resistance. However, some studies have also shown inhibition of flavonoid accumulation under severe drought stress [ 49 ]. In this study, both apigenin and mangiferin contents showed a decreasing trend with the extension of drought stress time, which might have affected some enzyme activities and thus led to the inhibition of flavonoids in the synthesis process. Plant cell membranes are the interface between the cell and the external environment directly and are more sensitive to environmental changes. Degradation of phospholipid molecules occurs when plants are subjected to drought stress, and PC, PE, and PG are the main targets of degradation [ 50 ]. The present study showed that the trends of phospholipid molecules within the root system of B. chinensis were not completely consistent under drought stress, suggesting that phospholipid molecules may respond to stress through different regulatory response pathways. Among them, the amount of PG was higher, suggesting that PG plays a major defense role in the plant root system under drought stress. In addition, abscisic acid is an essential hormone for maintaining root growth under drought conditions [ 51 ], and salicylic acid and jasmonic acid play a central role in plant defense against a wide range of adversities [ 52 ], and it has been shown that salicylic acid and jasmonic acid can reduce the level of peroxidation of plant cell membranes through the enhancement of the induced antioxidant system, removal of reactive oxygen species accumulated in cells that improves plant resistance [ 53 ]. The present study also found that abscisic acid, salicylic acid and jasmonic acid contents in the root system accumulated significantly with the duration of drought stress, suggesting that the hormone contents can be regulated to cope with the drought stress damage to plants. Relationships between metabolites and root growth parameters and physiological and biochemical indices under drought stress In recent years, WGCNAs have played an important role in plant stress tolerance research, serving as an association between co-expression modules and traits with higher reliability and biological significance [ 54 ]. Metabolites with similar expression patterns were divided into different modules by dynamic shearing, and metabolites in the same module had similar expression patterns and were functionally related to each other. In this study, a total of nine co-expression modules were constructed by screening 656 differential metabolites, among which three modules (Blue, Green, and turquoise) were significantly correlated with each phenotypic and physiological indicator traits, respectively. A total of 292 metabolites were obtained in the relevant modules (blue, green, and turquoise modules), with the highest number of compounds being flavonoids (60) and glycerophospholipids (27). Flavonoids are important secondary metabolites involved in plant growth and stress tolerance, and many studies have shown that flavonoids have biological activities including anti-allergic, anti-viral, anti-inflammatory, and vasodilatory effects, as well as other medicinal properties [ 55 ], Glycerol lipids are the most abundant lipids in plants and are involved in response and tolerance to abiotic conditions such as drought, high and low temperatures [ 56 , 57 ]. Li [ 58 ] observed that many glycerophospholipids accumulated in large quantities in hulled barley under drought stress, and another study found large accumulations of several glycerophospholipids in response to drought in ryegrass [ 59 ], suggesting that flavonoids and glycerophospholipids play important roles in drought response, and that the related metabolites may be related to the growth of the plant root system and the physiological and biochemical indicators. Although the physiological and biochemical response mechanisms and metabolomics analysis were conducted in this study on the root system of B. chinensis under drought stress, there are some limitations in this study, in which only the enzyme activities of SOD, POD and CAT and the contents of MDA, H 2 O 2 and O 2 - were measured, and the expression of key genes (antioxidant enzyme encoding genes) was not further verified Patterns. Therefore, the expression pattern of antioxidant enzyme genes such as SOD, POD, and CAT can be detected by qPCR in future studies to further explore the changes in antioxidant enzyme activities. Although the metabolite module was found to be phenotypically related in this study, no metabolism-phenotype regulatory network was constructed (e.g., how key metabolites affect root vigor or H 2 O 2 levels), and genes or enzymes (e.g., PAL, CHS) regulated by key metabolites (e.g., kaempferol) can be predicted based on the metabolism-phenotype correlations in a subsequent study. In this study, we systematically analyzed the phenotypic, physiological and metabolic characteristics of the drought response of the root system of B. chinensis. However, due to the limitations of the experimental conditions, there are some limitations, so further research on the physiological response mechanism of B. chinensis under drought stress should be conducted in the future in conjunction with multigenomics, validation of gene function, and field experiments. Conclusion In this study, two-year old B. chinensis seedlings were used as materials to investigate the effects of drought stress on their growth and metabolism through potting water-control experiments. The results showed that the diameter of root, diameter of the mid-column, thickness of cortex and diameter of the xylem of the B. chinensis under drought stress showed an increasing and then decreasing trend, while the relative water content and root activity showed a decreasing trend, and the root structure was severely damaged after 28 days, and partially recovered after re-watering. With the prolongation of stress, the relative electrical conductivity (REC), malondialdehyde (MDA), proline (Pro) and soluble protein (Sp) contents of roots increased significantly, while the activities of antioxidant enzymes were enhanced to maintain the balance of reactive oxygen species (ROS) and mitigate drought damage. Metabolomics analysis identified 656 root differential metabolites, mainly flavonoids, carboxylic acids and lipids, and KEGG enrichment showed that glycerophospholipid metabolism, amino acid metabolism and flavonoid biosynthesis pathways significantly responded to drought stress. WGCNA analysis further identified 292 key metabolites, among which flavonoids accounted for the highest proportion and were significantly correlated with stress indicators such as root activity, H₂O₂ and MDA, respectively, suggesting that flavonoids play a central role in regulating root growth and drought physiology. These findings provide a fundamental understanding of the drought stress response mechanisms in B. chinensis . Declarations Acknowledgements Not applicable. Authors' contributions HH and YQ conceived and designed the project. ML, XZ, and XL performed the experiments. ML wrote the manuscript. ML, SY and YX analyzed experimental results. ML and YX revised the manuscript. HH and YQ supervised the manuscript. All authors read and approved the manuscript. Funding This research was funded by the Talent Team Construction Platform Project of HAS (RC2023ZR01), Dual-Improvement Flying Goose Project of HAS (YZQY2023ZR01). Data availability All data generated or analyzed during this study are included in this article and its supplementary information files. All materials are available through corresponding authors upon reasonable request. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. 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Dynamic changes in membrane lipid composition of leaves of winter wheat seedlings in response to PEG-induced water stress. BMC Plant Biology 2020; 20(1): 1-15. https://doi.org/10.1186/s12870-020-2257-1. Ikegami K, Okamoto M, Seo M, Koshiba T. Activation of abscisic acid biosynthesis in the leaves of Arabidopsis thaliana in response to water deficit. Journal of Plant Research 2009; 122(2): 235-243. https://doi.org/10.1007/s10265-008-0201-9. Belt K, Huang S, Thatcher L F, Singh K B, Van Aken O, Millar A H. Salicylic acid-dependent plant stress signaling via mitochondrial succinate dehydrogenase. Plant Physiology 2017; 173(4): 2029-2040. https://doi.org/10.1104/pp.16.00060. Li M X, Guo R, Jiao Y, Jin X, Zhang H, Shi L. Comparison of salt tolerance in Soja based on metabolomics of seedling roots. Frontiers in Plant Science 2017; 8: 1011. https://doi.org/10.3389/fpls.2017.01101. Chou W C, Cheng A L, Brotto M, Chuang C Y. Visual gene-network analysis reveals the cancer geneco-expression in human endometrial cancer. BMC Genomics 2014; 15(1):1-2. https://doi.org/10.1186/1471-2164-15-300. Pietta P G. Flavonoids as antioxidants. Journal of Natural Products 2000; 63(7): 1035-1042. https://doi.org/10.1021/np9904509. Narayanan S, Prasad P V, Welti R. Alterations in wheat pollen lipidome during high day and night temperature stress. Plant, Cell&Environment 2018; 41(8):1749-1761. https://doi.org/10.1111/pce.13156. Margutti M P, Reyna M, Vilchez A C, Villasuso A L. Lipid profiling shows tissue-specific differences in barley for glycerolipid composition in response to chilling. Environmental and Experimental Botany 2019; 158: 150-160. https://doi.org/10.1016/j.envexpbot.2018.11.023. Li J, Li X, Han P, Liu H, Gong J, Zhou W, Xu L. Genome-wide investigation of bHLH genes and expression analysis under different biotic and abiotic stresses in Helianthus annuus L. 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Cite Share Download PDF Status: Published Journal Publication published 01 Oct, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 07 Aug, 2025 Reviews received at journal 06 Aug, 2025 Reviews received at journal 27 Jul, 2025 Reviewers agreed at journal 17 Jul, 2025 Reviewers agreed at journal 17 Jul, 2025 Reviewers invited by journal 17 Jul, 2025 Editor invited by journal 15 Jul, 2025 Editor assigned by journal 03 Jul, 2025 Submission checks completed at journal 02 Jul, 2025 First submitted to journal 02 Jul, 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6961345","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":487857900,"identity":"b7c62197-a67c-472c-9d70-9c7f5e08ffda","order_by":0,"name":"Yan Xiong","email":"","orcid":"","institution":"Heilongjiang Academy of Sciences (HAS)","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Xiong","suffix":""},{"id":487857901,"identity":"78a22da0-7e39-4cf4-a7b4-77106a3f48aa","order_by":1,"name":"Mingjing Li","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Mingjing","middleName":"","lastName":"Li","suffix":""},{"id":487857902,"identity":"f55f5fee-92f9-4357-a215-ef1210107784","order_by":2,"name":"Xiaoyu Zhang","email":"","orcid":"","institution":"Chifeng University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Zhang","suffix":""},{"id":487857903,"identity":"fee499a0-d0f7-447c-9260-49a0d1c55de3","order_by":3,"name":"Xue Lei","email":"","orcid":"","institution":"Youjiang Medical University for Nationalities","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Lei","suffix":""},{"id":487857904,"identity":"08a3d803-9d04-4237-b0c7-b7d05737fd5c","order_by":4,"name":"Shuchang Yang","email":"","orcid":"","institution":"Heilongjiang Academy of Sciences (HAS)","correspondingAuthor":false,"prefix":"","firstName":"Shuchang","middleName":"","lastName":"Yang","suffix":""},{"id":487857905,"identity":"bc5347a1-0dc5-40db-a542-df35ab252d17","order_by":5,"name":"Hui Han","email":"","orcid":"","institution":"Heilongjiang Academy of Sciences (HAS)","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Han","suffix":""},{"id":487857906,"identity":"4908e50d-4dfe-4f6a-a6c0-59d7980e29a7","order_by":6,"name":"Yanting Qu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYPACCQYGZubjPz5USMjxE6cjAaiFnS1BcsYZC2PJBuK0ADE/j4E0Z1tF4gZCWvhu5Bh+LvxhkSfvzGNgzDhPgnEDA/PDRzfwaJG8kWMsPSNBotjwMFtBcuE2CWZzBjZj4xw8Wgxu5BhI8yRIJG5sZt5weOY2CTbLBh42aQJajH9DtDAYNvPOkeAxOEBYixnYlvnMLMbMvA0SEgS1SJ55VmbNkyaRuIGZLY1xxjEJA8lmAn7hO568+TaPTV3i/P7Dxxg+1NTV97M3P3yMTwvDAQ4DiAsPwESY8SkHa2F/AKblGwipHAWjYBSMghELALHJSL0Idt8RAAAAAElFTkSuQmCC","orcid":"","institution":"Heilongjiang Academy of Sciences (HAS)","correspondingAuthor":true,"prefix":"","firstName":"Yanting","middleName":"","lastName":"Qu","suffix":""}],"badges":[],"createdAt":"2025-06-24 04:23:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6961345/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6961345/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07293-0","type":"published","date":"2025-10-01T15:57:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87218229,"identity":"620757a8-b285-4506-a401-fffb15bb6562","added_by":"auto","created_at":"2025-07-21 15:47:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":292360,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in growth status and water content of \u003cem\u003eB. chinensis \u003c/em\u003eroot system under drought stress\u003c/p\u003e\n\u003cp\u003eA, The growth status of roots in \u003cem\u003eB. chinensis \u003c/em\u003eunder drought and after re-watering; B, Root water content. Left is the control group; Right is the treatment group. Control, The control group; Treatment, The treatment group. 0-28 d is drought treatment, and 28-35 d is re-watering, different letters indicate significant differences (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). Three biological repeats.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/c41cc5fa1968d9e2e508018a.png"},{"id":87217572,"identity":"f0da6a1b-64d1-4418-9e45-7eeeefb6276e","added_by":"auto","created_at":"2025-07-21 15:39:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48812,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of root activity in\u003cem\u003e B. chinensis \u003c/em\u003eunder drought\u003c/p\u003e\n\u003cp\u003eDifferent letters indicate significant differences (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). Three biological repeats.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/1e1e8b875423fdb32428fcb8.png"},{"id":87217576,"identity":"f285ea47-3a0e-4661-8124-7fc205fa9dea","added_by":"auto","created_at":"2025-07-21 15:39:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":335763,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the cross-sectional of the root system in \u003cem\u003eB. chinensis \u003c/em\u003eseedlings under drought\u003c/p\u003e\n\u003cp\u003eStructure of transverse section of root system (×200); Ep, Epidermis; Ex, Exodermis; En, Endodermis; Pe, Pericycle; Ph: Primary phloem; Px, Protoxylem; Mx, Metaxylem; Pi, Medulla; a-f, The control group; a′-f′, The treatment group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/0f9de2bd8e3a07dde33e8b39.png"},{"id":87216366,"identity":"68177567-a2be-409c-b132-f9d05381eb9e","added_by":"auto","created_at":"2025-07-21 15:31:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":75821,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of conductivity and malondialdehyde contents of roots in\u003cem\u003e B. chinensis \u003c/em\u003eunder drought\u003c/p\u003e\n\u003cp\u003eA, Relative conductivity of roots; B, Malondialdehyde content of roots; Different letters indicate significant differences (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). Three biological repeats.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/9cff0309860245cc8eb33456.png"},{"id":87216369,"identity":"31f34e5b-13bd-4a09-9069-02da3b2331d6","added_by":"auto","created_at":"2025-07-21 15:31:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":76584,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of soluble protein and proline contents of roots in \u003cem\u003eB. chinensis \u003c/em\u003eunder drought\u003c/p\u003e\n\u003cp\u003eA, Soluble protein content of roots; B, Proline content of roots; Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05). Three biological repeats.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/4590c4ca4ba7e2d978327bb5.png"},{"id":87218230,"identity":"2a69b68e-938d-4c33-98a4-8f8918763ae0","added_by":"auto","created_at":"2025-07-21 15:47:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":73646,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of hydrogen peroxide and superoxide anion contents of roots in\u003cem\u003e B. chinensis \u003c/em\u003eunder drought\u003c/p\u003e\n\u003cp\u003eA, Hydrogen peroxide content of roots; B, Superoxide anion content of roots; Different letters indicate significant differences (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). Three biological repeats.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/cd11fbe8e8aeec6e328b2aa2.png"},{"id":87216375,"identity":"6b5144e8-6849-470a-af0b-3e5e44d360a0","added_by":"auto","created_at":"2025-07-21 15:31:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":143108,"visible":true,"origin":"","legend":"\u003cp\u003eTissue staining of roots in \u003cem\u003eB. chinensis \u003c/em\u003eseedlings under droughtr\u003c/p\u003e\n\u003cp\u003eDAB, the dye solution of Diaminobenzidine; NBT, The dye solution of Nitrotetrazolium Blue chloride; Left, the control group; Right, The treatment group.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/a750f8843e24e8060f9436c8.png"},{"id":87217578,"identity":"1940ece2-0931-4544-9a37-df8b7180d6b9","added_by":"auto","created_at":"2025-07-21 15:39:40","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":147451,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of superoxide dismutase, peroxidase, catalase, glutathione reductase, ascorbic acid peroxidase and monodehydroascorbic acid reductase activities of roots in\u003cem\u003e B. chinensis \u003c/em\u003eunder drought\u003c/p\u003e\n\u003cp\u003eA, Superoxide dismutase activity of roots; B, Peroxidase activity of roots; C, Catalase activity of roots; D, Glutathione reductase activity of roots; E, Ascorbate peroxidase activity of roots; F, Monodehydroascorbate reductase activity of roots; Different letters indicate significant differences (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). Three biological repeats.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/9451e0b290f5d82c43fed4c0.png"},{"id":87216381,"identity":"865b3804-0cd1-4439-9d73-d7da28ec4378","added_by":"auto","created_at":"2025-07-21 15:31:40","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":94631,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of reduced glutathione, oxidized glutathione, GSH+GSSG and GSH/GSSG ratio of roots in \u003cem\u003eB. chinensis \u003c/em\u003eunder drought\u003c/p\u003e\n\u003cp\u003eA, Reduced glutathione content of roots; B, Oxidative glutathione content of roots; C, GSH+GSSG content of roots ; D: Root GSH/GSSG; Different letters indicate significant differences (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). Three biological repeats.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/a050222edc56b450bd473000.png"},{"id":87218231,"identity":"3b1a5ae2-1dd9-4b7c-a3c6-e56a9e2cf5af","added_by":"auto","created_at":"2025-07-21 15:47:40","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":54937,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis of roots\u003cem\u003e \u003c/em\u003ein \u003cem\u003eB. chinensis \u003c/em\u003eunder drought\u003c/p\u003e\n\u003cp\u003eA, Principal component analysis of control groups; B, Principal component analysis of treatment groups.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/5e864b819687d1fefe37fabd.png"},{"id":87216388,"identity":"741437a4-5771-4c19-937f-b97eefc820e3","added_by":"auto","created_at":"2025-07-21 15:31:40","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":135342,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram and class diagram of differential metabolites\u003c/p\u003e\n\u003cp\u003eA, Venn diagram, venn diagram of differential metabolites in a multiple pairwise comparison\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/2c754ff7a785257fa78d40e6.png"},{"id":87219329,"identity":"7d12c912-0aea-481a-ab2c-c49742444bc9","added_by":"auto","created_at":"2025-07-21 16:03:40","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":355932,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of flavonoids content\u003c/p\u003e\n\u003cp\u003eA, Clustering heat map of flavonoid content; B, Contents of rutin, quercetin, hesperetin and gallocatechin. Significance levels are marked with asterisks, ns indicates no significant difference; * indicates \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ** indicates \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; *** indicates \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; The color from yellow to red means that the content gradually increases, and from yellow to blue means that the content gradually decreases.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/c8b89b5da1316a793d85b034.png"},{"id":87216378,"identity":"c89192a3-ee4e-48e2-a9e2-ba3e15988375","added_by":"auto","created_at":"2025-07-21 15:31:40","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":132198,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of content on amino acid compounds\u003c/p\u003e\n\u003cp\u003eA, Amino acid compound contents cluster heat map; B, Phenylalanine and valine contents. Significance levels are marked with asterisks, ns indicates no significant difference; * indicates \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; ** indicates \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; *** indicates \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/a39bf7442e66ed966329b2d5.png"},{"id":87217587,"identity":"b4fd44b1-0150-4199-9bea-1cf0b8894a8e","added_by":"auto","created_at":"2025-07-21 15:39:40","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":185468,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of content on phospholipids compounds\u003c/p\u003e\n\u003cp\u003eA, Heat map of phospholipid compounds content clustering; B, PG (17:0/19:0), PA (16:0/16:0) and PG (12:0/15:0) content. Significance levels are marked with asterisks, ns indicates no significant difference; * indicates \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ** indicates \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; *** indicates \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/4d0ef72ee05fd0eb0c87f711.png"},{"id":87216389,"identity":"c56de7bd-b859-429f-b590-788893eac3e8","added_by":"auto","created_at":"2025-07-21 15:31:40","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":86709,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of endogenous hormone content\u003c/p\u003e\n\u003cp\u003eA, Abscisic acid content; B, Jasmonic acid content; C, Salicylic acid content. Significance levels are marked with asterisks, ns indicates no significant difference; * indicates \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ** indicates \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; *** indicates \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/b9c8ebbdbe07868882d1ef71.png"},{"id":87216410,"identity":"93a54b07-ef76-414e-ab2d-303eb51bc8a3","added_by":"auto","created_at":"2025-07-21 15:31:41","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":157503,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of other metabolite content\u003c/p\u003e\n\u003cp\u003eA, Stearic acid; B: Arachic acid; C, Alpha-linolenic acid; D, Arachidonic acid; E, Nutriacholic acid; F, Vitamin D3; G, Phytol; H, Cinnamic acid; I, Adenine. Significance levels are marked with asterisks, ns indicates no significant difference; * indicates \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ** indicates \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; *** indicates \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"16.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/5a67e1f0ead4068049f3e155.png"},{"id":87216396,"identity":"4f6285c0-7a41-455b-91c0-451bea0f3d7b","added_by":"auto","created_at":"2025-07-21 15:31:40","extension":"png","order_by":17,"title":"Figure 17","display":"","copyAsset":false,"role":"figure","size":348567,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis of metabolite content\u003c/p\u003e\n\u003cp\u003e* \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05\u003c/p\u003e","description":"","filename":"17.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/1a76586681377f17ea69f3c2.png"},{"id":87217589,"identity":"1b3f17ab-c5f8-4d3c-9ca9-c07e0a518a9c","added_by":"auto","created_at":"2025-07-21 15:39:40","extension":"png","order_by":18,"title":"Figure 18","display":"","copyAsset":false,"role":"figure","size":299352,"visible":true,"origin":"","legend":"\u003cp\u003eEnrichment analysis of differential metabolite pathways\u003c/p\u003e","description":"","filename":"18.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/85e4ff6c7e56c81a904a50f6.png"},{"id":87216395,"identity":"37766c1c-fdfe-4aaf-ba1d-d37e1063b2b8","added_by":"auto","created_at":"2025-07-21 15:31:40","extension":"png","order_by":19,"title":"Figure 19","display":"","copyAsset":false,"role":"figure","size":337602,"visible":true,"origin":"","legend":"\u003cp\u003eThe analysis of differential metabolite network\u003c/p\u003e\n\u003cp\u003eNote: Red represents upward adjustment and blue represents downward adjustment; The solid line and the dotted line represent single step reaction and multi-step reaction respectively.\u003c/p\u003e","description":"","filename":"19.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/d05c41585e688ccdb0688cc2.png"},{"id":87216403,"identity":"12c67da4-a0cd-4dda-ae3c-68b3acc7717a","added_by":"auto","created_at":"2025-07-21 15:31:40","extension":"png","order_by":20,"title":"Figure 20","display":"","copyAsset":false,"role":"figure","size":224635,"visible":true,"origin":"","legend":"\u003cp\u003eHierarchical cluster tree of WGCNA analysis\u003c/p\u003e","description":"","filename":"20.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/200186a40692c689a00de775.png"},{"id":87216401,"identity":"c0fa4fac-b50b-4f31-9204-cfe44b674424","added_by":"auto","created_at":"2025-07-21 15:31:40","extension":"png","order_by":21,"title":"Figure 21","display":"","copyAsset":false,"role":"figure","size":318463,"visible":true,"origin":"","legend":"\u003cp\u003eAssociation analysis of metabolite co-expression network modules with physiological and biochemical traits\u003c/p\u003e\n\u003cp\u003eEach cell contains the corresponding correlation and \u003cem\u003eP-value\u003c/em\u003e, r \u0026gt; 0.65 and \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 were defined as modules positively correlated with the sample.\u003c/p\u003e","description":"","filename":"21.png","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/f26c3c5e3b3d803fe70a4248.png"},{"id":92884710,"identity":"12a7baf3-e706-491b-bfaf-10f9325517e3","added_by":"auto","created_at":"2025-10-06 16:13:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5213358,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6961345/v1/886b06f6-0b08-45e9-8246-a77c246c495d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The study of physiological response mechanism and metabolomics on B. chinensis under drought","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlants growth and development are affected by various biotic and abiotic stresses. Among them, abiotic stresses have a particularly effect on plant growth. Accordingly, plants are vulnerable to the threat of abiotic stresses such as drought, flooding, high temperature, low temperature, salinity, light, chemical and mechanical stresses. Among them, drought is a serious and complex abiotic stress, which can have a negative impact on plant growth and crop yield [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In general, the root system is an important organ for plants to extract soil water and nutrients, and is also one of the key organs for material and energy exchange with the external environment [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. When the plant root system is affected by drought stress, it will trigger a series of unfavorable effects such as impeded water uptake, cell structure and function damage, physiological and metabolic disorders, etc., which will ultimately lead to a decline in crop yields [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, some response mechanism of plant to drought stress is not clear.\u003c/p\u003e\u003cp\u003ePlants have developed a multifaceted network to withstand stressful environments [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Generally, when the root system senses the effects of environmental stress, chemical signals are generated and transported to the soil, which in turn cause a series of growth and physiological and biochemical responses of plants [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. During drought stress, unsaturated fatty acids in the cell membrane are affected by free radicals leading to lipid peroxidation, while malondialdehyde content and relative conductivity also increase [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Drought also leads to loss of cell expansion pressure and dehydration, so the accumulation of osmolytes is crucial for osmoprotection and osmoregulation, and plants accumulate large amounts of osmoregulatory substances to maintain normal physiological metabolism in the cell, prevent cell dehydration, and alleviate the damage of drought to the plant to a certain extent [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition, when plants are subjected to drought stress, a large amount of reactive oxygen species (ROS), including hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), superoxide anion radicals (O2\u003csup\u003e\u0026middot;-\u003c/sup\u003e), and hydroxyl radicals (-OH), etc [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In general, excessive accumulation of ROS can cause oxidative damage to proteins, nucleic acids, and lipids, and ultimately lead to plant cell death [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. At the same time, plants have sufficient antioxidant enzymes and non-enzymatic antioxidants to reduce excessive ROS accumulation in different organelles and reduce oxidative damage under drought conditions. In order to maintain a balance between ROS production and scavenging, plants have also evolved two different biological processes to cope with ROS, as well as to scavenge ROS through enzymatic and non-enzymatic processes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, to cope drought stress, plants have evolved complex system.\u003c/p\u003e\u003cp\u003ePlants response to adversity stress is a complex biological process, the most important of which is the alteration of metabolites. During drought stress, plants can maintain homeostasis by regulating a complex regulatory network consisting of genes-proteins-metabolites-phenotypes to achieve defense against drought stress, and thus metabolites can reflect the physiological state of plants during growth [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Under drought stress, the content of metabolites such as amino acids, flavonoids and phenolics increased significantly in plants [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Among them, metabolites such as flavonoids and phenolamines are widely involved in plant growth and development, physiological processes and plant stress response [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, sugars such as mannitol, sorbitol, sucrose, fructans, and amino acid metabolites such as proline act as osmotic factors in plants to resist drought. There are also many small molecules such as anthocyanins, carotenoids and glutathione metabolites that can protect the plants from the damage caused by drought stress by scavenging excess ROS [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, metabolites are the basic substances that constitute the life activities and are the concentration of multiple responses. In recent years, metabolomics has been widely used to study drought tolerance in plants with different drought tolerance ability. Therefore, some metabolites under drought stress can be identified, screened and analyzed by metabolomics to reveal the mechanism of drought tolerance in plants [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eBelamcanda chinensis\u003c/em\u003e L. is a perennial herb belonging to the genus \u003cem\u003eBelamcanda\u003c/em\u003e in the family Iridaceae, and is a cash crop with high medicinal and ornamental value [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Its rhizomes have important roles in antioxidant, anti-inflammatory and anti-tumor properties [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In addition, its drought-resistant characteristics make it widely used in parks and landscapes. However, the current study only illustrates the drought resistance of \u003cem\u003eB. chinensis\u003c/em\u003e under drought stress, but there is a lack of clarity about the mechanism of drought resistance. Therefore, it is important to understand the drought tolerance mechanism of \u003cem\u003eB. chinensis\u003c/em\u003e to safeguard its yield and quality. In this study, we analyzed the growth morphology and physiological and biochemical indexes of \u003cem\u003eB. chinensis\u003c/em\u003e under drought stress. At the same time, we also analyzed the differential metabolites of \u003cem\u003eB. chinensis\u003c/em\u003e under drought stress using metabolomics to provied a theoretical foundation for the elucidation of physiological and metabolic mechanism of \u003cem\u003eB. chinensis\u003c/em\u003e in response to drought stress.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and growing contitions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eB. chinensis\u003c/em\u003e L. seedlings used in this study were provided by Institute of Natural Resources and Ecology, Heilongjiang Academy of Sciences (HAS). Two-year old \u003cem\u003eB. chinensis\u003c/em\u003e L. seedlings with uniform growth were selected and planted in plastic nutrient pots (soil: perlite = 2:1). All the seedlings were divided into two groups including to the control and the treatment, and watered to reach 70%-80% of the soil water content before drought treatment. For the control group, the seedlings were fully irrigated every 7 d to saturate the soil and for the treatment group, the seedlings were continuously irrigated for 28 d, and re-watering was carried out on the 28 th day. The morphology was observed and photographed on the 0 d, 7 d, 14 d, 21 d, 28 d and after re-watering. Additionally, the seedlings roots were also selected in liquid nitrogen and stored in a refrigerator at -80℃ for further use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of roots phenotype and related physiological indexes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of basic morphological parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRelative water content was determined by drying method of Lang [21]. Root activity was determined by triphenyltetrazolium chloride (TTC) method of\u0026nbsp;Ruf\u0026nbsp;[22].\u0026nbsp;To observe root morphology, the roots were separated from the soil, the soil was carefully removed to avoid root damage and then the roots were scanned using a desktop scanner (Wanshen LA-S series plant image root analysis system software) . The observation of scanning electron microscopy was referred to the method of Shi [23].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of the degree of damage to the membrane system and osmotic regulatory substances\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe conductivity was determined by the conductivity meter method of Rhoades [24]. Malondialdehyde\u0026nbsp;(MDA) content was determined by TBA method of Mao [25]. Soluble proteins were determined by the G-250 method using the Thomas Blue method of Behnamnia [26]. Proline (Pro) was determined by the G-250 method using the Thomas Blue method of\u0026nbsp;Behnamnia\u0026nbsp;[26].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of reactive oxygen (ROS) content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSuperoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;-\u003c/sup\u003e) was determined by hydroxylamine oxidation Kono [27]. Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) was determined by the KI method of Wang [28]. Histochemical staining of\u0026nbsp;O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;-\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewas\u003csub\u003e\u0026nbsp;\u003c/sub\u003ecarried out by 3,3-diaminobenzidine (DAB) and nitinol Blue Tetrazolium (NBT), respectivly.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Antioxidant enzyme activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe peroxidase (POD) is determined by the method of Madamanchi [29]. Determination of Catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) were used the method of Deng [30]. The glutathione reductase (GR) activity is analyzed using the method of Behnamnia [26]. Monodehydroascorbate reductase (MDHAR) was determined by using the G0213F kit (Suzhou Grace Biotechnology Co.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of antioxidant content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe root samples were thoroughly ground. Then 3 mL of 5% sulfosalicylic acid solution was added and centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was used for the determination of antioxidant content. Then, the contents\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eof reduced glutathione (GSH) content and oxidized glutathione (GSSG)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewere determinated according to the method of\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eDeng [30].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolomics analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples were sent to Shanghai Boyun Biotechnology Co. for the extraction of sample metabolites. The supernatant was analyzed by liquid chromatography-tandem time-of-flight mass spectrometry UPLC-Q-TOF/MS. In addition, quality control (QC) samples were prepared by mixing all core samples. During instrumental analysis, a QC sample was inserted next to each of the six test samples to check the reproducibility of the analytical process.\u003c/p\u003e\n\u003cp\u003eThe samples were sent to Shanghai Boyun Biotechnology Co., Ltd. for the metabolomics of \u003cem\u003eB. chinensis\u0026nbsp;\u003c/em\u003eroots, and the raw data were integrated, corrected, peak-aligned, and normalized by the metabolomics processing software progenesis QI (Waters Corporation, Milford, USA), and the corresponding metabolite content was expressed as the chromatographic peak-area integral to ultimately obtain a data matrix.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are shown using the mean \u0026plusmn; standard deviation (SD) and were analyzed using SPSS 25 statistical software (Chicago, IL, USA). Differences among groups were compared using (One-way analysis of variance, ANOVA) followed by Duncan\u0026apos;s new multiple range test. \u003cem\u003eP\u003c/em\u003e-values\u0026nbsp;\u0026lt;\u0026nbsp;0.05 were considered significant. GraphPad Prism 8.0.2 software were used for graphing figures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffects of drought stress on phenotypic characterization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of drought stress on morphological parameters of \u003cem\u003eB. chinensis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared with the control, the number of lateral roots at 28 d of drought stress increased\u0026nbsp;\u003c/p\u003e\n\u003cp\u003esignificantly and became thinner, the degree of lignification increased after drought treatment, and the color gradually deepened from white to yellow-brown, and the morphology and color of the root system recovered after re-watering. Meanwhile, the root water content showed a decreasing trend with the prolongation of the stress time, which was the lowest at 28 d of stress, 66.22%, and lower than that of the control group. The root water content recovered after re-watering (Fig. 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInfluence of drought stress on growth parameters of \u003cem\u003eB. chinensis\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe root fresh weight and root dry weight of the control group gradually increased, the root fresh weight of the treatment group showed a trend of increasing and then slowly decreasing with the extension of drought stress, and the root fresh weight recovered after re-watering, but still lower than that of the control group (Table 1). The root dry weight of the treatment group showed a trend of increasing and then decreasing, and after re-watering, the root dry weight recovered, but did not recover to the level of the control group.\u003c/p\u003e\n\u003cp\u003eTable 1 Changes of biomass in \u003cem\u003eB. chinensis\u0026nbsp;\u003c/em\u003eunder drought\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 123px;\"\u003e\n \u003cp\u003eIndex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" style=\"width: 408px;\"\u003e\n \u003cp\u003edays\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 68px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 123px;\"\u003e\n \u003cp\u003eRoot fresh weight (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e3.4\u0026plusmn;0.1de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e4.2\u0026plusmn;0.2cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e4.8\u0026plusmn;0.3bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e5.2\u0026plusmn;0.4b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e5.3\u0026plusmn;0.5b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 68px;\"\u003e\n \u003cp\u003e6.1\u0026plusmn;0.5a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e3.8\u0026plusmn;0.8d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e3.6\u0026plusmn;0.1de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e4.2\u0026plusmn;0.7cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e2.9\u0026plusmn;0.5ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e2.2\u0026plusmn;0.6f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 68px;\"\u003e\n \u003cp\u003e2.7\u0026plusmn;0.7ef\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 123px;\"\u003e\n \u003cp\u003eRoot dry weight (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.6\u0026plusmn;0.1g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.8\u0026plusmn;0.1ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.9\u0026plusmn;0.1de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e1.0\u0026plusmn;0.1c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e1.2\u0026plusmn;0.1b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 68px;\"\u003e\n \u003cp\u003e1.6\u0026plusmn;0.1a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 63px;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.6\u0026plusmn;0.1g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.9\u0026plusmn;0.1def\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.9\u0026plusmn;0.1cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.8\u0026plusmn;0.1ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 68px;\"\u003e\n \u003cp\u003e0.8\u0026plusmn;0.1f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 68px;\"\u003e\n \u003cp\u003e0.8\u0026plusmn;0.1f\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eData are Mean \u0026plusmn; SE, different letters indicate significant differences (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of drought stress on root vigor of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eB. chinensis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe trend of root activity of the control group from 7 to 35 d was decreasing-rising-decreasing (Fig. 2). Under drought stress, the root activity of the shotguns decreased significantly, and the longer the duration of the stress, the lower the root activity was, and the difference was significant compared with that of the control group at 28 d. The root activity increased after the re-watering, but it was still lower than that of the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of drought stress on the structure of the root system of \u003cem\u003eB. chinensis\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe epidermal cells of the control root system were intact , and the thin-walled cells of the cortex were regular in shape and small in size, and closely and neatly arranged (Fig. 3). In the treatment group, there was no significant difference in the anatomical structure of the root system compared with the control at 7 d and 14 d of drought stress, and both grew well. At 21 d, the root system showed slight drought damage, and the thin-walled cells of the root cortex were more tightly packed and irregularly arranged, with larger cellular gaps, and at 28 d, the damage to the root system structure was more serious, with cortical breakage and other traits. The number of root conduits was less affected by drought stress at 0 d-7 d, the number of conduits did not change significantly, and the shape of conduits was fuller, at 14 d the number of conduits in each group increased faster, the conduits became thinner and increased, but the shape remained more intact, and with the prolongation of the time of drought stress, the conduits continued to become thinner and increase in number, and the shape was distorted, and the mid-columns appeared to be broken and shriveled deformed, and the order of the mid-columns were also broken. After re-watering, there was some recovery of root cortex breaks and some recovery of conduit and mesocolon morphology.\u003c/p\u003e\n\u003cp\u003eDiameter of root, diameter of the xylem, root cross-sectional area and diameter of the mid-column of the control group increased to a certain extent, the thickness of cortex was not significantly thickened, the mid-column structure was intact, and the morphology of the conduit was relatively full\u0026nbsp;(Table 2). The diameter of root of the treatment group showed a trend of increasing and then decreasing, and showed a downward trend at 14-28 d, with a significant difference compared with that of the control. The diameter of the mid-column showed an upward and then downward trend, and the diameter of the mid-column was 2,921.40 \u0026mu;m at 28 d of drought stress. The diameter of the mid-column was 2921.40 \u0026mu;m at 28 d of drought stress, which was significantly different from the control, but still lower than the control, the thickness of the cortex showed a trend of increasing and then decreasing under drought stress, which was increasing at 0-21 d and rapidly increasing at 7 d, and then decreasing at 21-28 d, which was 3346.47 \u0026mu;m at 28 d, which was significantly different from the control. The diameter of the xylem showed an increasing and then decreasing trend, gradually decreasing at 7 d and reaching a minimum value of 484.84 \u0026mu;m at 28 d, root cross-sectional area and mid-column area showed an increasing and then decreasing trend with the extension of drought stress, and the root cross-sectional area and mid-column area were 70.38\u0026times;10\u003csup\u003e6\u003c/sup\u003e \u0026mu;m\u003csup\u003e2\u003c/sup\u003e and 26.82\u0026times;10\u003csup\u003e6\u003c/sup\u003e \u0026mu;m\u003csup\u003e2\u003c/sup\u003e, respectively, at the time of 28 d of drought stress, and the differences were significant when compared with that of the control. All anatomical parameters recovered after re-watering, but not to control levels.\u003c/p\u003e\n\u003cp\u003eTable 2 Changes of structural parameters of stem root system under drought\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"607\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 98px;\"\u003e\n \u003cp\u003eIndex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 56px;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" style=\"width: 453px;\"\u003e\n \u003cp\u003edays\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 98px;\"\u003e\n \u003cp\u003eDiameter of Root (\u0026mu;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e3918.0\u0026plusmn;25.6h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e4110.0\u0026plusmn;49.9g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e4634.8\u0026plusmn;45.2f\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e5140.3\u0026plusmn;18.6c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e6275.3\u0026plusmn;22.9b\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e6576.1\u0026plusmn;26.9a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e3958.9\u0026plusmn;30.4h\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e4138.2\u0026plusmn;14.0g\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e5030.2\u0026plusmn;29.6d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e4813.2\u0026plusmn;29.6e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e4666.6\u0026plusmn;49.9e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e5013.3\u0026plusmn;79.9d\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 98px;\"\u003e\n \u003cp\u003eDiameter of the mid-column(\u0026mu;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e2119.2\u0026plusmn;9.8h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e2351.3\u0026plusmn;13.9g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e2938.9\u0026plusmn;24.4d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e3011.9\u0026plusmn;25.9c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e3237.2\u0026plusmn;30.0ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e3278.7\u0026plusmn;19.9a\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e2116.3\u0026plusmn;26.1 h\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e2356.5\u0026plusmn;17.3f\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e2882.2\u0026plusmn;47.9e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e2990.5\u0026plusmn;24.9c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e2921.4\u0026plusmn;21.5de\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e3209.9\u0026plusmn;27.5b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 98px;\"\u003e\n \u003cp\u003eThickness of cortex (\u0026mu;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e3138.5\u0026plusmn;13.4g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e3147.9\u0026plusmn;29.5g\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e3625.8\u0026plusmn;24.8e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e3960.9\u0026plusmn;28.9c\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e5138.4\u0026plusmn;14.9a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e5197.9\u0026plusmn;34.1a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e3168.5\u0026plusmn;33.9g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e3196.5\u0026plusmn;33.4fg\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e3609.1\u0026plusmn;11.2d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e3658.7\u0026plusmn;29.3d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e3346.5\u0026plusmn;24.2ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e4338.4\u0026plusmn;30.5b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 98px;\"\u003e\n \u003cp\u003eDiameter of the xylem (\u0026mu;m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e604.9\u0026plusmn;14.4d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e611.7\u0026plusmn;10.7cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e626.3\u0026plusmn;15.4bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e620.6\u0026plusmn;6.1cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e640.1\u0026plusmn;3.4ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e650.9\u0026plusmn;6.9a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e605.8\u0026plusmn;3.2d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e619.3\u0026plusmn;3.2cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e561.2\u0026plusmn;10.2e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e549.9\u0026plusmn;7.3ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e484.8\u0026plusmn;10.9g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e539.4\u0026plusmn;7.9ef\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 98px;\"\u003e\n \u003cp\u003eRoot cross-sectional area (10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003e\u0026mu;m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e48.1\u0026plusmn;0.1i\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e53.1\u0026plusmn;1.3h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e67.5\u0026plusmn;1.3f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e82.9\u0026plusmn;0.6c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e123.4\u0026plusmn;0.7b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e135.6\u0026plusmn;1.1a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e52.2\u0026plusmn;1.5h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e61.9\u0026plusmn;0.4g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e79.5\u0026plusmn;0.9d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e72.8\u0026plusmn;2.4e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e70.4\u0026plusmn;3.9ef\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e78.9\u0026plusmn;2.5d\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 98px;\"\u003e\n \u003cp\u003eMid-column area (10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003e\u0026mu;m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e14.5\u0026plusmn;0.6h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e16.9\u0026plusmn;0.9g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e27.1\u0026plusmn;0.4cde\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e28.5\u0026plusmn;0.5c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e32.3\u0026plusmn;1.7a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e33.5\u0026plusmn;1.5a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e13.8\u0026plusmn;0.6h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e19.5\u0026plusmn;0.8f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e26.1\u0026plusmn;0.9e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e28.1\u0026plusmn;0.5cd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e26.8\u0026plusmn;0.4de\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e30.6\u0026plusmn;0.5b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eData are Mean \u0026plusmn; SE, different letters indicate significant differences (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of drought stress on physiological and biochemical indexes in roots of \u003cem\u003eB. chinensis \u0026nbsp;\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of drought stress on cell membrane permeability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe relative conductivity of roots did not change significantly in the control group, and under drought stress, the relative conductivity of roots showed an increasing trend and was significantly higher than that of the control at 28 d of drought stress, and recovered after re-watering (Fig. 4). The changes of MDA content of root in the control group were basically stable, with the extension of drought stress time, the MDA content of root in the treatment group gradually increased, and the MDA content of root was significantly higher than that of the control group at 21 d. After re-watering, the MDA content of root decreased to the control level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of drought stress on osmoregulators\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe trend of soluble protein (Sp) content in the root in the control group was first increasing and then decreasing (Fig. 5).With the prolongation of drought stress, soluble protein (Sp) content in the treatment group gradually increased and was significantly higher than that of the control at 28 d of stress, and after re-watering, soluble protein (Sp) content in recovered, but did not return to the control level. Proline content is altered in plants under drought stress. The Proline content of the control group did not change significantly, and the Proline content of roots in the treatment group was higher than that of the control group at 28 d. The Proline content of roots decreased after re-watering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of drought stress on reactive oxygen species content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no significant change in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content of roots under normal conditions, and the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content of roots showed an increasing trend with the prolongation of drought stress, and it was significantly higher than that of the control group at 28 d (Fig. 6). At the same time, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content produced by the root system reached the maximum at 28 d of drought stress, and after re-watering, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content was restored to the level of the control. The O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;-\u0026nbsp;\u003c/sup\u003eproduction rate of the root system did not change significantly under normal conditions. With the prolongation of drought stress, the O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;-\u0026nbsp;\u003c/sup\u003eproduction rate of the root system showed an increasing trend, which was significantly higher than that of the control group at 28 d, and the production rate reached the maximum value, which could be recovered to a certain extent after re-watering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of drought stress on reactive oxygen species staining profiles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to detect the reactive oxygen content more intuitively, DAB and NBT staining were used to detect the changes of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;-\u0026nbsp;\u003c/sup\u003econtent under different days of drought stress in \u003cem\u003eB. chinensis\u003c/em\u003e, respectively.The reaction between H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and DAB can produce brown precipitate, and the degree of staining can indicate the accumulation of hydrogen peroxide. The DAB staining of the roots in the treatment group did not change significantly at 0-14 d compared with the control, indicating that the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content accumulated less (Fig. 7). the staining area of the roots increased and the color became darker at 21 d, and the staining area of the roots was large and the staining was aggravated at 28 d, which indicated that the highest amount of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was produced at this time. The reaction of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;-\u003c/sup\u003e with NBT can form a blue polymeric precipitate, which can indicate the accumulation of superoxide under stress. The NBT staining of the root system of the treatment group was light blue at 7 d, indicating that the O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;-\u0026nbsp;\u003c/sup\u003econtent accumulated less. the staining area of the root system increased and deepened from 14 d to 21 d, and then increased and deepened at 28 d. The NBT staining of the root system of the treatment group was light blue at 7 d, indicating that the O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;-\u0026nbsp;\u003c/sup\u003econtent accumulated less. The degree of DAB and NBT staining in the root system recovered after re-watering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of drought stress on antioxidant enzymes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SOD activity of roots in the control group basically remained unchanged before 28 d, and the activity increased at 28-35 d (Fig. 8). The SOD activity of roots in the treatment group showed an increasing trend at 7-21 d of stress, and then showed a decreasing trend with the prolongation of the stress time, and the SOD activity reached the maximum value at 21 d of stress, which was significantly different from that of the control. In the control group, the POD activity of roots basically remained unchanged, in the treatment group, the POD activity of roots showed an increasing trend with the prolongation of drought stress, and reached the maximum value at 28 d, with a significant difference compared with the control. In the control group, the CAT activity of roots showed a decreasing-rising-decreasing trend, whereas in the treated group, the CAT activity of root basically remained unchanged from 0 to 14 d, and then increased with the prolongation of the stress time, and reached the maximum value at 28 d, with a significant difference compared with that of the control. The GR activity of roots in the control group basically remained unchanged, and the GR activity of roots under drought stress showed an increasing trend. The APX activity of roots in the control group showed a decreasing and then increasing trend, and the APX activity of roots in the treatment group gradually increased with the prolongation of the drought stress time, there was no significant change in the MDHAR activity of roots in the control group, and it gradually increased with the prolongation of the drought stress time. The enzyme activities at 28 d of drought stress were all significantly higher than the control (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and the POD, CAT, GR , APX and MDHAR activities of roots increased 1.8, 2.4, 4.2, 2.0 and 6.4 times, respectively, compared with the control, and the POD, CAT, GR, APX and MDHAR activities of roots decreased significantly after re-watering. The POD, CAT and GR activities of roots recovered but not to the control level, and the SOD, APX and MDHAR activities of roots recovered to the control level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of drought stress on antioxidants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGR catalyzes the reduction of GSSG to GSH, thereby maintaining GSH levels and the GSH/GSSG ratio. The GSH, GSSG and GSH+GSSG content of roots in the control group remained basically unchanged, while the GSH, GSSG and GSH+GSSG content of roots in the treatment group showed an increasing trend, and the GSH, GSSG and GSH+GSSG content of roots in the treatment group reached the maximum at 28 d. GSH/GSSG in the root system of the control group showed an increasing and then decreasing trend from 0 to 21 d, and the change was not obvious from 21 to 35 d. The trend of GSH/GSSG in the root system of the treatment group basically remained unchanged, and the root system GSH/GSSG were lower than that of the control group. After re-watering, the GSH content of roots recovered to the control level, and both the GSSG content and GSH+GSSG recovered, but not to the control level, and the GSH/GSSG of the root system recovered to the control level (Fig. 9).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolomics analysis \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrincipal component analysis of the sample\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe two principal components PC1 and PC2 explained 29.3% and 13.1% of the variability in the control group, and the two principal components PC1 and PC2 explained 21.9% and 13.7% of the variability in the treatment group, and the six biological replicates of each sample could be clustered well, indicating that the differences between core samples within the group were small, and the experiment was reproducible and reliable. In addition, there was a clear trend of separation of core samples between the drought treatments, indicating significant metabolic differences in the drought treatments (Fig. 10).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential metabolite screening and identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eP\u003c/em\u003e-value of the t-test combined with the variable importance projection (VIP) value of the OPLS-DA model was adopted to screen for differential metabolites between the different groups, and the criteria for screening were \u003cem\u003eVIP\u0026nbsp;\u003c/em\u003e\u0026gt; 1.0 and significance at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening for differential metabolites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith 58 significantly different metabolites between C7 and T7, 150 significantly different metabolites between C14 and T14, 238 significantly different metabolites between C21 and T21, 272 significantly different metabolites between C28 and T28, and 172 significantly different metabolites between C35 and T35 (Fig. 11). Thus, it can be found that the number of differential metabolites increased with the increase of drought stress time. In addition, the differential metabolites in five groups, C7-vs-T7, C14-vs-T14, C21-vs-T21, C28-vs-T28, and C35-vs-T35, were categorized into 13 classes, mainly consisting of 172 flavonoids, 48 fatty acyls, 39 steroids and steroid derivatives, 29 glycerophospholipids, and 21 Benzene and substituted derivatives, 23 Prenol lipids, 20 carboxylic acids and derivatives, 18 organoxygen compounds, 6 guanosine monophosphate, 6 coumarins and derivatives, 3 phenols, 2 phenylpropanoic acids and 274 other compounds. In order to determine the correlation between different metabolites and drought stress at different times, the analysis of the amount of change was carried out. Differential metabolites were clustered by means, and the results are shown in FigS 1. Differential metabolites were classified into 16 different clusters (C1-C16), in which the metabolites in the treatment groups C2, C3, C4, C9, C10, C12, C15, and C16 showed a decreasing and then increasing trend, and their contents recovered after re-watering, while the contents of the metabolites in C11 and C14 showed an increasing trend in drought stress for 21 d. The metabolites in C1 and C13 showed a continuous decreasing trend, while the contents of the metabolites in C1 and C13 showed a recovery after re-watering. The metabolites in C11 and C14 showed an increasing trend at 21 d of drought stress, while the metabolites in C1 and C13 showed a continuous decreasing trend and recovered after re-watering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChanges in differential metabolite content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChanges of flavonoids content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe contents of metabolites such as naringenin, lignocerosin 5-galactoside and rutin increased with the extension of drought stress and recovered after re-watering, whereas the contents of flavonoids such as apigenin, mangostin and naringenin 4-O-glucosidic acid showed a decreasing tendency under drought stress and still did not recover after re-watering. Another part of flavonoids such as apigenin 7-lactic acid, gallocatechin, naringenin 3-O-glucosiduronic acid, etc. showed significant increase in content at 14 and 21 d of drought, but showed a decreasing trend at 28 d of drought, and did not recover after re-watering. The contents of kaempferol, lignocerotoxin, hesperidin, quercetin, apigenin 7-xyloside, naringenin-5-O-glucosiduronic acid, lignocerotoxin 5-galactoside, and hesperidin 7-O-glucosiduronic acid did not change significantly at the early stage of the drought stress, and showed an increasing trend with the prolongation of the drought stress time, and then declined after re-watering (Fig. 12).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChanges of content on amino acid compounds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe contents of threonylvaline, glutamyltryptophan, isoleucine, phenylalanine and tryptophan increased at 28 d of drought stress and decreased after re-watering (Fig. 13). In addition, phenylalanine, as a precursor substance for the synthesis of phenolic compounds, was associated with changes in the content of its downstream compounds. The content of phenylalanine increased after drought stress and was always higher than that of the control, and the content of phenylalanine was lower than that of the control after re-watering. The contents of histidine, phenylalanyl-alanine, histidinol, alanine tryptophan, asparagine, aspartic acid and valine were the highest at 14 d of drought, and decreased with the increase of drought stress time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChanges of endogenous hormone content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ABA content of the control group did not change significantly from 0 to 35 d, and the ABA content of the control group was maintained at a low level, while the ABA content of the treatment group basically remained unchanged at the beginning of the drought stress, but with the prolongation of the drought stress time, especially at 28 d of the drought stress, the ABA content of the roots was significantly higher than that of the control, and the ABA content of the roots significantly declined after the re-watering (Fig. 15). The JA content of the control group remained basically unchanged from 0-28 d and showed an increasing trend at 35 d. In the treatment group, at 0-14 d of drought stress, the JA content of the root system was significantly not different from that of the control group, and with the extension of the stress time, the JA content in the root system was significantly higher than that of the control at 28 d and differed significantly from that of the control, and the JA content of the root system decreased after re-watering and was significantly lower than that of the control. The content of salicylic acid was higher than that of abscisic acid and jasmonic acid under drought stress. The SA content of control roots gradually increased from 0-35 d. The SA content of treated roots showed an increasing trend until 21 d, and was significantly higher than that of control at 21 d of stress, whereas the SA content decreased from 21 to 28 d of stress, and there was no significant difference between the SA content of roots and that of control at 28 d of stress, and the SA content of roots recovered after re-watering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChanges of other metabolite content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder drought stress, stearic acid, linolenic acid and arachidonic acid content were all in a decreasing trend and significantly lower than the control, arachidonic acid content content of the control group did not change significantly, with the extension of drought stress time, arachidonic acid content showed a trend of increasing and then decreasing, with the highest content in drought for 21 d, and the treatment groups were all higher than the control group, and the content of nutriacholic acid showed an increasing trend with the extension of drought stress time. The content of vitamin D\u003csub\u003e3\u003c/sub\u003e and phytol showed an increasing and then decreasing trend with the prolongation of drought stress, and the difference was significant at 21 d. The content of cinnamic acid showed a decreasing trend and was lower than that of the control group, while the content of adenine showed an increasing trend, and the difference of the content was more significant at 28 d, and it was higher than that of the control group (Fig. 16).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation analysis of metabolite content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRutin was positively correlated with the content of hesperidin with a correlation coefficient r of 0.68, and negatively correlated with the content of stearic acid with a correlation coefficient r of -0.53. PA (16:0/16:0) was negatively correlated with the content of Alpha-linolenic acid with a correlation coefficient r of -0.54. PG (12:0/15:0) was negatively correlated with PG (17:0/19:0) content, with a correlation coefficient r of -0.66, and positively correlated with arachidonic acid and stearic acid content, with correlation coefficients r of 0.53 and 0.59, respectively. Phenylalanine was positively correlated with valine content with a correlation coefficient r of 0.50. Arachidic acid was positively correlated with phytol content with a correlation coefficient of 0.77, and negatively correlated with Alpha-linolenic acid, arachidonic acid and stearic acid content with correlation coefficients r of -0.53, -0.58 and -0.59, respectively. Arachidonic acid content was negatively correlated with phytol content with a correlation coefficient of -0.72, and positively correlated with stearic acid content with a correlation coefficient r of 0.53. Quercetin content had no correlation with myristic acid content or with any of the other metabolites (Fig. 17).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolic pathway analysis of differential metabolites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKEGG enrichment analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to better explain the role of metabolites in metabolic pathways, the differential metabolites were analyzed by KEGG pathway enrichment. The metabolites were mainly involved in a total of 49 metabolic pathways, of which the key metabolic pathways were amino acid biosynthesis, arachidonic acid metabolism, flavonoid and flavonol biosynthesis, glycerophospholipid metabolism, steroid biosynthesis, tyrosine metabolism, zeatin biosynthesis, monoterpene biosynthesis, and fatty acid biosynthesis pathways.\u003c/p\u003e\n\u003cp\u003eThe KEGG metabolic pathway of C7-vs-T7 is dominated by the pathways of glycerophospholipid metabolism, fatty acid biosynthesis, unsaturated fatty acid biosynthesis, histidine metabolism, aminoglycan and nucleotide sugar metabolism, and biosynthesis of secondary metabolites (Fig. 18). The KEGG metabolic pathways of C14-vs-T14 are mainly cysteine and methionine metabolism, arachidonic acid metabolism, alanine, phytohormone signaling, aspartate and glutamate metabolism, amino acid metabolism, degradation of valine, leucine, and isoleucine, biosynthesis of valine, leucine, and isoleucine, pantothenic acid and coenzyme a biosynthesis, diterpene biosynthesis, and arginine biosynthesis synthesis and other pathways enriched. The KEGG metabolic pathway of C21-vs-T21 focuses on the pathways of zeatin biosynthesis, fatty acid biosynthesis, degradation of valine, leucine, and isoleucine, valine, leucine, and isoleucine biosynthesis, oleuropein steroid biosynthesis, and carotenoid biosynthesis. The KEGG metabolic pathway of C28-vs-T28 mainly focuses on the pathways of sesquiterpene and triterpene biosynthesis, fatty acid biosynthesis, flavonoid and flavonol biosynthesis, tryptophan metabolism, fatty acid degradation, sphingolipid metabolism, isoflavone biosynthesis, ether lipid metabolism, pyrimidine metabolism, terpenoid biosynthesis, and indole alkaloid biosynthesis.The KEGG pathway of C35-vs-T35 KEGG metabolic pathways were mainly focused on histidine metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, \u0026beta;-alanine metabolism, monoterpene biosynthesis, and carotenoid biosynthesis. Among them, KEGG metabolic pathways at 28 d of drought were more involved, mainly including more than ten metabolic pathways.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolic network analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhospho-6-glucose is converted to acetyl coenzyme A and phospho-6-lactose, respectively (Fig. 19). Acetyl coenzyme A enters fatty acid metabolism and the metabolites involved in its pathway are stearic acid, \u0026alpha;-linolenic acid, linolenic acid, \u0026gamma;-linolenic acid, 17-hydroxylinolenic acid, arachidonic acid and jasmonic acid. Among them, the contents of \u0026alpha;-linolenic acid, \u0026gamma;-linolenic acid and stearic acid decreased, and the contents of arachidonic acid and jasmonic acid increased significantly with the prolongation of drought stress. Phospho-6-lactose enters the phenylalanine pathway by generating pyruvate, and involved in this metabolic pathway are asparagine, aspartic acid, valine, isoleucine, proline, histidine, and serine. The isoleucine and proline contents increased with the duration of stress, and after re-watering, the contents returned to the control level, implying that proline plays a crucial role in coping with drought stress. Amino acids and their derivatives are major factors in plant response to drought stress, and specific amino acids can retard protein degradation under drought stress. The contents of asparagine, aspartic acid and valine were highest at 7 d of drought stress, and the content of histidine decreased with the duration of drought stress. The large accumulation of amino acids and derivatives regulates plant drought stress tolerance through osmotic homeostasis and maintenance of the stability of the cell membrane structure. Phospho-6-lactose is converted to phenylalanine, a precursor of many key secondary metabolic pathways that directly affect cellular osmoregulation and improve plant drought tolerance, and subsequently to flavonoid metabolism. Flavonoids are an important class of secondary metabolites in plants with protective roles in plant development and in response to biotic and abiotic stresses. We identified nine metabolites associated with flavonoid metabolism including: hesperidin, naringenin, apigenin, lignan, kaempferol, quercetin, populin, epicatechin and rutin. The contents of the nine metabolites did not change significantly under normal conditions, and at 28 d of drought stress, naringenin, apigenin, lignans, kaempferol, rutin and epicatechin contents increased, while the changes of hesperidin and populin were not significant, suggesting that flavonoids have a key role in coping with drought stress. Vitamin D, tyrosine, homovanillic acid and 3-methoxytyramine were also detected in this study. Vitamin D3, Homovanillic acid and 3-methoxytyramine content increased at 28 d under drought stress, while tyrosine and sphingosine content decreased.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWeighted gene co- expression network analysis\u003c/strong\u003e\u003cstrong\u003e，\u003c/strong\u003e\u003cstrong\u003eWGCNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHierarchical clustering tree\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWeighted gene co-expression network analysis is a biological method for analyzing patterns of metabolite associations between different samples, allowing clustering of metabolites with similar expression patterns and analyzing associations between modules and specific traits and phenotypes. To further elucidate the complex regulatory network of\u0026nbsp;\u003cem\u003eB. chinensis\u003c/em\u003e in response to drought stress, total differential metabolites from the root system were used for weighted gene co-expression network analysis. A co-expression network was constructed using WGCNA to further explore the relationship between the 656 differential metabolites screened in the root system and root growth parameters and physiological and biochemical index parameters, and a total of nine metabolite modules were identified, with metabolites that did not belong to these modules indicated in gray (Fig. 20).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation analysis of modules with the matrix of physiological and biochemical traits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo delve into the modules associated with drought stress, the metabolites characterizing the metabolite accumulation profiles contained in each module were calculated and, in addition, each co-expressed module was correlated with growth and physiological and biochemical parameters by means of Pearson\u0026apos;s correlation coefficient. The Meblue (blue) module is the key module positively correlated with root activity, the Megreen (green) module is the module positively correlated with catalase, and the Meturquoise (turquoise) module is the key module positively correlated with malondialdehyde, catalase, soluble proteins, and relative conductivity (Fig. 21).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003eEffect of drought stress on phenotypic parameters of the root system of\u003c/b\u003e \u003cb\u003eB. chinensis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe effects of drought stress on phytomass are mainly manifested in plant growth and development. Normally, under drought stress, the number of lateral roots in the root system of \u003cem\u003eB. chinensis\u003c/em\u003e increased significantly and became thinner, and the color changed from white to yellow-brown, and the morphology recovered after re-watering, suggesting that the plasticity of the root system is a kind of adaptive mechanism for \u003cem\u003eB. chinensis\u003c/em\u003e to cope with the environmental stresses. Relative water content is an important measure of the water status of all parts of the plant and a reflection of the plant's ability to hold water. In this study, with the prolongation of the drought stress time, the relative water content of roots of \u003cem\u003eB. chinensis\u003c/em\u003e showed a decreasing trend, and after re-watering, it could be regulated by itself by different degrees of recovery, and this result was similar to the trend of the relative water content of the root system of subtropical conifers under drought stress [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDrought stress significantly affected the accumulation and distribution of plant biomass [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Shan [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] found significant changes in physiological traits and biomass allocation of Reaumuria soongorica root system under drought stress compared to control, similar results were obtained in this study. In this study, with the prolongation of drought stress, the root dry weight showed an increasing and then decreasing trend, while the dry weight of the aboveground part showed an overall increasing and then stabilizing trend. Under drought stress, the plant growth index's will also be changed accordingly, and the plant root system usually shows the corresponding morphological structure and physiological characteristics with the change of environment [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this study the diameter of root, the diameter of the mid-column, the thickness of the cortex, and the diameter of the xylem all showed an upward trend and then a downward trend, the root structure was impaired, the cortex was not arranged and ruptured, which could be attributed to the fact that in the early stage of the drought, the \u003cem\u003eB. chinensis\u003c/em\u003e enhanced the water uptake capacity by actively adjusting the root morphology (e.g., increasing the diameter of root and the cortex thickness), and that persistent drought resulted in the disorder of the osmotic regulation capacity, the cellular water loss increased, protoplasts contracted, and ultimately led to the damage of root system structure.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of drought stress on physiological and biochemical indexes of the root system of\u003c/b\u003e \u003cb\u003eB. chinensis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWhen plants are under drought stress, ROS production and removal will be out of balance. Drought leads to an increase in reactive oxygen radicals, subjecting plant cells to oxidative stress. When ROS exceed the capacity of the ROS scavenging system, it will cause the accumulation of MDA and oxidative damage, greater cell membrane permeability, and loss of intracellular substances, which will cause an increase in REC [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, MDA content and REC in roots gradually increased under drought stress and recovered after re-watering, which was similar to the findings of Filippou [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and Liu [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Plants subjected to drought stress reduce damage to themselves by increasing the levels of osmoregulators such as Sp and Pro. It was found that drought stress significantly increased the content of osmoregulatory substances in plants such as lily and Acorus calamus [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this study, Sp and Pro contents in the root system of \u003cem\u003eB. chinensis\u003c/em\u003e increased with the prolongation of drought stress, suggesting that the plant root system can protect itself from dehydration damage by accumulating large amounts of osmoregulators under drought stress.\u003c/p\u003e\u003cp\u003eUsually, to reduce the damaging effects of ROS, plants can regulate their drought tolerance through enzymatic reactions of antioxidant enzymes and the content of antioxidants in the ascorbate-glutathione cycle (ASA-GSH). Among them, the antioxidant enzymes SOD, POD, and CAT scavenge ROS, and in general, increasing enzyme activities under drought conditions are indicative of strong drought tolerance [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this study, it was found that SOD activity in the root system of \u003cem\u003eB. chinensis\u003c/em\u003e showed an increasing and then decreasing trend with drought stress time, and both POD and CAT activities in the root system showed an increasing trend, which was similar to the performance of the results of the study by Iqbal [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and both enzyme activities recovered after re-watering. Thus, the AsA-GSH cycle plays an important role in the antioxidant protection of plants under drought stress. In this study, the ASA-GSH cycle and the antioxidant enzyme activities that maintain the cycle homeostasis showed a gradual increase, and the results were similar to the findings of Shan [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] on drought tolerance in wheat.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of drought stress on metabolites in the root system of\u003c/b\u003e \u003cb\u003eB. chinensis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUnder adversity stress, plants regulate the content of multiple metabolites through a complex metabolic network, thus alleviating the damage caused by adversity stress. Meanwhile, the changes of metabolites in plants can be analyzed by metabolomics as a way to understand the response mechanism of plants to adversity stress [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].In this study, a total of 656 differential metabolites were screened by LC-MS metabolomics, and the changes of metabolites under drought stress were characterized, as well as the important metabolic pathways that might be involved in the process of adapting to the arid environment.\u003c/p\u003e\u003cp\u003eIt has been shown that amino acid accumulation can regulate plant defense against drought stress through osmotic homeostasis and maintenance of cell membrane structural stability [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. It was found that the amino acid content in the root system of wheat [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and sorghum [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] tended to increase after environmental stresses, e.g., drought stress increased the content of several free amino acids in the root system of sorghum. In this study, isoleucine, phenylalanine and tryptophan contents within the root system of \u003cem\u003eB. chinensis\u003c/em\u003e showed a significant increasing trend under drought stress, while histidine, valine, aspartic acid and asparagine contents all showed an increasing and then decreasing trend. This may be due to the disruption of cell membrane structure when the roots were subjected to severe drought stress, which led to an increase in amino acid content, suggesting that \u003cem\u003eB. chinensis\u003c/em\u003e roots can maintain the osmotic balance required for root growth by increasing isoleucine, phenylalanine and tryptophan content.\u003c/p\u003e\u003cp\u003eFlavonoids, as the most important secondary metabolites in plants, act as antioxidants in vivo and to some extent mitigate the damage caused by drought in plants [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In this study, flavonoids such as kaempferol, lignans, hesperidin, quercetin, lignans, naringenin, and rutin were significantly accumulated under drought stress.The results of the this study were similar to those of Rao [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and Hodaei [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], which indicated that the antioxidant capacity of the root system of \u003cem\u003eB. chinensis\u003c/em\u003e was increased, thereby balancing the excess free radicals produced by the drought stress. After re-watering, it recovered to the control level, showing the sensitivity of metabolites to drought stress, which reconfirmed the positive regulatory effect of flavonoids on drought resistance. However, some studies have also shown inhibition of flavonoid accumulation under severe drought stress [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In this study, both apigenin and mangiferin contents showed a decreasing trend with the extension of drought stress time, which might have affected some enzyme activities and thus led to the inhibition of flavonoids in the synthesis process.\u003c/p\u003e\u003cp\u003ePlant cell membranes are the interface between the cell and the external environment directly and are more sensitive to environmental changes. Degradation of phospholipid molecules occurs when plants are subjected to drought stress, and PC, PE, and PG are the main targets of degradation [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The present study showed that the trends of phospholipid molecules within the root system of \u003cem\u003eB. chinensis\u003c/em\u003e were not completely consistent under drought stress, suggesting that phospholipid molecules may respond to stress through different regulatory response pathways. Among them, the amount of PG was higher, suggesting that PG plays a major defense role in the plant root system under drought stress. In addition, abscisic acid is an essential hormone for maintaining root growth under drought conditions [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], and salicylic acid and jasmonic acid play a central role in plant defense against a wide range of adversities [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], and it has been shown that salicylic acid and jasmonic acid can reduce the level of peroxidation of plant cell membranes through the enhancement of the induced antioxidant system, removal of reactive oxygen species accumulated in cells that improves plant resistance [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The present study also found that abscisic acid, salicylic acid and jasmonic acid contents in the root system accumulated significantly with the duration of drought stress, suggesting that the hormone contents can be regulated to cope with the drought stress damage to plants.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRelationships between metabolites and root growth parameters and physiological and biochemical indices under drought stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn recent years, WGCNAs have played an important role in plant stress tolerance research, serving as an association between co-expression modules and traits with higher reliability and biological significance [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Metabolites with similar expression patterns were divided into different modules by dynamic shearing, and metabolites in the same module had similar expression patterns and were functionally related to each other. In this study, a total of nine co-expression modules were constructed by screening 656 differential metabolites, among which three modules (Blue, Green, and turquoise) were significantly correlated with each phenotypic and physiological indicator traits, respectively. A total of 292 metabolites were obtained in the relevant modules (blue, green, and turquoise modules), with the highest number of compounds being flavonoids (60) and glycerophospholipids (27). Flavonoids are important secondary metabolites involved in plant growth and stress tolerance, and many studies have shown that flavonoids have biological activities including anti-allergic, anti-viral, anti-inflammatory, and vasodilatory effects, as well as other medicinal properties [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], Glycerol lipids are the most abundant lipids in plants and are involved in response and tolerance to abiotic conditions such as drought, high and low temperatures [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Li [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] observed that many glycerophospholipids accumulated in large quantities in hulled barley under drought stress, and another study found large accumulations of several glycerophospholipids in response to drought in ryegrass [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], suggesting that flavonoids and glycerophospholipids play important roles in drought response, and that the related metabolites may be related to the growth of the plant root system and the physiological and biochemical indicators.\u003c/p\u003e\u003cp\u003eAlthough the physiological and biochemical response mechanisms and metabolomics analysis were conducted in this study on the root system of \u003cem\u003eB. chinensis\u003c/em\u003e under drought stress, there are some limitations in this study, in which only the enzyme activities of SOD, POD and CAT and the contents of MDA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003ewere measured, and the expression of key genes (antioxidant enzyme encoding genes) was not further verified Patterns. Therefore, the expression pattern of antioxidant enzyme genes such as SOD, POD, and CAT can be detected by qPCR in future studies to further explore the changes in antioxidant enzyme activities. Although the metabolite module was found to be phenotypically related in this study, no metabolism-phenotype regulatory network was constructed (e.g., how key metabolites affect root vigor or H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels), and genes or enzymes (e.g., PAL, CHS) regulated by key metabolites (e.g., kaempferol) can be predicted based on the metabolism-phenotype correlations in a subsequent study. In this study, we systematically analyzed the phenotypic, physiological and metabolic characteristics of the drought response of the root system of \u003cem\u003eB. chinensis.\u003c/em\u003e However, due to the limitations of the experimental conditions, there are some limitations, so further research on the physiological response mechanism of \u003cem\u003eB. chinensis\u003c/em\u003e under drought stress should be conducted in the future in conjunction with multigenomics, validation of gene function, and field experiments.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, two-year old \u003cem\u003eB. chinensis\u003c/em\u003e seedlings were used as materials to investigate the effects of drought stress on their growth and metabolism through potting water-control experiments. The results showed that the diameter of root, diameter of the mid-column, thickness of cortex and diameter of the xylem of the \u003cem\u003eB. chinensis\u003c/em\u003e under drought stress showed an increasing and then decreasing trend, while the relative water content and root activity showed a decreasing trend, and the root structure was severely damaged after 28 days, and partially recovered after re-watering. With the prolongation of stress, the relative electrical conductivity (REC), malondialdehyde (MDA), proline (Pro) and soluble protein (Sp) contents of roots increased significantly, while the activities of antioxidant enzymes were enhanced to maintain the balance of reactive oxygen species (ROS) and mitigate drought damage. Metabolomics analysis identified 656 root differential metabolites, mainly flavonoids, carboxylic acids and lipids, and KEGG enrichment showed that glycerophospholipid metabolism, amino acid metabolism and flavonoid biosynthesis pathways significantly responded to drought stress. WGCNA analysis further identified 292 key metabolites, among which flavonoids accounted for the highest proportion and were significantly correlated with stress indicators such as root activity, H₂O₂ and MDA, respectively, suggesting that flavonoids play a central role in regulating root growth and drought physiology. These findings provide a fundamental understanding of the drought stress response mechanisms in \u003cem\u003eB. chinensis\u003c/em\u003e .\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHH and YQ conceived and designed the project. ML, XZ, and XL performed the experiments. ML wrote the manuscript. ML, SY and YX analyzed experimental results. ML and YX revised the manuscript. HH and YQ supervised the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Talent Team Construction Platform Project of HAS (RC2023ZR01), Dual-Improvement Flying Goose Project of HAS (YZQY2023ZR01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article and its supplementary information files. All materials are available through corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang Z, Luo R, Wen Q, Liang X, Zhao H, Zhao Y, Huang F. Screening and functional verification of drought resistance-related genes in castor bean seeds. BMC Plant Biology 2024; 24(1): 493. https://doi.org/10.1186/s12870-024-04997-7.\u003c/li\u003e\n\u003cli\u003eYildirim K, Yagci A, Sucu S, Tun\u0026ccedil; S. 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Salinity and Water Stress 1990; 12: 113-146. https://doi.org/10.1007/978-1-4020-9065-3_5.\u003c/li\u003e\n\u003c/ol\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":"B. chinensis, Drought stress, Physiology, Response mechanism, Metabolome","lastPublishedDoi":"10.21203/rs.3.rs-6961345/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6961345/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eDrought has become an increasingly important environmental problem worldwide and is one of the major abiotic stress factors affecting crop growth and development. Therefore, elucidating the mechanism of drought resistance in plants is of great significance to improve the drought resistance of plants and to cultivate drought-resistant varieties. \u003cem\u003eB. chinensis\u003c/em\u003e is an important medicinal plant and garden plant in China, and drought is one of the factors limiting the growth of \u003cem\u003eB. chinensis\u003c/em\u003e, so it has become a research hotspot to investigate the physiological response mechanism of \u003cem\u003eB. chinensis\u003c/em\u003e to drought.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIn this study, we investigated the physiological and biochemical response mechanisms of the root system of \u003cem\u003eB. chinensis\u003c/em\u003e using \u003cem\u003eB. chinensis\u003c/em\u003e seedlings as experimental materials, and conducted metabolomics analysis of the root system of \u003cem\u003eB. chinensis\u003c/em\u003e using liquid chromatography-mass spectrometry (LC-MS) to verify the mechanisms of \u003cem\u003eB. chinensis\u003c/em\u003e in response to drought stress after 0, 7, 14, 21, 28 d of drought and after re-watering.The results showed that: (1) with the prolongation of drought stress, the relative water content gradually decreased, the diameter of root, the diameter of the mid-column, the thickness of the cortex, and the diameter of the xylem all showed an upward trend and then a downward trend, the root activity gradually declined, the root structure was impaired, the cortex was not arranged and ruptured. (2) With the prolongation of drought stress, the cell membrane function was impaired, the relative conductivity and malondialdehyde content of roots tended to increase, the content of osmoregulatory substances such as soluble proteins and free proline also accumulated in large quantities in the roots, and the content of hydrogen peroxide and superoxide anion gradually increased. Meanwhile, the activities of peroxidase, catalase, glutathione reductase, ascorbate peroxidase and monodehydroascorbate reductase in the roots reached the maximum value after 28 d of drought, and all of them recovered to a different extent after re-watering. (3) A total of 656 differential metabolites were screened in the root system of \u003cem\u003eB. chinensis\u003c/em\u003e under drought stress. In addition, KEGG enrichment analysis revealed that the metabolic pathways involved in the differential metabolites were mainly glycerophospholipid metabolism, sphingolipid metabolism, amino acid metabolism, tyrosine metabolism, arachidonic acid metabolism, flavonoid biosynthesis, steroid biosynthesis and tyrosine metabolism. Among them, the differences in flavonoid biosynthesis varied greatly, suggesting that flavonoids play an important role in the response to drought stress in \u003cem\u003eB. chinensis\u003c/em\u003e. (4) The weighted gene co-expression network analysis WGCNA revealed high correlations between the three metabolite modules and root growth parameters and physiological and biochemical index parameters. Among them, the Meblue module was positively correlated with root activity, the Megreen module was positively correlated with catalase, and the Meturquoise module was positively correlated with malondialdehyde, catalase, soluble proteins, and relative conductivity.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study reveals the defense response mechanism of \u003cem\u003eB. chinensis\u003c/em\u003e under drought, and also analyzes the differential metabolites of \u003cem\u003eB. chinensis\u003c/em\u003e under drought stress according to the changes in the quantity of related metabolites and explores the key metabolic pathways related to drought resistance, with a view to laying a theoretical foundation for physiological and metabolic studies revealing \u003cem\u003eB. chinensis\u003c/em\u003e response to drought stress.\u003c/p\u003e","manuscriptTitle":"The study of physiological response mechanism and metabolomics on B. chinensis under drought","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-21 15:31:35","doi":"10.21203/rs.3.rs-6961345/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-07T17:14:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-06T14:35:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-27T17:02:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287257096009876102427012687420127804154","date":"2025-07-17T13:32:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138260558220470775308410996403075605958","date":"2025-07-17T07:47:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-17T06:52:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-15T06:01:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-03T09:12:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-02T08:29:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-07-02T08:22:57+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":"55c44294-54d0-438f-aea4-cf693253a62c","owner":[],"postedDate":"July 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:12:23+00:00","versionOfRecord":{"articleIdentity":"rs-6961345","link":"https://doi.org/10.1186/s12870-025-07293-0","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-10-01 15:57:09","publishedOnDateReadable":"October 1st, 2025"},"versionCreatedAt":"2025-07-21 15:31:35","video":"","vorDoi":"10.1186/s12870-025-07293-0","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07293-0","workflowStages":[]},"version":"v1","identity":"rs-6961345","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6961345","identity":"rs-6961345","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}