Integrated transcriptomic and metabolomic analyses revealed that AmASMT positively regulates drought tolerance in Agropyron mongolicum by modulating melatonin biosynthesis

Integrated transcriptomic and metabolomic analyses revealed that AmASMT positively regulates drought tolerance in Agropyron mongolicum by modulating melatonin biosynthesis | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 16 April 2025 V1 Latest version Share on Integrated transcriptomic and metabolomic analyses revealed that AmASMT positively regulates drought tolerance in Agropyron mongolicum by modulating melatonin biosynthesis Authors : Jing Wang 0009-0004-2917-1409 , Jinqing Zhang , Shuxia Li , Shoujiang Sun , Wenxue Song 0009-0002-3102-6038 , Xing Wang , Xiaocong Li , Juhui Yan , Xueqin Gao , and Bingzhe Fu [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174479111.13749463/v1 Published Plant Physiology and Biochemistry Version of record Peer review timeline 168 views 127 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Global climate change has exacerbated drought stress episodes, which are emerging as a serious threat to plant growth and productivity worldwide. In this context, melatonin has emerged as a potential signaling molecule for improved drought tolerance in plants, primarily through enhanced antioxidant defenses. Here, physiological, transcriptome, and metabolome analyses were used to investigate the physiological and molecular mechanisms of melatonin in drought stress mitigation in A. mongolicum with both drought-tolerant and drought-sensitive genotypes. Physiological results suggest that melatonin improves drought tolerance in A. mongolicum primarily by enhancing the antioxidant enzyme system. Integrated transcriptomic and metabolomic analyses have demonstrated that the tryptophan metabolic pathway plays a crucial role in melatonin-mediated enhancement of drought resistance. Notably, we report on the drought-related gene AmASMT , which encodes a melatonin biosynthesis enzyme and contributes to drought stress tolerance in A. mongolicum . We found that the AmASMT overexpressing rice lines exhibited higher endogenous melatonin levels and increased tolerance to drought stress by promoting antioxidant systems. Our findings indicate that the AmASMT plays a crucial role in regulating melatonin biosynthesis A. mongolicum while facilitating protection against drought stress. These results provides a basis for exploiting melatonin-mediated mechanisms and genetic engineering approaches to enhance plant drought tolerance. Integrated transcriptomic and metabolomic analyses revealed that AmASMT positively regulates drought tolerance in Agropyron mongolicum by modulating melatonin biosynthesis Jing Wang 1 , Jinqing Zhang 1 , Shuxia Li 1,2 , Shoujiang Sun 1 , Wenxue Song 1 , Xing Wang 1 , Xiaocong Li 1 , Juhui Yan 1 , Xueqin Gao 1,2* and Bingzhe Fu 1,2,3* 1 College of Forestry and Pratacuture, Ningxia University, Yinchuan, Ningxia, China 2 Ningxia Grassland and Animal Husbandry Engineering Technology Research Center, Yinchuan, Ningxia, China 3 Key Laboratory for Model Innovation in Forage Production Efficiency, Ministry of Agriculture and Rural Affairs, Yinchuan, Ningxia, China * Correspondence: Bingzhe Fu; Xueqin Gao [email protected] ; [email protected] Abstract Global climate change has exacerbated drought stress episodes, which are emerging as a serious threat to plant growth and productivity worldwide. In this context, melatonin has emerged as a potential signaling molecule for improved drought tolerance in plants, primarily through enhanced antioxidant defenses. Here, physiological, transcriptome, and metabolome analyses were used to investigate the physiological and molecular mechanisms of melatonin in drought stress mitigation in A. mongolicum with both drought-tolerant and drought-sensitive genotypes. Physiological results suggest that melatonin improves drought tolerance in A. mongolicum primarily by enhancing the antioxidant enzyme system. Integrated transcriptomic and metabolomic analyses have demonstrated that the tryptophan metabolic pathway plays a crucial role in melatonin-mediated enhancement of drought resistance. Notably, we report on the drought-related gene AmASMT , which encodes a melatonin biosynthesis enzyme and contributes to drought stress tolerance in A. mongolicum . We found that the AmASMT overexpressing rice lines exhibited higher endogenous melatonin levels and increased tolerance to drought stress by promoting antioxidant systems. Our findings indicate that the AmASMT plays a crucial role in regulating melatonin biosynthesis A. mongolicum while facilitating protection against drought stress. These results provides a basis for exploiting melatonin-mediated mechanisms and genetic engineering approaches to enhance plant drought tolerance. Keywords Drought, melatonin, Agropyron mongolicum , transcriptome, metabolome, AmASMT . Introduction With rising global temperatures, drought stress is becoming one of the major environmental constraints that detrimentally affects plant growth and productivity worldwide (Cui et al., 2017). Drought stress triggers multiple physiological and metabolic changes, such as inhibition of photosynthesis, interruption of carbohydrate metabolism, protein degradation, lipid peroxidation, and dysregulation of osmolarity, by disrupting the water balance and decreasing the efficiency of plant water utilization (Liang et al., 2018). Meanwhile, the molecular changes under drought stress lead to the expression of drought-responsive genes in plants, such as functional genes and regulatory genes related to osmotic adjustment, specific protein synthesis, signal transduction, transcription factors (Yang et al., 2021). Interestingly, plants have evolved different strategies to alleviate the deleterious effects of drought and to defend themselves against the damage of excessive reactive oxygen species (ROS), such as increasing the content of antioxidants, osmotic regulators, and plant growth substances (Naudts et al., 2014). However, this inherent defense system of the plant is activated only at a certain tolerance level. Natural defenses are disrupted under severe and prolonged drought stress conditions, ultimately resulting in death. Therefore, several studies have been carried out by previous researchers to improve the drought resistance of plants (Begum et al., 2019). These studies mainly include the application of phytoprotectant, such as osmoprotectants, antioxidant compounds, and growth promoters, which are believed to be effective in enhancing plant adaptability to drought stress (Farooq et al., 2009; Kosar et al., 2021). Melatonin (MT) is a naturally occurring indolamine that is emerging as a versatile phytoprotectant biomolecule in mitigating the abiotic and biotic stresses of plants (Tiwari et al., 2020). Since its discovery in plants in the 1990s, MT has been implicated in multiple processes of plant development, such as promoting photosynthesis, participating in root regeneration, regulating circadian rhythms, regulating floral development and fruit ripening, and delaying leaf senescence. Moreover, MT is a well-known antioxidant in plants, which is documented to enhance the tolerance of drought, saline and alkaline ions, heavy metals, ultraviolet radiation, chilling, heat stress, and viral and fungal infections (Arnao and Hernandez-Ruiz, 2015). Remarkably, MT strengthens antioxidant defenses and improves drought tolerance in multiple plant species. In the recent past, extensive studies have been conducted to unravel the role of exogenous and endogenous MT in the drought stress mitigation in plants. These studies demonstrated that the pretreatment of MT as a root dipping and foliar spray regulates physiological processes such as germination, osmoregulation, photosynthesis, hormonal cross-talks, and antisenescence primary and secondary metabolism in plants under drought stress (Cui et al., 2017; Liang et al., 2019). Meanwhile, research of the molecular mechanism of MT in mitigating drought stress in plants indicated that MT reinforces drought tolerance in plants primarily through regulating the hormone metabolism, MAPK pathway, flavonoid biosynthesis, photosynthetic system, carbohydrate metabolism, and ascorbate-glutathione cycle (Cui et al., 2017; Moustafa-Farag et al., 2020). Furthermore, exogenous MT promotes drought tolerance by regulating the metabolic homeostasis and the extensive transcriptional reprogramming of genes related to drought stress tolerance, including transcription factors, stress receptors, and genes involved in hormone signaling pathways linked to drought tolerance (Shi et al., 2015). Thus, the exogenous addition of MT can be an effective strategy to enhance drought tolerance in plants. MT is biosynthesized within the chloroplasts and mitochondria of plants (Tan and Reiter, 2020). Despite distinct enzymes and reaction stages have been identified in the MT biosynthesis pathways of plants and animals (Back et al., 2016), tryptophan has been universally confirmed as the sole precursor for MT biosynthesis across all organisms (Schmid and Amrhein, 1995). The MT biosynthesis pathway in plants involves four different enzymes: tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), serotonin N-acetyltransferase (SNAT) and N-acetylserotonin methyltransferase (ASMT) (Liu et al., 2017). There is compelling evidence to suggest that ASMT is a major rate-limiting enzyme for MT biosynthesis in plants (Kang et al., 2011; Park et al., 2013; Arnao and Hernández-Ruiz, 2014; Wang et al., 2014; Cen et al., 2020). Liu et al. (2017) systematically characterized the tomato ASMT gene family and elucidated its functional roles in abiotic stress responses. Subsequent genetic studies have further confirmed the involvement of the ASMT gene in the stress tolerance mechanism. Transgenic tomato plants overexpressing the ovine ASMT gene demonstrated elevated MT accumulation and enhanced tolerance to drought stress conditions (Wang et al., 2014). Zuo et al. (2014) successfully cloned the ASMT gene from apple rootstock ( Malus zumi Mats ) and introduced it into Arabidopsis, demonstrating that ASMT overexpression enhanced endogenous MT levels and improved drought stress tolerance. Collectively, ASMT plays a crucial role in plant developmental processes and in mediating responses to diverse abiotic stresses. Agropyron mongolicum is an important perennial herb in forage production and ecological construction (Che and Li, 2007). The ecological amplitude of A. mongolicum is very extensive, and it is distributed mainly in the desert steppes of central and western China. A. mongolicum is required to become more drought-tolerant as a critical species for the improvement of deteriorated grasslands and the reseeding of desert grasslands. Therefore, it is particularly necessary to effectively improve the drought tolerance of A. mongolicum to adapt to the harsh growing conditions (Zhao et al., 2018). The role of MT in plant drought stress has been well reported. However, the underlying mechanism by which MT mitigates drought stress in A. mongolicum remains largely unknown. In the present study, physiological and molecular mechanisms of MT mitigation of drought stress in A. mongolicum with drought-tolerant and drought-sensitive genotypes were investigated using physiological, transcriptomic, and metabolomic analyses, providing potential target metabolites and genes for MT enhancement of drought tolerance in A. mongolicum . In addition, we further clone the key gene for MT synthesis, AmASMT , from A. mongolicum and elucidate its potential function under drought stress. These findings help us to better reveal the molecular mechanisms by which MT mitigates drought stress in A. mongolicum and provide a reference for the exploitation and utilization of MT and the genetic modification for drought tolerance in plants. Materials and methods Plant materials and treatments In a preliminary experiment, we analyzed the growth and physiological properties of 88 genotypes of A. mongolicum under drought stress. These genotype accessions were originally procured from the Institute of Grassland Research, Chinese Academy of Agricultural Sciences, and are being actively maintained at Ningxia University. Through the utilization of a subordinative function method, we identified drought-tolerant genotype ‘A84’ (T) and drought-sensitive genotype ‘A72’ (S) (Supplementary Table S1, S2). In the current study, the T and S genotypes were used as experimental material. The seeds of the T and S genotypes seeds were sterilized with 10% sodium hypochlorite for 10 minutes, and sowed in a nutrient bowl (7×7×8 cm) containing fine sand (121℃, high-temperature sterilization for 20 min). The nutrient bowl was placed in a hydroponic box (25×15×10 cm) and then cultivated in an incubator (photoperiod of 12 h /12 h (light/dark), and light intensity of 5000 lx, temperature of 25±1℃ (day) / 20±1℃(night), humidity of 60%-70%). The seeds were watered daily to ensure their normal germination. After 10 days, the seedlings were thinned, and eight seedlings with uniform growth and distribution were retained in each bowl. Afterwards, 1.5L of Hoagland nutrient solution is poured into each hydroponic box to ensure normal growth of the seedlings. When the seedlings were grown for 30 days, the leaves were pre-sprayed with MT solution for 7 days. Because MT decomposes easily under light, it needs to be sprayed at night. Each hydroponic box was sprayed with 50 mL of 0, 1, 10, 50, 100, 150, 200 mg·L -1 MT every day. After pre-spraying for 7 days, the Hoagland nutrient solution containing 25% polyethylene glycol 6000 (PEG) was used to simulate drought stress. At the same time, the leaves were sprayed with distilled water for 7 days and then cultured in normal Hoagland nutrient solution as a control treatment. After that, the plant leaves were sampled on the 7th day of drought stress treatment and immediately frozen in liquid nitrogen and stored at -80℃ for physiological characteristics analysis. The leaves of the T and S genotypes at the optimal concentration of MT were selected for transcriptome and metabolome determination. Thus, the following treatments were studied and analyzed: (1) The T/S genotype was pre-sprayed with distilled water for 7 days and then cultured with Hoagland nutrient solution for 7 days (T-C/S-C); (2) The T/S genotype was pre-sprayed with distilled water for 7 days and then cultured in Hoagland nutrient solution containing 25% PEG for 7 days (T-D/S-D); (3) The T/S genotype was pre-sprayed with 100 mg·L -1 MT for 7 days and then cultured in Hoagland nutrient solution containing 25% PEG for 7 days (T-MD/S-MD). Growth parameter The plant height was measured with a ruler; The aboveground part of each seedling was sampled to determine its fresh weight; The relative water content (RWC) of leaves was determined by the saturated weighing method weighing method (Mullan and Pietragalla, 2012). Determination of physiological characterization Chlorophyll was extracted from 0.1g fresh leaf tissue and determined by measuring the absorbance at 663 nm and 645 nm using Victor Nivo Multimode Plate Reader (PerkinElmer LLC, America) as described previously (Wellburn and Lichtenthaler, 1984). The relative electrical conductivity (REC) of leaves was determined using the method described by Wu et al (Wu et al., 2017). The malondialdehyde (MDA) content was determined by thiobarbituric acid method with MDA Assay Kit (BC0025; Solarbio, Beijing, China); The proline (Pro) content was determined by the acid-ninhydrin method with Pro Assay Kit (BC0295; Solarbio, Beijing, China). Endogenous reactive oxygen species (ROS) including hydrogen peroxide (H 2 O 2 ) and superoxide radical (O 2 - ), which were quantified using the H 2 O 2 Assay Kit (BC3595; Solarbio, Beijing, China) and O 2 - Assay Kit (BC1295; Solarbio, Beijing, China). The antioxidant enzyme activities including ascorbate peroxidase (APX), superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), which were quantified using APX activity Assay Kit (BC0220; Solarbio, Beijing, China), SOD activity Assay Kit (WST-1) (BC5165; Solarbio, Beijing, China), POD activity Assay Kit (BC0090; Solarbio, Beijing, China) and CAT activity Assay Kit (BC0200; Solarbio, Beijing, China), respectively, according to the manufacturer’s instruction. Endogenous MT quantification The content of MT was determined by high performance liquid chromatography (HPLC) (S6000, Acchrom, China). About 0.2 g of the sample was weighed and 1 mL of 80% methanol was added. The sample was ground and extracted overnight and then centrifuged to obtain the supernatant. The mobile phase was prepared by a 4:6 ratio of methanol to a 0.1% formic acid solution. After setting the parameters, the moving phase was used to cross the column and the samples were added after the baseline was stabilized. RNA extraction and sequencing Total RNA was extracted from the leaves of T and S genotypes after exogenous addition of MT under drought stress with Trizol reagent (TRI reagent, Sigma-Aldrich, USA). Subsequently, the total RNA was identified and quantified using a Qubit fluorescence quantifier (Qubit 4.0, Thermo Fisher, America) and a high-throughput biofragment analyzer (Qsep400, Guangding Biological, China). In the library construction, the mRNAs with polyA tail enriched by Oligo (dT) magnetic beads were randomly disrupted into small fragments, and then the fragmented mRNAs were used as a template to synthesize cDNAs. The purified double-stranded cDNAs were again screened for PCR amplification and PCR product purification to finally obtain the library. After the construction of the library, the concentration of the library is accurately quantified by qRT-PCR. Finally, the Illumina HiSeqTM 2000 system (Illumina, San Diego, CA, USA) was used for sequencing. The clean reads were obtained after filtering the raw data using fastp v 0.19.3. Transcriptome assembly of clean reads was performed using Trinity (https://github.com/trinityrnaseq/trinityrnaseq). The expression level of genes was calculated using RSEM software, and the fragments per kilobase of transcripts per million (FPKM) of each gene was calculated. FDR<0.05 and |log 2 Fold Change|≥1 were used as the threshold for significant differential expression genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed based on the hypergeometric test. For Gene Ontology (GO), it was performed based on the GO term. Metabolites analysis by LC-MS/MS The stored leaves samples were freeze-dried and ground to a fine powder, and sample powder was dissolved in 70% methanol for extraction. The obtained filtrates were was used for the determination of broadly targeted metabolites. For the relative quantification of metabolites, a pre-determined multiple reaction monitoring method was used. To ensure the reliability of the data, three biological replicas were set for each sample. Differentially expressed metabolites (DEMs) between comparison groups were determined by Variable Importance in Projection (VIP)>1, fold change≥2 and fold change≤0.5. VIP values were extracted from Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) results. The identified metabolites were annotated using the KEGG Compound database and mapped to the KEGG Pathway database. Then the pathway mapped by DEMs were input into the metabolite set enrichment analysis (MSEA), and its significance was determined by the p-value of the hypergeometric test. Subcellular localization of AmASMT The full-length open reading frame (ORF) of the Cluster-43602.0 ( AmASMT ) gene sequence was cloned. A recombinant vector, pBWA(V)HS- AmASMT -GFP, was constructed and transformed into rice protoplast. Primers used are listed in Table S3. After 12 to 20 h of culture, the location of the fluorescence signal was captured using a confocal laser scanning microscope (LSM710, Zeiss, Germany). Overexpression AmASMT of rice The overexpression vectors pBWA(V)HS- AmASMT- GFP was constructed and then transformed into wild type (WT) Nipponbare rice plants mediated by EHA105. Primers used are listed in Table S3. The positive T1 generation plants were treated with drought stress. Rice calluses were inoculated on MS media containing hygromycin to select T0 generation transgenic plants. The overall process consisted of precultivation, agrobavterium infection, screening, differentiation, and rooting. After that, the positive T0 generation seeds were sown and cultured to obtain positive T1 generation plants, and the seedlings were treated with drought stress. Quantitative real-time PCR analysis Total RNA was extracted from the A. mongolicum leaves using an RNA extraction Kit (Tiangen, Beijing, China) following the manufacturer’s instructions. cDNA was synthesized from RNA with the ReverTra Ace qPCR RT Kit (Toyobo, Tokyo, Japan). The qRT-PCR analyses were performed using a C1000 TouchChihermal Cycler system (Bio-Rad) with BioEasy Master Mix (SYBR Green) Kit (Bioer, Hangzhou, China). Gene-specific primers are listed in Supplementary Table S3. To normalize gene expression levels, TLF in A. mongolicum and OsActin1 in rice were used as their internal reference genes, respectively, and relative gene expression was calculated based on previous studies(Tian et al., 2016; Sahoo et al., 2022). Effects of exogenous MT on physiological properties of A. mongolicum under drought stress To determine whether MT can alleviate the damage in A. mongolicum seedlings under drought stress, PEG and MT treatments were performed on both T and S genotypes and their phenotypic and physiological characteristics were analyzed. The preliminary tests for PEG concentration screening (0, 10%, 15%, 20%, 25%, 30% PEG) showed that with the increase of PEG concentration, the aboveground fresh weight, aboveground dry weight, chlorophyll content and RWC of T and S genotypes showed a significant downward trend, while the REC showed a significant upward trend. However, the change range of each index is different under various PEG concentration gradients, and when the PEG concentration reaches 25%, the change range is the largest (Supplementary Figure S1). Therefore, 25% PEG was selected to simulate drought stress treatment in subsequent experiments. At the same time, the RWC of the T and S genotypes at 25% PEG stress was also dynamically monitored. During the first 7 days of drought stress treatment, the RWC of the seedling leaves decreased significantly as the number of treatment days increased, but tended to stabilize later in the drought stress treatment period. The 7th day was the “dividing ridge” of this phenomenon (Supplementary Figure S2), so the 7th day was selected as the time node of drought stress treatment. Furthermore, to investigate the response of A. mongolicum seedlings to MT under drought stress, the physiological characteristics of the T and S genotypes of A. mongolicum seedlings treated with different concentrations of MT under drought stress were studied. As shown in Figure 1A-L, drought stress significantly suppresses the growth of A. mongolicum seedlings, with the S genotype being more suppressed than the T genotype. However, MT application dramatically mitigates the damage caused to seedlings by drought stress. Exogenous addition of different concentrations of MT significantly increased the aboveground fresh weight, chlorophyll content, and RWC, and decreased REC, MDA, H 2 O 2 , and O 2 - content in the T and S genotypes under drought stress. Moreover, the activities of four key antioxidant enzymes consisting of APX, SOD, POD, CAT and the content of osmotic adjustment substance of Pro were significantly increased under drought stress, and were further increased by different MT concentrations, and reached the highest level at 100 mg·L −1 MT. After MT treatment, the above indexes were different between T and S genotypes, especially the content of MDA, Pro, H 2 O 2 and O 2 - were significantly different between the two genotypes with different drought resistance. These results indicated that the addition of various concentrations of MT could alleviate the damage of A. mongolicum to drought stress to a certain extent, and 100 mg·L −1 MT had the most significant mitigation effect on drought stress of T and S genotype. Effects of exogenous MT on phenotype and endogenous MT content of A. mongolicum under drought stress Exposure of the T and S genotypes to drought stress resulted in obvious foliation damage, manifested in large-scale leaf wilting and necrosis, with drought stress causing more severe damage to the S genotype. In turn, obvious mitigating effects were observed in 100 mg·L -1 MT-pretreated plants exposed to drought stress (Figure 1M). MT content in the leaves of the T and S genotypes under drought stress was not significantly different from the control. The addition of 100 mg·L -1 MT treatment significantly increased the endogenous MT content in the leaf compared to drought stress, with a higher increase in S genotype than in T genotype (Figure 1N). Transcriptome profile of MT in response to drought stress in A. mongolicum To investigate the transcriptional changes of MT in alleviating drought stress in A. mongolicum , 18 cDNA libraries were constructed from T-C, T-D, T-MD, S-C, S-D, S-MD treated leaves of T and S genotypes. More than 5G clean reads were generated from each sample after removing low-quality reads as well as those containing poly-N and adaptors. The percentage of GC was 55.18 to 57.04%, and the Q30 base was above 93.87% for each sample, indicating high data quality and purity for transcriptome sequencing (Supplementary Table S4). Differential analysis showed that a total of 7034 (4611 up- and 2423 down-regulated), 7856 (2568 up- and 5288 down-regulated), 6900 (3817 up- and 3083 down-regulated) and 6833 (1947 up- and 4886 down-regulated) genes were differentially expressed in T-D vs T-C, T-MD vs T-D, S-D vs S-C, S-MD vs S-D comparisons, respectively (Figure 2A, Supplementary Table S5). In all, 329 DEGs overlapped among these four comparisons, while 2508 DEGs were shared between T-D vs T-C and T-MD vs T-D comparisons, and 2200 DEGs were shared between S-D vs S-C and S-MD vs S-D comparisons (Figure 2B). GO and KEGG enrichment analysis To further clarify the functions and characteristics of these DEGs in response to MT, GO and KEGG enrichment analyses were performed on these genes (Supplementary Table S6). GO analysis shows the top 20 enrichment categories for biological processes, cellular components, and molecular functions. When the addition of MT under drought stress, the largest proportion of DEGs was enriched for biological processes such as “cinnamic acid biosynthetic process”, “cinnamic acid metabolic process”, “positive regulation of defense response to bacterium”, “L-phenylalanine catabolic process and erythrose 4-phosphate/phosphoenolpyruvate family amino acid catabolic process” (Supplementary Figure S3). KEGG enrichment analysis revealed the key biological pathway of MT in A. mongolicum that alleviates drought stress. Figure 2C shows 18 pathways that are significantly enriched in the leaves of the T and S genotypes treated with MT under drought stress. The “phenylpropanoid biosynthesis”, “phenylalanine metabolism”, “MAPK signaling pathway-plant” pathways were enriched in the T-MD vs T-D group. At the same time, the “phenylpropanoid biosynthesis”, “starch and sucrose metabolism”, “plant-pathogen interaction”, “MAPK signaling pathway-plant” pathways were significantly enriched in the S-MD vs S-D group. To further characterize the molecular interaction between DEGs, we screened five KEGG pathways that were significantly enriched in the T-MD vs T-D and S-MD vs S-D comparison groups, and showed the six key genes with the largest |log 2 FC| in each pathway (Figure 2D, E). The detailed information on key genes is shown in Supplementary Table S7. Analysis of transcription factors Changes in gene expression ultimately regulate the response mechanism of A. mongolicum leaves to MT under drought stress. This research screened 4323 transcription factors (TFs) from 90 different families. After adding MT treatment under drought stress, T and S genotypes were annotated with more genes in TFS such as AP2/ERF, WRKY, NAC, bHLH, C2H2 and FAR1 (Supplementary Table S8, Figure S4). These TFs are involved in a variety of biological pathways such as plant growth and development, secondary metabolism and synthesis, and stress resistance. Metabolome profile of MT in response to drought stress in A. mongolicum Widely targeted metabolomes were used to obtain MT responses to drought stress in leaves of T and S genotypes. The PCA plots show a clear separation between the different treatments for the two genotypes, indicating that the leaf metabolites of the two genotypes chang between the different treatments, with a small separation between the three biological replicas. In addition, the metabolism of T-MD and S-MD treatments was significantly separated in the PC1, demonstrating that the effect of MT on the metabolism of T and S genotypes under drought stress was obvious (Figure 3A). A total of 999 DEMs were detected in all samples, which were mainly classified into flavonoids (232), amino acids and derivatives (161), alkaloids (130), phenolic acids (122), lipids (115), organic acids (43), lignans and coumarins (42), terpenoids (34), nucleotides and derivatives (21), quinones (6), Tannins (4) and others (89) (Figure 3B, Supplementary Table S9). Differential analysis showed that the number of metabolites in T-D vs T-C, T-MD vs T-D, S-D vs S-C and S-MD vs S-D comparisons were 435 (377 up- and 58 down-regulated), 167 (62 up- and 105 down-regulated), 529 (364 up- and 165 down-regulated), and 367 (160 up- and 207 down-regulated), respectively. In all, 27 DEMs were overlapped among these four comparisons. (Figure 3C, Supplementary Table S10). KEGG enrichment analysis of DEMs showed that the “flavonoid biosynthesis”, “luteolin aglycone biosynthesis”, “flavone and flavonol biosynthesis” and “tryptophan metabolism” pathways were significantly enriched in the T-D vs T-MD and S-D vs S-MD comparisons. In addition, “plant hormone signal transduction”, “biosynthesis of secondary metabolites” and “isoflavonoid biosynthesis” were significantly enriched in T-D vs T-MD, while “chrysoeriol aglycones biosynthesis”, “tryptophan metabolism” and “flavone and flavonol biosynthesis” were significantly enriched in S-D vs S-MD (Figure 3D). Thus, these metabolites involved in the aforementioned metabolic pathways may play an important role in MT alleviating drought stress in A. mongolicum . Integrated analysis of the transcriptome and metabolome of A. mongolicum treated with MT under drought stress To gain a comprehensive understanding of the changes in the DEGs and DEMs of the two genotypes of A. mongolicum after adding MT under drought stress, we constructed an overall pathway network based on the literature and pathway databases. And we focus on the tryptophan metabolism pathway by performing an integrated analysis of the transcriptome and metabolome (Supplementary Table S11). KEGG enrichment analysis indicated that multiple DEGs and DEMs were significantly enriched in tryptophan metabolism (Supplementary Table S12). Tryptophan metabolism genes were largely up-regulated by drought stress, but largely down-regulated after the addition of MT (Figure 4A). In the T genotype, two DEGs encoding amidase (AMD) and two DEGs encoding indole-3-pyruvate monooxygenase (YUCCA) were significantly up-regulated under drought stress, but MT treatment down-regulated the expression of these genes. However, the expression of one DEG encoding acetyl-CoA C-acetyltransferase (ACAT), one DEG encoding YUCCA and one DEG encoding acetylserotonin O-methyltransferase (ASMT) was the opposite. They were all significantly down-regulated under drought stress, and expression of these genes was up-regulated after the addition of MT. In the S genotype, one DEG encoding aldehyde dehydrogenase (ALDH), one DEG encoding tryptamine 5-hydroxylase (T5H), one DEG encoding AMD, one DEG encoding tryptophan decarboxylase (TDC) and five DEGs encoding YUCCA were significantly up-regulated under drought stress, while MT treatment down-regulated the expression of these genes. However, the expression for one DEG encoding AMD, one DEG encoding YUCCA, and two DEGs encoding ASMT are reversed. They were all significantly down-regulated under drought stress treatment, and the expression of these genes was up-regulated after adding MT. Interestingly, one DEG encoding ACAT and one DEG encoding YUCCA in both T and S genotypes were significantly up-regulated after MT treatment, while seven DEGs encoding AMD, five DEGs encoding TDC and six DEGs encoding YUCCA were significantly down-regulated after MT treatment in both T and S genotypes. Metabolites involved in tryptophan metabolism, such as indole, tryptamine, N-hydroxytryptamine, N-methyltryptamine and N-methylserotonin, were increased by drought stress treatment in both the T and the S genotypes, but decreased after MT treatment (Figure 4B). However, N-acetylisatin, N-acetylserotonin, melatonin and 6-hydroxymelatonin were all increased by MT treatment in both the T and the S genotypes. In addition, picolinate and tryptophan were significantly increased under drought stress in both T and S genotypes, while only in S genotype there was a significant decrease after MT treatment compared to drought stress treatment. These results suggest that tryptophan metabolism is involved in the MT response to drought stress in A. mongolicum . AmASMT overexpression increase MT accumulation and improves drought tolerance in rice Through an integrated analysis of the transcriptome and metabolome, we identified tryptophan metabolism as a critical pathway in MT regulation of drought tolerance in A. mongolicum . ASMT is a key enzyme in the tryptophan metabolic pathway responsible for MT synthesis, so we investigated the role of the gene involved in MT synthesis, AmASMT , in drought resistance regulation in rice using heterologous overexpression. The AmASMT sequence was successfully amplified by PCR to a size of 1089 bp (Figure 5A). To characterize the function of AmASMT in rice, we performed subcellular localization. Transient expression of pBWA(V)HS- AmASMT -GFP fusion protein in rice protoplasts showed that AmASMT protein was localized in the nucleus and cytoplasm (Figure 5B). To investigate the role of AmASMT in drought conditions, we created transformants that overexpress AmASMT . The qRT-PCR results showed a significant increase in the levels of AmASMT transcripts in AmASMT overexpressed rice compared to WT plants (Figure 5C). And we selected lines with high AmASMT gene expression (OE2, OE3 and OE6) for further analysis. Next, we analyze the phenotypes of the AmASMT -OE lines before and after drought treatment. Morphologically, there was no apparent difference between the WT and AmASMT -OE under normal conditions. However, under drought stress, the WT plants displayed noticeable wilting of leaves compared with those of the AmASMT -OE lines (Figure 5D), indicating that overexpressing AmASMT enhances drought tolerance. AmASMT expression can be significantly induced by drought stress, and the expression of AmASMT is significantly higher in AmASMT -OE lines than in WT plants (Figure 5E). Since AmASMT is a critical enzyme in the MT biosynthesis, we next measured the endogenous MT content in rice AmASMT -OE lines. Both WT plants and AmASMT -OE lines showed significant increases in MT levels under drought stress, with the AmASMT -OE lines showing significantly higher MT levels under normal conditions and drought stress than the WT plants, suggesting that increased AmASMT transcript levels enhance the MT accumulation in rice (Figure 5F). Analysis of growth and physiological characteristics showed that, in alignment with the phenotype, the AmASMT -OE lines showed higher plant height and fresh weight than the WT under drought treatment (Figure 6A, B). In addition, under drought stress, the relative water content of the AmASMT -OE lines increases and the relative electrical conductivity decreases compared to WT plants (Figure 6C, D). AmASMT -OE lines had lower levels of MDA and H 2 O 2 , higher levels of Pro, and higher activity of SOD and CAT than WT plants (Figure 6E-I). These results suggest that rice plants that overexpress the AmASMT gene grow better, have a stronger ability to scavenge ROS, and are more drought tolerant. qRT-PCR validation To validate the RNA-seq results, qRT-PCR was selected and used to quantify some crucial genes. The results showed that the expression levels of most genes were consistent with the RNA-seq data (Supplementary Figure S5), which verified the reliability and reproducibility of the RNA-seq data. Discussion Physiological characteristics of exogenous MT alleviating drought stress in A. mongolicum Plants are immobile organisms that are exposed to a variety of environmental conditions, including biotic and abiotic stresses. The antioxidant and tolerance mechanism at the cellular and molecular level regulates these stresses in plants (Tiwari et al., 2020). Drought stress compromises the integrity and function of plant cellular membranes, disrupting redox homeostasis and subsequently altering physiological and biochemical processes (Sattar et al., 2020). However, plants have developed a variety of defense mechanisms, such as the osmoregulatory system and the antioxidant defense system, which may lead to cellular homeostasis and adaptation when exposed to mild drought stress, or to programmed cell death when exposed to severe long-term drought stress. Exogenously applied MT has been recently considered to exert priming effects on some plants exposed to drought, as well as other stresses, as MT treated plants indicated higher biomass production, and photosynthetic capacity, compared to stressed plants without MT treated (Antoniou et al., 2017). Moreover, MT treatment enhances drought tolerance by activating the antioxidant defense system, functioning both as a direct ROS scavenger and as a regulator of enzymatic and non-enzymatic antioxidant mechanisms (Wang et al., 2013). In this study, drought stress treatment significantly inhibited the growth of A. mongolicum seedlings and had a greater effect on the S genotype was greater than that of the T genotype. Application with 100 mg·L -1 MT mitigated detrimental effects of drought stress in T and S genotypes, as evidenced by increased aboveground fresh weight, RWC and chlorophyll content, less ROS accumulation, reduced REC and MDA contents, and enhanced antioxidant and osmotic adjustment capacity in MT-treated seedlings compared to drought stress treatment seedlings (Figure 1A-L). Consistent with our findings, the mitigation role played by MT against drought stress has been demonstrated in various important plants, such as alfalfa ( Medicago sativa L.) (Antoniou et al., 2017), wheat ( Triticum aestivum L.) (Cui et al., 2017), maize ( Zea mays L.) (Zhao et al., 2021), naked oat ( Avena nuda L.) (Gao et al., 2019), and apple ( Malus domestica Borkh.) (Liang et al., 2018). Furthermore, the induction of endogenous MT levels in drought-stressed A. mongolicum revealed MT synthesis in response to drought. The exogenous application of MT significantly increased the endogenous MT content in the T and S genotypes compared to the control, suggesting the absorption of exogenous MT and thus the successfully utilizing of the compound. And the increase of endogenous MT content in the S genotype was higher than that in the T genotype, which indicated that the S genotype was more sensitive to MT (Figure 1N). A recent report has demonstrated that drought resulted in increased endogenous MT content in soybean ( Glycine max L.) (Cao et al., 2023). It is worth noting that relevant MT contents in drought-stressed plants of the two researches are not directly comparable, partly due to the degree of drought stress in the respective experimental, while monocotyledonous are also known to be more sensitive to MT than dicotyledonous or woody (Zhang et al., 2016). In summary, the exogenous addition of an appropriate concentration of MT can alleviate the growth inhibition of two A. mongolicum seedlings with different drought tolerance under drought stress by affecting physiological effects. Exogenous MT enhances drought tolerance by modulating tryptophan metabolism Drought stress perturbs plant metabolism and provokes diverse cellular responses, manifesting as alterations in the plant transcriptome and metabolome that ultimately lead to significant changes in chemical composition (Zhang et al., 2014). Integrated analyses of the transcriptome and metabolome provide a more precise link between gene regulation and metabolite production for understanding how plants respond to changing environments. In the present study, through a integrated analysis of genes and metabolites, we identified the tryptophan metabolism pathway as the critical pathways for MT to alleviate the drought stress of A.mongolicum . The tryptophan metabolism pathway plays an important role in plant growth and development, adversity stress, and secondary metabolism. Intensive studies of plant tryptophan metabolism pathways have been helpful in shedding light on the molecular mechanisms of plant growth and development and adversity adaptation. Tryptophan is an essential amino acid and a component of different proteins, and MT biosynthesis begins with tryptophan (Back et al., 2016). The first step of the MT biosynthesis process in plants is related to the formation of serotonin from tryptophan, and may involve two different pathways. The first pathway is the decarboxylation of tryptophan into tryptamine by TDC, which is then is hydroxylated into serotonin by T5H. On the other hand, another possibility is that tryptophan is hydroxylated into 5-hydroxytryptophan by TPH, which is then 5-hydrotryptophan is decarboxylated into 5-hydroxytryptamine by TDC. However, it has been demonstrated that decarboxylation is more frequent than hydroxylation as the first step in plants (Back et al., 2016). The results of this study showed that in the first way in which TDC catalyzed the conversion of tryptophan to serotonin, tryptophan and intermediate metabolite tryptamine were consistent with most of the gene changes that regulated TDC, which were up-regulated under drought stress and down-regulated after the addition of MT (Figure 4). T5H is responsible for adding a hydroxyl group to the 5-position of tryptamine. This reaction causes the formation of 5-hydroxytryptamine (Fujiwara et al., 2010). The expression pattern of a gene that regulates T5H in our study is consistent with that of most TDCs. Previous studies of MT biosynthesis genes in apples have also shown that TDC and T5H are up-regulated after drought treatment. In contrast to the enhanced MT production, overexpression of the T5H gene in rice plants reduced MT synthesis, which is similar to the results of the present study in which T5H was down-regulated to increase MT synthesis (Park et al., 2013) (Figure 4). In addition, the downstream metabolites of tryptamine, N-hydroxytryptamine and N-methyltryptamine, increased under drought stress, while decreased after adding MT. The second step of MT biosynthesis is from serotonin to N-acetylserotonin by SNAT, and N-acetylserotonin to MT by ASMT. ASMT is an enzyme that catalyzes the final reaction of MT biosynthesis. In this study, two ASMT genes were significantly up-regulated in each of the T and S genotypes after the addition of MT under drought stress. Meanwhile, the downstream MT and its metabolite 6 -hydroxymelatonin content were both significantly increased by MT addition, and the increase was more in S genotypes than in T genotypes, which again suggests that S genotypes are more sensitive to MT (Figure 4). Similarly, the application of exogenous MT also increases MT production in the leaves of plants such as soybeans (Cao et al., 2023) and loquats ( Eriobotrya japonica Lindl.) (Wang et al., 2021), and enhances the ability of plants to withstand drought conditions. In addition, the production of 6-hydroxymelatonin in the leaves of T and S genotypes was significantly increased by exogenous MT addition. Surprisingly, 6-hydroxymelatonin was not detected in rice seedlings, suggesting differences in MT catabolism pathways between plants (Lee et al., 2016). In summary, the addition of exogenous MT causes the expression of genes for the tryptophan metabolic pathway, promotes the synthesis of endogenous MT, and increases the activity of antioxidant enzymes, thereby improving the resistance of A.mongolicum to drought stress. Overexpression of AmASMT enhances drought tolerance in transgenic rice by promoting MT biosynthesis With the development of molecular biology, gene overexpression has become one of the important means of studying gene function, and this method has been widely used for genetic improvement in plants. Previous studies have indicated that the transcripts of vital catalytic enzymes responsible for MT biosynthesis were enhanced by drought treatment in various species, such as wheat TaCOMT (Yang et al., 2019), cucumber CsTDC2 (Zhang et al., 2024) and maize ZmSNAT1 and ZmSNAT1 (Guo et al., 2024). The last critical gene in the tryptophan metabolic pathway for MT synthesis is ASMT , which catalyzes the production of MT from N-acetylserotonin (Yu et al., 2018). Several studies have revealed that overexpression of endogenous or exogenous ASMT genes, or even animal ASMT in transgenic plants, improves tolerance to abiotic stress (Zuo et al., 2014; Xu et al., 2016; Choi et al., 2017). Based on the results of an integrated analysis of the transcriptome and metabolome, the gene AmASMT was found to be involved in the tryptophan metabolism pathway and to be upregulated after MT and drought stress therapy. Similar to the Hypericum perforatum ASMT , AmASMT was also identified to be distributed in the nucleus and cytoplasm (Zhou et al., 2021). To explore the role of the AmASMT gene in response to drought stress, the AmASMT gene was overexpressed and its relative expression was analyzed after drought stress treatment. The results show that AmASMT can be significantly induced by drought stress, suggesting that the gene plays an important role in the response of A. mongolicum to drought stress. Drought-induced plant damage occurs largely via oxidative stress, making ROS scavenging and oxidative stress reduction pivotal for enhancing abiotic stress tolerance (Apel and Hirt, 2004). MT is an important antioxidant, which can reduce oxidative stress and protect plants from drought stress. Previous studies have stabilized the MzASMT1 gene from Malus spectabilis into Arabidopsis thaliana , revealing that MT levels are significantly higher in overexpressed plants under drought stress than in WT plants, while ROS levels are significantly lower. And the transgenic lines showed greater drought tolerance than the WT line, suggesting that enhanced drought tolerance in transgenic Arabidopsis is closely related to MT antioxidant capacity (Zuo et al., 2014). In the present study, transgenic rice had higher accumulation levels of MT, lower levels of membrane lipid peroxidation, and higher levels of Pro and antioxidant enzyme activity than WT plants, suggesting that AmASMT may be a key factor in regulating plant response to drought stress by promoting the production of endogenous MT to enhance antioxidant capacity (Figure 7). Conclusion In this study, physiological, transcriptome, and metabolome analyses were used to investigate the physiological and molecular mechanisms of MT in drought stress mitigation in A. mongolicum with both drought-tolerant and drought-sensitive genotypes. We found that the exogenous addition of MT improved the drought tolerance of A. mongolicum . Further investigation revealed that MT pretreatment modulates the tryptophan metabolism pathway under drought stress. Notably, the AmASMT involved in the tryptophan metabolic pathway is induced by drought and exogenous MT. Functional characterization revealed that rice lines overexpressing AmASMT exhibit higher endogenous MT levels, leading to enhanced tolerance to drought stress by promoting the antioxidant defense system, suggesting the pivotal role of AmASMT in conferring drought stress resistance. These findings provide new insights into the mechanisms of MT biosynthesis in response to drought stress and could potentially be used to genetically modify drought tolerance in plants. Accession numbers The raw transcriptome data were submitted to NCBI SRA (https://www.ncbi.nlm.nih.gov/sra/PRJNA1138468). The other supporting data are available in Supplementary Tables S1–S12. Supplementary data Supplementary Figure S1. Effects of different concentrations of PEG on seedling growth of T and S genotypes of A.mongolicum . Supplementary Figure S2. Effects of different drought stress treatment days on the changes of relative water content of T and S genotypes of A.mongolicum . Supplementary Figure S3. GO enrichment analysis of DEGs in different comparisons. Supplementary Figure S4. TF faminy classification. Supplementary Figure S5. Validation of differentially expressed genes in the RNA-Seq database by quantitative realtime PCR. Supplementary Table S1. Statistical analysis of indexes of 88 genotypes of A.mongolicum under drought treatment and control groups. Supplementary Table S2. The value of each germplasms’comprehensive index, subordinative function, D value, and rank. Supplementary Table S3. Primers used in this study. Supplementary Table S4. Functional annotation information of DEGs in the database. Supplementary Table S5. DEGs in T-D vs T-C, T-MD vs T-D, S-D vs S-C and S-MD vs S-D comparisons Supplementary Table S6. GO and KEGG enrichment analysis of DEGs. Supplementary Table S7. Detailed information on key genes of KEGG enrichment chord diagram. Supplementary Table S8. TFs in T-MD vs T-D and S-MD vs S-D. Supplementary Table S9. Details of all detected DEMs. Supplementary Table S10. DEMs in T-D vs T-C, T-MD vs T-D, S-D vs S-C and S-MD vs S-D comparisons. Supplementary Table S11. KEGG enrichment analysis of DEGs and DEMs. Supplementary Table S12. DEGs and DEMs related to tryptophan metabolism pathway. Acknowledgments We thank Metware Biotechnology Co., Ltd., for providing technical support. Author contributions B.Z.F, J.Q.Z, and J.W planned and designed the experiments. J.W, W.X.S, X.W, X.C.L, J.H.Y performed the experiment. J.W, J.Q.Z, S.J.S and S.X.L analyzed the data and wrote the paper. B.Z.F, J.Q.Z, S.J.S and S.X.L provided ideas and revised the paper. All authors contributed to the article and approved the submitted version. Conflict of Interest The authors declare no conflicts of interest. Funding This work was supported by the National Natural Science Foundation of China (32260349), and Ningxia Hui Autonomous Region Agricultural Breeding Special Project ( 2019NYYZ0403). 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(A) Aboveground fresh weight, (B) Chlorophyll content, (C) Relative water content, (D) Relative electrical conductivity, (E) MDA content, (F) Pro content, (G) H 2 O 2 content, (H) O 2 − content, (I) APX activity, (J) SOD activity, (K) POD activity, (L) CAT activity. CK: 0 mg·L -1 MT+0% PEG; M 0 : 0 mg·L -1 MT+ 25% PEG; M 1 : 1 mg·L -1 MT+25% PEG; M 10 : 10 mg·L -1 MT+25% PEG; M 50 : 50 mg·L -1 MT+25% PEG; M 100 : 100 mg·L -1 MT+25% PEG; M 150 : 150 mg·L -1 MT+25% PEG; M 200 : 200 mg·L -1 MT+25% PEG. (M) Effect of 100 mg·L -1 MT on the phenotypes of A. mongolicum ; (N) MT content in the leaves of A. mongolicum . The two small figures represent the MT content in the leaves for the CK and D treatments in the T and S genotypes, respectively. Mean values followed by different lowercase letters were significantly different by Duncan’s test ( P <0.05). Figure 2 DEGs analysis of MT alleviation of drought stress in T and S genotypes. (A) Number of DEGs. (B)Venn diagrams of DEGs. (C) KEGG pathway enrichment analysis of DEGs. The size of the point represents the number of DEGs enriched to the pathway, and the color of the point represents the p-value of the enrichment to the pathway. (D)~(E) KEGG enrichment chord diagram of DEGs in T-MD vs T-D and S-MD vs S-D comparisons. The left side of the figure is the six genes with the largest |log 2 FC| in each pathway. The right side of the figure is the five pathways with the most significant enrichment, and the middle line shows the correspondence between the pathway and the gene. Figure 3 Metabolite analysis of MT alleviation of drought stress in T and S genotypes. (A) PCA analysis of metabolites. (B) Category and number of the DEMs. (C) Venn diagram of DEMs. (D) KEGG pathway analysis of DEMs. The color in the heat map represents the p-value (gray indicates undetected). Figure 4 Integrated analysis of DEGs and DEMs in tryptophan metabolism. (A) DEGs and DEMs are involved in tryptophan metabolism in response to MT under drought stress. The blue and orange patterns represent metabolite and gene changes after the addition of MT under drought stress, respectively. The rectangle is divided into four equal parts: from left to right represent the DEGs or DEMs in T-D vs T-C, T-MD vs T-D, S-D vs S-C and S-MD vs S-D, respectively. The color in the rectangle represents the genes or metabolites of T and S genotypes regulated by the exogenous addition of MT under drought stress (red indicates up-regulation; blue indicates down-regulation). (B) Effects of MT on DEMs in the tryptophan metabolism pathway in A. mongolicum leaves under drought stress. Mean values with different letters are significantly different at P <0.05. Figure 5 AmASMT overexpression increase MT accumulation and improves drought tolerance in rice. (A) Cloning of the AmASMT gene in A. mongolicum . 1 represents the PCR amplification product of AmASMT gene, and M represents marker DL5000. (B) Subcellular localization of AmASMT . GFP, green fluorescent protein. Bars=10 µm. (C) The expression level of AmASMT in transgenic rice. WT represents wild type line, OE1-10 are ten transgenic lines. (D) Phenotypes of AmASMT -OE rice under control and drought stress. Control: plants were grown under normal growth conditions. Drought: plants were treated with 15% PEG for 7 days. Bars=10 cm. (E) Relative expression levels of AmASMT in AmASMT -OE rice lines. (F) MT content in AmASMT -OE rice lines. Data are presented as means SD (n=3). Duncan’s analysis using SPSS 26 software indicates that different letters represent statistically significant differences ( P <0.05). Figure 6 Measurements of growth and physiological indicators of drought stress response in AmASMT-OE rice. (A) Plant height, (B) Fresh weight, (C) Relative water content, (D) Relative electrical conductivity, (E) MDA content, (F) H 2 O 2 content, (G) Pro content, (H) SOD activity, (I) CAT activity. Control: plants grown under normal growth conditions. Drought: treatment with 15% PEG for 7 days. Data were means SD (n=3). Duncan’s analysis with SPSS 26 software; different letters represent statistically significant differences, P <0.05. Figure 7 Regulatory pathways of AmASMT enhancing drought tolerance of transgenic rice. WT plants represent wild type plants, TG plants represent transgenic plants; OE indicates overexpression. The size of the red circular symbols represents the accumulation levels. 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Keywords agropyron mongolicum amasmt drought hormones melatonin transcriptome Authors Affiliations Jing Wang 0009-0004-2917-1409 Ningxia University View all articles by this author Jinqing Zhang Ningxia University View all articles by this author Shuxia Li Ningxia University View all articles by this author Shoujiang Sun Ningxia University View all articles by this author Wenxue Song 0009-0002-3102-6038 Ningxia University View all articles by this author Xing Wang Ningxia University View all articles by this author Xiaocong Li Ningxia University View all articles by this author Juhui Yan Ningxia University View all articles by this author Xueqin Gao Ningxia University View all articles by this author Bingzhe Fu [email protected] Ningxia University View all articles by this author Metrics & Citations Metrics Article Usage 168 views 127 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jing Wang, Jinqing Zhang, Shuxia Li, et al. Integrated transcriptomic and metabolomic analyses revealed that AmASMT positively regulates drought tolerance in Agropyron mongolicum by modulating melatonin biosynthesis. Authorea . 16 April 2025. DOI: https://doi.org/10.22541/au.174479111.13749463/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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