Muti-omics revealed the mechanisms of MT-conferred tolerance of Elymus nutans Griseb. to low temperature at XiZang

doi:10.21203/rs.3.rs-4140249/v1

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Research Article

Muti-omics revealed the mechanisms of MT-conferred tolerance of Elymus nutans Griseb. to low temperature at XiZang

https://doi.org/10.21203/rs.3.rs-4140249/v1

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published 30 Sep, 2024

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Background

Low temperature seriously limited the development of grass and crops in plateau. Thus, it is urgent to develop an effective management strategy for improving the plant cold tolerance and elucidate the underlying mechanisms.

Results

we found that MT improved could tolerance of ENG via enhancing antioxidant enzymes and accumulating flavonoids. Further integration of transcriptome and metabolome profiles showed that both cold exposure (CE) and MT reprogrammed the transcription pattern of galactose and flavonoids biosynthesis, leading to changes in compositions of soluble sugar and flavonoids. Additionally, CE inhibited the photosynthesis, and destroyed the antioxidant system of ENG, leading to accumulation of oxidant radicals represented by MDA. Of note, MT improved the activity of antioxidant system, reduced the MD content and maintained normal expression of photosynthesis-related genes of ENG even under CE. Importantly, MT mainly enhanced cold tolerance of ENG via activating zeatin signal to regulate flavonoid biosynthesis in vivo. Numerous transcription factors (WRKY17, WRKY11 and MYB59) involved in regulating zeatin synthesis were activated by MT in ENG.

Conclusions

our research gain insight into the global mechanisms of MT in promoting cold tolerance of ENG, then provided guidance for protecting plant from cold stress in plateau.

Campeiostachys nutans

low temperature stress

multi-omics

adaption mechanisms

regulation network

Plants are often challenged by numerous adverse abiotic stresses, including drought, high salinity, and especially cold stress (i.e., low temperature stress) [1]. Of note, low temperature often determines the geographical distribution, growth and development of plants, eventually affecting the crop yield and fruit quality [2]. Numerous crops represented by corn (Zea mays L.), Sorghum (Sorghum bicolor), rice (Oryza sativa L.), tomato (Solanum lycopersicum) and cucumber (Cucumis sativus L.) have been documented to be adversely affected by cold stress[3]. Statistically, low temperature damage has caused serious damages to the global economics and crop yield. Cold stress occurs frequently in early spring and late autumn, leading to a series of reactions such as freezing of plant cell membranes, which directly or indirectly inhibit plant growth and cause irreversible damage[4–6]. In addition, low temperature stress can change the membrane permeability and cause lipid peroxidation of plant cell membrane, then leading oxidative stress to plants[7]. Additionally, low temperature could destroy the function of chloroplast and mitochondria to result in excessive accumulation of ROS in plants [8]. Moreover, numerous researches found that cold exposure could also affect the photosynthesis in plants, ultimately causing irreversible damage to plants[9–11]. Typically, low temperature stress can inhibit the synthesis and repair of D1 protein in photosystem II (PSII) reaction center, and affect the photochemical efficiency[9]. Thus, development of novel strategy for managing the damage to crops caused by cold stress is urgent and of great significant for agricultural development in future.

Plants will make a variety of physiological changes to adapt low temperature environment, such as altering metabolic compositions (glucose and flavonoid), activating antioxidant system and hormone signaling [12, 13]. Additionally, the increase of soluble sugar content under cold stress also can reduce the damage to plants caused by low temperature[14]. Facing low temperature stress, a large amount of reactive oxygen species accumulated in plants and induce membrane peroxidation, which will destroy the integrity of the structure and function of the plant cell [15]. Malondialdehyde (MDA) is the product of membrane lipid peroxidation, and its content can reflect the damage degree of plant cells under adverse conditions, especially low temperature [16, 17]. In response, plant could deploy its antioxidant enzymes and metabolites to alleviate oxidate stress caused by cold exposure [18–20]. Melatonin (n-acetyl-5-methoxytryptamine), a small molecule belonging to the indole amines, is widely distributed in plants, where chloroplasts and mitochondria are the main sites of melatonin synthesis[21]. It was found that exogenous application of melatonin can alleviate the damage to plants caused by various abiotic stresses, especially low temperature[21–23]. Typically, melatonin could enhance plant cold tolerance via promoting the activity of antioxidant enzymes and reducing the loss of mineral elements caused by cold stress[24]. Despite these processes, the detailed mechanisms of melatonin in improving cold tolerance of plants remain unclear.

Elymus nutans Griseb., which is widely distributed in cold habitat, exerted cold tolerance, and could be used as a model materials to investigate plant responses to cold exposure. In this study, we used Elymus nutans Griseb. to investigate the molecular mechanism of melatonin in improving plant resistance to low temperature. We performed integrated metabolome and transcriptome to systematically analyzed the changes in plants following MT and cold treatments. It was found that MT improved plant cold tolerance via improving physiological responses, and activating zeatin signal to reconstruct the metabolic composition in plants. Overall, our research provided novel insight into the mechanisms of MT-enhanced cold tolerance of plants, which contributed to the development and usage of MT in protecting crops from cold stress in filed, especially in cold plateau.

Sample collection and preparation

The Elymus nutans Griseb. (ENG) named by Yang[25] used in this study were collected from Yangbajing (30° 28.535’N, 91° 06.246’E, altitude 4618 m), Dangxiong County, Lhasa, XiZang. The seeds were disinfected with 0.1% (W/V) sodium hypochlorite solution, and washed with distilled water. Then, the seeds were incubated in an artificial climate chamber for seedling growing to about 2 cm. The healthy plants were selected and transferred to the culture bowl, and the seedlings were cultured in the artificial climate chamber using sand culture. During the this period, the Hoagland nutrient solution was changed every 3 days. The seedlings pre-cultured for 21 days were collected for further experiments. The experiment contained 4 treatments: CK (25℃, Hoagland nutrient solution), low temperature treatment (4°C, Hoagland nutrient solution), CK + MT treatment (25°C with melatonin), low temperature + MT(4°C with melatonin) and zeatin (at 25°C). The experiment were repeated every 3 times, and each time contained 3 biological replicates at least.After being treated for 5 days, all samples were collected via liquid nitrogen, and stored at -80°C for later use.

Determination of physiological parameters

The content of malondialdehyde in ENG from different experimental treatment was extracted using 5 ml of 5% trichloroacetic acid and detected following Huang et al., (2020) [50]. After centrifugation at 3000 r/min, the supernatant was determined at 450 nm, 532 nm and 600 nm. The CAT, SOD and POD activities were analyzed according to Du et al., (2024) [25], and the sugar content in ENG was determined following Xu[26].

RNA extraction

Total RNA of Elymus nutans Griseb. (ENG) with three replicates from each experimental group was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions [27]. After determination of RNA purity and integrity number, all samples were collected for Illumina RNA sequencing by Biotree company.

Transcriptome sequencing and analysis

A total amount of 1.5 µg RNA per sample was used as samples for libraries construction, and the sequencing libraries of cDNA were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. And we used AMPure XP system (Beckman Coulter, Beverly, USA) to obtain 250 ~ 300 bp cDNA for further sequencing, and Agilent Bioanalyzer 2100 system to evaluate the library quality. Raw data (raw reads) of fastq format were firstly processed to obtain high quality clean data (clean reads). Further transcriptome assembly was performed using Trinity.

RSEM was used for analyzing the gene expression levels by DESeq R package (1.10.1), and genes with an adjusted P-value < 0.05 as well as Foldchange > 2.0 was recognized as differentially expressed genes (DEGs). Further Gene Ontology (GO) and KEGG pathway enrichment analysis on DEGs was performed by GOseq R packages an KOBAS software based Wallenius non-central hyper-geometric distribution, respectively.

Metabolite Extraction

Three biological replicates of ENG from different altitudes were collected for metabolite extraction to ensure the validity of altitude-induced metabolic changes in ENG. Totally, 20 mg of each ENG sample from CK, CKMT, TW and TWMT groups was used and extracted by 1000 µL extract solution (methanol: water = 3: 1, with isotopically-labelled internal standard mixture) via ultrasonic extraction method for 3 times. After centrifugation, the supernatant from quality control (QC) sample and each experimental sample was collected for further LC-MS/MS analysis [28].

LC–MS/MS Analysis and Data Processing

LC-MS/MS analysis was performed following Du et al., (2023) [51]. In detail, UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm) coupled to Orbitrap Exploris 120 mass spectrometer (Orbitrap MS, Thermo) was used for LC-MS/MS analysis, with 5 mmol/L ammonium acetate and 5 mmol/L acetic acid in water (A) and acetonitrile (B) as mobile phase. The temperature and injection volume were set as 4°C and 2 µL, respectively. Information-dependent acquisition (IDA) mode was deployed to get MS/MS spectra and evaluate the full scan MS spectrum. The ESI source conditions were set as following: capillary temperature 320°C, full MS resolution as 60000, MS/MS resolution as 15000 collision energy as 10/30/60 in NCE mode, spray Voltage as 3.8 kV (positive) or -3.4 kV (negative), respectively [29].

Then, the raw data of all samples were processed by an in-house MS2 database (BiotreeDB) for metabolite annotation [30]. The cutoff for annotation was set at 0.3. Typically, p < 0.05 and fold change > 2.0 were used to identify differential metabolites. Further metabolomics analysis was performed using Metaboanalyst 3.0, including PCA, OPLS-DA, PLS-DA algorithms and KEGG pathway enrichment analysis.

Melatonin (MT) treated Elymus nutans Griseb. (ENG) exhibited stronger physiological characteristics to cold exposure.

To investigate the physiological responses of Elymus nutans Griseb. (ENG) to cold exposure (CE), we determined numerous physiological parameters of plants from different treatments. The results showed that 4°C (TW/CE) did not affected the sugar contents in ENG, and significantly promoted the accumulation of MDA in vivo (Fig. 1A, B). By contrast, melatonin (MT) treatment reduced the accumulation of MDA in ENG under CE (Fig. 1A, B), suggesting that MT might promote the antioxidant capacity of ENG. Then, we determined the activities of antioxidant enzymes of ENG from each experiment group, including SOD, CAT and POD (Fig. 1C-E). The results showed that the activity of CAT and SOD in vivo was not affect by CE and MT (Fig. 1C, E). Of note, the activity of POD in TW (CE) was significantly lower than that in CK, suggesting that CE damaged the antioxidant system of ENG (Fig. 1D), then leading accumulation of MDA. Typically, MT increased the bioactivities of POD and SOD in ENG even under CE (Fig. 1D, E), suggesting that MT could enhance plant antioxidant capacity. Additionally, secondary metabolites represented by flavonoid also involved in the antioxidant system of plants[31]. Here, we found that the flavonoid content in ENG from TW/CE was significantly lower than that in CK, whereas MT increased the flavonoid content in ENG even under CE (Fig. 1F). Taken together, CE could damage the antioxidant system of plant, while MT improved this adverse effects, then enhanced ENG cold tolerance.

Cold stress altered the metabolic and transcriptomic patterns of Elymus nutans Griseb. (ENG).

To investigate the effects of cold stress on ENG, three biological replicates for ENG from both TW/CE and CK treatments were collected for transcriptome sequencing. Totally, 548970 M clean reads were generated from all samples with high-throughput sequencing, and 142,219 genes were identified in ENG. Then, we performed principal component analysis (PCA) on all transcriptome profiles to investigate the transcriptome pattern of ENG under CE (Fig. 2A). PCA plot was constructed using principal component 1 (PC1) and PC2, which explained 32% and 23% variations between all samples (Fig. 2A). The PCA plot showed that the samples from TW/CE clearly distributed at a single region and separated with CK (Fig. 2A), suggesting that low temperature significantly altered the transcriptome pattern of ENG. Typically, the differences between TW/CE and CK were mainly represented by PC2 (Fig. 2A). Meanwhile, Pearson correlation analysis showed an obvious cluster containing all samples of TW/CE, and separated with CK, supporting the PCA results and the credibility of our transcriptome data (Figure S1A). Then, 12,626 DEGs were identified, with 8794 downregulated DEGs and 3832 upregulated DEGs in TW vs. CK comparison based on FDR < 0.05 and Foldchange > 2.0 (Fig. 2B). Further Go enrichment analysis on all DEGs showed that these DEGs were significantly classified into 161 Go terms (FDR < 0.05), including 83 terms relevant to biological progress, 40 terms of cellular component and 39 molecular functions (Table S1). Typically, these genes mainly functioned in chloroplast, plastid, thylakoid, plastid stroma, photosynthetic membrane and plastid envelope, with numerous functions, such as chlorophyll binding, cofactor binding, oxidoreductase activity, carotenoid dioxygenase activity, linoleate 13S-lipoxygenase activity, oxidoreductase activity and ATPase (Table S1). And these genes mainly involved in Photosynthesis, plastid organization, oxidation-reduction process, response to toxic substance, pigment biosynthetic process, tetraterpenoid metabolic process, carotenoid metabolic process and flavonoid metabolic process of ENG (Table S1). Further KEGG pathway enrichment results showed that TW treatment mainly affected the expression of genes involved in Photosynthesis, Galactose metabolism, Linoleic acid metabolism, Phenylpropanoid biosynthesis, Starch and sucrose metabolism, Peroxisome, Glutathione metabolism, hormone signaling pathway and Flavonoid biosynthesis (Fig. 2C). Typically, numerous pathways relevant to antioxidant capacity of plants were significantly enriched in TW vs. CK comparison, especially Phenylpropanoid biosynthesis, Peroxisome, Glutathione metabolism and Flavonoid biosynthesis (Fig. 2C), supporting that low temperature affected the plant antioxidant system. Although the total content of sugar was not affected by cold stress in ENG, the genes involved in soluble sugar synthesis represented by Galactose metabolism were significantly differentially expressed in ENG under cold treatment (Fig. 2C). These results suggested that low temperature could altered the transcription pattern relevant to metabolisms of ENG.

Subsequently, we performed metabolomics analysis to investigate the metabolic composition of ENG under different temperature. Totally, 603 metabolites were identified in ENG, and the differentially expressed metabolites (DEMs) in TW vs. CK comparison were further filtered using P < 0.05 and Foldchange > 2.0 (Fig. 2E). The results showed that TW reduced the contents of 7 metabolites, and increases 39 metabolite contents in ENG (Fig. 2E). Further PCA and OPLS-DA analysis on metabolic profiles showed that TW/CW significantly altered the metabolic compositions of ENG (Fig. 2D; Figure S2), reaching a consensus with the transcriptome profiles that low temperature changed the metabolic compositions of ENG. Further KEGG enrichment analysis on these DEMs showed that these DEMs were significantly categorized in 8 pathways, including Glycine, serine and threonine metabolism, Aminoacyl-tRNA biosynthesis, Arginine and proline metabolism, Lysine biosynthesis, beta-Alanine metabolism, Sphingolipid metabolism, Nitrogen metabolism, Glutathione metabolism, Starch and sucrose metabolism and Flavonoid biosynthesis (Fig. 2F). Of note, Phenylpropanoid and Flavonoid biosynthesis were also significantly enriched against metabolic profiles (Fig. 2F). Typically, we found that the contents of phenylpropanoids and flavonoids varied among CK and TW groups (Fig. 2G). Thus, these results suggested low temperature could alter the transcriptome and metabolic pattern of ENG, especially leading changes in compositions of flavonoids.

MT treatment altered the transcriptome response of Elymus nutans Griseb. to cold stress.

Numerous researches documented the effects of MT in improving cold tolerance of plants [21–22]. Here, we found that MT promoted the physiological characteristic of ENG in response to low temperature. Then, we further compared the transcriptome profiles of ENG after MT treatment under different temperature environment. The PCA results showed distinct variations in CKMT vs. CK and TWMT vs. TW comparisons (Fig. 2A), suggesting that MT altered the transcriptome pattern of ENG. Of note, the CKMT and TW samples partially overlapped with each other, suggesting that MT could affected some responses of ENG to cold challenge (Fig. 2A). Totally, 2310 (1465 downregulated DEGs and 845 upregulated DEGs) and 4902 (1856 downregulated DEGs and 3046 upregulated DEGs) DEGs were identified in CKMT vs. CK and TWMT vs. TW comparisons (Fig. 3A). Further Go enrichment analysis on DEGs from CKMT vs. CK showed that these genes significantly involved in 65 Go terms, such as NAD(P)H dehydrogenase complex assembly, cell wall modification, L-phenylalanine catabolic process, phenylpropanoid metabolic process, oxidation-reduction process, tyrosine catabolic process, ATPase inhibitor activity, dioxygenase activity, glucuronokinase activity, cell periphery, extracellular region and oxidoreductase complex (Table S1). It has been found that flavonoids accumulation and cell wall modification involved in the cold tolerance of plants[19]. Thus, these results implied that MT could alter the metabolic composition of ENG to enhance cold tolerance. In parallel, DEGs from TWMT vs. TW comparison mainly involved in photosynthesis, oxidation-reduction process, NAD(P)H dehydrogenase complex assembly, Thylakoid, photosynthetic membrane, chloroplast, plastid, hydrolase activity, oxidoreductase activity, glycerophosphocholine phosphodiesterase activity and glutathione hydrolase activity (Table S1). Cold exposure will damage the photosynthesis of plant to affect plant normal growth [9]. Of note, numerous terms relevant to photosynthesis were significantly enriched in TWMT vs. TW (Table S1), implying that MT might help plants maintain normal photosynthesis under low temperature condition.

Subsequently, we performed KEGG pathway enrichment analysis on both sets of DEGs (Fig. 3B). For DEGs from CKMT vs. CK, KEGG results showed that these DEGs mainly involved in Photosynthesis, ABC transporters, Phenylpropanoid biosynthesis, Glutathione metabolism, Plant hormone signaling, Zeatin biosynthesis and Flavonoid biosynthesis (Fig. 3B). It reached a consensus with Go results that MT could affect the transcriptome pattern of flavonoid biosynthesis in ENG under normal condition (Fig. 3B, C; Table S1). Additionally, we noted the enrichment of zeatin biosynthesis in CKMT vs. CK (Fig. 3B, C), implying that hormone zeatin might be responsible for regulating responses of ENG to cold stress. In addition, KEGG pathway enrichment analysis on DEGs from TWMT vs. TW showed that 17 biosynthesis pathways were significantly enriched, including Photosynthesis, Starch and sucrose metabolism, Galactose metabolism, Flavonoid biosynthesis, Phenylpropanoid biosynthesis, Tyrosine metabolism, Phenylalanine, tyrosine and tryptophan biosynthesis and Zeatin biosynthesis (Fig. 3B). Of note, Flavonoid biosynthesis, Zeatin biosynthesis and Photosynthesis were also significantly enriched in TWMT vs. TW comparison (Fig. 3B, C), supporting that MT could affect these processes to enhance ENG cold tolerance. Importantly, the pathways affected by both low temperature and MT treatments were highly consistent in our transcriptome profiles of ENG (Fig. 3C). Then, we analyzed the detailed expression pattern of genes involved in Flavonoid and Zeatin biosynthesis (Fig. 3D). As shown in Fig. 3D, the expression pattern of genes relevant to flavonoid biosynthesis were reprogrammed by MT, leading to accumulation of specific metabolites in ENG. Additionally, most of genes relevant to zeatin signal were upregulated by cold and MT treatments (Fig. 3E), supporting the involvement of zeatin in regulating MT-enhanced cold tolerance of ENG.

Taken together, we proposed that MT could enhanced cold tolerance of ENG via promoting accumulation of specific flavonoids and maintaining normal photosynthesis, and this effect of MT were mediated by zeatin signal.

MT treatment altered the metabolic compositions associated with cold environment in Elymus nutans Griseb.

Here, we further compared the metabolic profiles of ENG rom CKMT vs. CK and TWMT vs. TW comparisons. To identify the overview of changes in metabolic pattern of ENG following MT treatment under different temperature, we performed PCA analysis on all metabolic profiles of ENG (Fig. 2D). The PCA plots constructed by PC 1 and 2 showed significant variations in metabolic patterns among all groups, while PC1 and PC2 explained 19.5% and 11.9% variations associated with experimental treatments (Fig. 2D). Typically, PC1 mainly explained the variations between TWMT and TW comparison (Fig. 2D), suggesting that MT altered the metabolic composition of ENG under low temperature. Then, 22 (9 upregulated DEMs and 13 downregulated DEMs) and 46 (41 upregulated DEMs and 5 downregulated DEMs) DEMs were identified in CKMT vs. CK and TWMT vs. TW, respectively, based on P < 0.05 and Foldchange > 2.0 (Fig. 4A). We cauterized all these DEMs using KEGG pathway analysis, and found that these DEMs were mainly synthesized from beta-Alanine metabolism, Citrate cycle (TCA cycle), Alanine, aspartate and glutamate metabolism, Flavonoid biosynthesis, Phenylpropanoid biosynthesis, Starch and sucrose metabolism and Zeatin biosynthesis (Fig. 4B). Of note, MT treatment also reprogrammed the compositions of flavonoids and soluble sugars in ENG (Fig. 4B). Typically, we found that numerous flavonoids were induced to increase in ENG by MT and low temperature treatments, especially neoglycyrol, daidzein-4'7-diglucoside, delphinidin, pelargonidin-3-O-glucoside, butein and phillyrin (Fig. 4C). Considering the effects of flavonoids in plant response to cold stress, these metabolites were identified as candidate compounds that involved in the mechanisms of MT-enhanced cold tolerance of ENG. Additionally, we noted the accumulation of zeatin in TW, and its content were further induced to higher levels in ENG by MT (Fig. 4D). Thus, we proposed that zeatin might involve in regulating MT-enhanced cold tolerance of ENG. To test this possibility, we detected the expression levels of numerous genes relevant to flavonoid biosynthesis in ENG after exogenous zeatin treatment using RT-qPCR (Fig. 4E). The results showed that zeatin treatment significantly induced the expression levels of PAL, CHS, 4CL and FLS (Fig. 4E), suggesting that the MT- and cold-induced accumulation of flavonoids in ENG were mediated by zeatin signal. Overall, these results suggested that MT mainly promoted the cold tolerance of ENG via increasing flavonoid content, which was mediated by zeatin signal.

Active regulation network of zeatin synthesis involved in regulating MT-conferred cold tolerance of ENG.

Considering the effects of zeatin in regulating MT-enhanced cold tolerance of ENG, we further constructed the co-expression network of genes relevant to zeatin synthesis (zeatin1, zeatin2, zeatin3 and UGT73C3) using WGCNA based on all transcriptomic profiles of ENG (Fig. 5). The expression data of all transcription factors of ENG were selected via Nr annotation for WGCNA construction. We set the soft threshold to 22 (R2 = 0.75) to construct a scale-free network (Fig. 5A). Then, 14 modules were identified by hierarchical clustering and the dynamic branch cutting (Fig. 5B, C), each module was assigned a unique color as an identifier (Fig. 5C, D). Then, the modules highly correlated with the related traits were filtered out for further construction of regulation network of zeatin synthesis in ENG (Fig. 5D). Based on the WGCNA results, we identified that MEbrown, MEmagenta, MEyellow and MEturquoise were highly related to genes relevant to zeatin synthesis (Fig. 5D). Among genes from these modules, we constructed the co-expression regulation networks for zeatin synthesis (Fig. 5E). Totally, we identified that 170 transcription factors were responsible for positively regulating zeatin synthesis in vivo (P < 0.05; Fig. 5E; Table S3). As shown in Fig. 5E, NAM, GT-3a, GHD7, WRKY17, WRKY11, MYB59 and RAP2 (Accession number: TRINITY_DN1111_c0_g2, TRINITY_DN17254_c0_g1, TRINITY_DN20832_c0_g1, TRINITY_DN25531_c0_g1, TRINITY_DN30587_c0_g1, TRINITY_DN37908_c0_g1 and TRINITY_DN5309_c0_g1) were the hub genes involved in the regulation network of zeatin synthesis in ENG, and it was positively correlated with the expression of zeatin-related genes (Fig. 5E; Table S3). As shown in Fig. 5F, most of these genes were upregulated to higher expression levels in ENG by MT and low temperature treatments, suggesting that MT could activate the regulation network of zeatin synthesis to increase zeatin content in ENG. Further RT-qPCR assay confirmed that transcription factors including WRKY17, WRKY11 and MYB59 were activated to upregulate in ENG by MT and low temperature treatments (Fig. 5G). Taken together, these results suggested that MT could activate zeatin signal to promote flavonoid accumulation in ENG, then promoted the cold tolerance of ENG.

In the cold region, especially plateau, low temperature disaster has become a nonnegligible obstacle to the development of grass. Thus, it is of great scientific value and practical significance to develop an effective management strategy of improving the cold tolerance of plants for guiding the development of grass industry. MT is known to promote plant growth and improve plant resistance to adapt abiotic stress in field [21, 22]. Presently, we found that cold stress reprogrammed the expression pattern of genes involved in galactose and flavonoids biosynthesis, leading to dramatic changes in the metabolic compositions of ENG, typically altering the composition of soluble sugar and flavonoids. Additionally, cold stress inhibited the photosynthesis, and destroyed the antioxidant system of ENG, leading accumulation of oxidant radicals represented by MDA in ENG. Of note, we found that MT treatment effectively promoted cold tolerance of ENG via increasing the activity of antioxidant system of ENG even under cold condition, leading to decreases in MDA content. Moreover, transcriptome profiles showed that MT altered the transcription responses of ENG to cold stress, especially maintaining normal expression of photosynthesis-related genes and reprogramming the synthesis pattern of flavonoid and soluble sugar. Further metabolome showed that MT promoted the accumulation of specific flavonoids and soluble sugars. Importantly, we noted that MT mainly enhanced cold tolerance of ENG via activating zeatin signal to regulate flavonoid biosynthesis in vivo. Meanwhile, coexpression network analysis showed that MT could activate expression of numerous transcription factors (WRKY17, WRKY11 and MYB59) to promote zeatin synthesis in ENG. Overall, our research investigated the detailed global mechanisms of MT-promoted cold tolerance of ENG, which provided guidance for usage of MT in managing damage to plant caused by cold stress in plateau.

Cold stress always caused excessive accumulation of reactive oxygen species (ROS) in plants [32]. ROS accumulation aggravated membrane lipid peroxidation and destroyed the membrane integrity, then negatively affected plant development [33]. In response, plants deployed ROS scavenging systems consisted with SOD, CAT and POD enzymes to scavenge ROS in vivo [34]. Here, we found that these antioxidant enzymes of ENG were inhibited by cold stress, leading to accumulation of oxidant radical, especially MDA in vivo. Interestingly, MT effectively increases the activity of antioxidant enzyme of ENG even under cold conditions, leading to decreases in MDA content. Additionally, numerous research found that cold stress significantly destroyed photosystem II (PSII) and photosystem I (PSI), leading to the degradation of chloroplasts in severe cases [35, 36]. Chloroplast degradation reduces the APX activity, then blocking the scavenging of H2O2 and causing changes in plant metabolism [33]. Moreover, disruption of photosynthesis leads to a decrease in soluble sugars of plants. Contents of soluble sugar are important for plants under cold stress, and their accumulation enhanced plant cold resistance through increasing the concentration of cell fluid to reduce the freezing point of cytoplasm [37–41]. Here, our transcriptome profiles also showed downregulation of photosynthesis-related genes in ENG following cold challenge.This phenomenon has been documented in a range of plants under cold stress [39–40, 42]. However, MT treatment increased their expression levels in ENG under cold conditions, suggesting that MT could alleviate the obstacle of photosynthesis in plants, then maintaining normal growth of plant under cold conditions. Additionally, we found that genes involved in biosynthesis of flavonoid and soluble sugars were also significantly affected by cold stress. Flavonoids and their derived anthocyanins have antioxidant activities and could scavenge ROS in vivo [43]. It was found that the reprogrammed pattern of flavonoids in vivo was a response of plant to cold stress [44, 45]. Typically, accumulation of flavonoids will enhance the cold tolerance of plants [44, 45]. Here, our integrated transcriptome and metabolome of ENG showed that MT effectively reprogrammed the biosynthesis of flavonoids in ENG under cold condition, leading to accumulation of numerous metabolites, including neoglycyrol, daidzein-4'7-diglucoside, delphinidin, pelargonidin-3-O-glucoside, butein and phillyrin. Thus, these results supported our hypothesis that MT could enhance cold resistance in ENG through preventing decreases in related metabolites to promote capacity of antioxidant system in plants. Importantly, we found that MT mainly triggered zeatin synthesis to promote flavonoid biosynthesis in ENG. Numerous transcription factors represented by WRKYs were identified to be responsible for regulating MT-triggered zeatin synthesis in ENG. It has been well documented that WRKYs transcription factors performed important function in plant resistance to cold stress [46–49]. Thus, we conclude that MT mainly enhanced ENG cold tolerance via increasing antioxidant system, maintaining photosynthesis and promoting zeatin-mediated flavonoid biosynthesis.

In the present study, we deciphered the global mechanisms of MT-enhanced cold tolerance of ENG via integrating metabolome and transcriptome. We found that exogenous MT could promoted the cold resistance in ENG. Typically, MT mainly enhanced ENG cold tolerance via increasing antioxidant system, maintaining photosynthesis and promoting zeatin-mediated flavonoid biosynthesis. Moreover, numerous transcription factors represented by WRKYs were identified to involve in regulating MT-triggered zeatin synthesis for high cold tolerance of ENG through our MT-cold regulation network. Although the function of these WRKYs were not investigate in present work, we fully demonstrated that MT could be applied to improve cold resistance of crops in plateau. Overall, the present study provides a novel and valuable strategy for protecting ENG from cold stress in plateau through the application of MT.

C. nutans:Campeiostachys nutans; PSII: photosystem II; RuBisCO: ribulose bisphosphate carboxylase oxygenase; MDA: Malondialdehyde; ROS: reactive oxygen species; CAT: Catalase；MT: CK+melatonin; SOD: superoxide dismutase; POD: peroxidase; GO: Gene Ontology; DEGs: differentially expressed genes; IDA: information-dependent acquisition; PCA: The Principal Component Analysis; KEGG: Kyoto Encyclopedia of Genes and Genomes; TCA cycle: Citrate cycle.

Funding Declaration

This project was supported by the Second Xizangan Plateau Scientific Expedition and Research (STEP) program (2019QZKK0502), China Foraga and Grass Research System Lhasa comprehensive Test Station (CARS-34).

Declaration of competing interest

The authors declare no conflict of interest.

Ethics approval and consent to participate

The collection of plant materials used in our study complied with permission of related institutions, and complied with national or international guidelines and legislation. The experiments did not involve endangered or protected species.

Consent for publication

Not applicable.

Competing Interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Availability of data and materials: The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA014891) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

Acknowledgments: We thank Jinchao Zhou and Shanghai Biotree Biomedical Technology for RNA-sequencing and metabolite annotation.

Author Contribution: Conceived and designed the experiments: PBZM and DJTD Performed the experiments: PBZM, TDQP and SD. Analyzed the data: PBZM and FJJ. Contributed reagents/materials/analysis tools: TDQP. Wrote the paper: PBZM and DJTD. All authors have read and approved the final manuscript.

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Muti-omics revealed the mechanisms of MT-conferred tolerance of Elymus nutans Griseb. to low temperature at XiZang

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Background

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Introduction

Materials and Methods

Sample collection and preparation

Determination of physiological parameters

RNA extraction

Transcriptome sequencing and analysis

Metabolite Extraction

LC–MS/MS Analysis and Data Processing

Results

Discussion

Conclusion

Abbreviations

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