Exogenous melatonin improved cold and drought tolerance in perennial ryegrass
The exogenous application of melatonin reduced the inhibition of plant growth under both cold and drought stress in a dose and stress treatment duration-dependent manner, while no significant differences were observed in non-treated and plants pre-treated with different concentrations of melatonin under normal conditions (Fig. 1). Pretreatment with 20, 100 and 1,000 µM melatonin resulted in a higher fresh weight and lower REL when the plants were exposed to 3 d of cold stress. Treatment with 100 µM was the most effective. Along with the extension of cold stress, the pre-treatment of plants with a higher concentration of mealtonin (100 and 1,000 µM) grew better, whereas a lower fresh weight and higher REL were found in the plants treated with 20 µM of melatonin (Figs. 1a, c). A similar changing patterns of fresh weight and REL was observed in the melatonin-treated plants under drought conditions (Figs. 1b, d). As expected, 100 µM melatonin significantly alleviated the stress damage caused by cold and drought treatment, as indicated by less severe phenotypes (Fig. 1e).
Systematic transcriptome shifts modulation by melatonin in perennial ryegrass
Treatment with melatonin causes a dramatic change in gene expression in perennial ryegrass under cold and drought stress. In total, 11,588 differentially expressed genes (DEGs) were identified in at least one sample of plants subjected to cold and drought or treatment with melatonin compared with their respective controls. Among these, a number of 5,279 (2,848 up- and 2,431 down-regulated), 5,105 (2,307 up- and 2,798 down-regulated), 2,249 (727 up- and 1,522 down-regulated), and 7,162 (3,943 up- and 3,218 down-regulated) DEGs were obtained in cold, drought, cold plus melatonin, and drought plus melatonin treated seedlings, respectively (Supplementary Fig. S1a, b).
A GO enrichment analysis of the DEGs showed that 347 GO terms were notably enriched in at least one of the treatments (FDR < 0.05; Supplementary Table S2). An overrepresentation of genes in the GO biological processes related to abiotic stress responses, redox and osmotic homeostasis (i.e., AsA-GSH cycle, glutathione, polyamine and proline metabolism), regulation of phytohormones (i.e., ABA, brassinosteroid, melatonin, ethylene, jasmonic acid, and salicylic acid), photosynthesis (i.e., chloroplast organization, chlorophyll biosynthesis, and photosystem I/II assembly), carbohydrate metabolism (i.e., trehalose, galactose, raffinose, maltose, and sucrose), lipid metabolism (i.e. sterol, phospholipid, and wax), and the phenylpropanoid pathway, i.e., flavonoids. Moreover, signal transduction (i.e., MAPK signaling, phosphorylation, and ubiquitination), protein folding, translation and transcriptional regulation were closely related to the response of melatonin-mediated cold and drought stress (Supplementary Table S2). Several transcription factors, including DREB1A, ERF1, ERF109, MYB108, MYB4, WRKY30, WRKY53, bZIP73, ZAT6, and MADS57, were induced by melatonin under cold and drought conditions. Interestingly, the genes that encode dehydrin, such as DHN1, DHN3, RAB15, CS66, COR47, and COR413PM1, were upregulated by melatonin under abiotic stress (Supplementary Table S3). Nineteen differentially expressed transcripts from RNA-Seq were used to validate the RNA-Seq data using qPCR analysis. The qPCR measurements were highly correlated with the transcript abundance from RNA-Seq analysis (y = 0.9368x + 0.1062, r = 0.8992) (Supplementary Fig. S1c).
The significantly enriched KEGG pathways included brassinosteroid biosynthesis, tryptophan metabolism, phenylalanine metabolism, biosynthesis of unsaturated fatty acids, alpha-linolenic acid metabolism, glutathione metabolism, starch and sucrose metabolism, galactose metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, MAPK signaling, and ubiquitin mediated proteolysis. Additionally, melatonin changed the citrate cycle and glycolysis/gluconeogenesis processes (Supplementary Table S2).
ROS producing and scavenging processes are mediated by melatonin
A total of 43 genes related to production of H2O2 were substantially activated under cold and drought stress, including RBOHB, RBOHC, and DGS1 (Supplementary Fig. S2). Melatonin induced the expression of genes for RBOHB, RBOHC, and DGS1 following exposure to short-term cold and drought stress but reduced their levels of expression and accumulation of H2O2 under long-term stress (Fig. 2). Notably, the upregulation of melatonin genes was related to the mitochondrial respiratory chain complex and chloroplast respiratory chain (Supplementary Fig. S2). Polyamine oxidase-related genes, which catalyze the degradation of polyamines to induce the accumulation of H2O2, i.e., PAO2, PAO4, and PAO5 were induced by cold or drought stress but repressed by treatment with melatonin. The genes that encode enzymes involved in spermine and proline biosynthesis were activated by melatonin, including SPMS, SAMDC, SPDSYN1, P5CS, and P5CR (Supplementary Fig. S2).
The AsA-GSH cycle and glutathione metabolism also participate in melatonin-mediated cold and drought tolerance. A total of 51 genes were involved in glutathione metabolism, including glutathione S-transferases (GSTs) and glutaredoxin that were markedly induced by melatonin under cold and drought stress. Seventeen up-regulated genes related to the AsA-GSH cycle, including MDAR2, MDAR5, APX7, and APX8, were found in plants that were treated with melatonin. Superoxide dismutase, peroxidases, catalase, and thioredoxin, as well as ferredoxin, were also differentially regulated by melatonin (Supplementary Fig. S3). The physiological experiments also revealed that melatonin significantly increased the ratio of AsA/DHA and GSH/GSSG and enhanced the activities of APX, GR, GST, and CAT following exposure to 5 d of cold and 10 d of drought stresses (Supplementary Fig. S4).
Brassinosteroid biosynthesis and the signaling pathway are induced by melatonin
Many hormone-related genes were differentially regulated by melatonin in perennial ryegrass and were assigned to nine major categories of phytohormones (Supplementary Table S3). Among them, 10 genes that encod the biosynthessis of melatonin were significantly induced by melatonin. More importantly, 37 genes related to the biosynthesis, response, and signal transduction of brassinosteroid were substantially induced by melatonin, particularly SERK1 and IWS1, the genes involved in brassinosteroid-mediated signaling transduction, and CYP750A1, CYP707A5, CYP707A7, CYP87A3, and CYP90D2, the genes involved in biosynthesis of brassinosteroid (Figs. 3a-k). The expression of ASMT1 gene that encodes the key biosynthetic enzyme in melatonin biosynthesis was upregulated after 1 h of cold or drought stress. The expression of OMT2 and ASMT3 under drought and cold stress was continually higher than that of the control. All three genes, ASMT1, ASMT3, and OMT2, were gradually induced by melatonin and reached a maximum at 3 or 6 h following the treatment of leaves with cold or drought. The level of expression of CYP87A3, CYP707A7, and CYP750A1, continuously increased when subjected to stress and reached their maximal levels at 6 h of cold and drought, respectively. CYP90D2 and CYP707A5 were more highly expressed following 1 and 3 h of drought stress, respectively. Cold stress also induced the expression of CYP707A5 and CYP90D2 genes with a higher expression at 1 h. Pretreatment with melatonin significantly enhanced the transcription levels of the CYP90D2, CYP707A5, and CYP707A7 genes during the whole period of stress, while a slight change was observed in the level of expression of CYP87A3 and CYP750A1 genes at 1 and 3 h of cold or drought stress compared with nontreated stressed plants, respectively. Additionally, IWS1 and SERK1 were up-regulated by melatonin following exposure to cold or drought stress (Figs. 3e-k). As expected, cold and drought stress increased the content of melatonin in leaves after 1 h with leaf brassinosteroids that showed a similar tendency. Melatonin pretreatment improved the levels of endogenous melatonin and BRs (Figs. 3i, m).
BRs play a partial role in the improvement of cold and drought tolerance of perennial ryegrass by melatonin
Our GO and KEGG enrichment analysis showed that a total of 147 genes involved in photosynthesis were identified after melatonin treatments (Supplementary Fig. S5). The expression of seven genes that encode the assembly of photosystem II (PS II), including PsbM, PsbE, PsbB, PsbA, LTO1, SEP1 and EGY1, were induced by melatonin. Melatonin also markedly induced the expression of genes that encode the PSII-associated light-harvesting complex and oxygen evolving complex, i.e. PSBR, PSBS, PSBO, PPD3, and PNSB5. Genes related to the chlorophyll a-b binding protein involved in light harvesting in photosystem I were upregulated by melatonin treatment following exposure to cold and drought stress. These genes included eight WHAB1.6, six CAB2R, three CAB3A, four CAB2R, two CAB2, two LHBC. Exogenous melatonin increased the content of chlorophyll in the leaves of perennial ryegrass exposed to cold and drought stress, which was consistent with the upregulation of genes that encode chlorophyll biosynthesis and the downregulation of the genes involved in the catabolism of chlorophyll (Fig. 4; Supplementary Fig. S5). Furthermore, the genes involved in PSII repair were increased by melatonin. To confirm this, we measured Fv/Fm, Pn, and Chl content. Plants treated with melatonin or EBL had a higher Fv/Fm and lower REL and accumulation of H2O2 after 5 d of cold or 10 d of drought stress. However, pretreatment with PPZ substantially inhibited the induction of cold or drought tolerance by melatonin as indicated by a lower Fv/Fm and higher REL and accumulation of H2O2 (Figs. 4a-d). Decreased activitives of CAT, POD, APX, GR, and GST and a lower ratio of AsA/DHA and GSH/GSSG were observed in plants treated with melatonin plus PPZ (Supplementary Fig. S4).
To further confirm whether BR signaling was involved in the mediation of cold and drought tolerance by melatonin, we analyzed the effects of exogenous melatonin on morphologies in A. thaliana bri1.9 and bak1 mutants. The bri1.9 and bak1 mutants were more sensitive to cold and drought stress, and pretreatment with melatonin could alleviate the damage caused by stress in the WT and bak1 plants but exhibited no obvious improvement in the bri1.9 mutants (Fig. 5a).
Consistent with the phenotypic analysis, a stronger DAB staining density of H2O2 was detected in the leaves of bak1 mutants exposed to cold and drought stress compared with the WT plants (Fig. 5b). Pre-treatment with melatonin decreased H2O2 accumulation in both genetypes under cold or drought stress, while the accumulation of H2O2 in the bak1 mutants was maintained at a high level. This indicates that signaling by BRs plays key role in the regulation of cold and drought tolerance coupled with H2O2 levels regulated by melatonin.
An appropriate accumulation of H2O2 is required for melatonin- and BRs-induced plant growth
To determine the important role of H2O2 signaling in the mediation of responses to cold and drought stress by melatonin in perennial ryegrass. We utilized an H2O2 scavenger DMTU to analyze the effects of melatonin and BRs on seed germination and seedling growth under cold and drought stress. Both cold and drought stress lowered the rate of seed germination of perennial ryegrass, while the application of 100 µM melatonin and 0.1 µM BRs significantly improved seed germination under the stresses but delayed seed germination at a higher concentration (1,000 µM melatonin and 1 µM BRs). Interestingly, the plants pretreated with melatonin and DMTU (0.5 and 5 mM, respectively) exhibited a lower rate of seed germination than those treated with melatonin exposed to both cold and drought stress. A lower concentration of DMTU consistently led to an increase in the germination rate when the seeds were treated with BRs under cold and drought stress compared with treatment with BRs alone, while the germination of seeds pretreated with BRs and a higher dose of DMTU level was inhibited (Fig. 6; Supplementary Fig. S6).
The application of melatonin alleviated the inhibition of roots induced by cold or drought (Fig. 7a). In plants pretreated with melatonin, the root length, surface area, and volume were 111.32%, 68.12%, 30.58% and 65.64%, 52.59%, 17.24% (P < 0.05) higher after exposure to drought and cold stress compared with non-melatonin treated plants, respectively. Pretreatment with DMTU and melatonin inhibited the effect of melatonin on the growth of perennial ryegrass seedling roots under cold and drought stress (Figs. 7b-d).
Interaction with BR and RBOH-mediated H2O2 signaling plays a critical role in melatonin-induced plant growth and stress tolerance
To further clarify the interactive role of H2O2 and BR signalings in the regulation of cold and drought responses by melatonin, the growth phenotypes of the WT and bak1 and bri1.9 mutants were analyzed following treatment with DMTU and melatonin. We found that the appication of DMTU and melatonin inhibited the growth of WT, bak1 and bri1.9 mutants under cold and drought stress compared with plants treated with melatonin, but the bak1 and bri1.9 mutants were only slightly sensitive (Fig. 5a).
Moreover, the effects of melatonin on the expression of BR biosynthesis and signaling-related genes, DWF4, BZR1, BRI1 were inhibited in the rbohC and rbohF mutants. Melatonin and BRs induced the expression of stress-responsive genes in WT, but this induction was impaired in the rbohC and rbohF mutants. Pretreatment with H2O2 effectively restored the expression of these stress-related genes in rbohC and rbohF mutants (Fig. 8).