| Annual ryegrass(Lolium multiflorum L.)is an important commercial forage grass,which has been widely used in the production of hay and silage all over the world.Although the powerful and intense root system of annual ryegrass contributes to its good drought-tolerant in comparison to other grass species,drought has been a severe environmental factor that impairs plant growth and forage productivity in annual ryegrass.Therefore,reliable identification of tolerant variety of annual ryegrass and the understanding of molecular mechanism of drought tolerance are urgently needed.Nevertheless,stress tolerance is a complex quantitative trait.Muti-omics technologies can be powerful tools to produce comprehensive datasets on changes in gene expression,protein profiles and differential metabolites that the results from plant suffered from drought stress.By comparing transcriptome,proteome,metabolome between the drought-tolerant and drought-susceptible L.multiflorum genotypes,the differentially expressed genes and their corresponding differentially abundant proteins,and metabolites were identified,which were mapped onto multiple metabolic pathways.The difference of core regulatory pathways can be revealed at the cellular level to help us to better understand how drought-tolerant L.multiflorum seedlings adapt to drought stress.To get the tolerant L.multiflorum genotype,25% PEG-6000 was used to simulate drought condition without causing direct physiological damage to the seedlings of annual ryegrass.The different types of physiological and biological parameters were measured,such as seedling height(SH),root-shoot ratio(RSR),germination potential(GP),relative electrical conductivity(REC),move transfer rate(MTR)and relative water content(RWC).After data normalization,according to D value calculated by processing membership function(MF)and principal components analysis(PCA),the drought-resistant coefficient of each genotype was evaluated and ranked,the two L.multiflorum genotypes,“Abundant” and “Aderenalin”,were identified as the drought-tolerant and drought-susceptible genotypes.The pot experiment was designed for the phenotypic identification of drought tolerance in the two L.multiflorum genotypes.The drought-treated seedlings of annual ryegrass were grown at non-watered condition for 5 weeks,with 30% water capacity(WC)per pot.The control was simultaneously watered up to 70% WC.The difference in morphologic changes between drought-tolerant genotype and drought-susceptible genotype was observed.There is no difference presented in SH compared with the control in the two L.multiflorum genotypes.However,RSR highly increased in seedlings of drought-tolerant genotypes in comparison to drought-susceptible genotype.Higher levels of antioxidant enzyme such as CAT and SOD were found among the seedlings of drought-tolerant genotype,than those of drought-susceptible genotypes in response to long-term drought.Due to the above results of the pot experiment,the two L.multiflorum genotypes,drought-tolerant “Abundant” and drought-susceptible “Adrenalin”,were used for multi-omic studies.LC-MS-based untargeted metabolomics was used to identify differential metabolites of two L.multiflorum genotypes when exposed to drought of 24 h.After data filtering and normalization,a total of 2,001 and 1,017 features were detected by positive and negative ionization modes of liquid chromatography/mass spectrometry(LC/MS).Only the metabolite with a VIP>1 and a p-value <0.05 were defined as differential metabolite.Different types of the identified metabolites were detected,including lipids,amino acids,organic acids,carbohydrates and carbohydrates conjugates,nucleosides,nucleotides and other analogs,indoles and their derivatives,alkaloids and their derivatives,amine compounds,pyridine and their derivative.Metabolite-metabolite correlation analysis showed that lipids levels exhibited the greatest number of significant correlation with other metabolites,followed by amino acids and organic acids.A total of 168 differential metabolites exhibited contrasting expression levels between the drought-tolerant genotype and drought-susceptible genotype.Using KEGG pathway enrichment analysis,the differential metabolites were mapped onto multiple biological processes,including valine,leucine,and isoleucine biosynthesis;Valine,leucine,and isoleucine degradation;?-linolenic acid metabolism;Biotin metabolism;Phenylalanine,tyrosine,and tryptophan biosynthesis;tropane,piperidine,and pyridine alkaloid biosynthesis;Phenylalanine metabolism;Sphingolipid metabolism;Fatty acid biosynthesis;Phenylpropanoid biosynthesis;Glycerolipid metabolism;Pantothenate and Co A biosynthesis;Histidine metabolism;Tryptophan metabolism;Galactose metabolism;purine metabolism;Cysteine and methionine metabolism;Fatty acid metabolism;Arginine and proline metabolism;2-Oxocarboxylic acid metabolism;Cutin;suberin and wax biosynthesis;Biosynthesis of amino acids;and Citrate cycle.Stress-induced proteins were captured in the proteome of two L.multiflorum genotypes treated with drought for 0,1,2,and 24 h by using i TRAQ-based quantitative analysis and LC-ESI-MS/MS analysis.A total of 26,189 unique peptides matching to 8,224 proteins were detected with a high level of confidence(all with 1% FDR).Only the identified proteins with p-value<0.05 and fold changes>1.2 were considered as differential abundant proteins.Comparative proteomic analysis revealed that 667 proteins were up-regulated and 718 proteins were down-regulated in the two L.multiflorum genotypes at different time points.The KEGG pathway enrichment analysis was performed on the drought-induced proteins,and the results showed that the number of drought-induced proteins varied from 21 metabolic pathways.At 1h of drought treatment,more than twenty proteins involved in“phenylpropanoid biosynthesis,” whereas the decreasing number of proteins participated in the pathway at 24 h of drought stress.Additionally,a comparison of differential accumulated proteins revealed that 51 drought-induced proteins had same or opposite change trends(up-regulated or down-regulated)in response to drought stress.An interaction network was built with 76 proteins identified by Cytoscape software,the majority of responsive proteins were mapped on carbohydrate metabolism-related pathways,amino acid metabolism-related pathways.Drought transcriptional profiles of the resistant and susceptible L.multiflorum genotypes were detected to understand the molecular mechanism of drought tolerance in annual ryegrass by treating plants with drought for 0,1,2 and 24 h.After sequencing reads filtering,approximately 137,708 unigenes were assembled.These annotated unigenes were used to search various functional databases.Using a FDR<0.001 and a fold change value>=2 as threshold,a total of 59,060 genes exhibited significantly differential expression in the tolerant-and susceptible-genotypes at different time points.More up-regulated genes associated with drought response were found in the resistant genotypes compared with the susceptible genotypes.The number of differentially expressed genes(DEGs)enriched in KEGG pathways varied from different time points in the two annual ryegrass genotypes.In comparsion to the drought-susceptible genotype,the expression of differentially expressed genes was significantly increased in tolerant genotype after 1h of drought,including those associated with “nitrogen metabolism,” “cysteine and methionine metabolism,” “citric acid cycle(TCA cycle),” “biotin metabolism,” “arginine and proline metabolism,” and“2-oxocarboxylic acid metabolism”.To futher confirm the results of RNA-seq,26 amino acid-related and lipid metabolism-related genes were chosen for q RT-PCR.The expression trends of these genes were consistent with RNA-seq data in the two L.multiflourm genotypes.Correlation analysis were performed among metabolites,proteins and genes.We first matched all transcripts with their corresponding proteins,and an intermediate Pearson correlation coefficient of r=0.724 was calculated.The DEGs and corresponding differentially accumulated proteins were divided into different groups exhibiting up-regulation or down-regulation.A total of 47 DEGs displaying the same trends that matched those of their corresponding proteins in the resistant-and susceptible-genotypes treated with drought for 1,2,and 24 h.These down-regulated DEG and their corresponding proteins were involved in“phenylalanine,tyrosine,and tryptophan biosynthesis,” “tropane,piperidine,and pyridine alkaloid biosynthesis,” “phenylalanine metabolism,” “phenylpropanoid biosynthesis,”“biosynthesis of amino acids,” “2-oxocarboxylic acid metabolism,” and “glycerolipid metabolism.” Similarly,51 DEGs had contrasting expression changes with their corresponding proteins under drought stress,including genes involved in “valine,leucine,and isoleucine degradation” and “biosynthesis of amino acids.” In addition,a large number of transcripts or proteins that had no significant regulation were also observed in metabolic pathways.Multiple co-inertia analysis(MCo A)was performed to demonstrate the relationship of transcripts,proteins,and metabolites.A strong correlation was observed among them.The first and second MCo A components accounted for more than 80% of total variation,the drought-tolerant and drought-susceptible genotypes were clearly separated,implying that genetic background and drought treatment underlie considerable variation in the data.In addition,the pre-ranked analysis was performed,in which variables were ranked according to their loadings.The loading vectors for component 1 were significantly and positively correlated with amino acid metabolism and lipid metabolism(p<0.001).Protein-gene-metabolite correlation networks were generated to visualize the relationship among transcripts and their corresponding proteins and metabolites which were mapped into21 metabolic pathways.The integrated metabolic networks were used to completely link activity changes in enzymes with corresponding the levels of their metabolites such as amino acids,lipids,carbohydrate conjugates,nucleosides,alkaloids and their derivatives,and pyridines and their derivatives.For example,the higher metabolic levels of ?-galactosidase(EC 3.2.1.22),?-glucosidase(EC 3.2.1.20),and ?-fructofuranosidase(EC 3.2.1.26)indicated a stronger metabolism of galactose.Moreover,colnelenic acid,traumatic acid,stearidonic acid,?-linolenic acid,and 13(S)-HPOT-(9Z,11 E,15Z)-(13S)-13-Hydroperoxyoctadeca-9,11,15-trienoic acid)are all involved in ?-linolenic acid metabolism,in which genes encoding enzymes,such as phospholipase A1(EC 3.1.1.32),lipoxygenase(EC 1.13.11.12),and alcohol dehydrogenase class-P(EC 1.1.1.1)were each either strongly downregulated or upregulated.To develop whole genome-wide distributed SSR markers for annual ryegrass breeding,a total of 11,254 EST-SSR were detected from transcriptome datasets of annual ryegrass subjected to drought stress.Fifty EST-SSR markers were selected to test amplification and polymorphism rate in 23 accessions of Lolium and Festuca diploid species.The results showed that all of the 50 functional relevant markers were polymorphic,with allele number and polymorphism content averaging 7.4 and 0.839,respectively and fully transferable across23 accessions of Lolium and Festuca diploid species.A number of EST-SSRs were mapped to genome sequences of Lolium perenne and chromosomes of Brachypodium distachyon.Among them,973 and 1,375 Lolium SSR markers were located on the five chromosomes of B.distachyon and L.perenne scaffolds,respectively.In addition,we confirmed that EST-SSRs as well as SRAP markers could clearly identify varieties of Lolium and Festuca species,and the two sets of molecular markers had a significant correlation(r = 0.74).This set of EST-SSR markers are valuable genetic tools for genetic diversity,evolutionary,and association mapping studies in Lolium,Festuca and many other grass species. |
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