| Reaumuria trigyna,an endangered recretohalophyte,is a small halophytic shrub belonging to Reaumuria in Tamaricaceae.R.trigyna is a unique species in China,which has unique adaptability to arid and saline environments in desert grasslands.In recent years,our group has conducted intensive studies on the morphological structure,physiological and ecological characteristics,and molecular mechanism of salt tolerance in Reaumuria trigyna.However,a systematic bioinformatics study is still lacking for the mechanism of gene expression regulation and metabolite dynamic accumulation induced by salt stress.In this study,transcriptome sequencing and widely-targeted metabolomics analysis were carried out for the first time under neutral salt and alkaline salt stress to compare the gene transcription levels and metabolite accumulation mechanisms.Meanwhile a preliminary validation of the transcription factor RtMYBC1 involved in the regulation of the plant flavonoid synthesis pathway was performed based on transcriptome data.These findings are of great theoretical significance and practical value for dissecting the stress resistance mechanisms of plants and for mining engineering genes that can be used for genetic improvement in crops and forages.The main results are as follows:1.Transcriptome sequencing analysis of Reaumuria trigyna under neutral salt and alkaline salt stress resulted in 359045 unigenes with an average length of 803,and 12151 and 9288 differentially expressed genes(DEGs)were identified,of which 7059 and 5092 DEGs were significantly up and down regulated under neutral salt stress and 5241 and 4047 DEGs were significantly upregulated and downregulated under alkaline salt stress respectively;A total of 4255 DEGs were obtained under the two kinds of stresses,among which 1734 DEGs were significantly up-regulated and2521 DEGs were significantly down-regulated.GO analysis revealed that DEGs enriched to cellular processes and metabolic processes,cell and cell part,binding and catalytic activitiy;KEGG enrichment analysis revealed that DEGs in both groups were significantly enriched in photosynthesisrelated signaling pathway,MAPK signaling pathway and carotenoid biosynthesis pathway had the highest enrichment.2.In the cluster analysis of differentially expressed genes(DEGs)identified 451 and 385,222 and 177 DEGs enriched in transcription factors,phytohormone signaling pathways in Reaumuria trigyna under neutral salt and alkaline stresses respectively.Among them,the differentially expressed transcription factors belong to 35 transcription factor families,and AP2 / ERF,MYB,WRKY,NAC,b ZIP,b HLH and other families account for a relatively high proportion.The hormone signaling pathways showed that the down-regulated expression of auxin and cytokinin signaling pathways in somatotropin was most common,which may play an important role in regulating plant growth inhibition.Some key kinases and transcription factors in abscisic acid,ethylene and jasmonic acid signaling pathways of stress hormones were up-regulated,which were involved in plant stress regulation.Overall,there were more differentially expressed genes for transcription factors and plant hormone signaling pathways in the neutral salt stress group than in the alkaline salt stress group.3.There were 133 and 123,158 and 145,133 and 74 DEGs enriched in photosynthetic system,starch and sucrose metabolism pathway and phenylpropanoid metabolism pathway,and there were more differentially expressed genes in neutral salt stress than those in alkaline salt stress.Among them,DEGs involved in the photoreaction stage,such as photosystem I,photosystem II,F-type ATP synthase,and chlorophyll synthesis,were mainly down regulated,which demonstrated that these salt stress completely inhibited the photosynthesis of Reaumuria trigyna.Furthermore,among the metabolic pathway of starch and sucrose,the DEGs involved in the synthesis of monosaccharides such as glucose and fructose,as well as key enzymes(α-amylase and β-amylase)involved in starch degradation were mainly up-regulated,while DEGs related to starch synthase were down-regulated,which indicated that Reaumuria trigyna resists two kinds of salt stress by upregulating starch degradation and monosaccharide synthesis genes,as well as downregulating the expression of starch synthesis genes,promoting the accumulation of soluble sugars when photosynthesis was inhibited.DEGs involved in phenylpropanoid metabolic pathways were mainly annotated to the lignin synthesis-related pathways,which indicated that lignin metabolic pathways were active.4.Metabolome analysis results of Reaumuria trigyna under neutral and alkaline salt showed that a total of 541 metabolites of 10 classes were detected,including 76 amino acid metabolites,40 nucleotide metabolites,51 lipids metabolites and 43 organic acids.Secondary metabolites included:124 phenolic acids,139 flavonoids,14 lignans and coumarins,11 tannins,9 terpenoids and 34 other metabolites.5.The primary metabolites were analyzed under the stress neutral salt and alkaline salt stresses,and 76 kinds of amino acids,40 kinds of nucleotides and 51 kinds of their derivatives were detected.There were 34 and 23 kinds of amino acid metabolites,24 and 21 kinds of nucleotide metabolites and 25 and 33 kinds of lipid metabolites respectively.The amount of amino acid metabolites decreased was much more than that increased,which indicated that both the two kinds of salt stress could cause partial amino acid degradation of Reaumuria trigyna.Meanwhile S-allyll-cysteine specifically accumulated under the two kinds of salt stresses,the accumulation of N-(3-Indolylacetyl)-L-alanine,N-Acetyl-DL-tryptophan,S-(5’-Adenosy)-L-homocysteine,acetyltryptophan and L-tyramine increased most under neutral salt stress.The accumulation of amino acids such as 1,2-N-methylpipecolic acid,L-pyroglutamic acid,N-acetyl-DLtryptophan and L-tyramine increased the most under alkaline salt stress.The accumulation of these amino acids may play an important role in response to the two kinds of salt stresses;Among nucleotides and their derivatives,c AMP and c GMP were upregulated most significantly,increasing by 9.6 and 10.7,4.1 and 5.5 times respectively;The amounts of2’-deoxyinosine,deoxycytidine,and deoxyadenosine were significantly higher in the neutral salt group than in the alkaline salt group.It was speculated that these nucleotide metabolites play an important role in response to neutral and alkaline salt stress.There were a variety of lysophospholipids that significantly increased in content under both salt stresses,and that were much greater in the neutral salt group than in the alkaline salt group.6.A total of 31 and 34 organic acid metabolites,57 and 55 phenolic acid metabolites and 45 and 51 flavonoids were detected when analyzing the secondary metabolites of organic acids and phenylpropanoid in Reaumuria trigyna under neutral salt and alkaline salt stresses respectively.Among them,there were 11 and 8 kinds of organic acids with specific changes in the neutral and alkaline salt groups,and 8 kinds of organic acids with common changes.The content of shikimic acid increased only under neutral salt stress,while the content of shikimic acid increased under alkaline salt stress.Shikimic acid is one of the key metabolites of shikimic acid pathway,which may play a role in the reconfiguration of carbon sources to a certain extent.The accumulation of caffeoylbenzoyltartaric acid and 3,5-Di-O-galloylshikimic acid increased significantly after the two kinds of salt stress,suggesting that these two phenolic acids play a positive role in the response of the neutral and alkaline salt stress;The contents of galloyl methyl gallate,3,4-dimethoxycinnamic acid and brevifolin carboxylic acid in alkaline salt stress group were much higher than those in neutral salt stress group,which may play a positive role in promoting the absorption of nutrients in Reaumuria trigyna under alkaline stress.Among the flavonoids,chrysoeriol-O-glucuronic acid,diosmetin-7-O-glucuronide and 3-O-acetylpinobanksin were specifically accumulated under two kinds of salt stresses.Three flavonoids with myricetin as the framework(myricetin-O-glucuronate,myricetin,and myricetin-3-Orhamnoside-7-O-ramnoside)and syringetin were upregulated under both salt stresses.7.The function preliminary verification of RtMYBC1 demonstrated that overexpression of RtMYBC1 enhanced the tolerance of Arabidopsis to drought and salt.Specifically,the phenylpropane pathway functional genes At PAL,At CHS,and At F3 H were significantly induced,which directly or indirectly regulated the antioxidant enzyme gene and proline synthesis gene,increasing the antioxidant enzyme activity and proline content. |
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