Candidate Genes Analysis For Yellow-green Leaf Color Mutant Ygm In Common Wheat (Triticum Aestivum L.) According To Transcriptome Profiling

Leaf color mutants are special materials to investigate chlorophyll biosynthesis,chloroplast development,photosynthesis and cytoplasm-nucleus interaction.The molecular mechanism underlying leaf color variation have been the research hotspot.Ygm,a spontaneous yellow-green leaf color mutant was derived from the common wheat(Triticum aestivum L.)cultivar Xinong1718 that has been maintained and selected by 14 successive generations of self-crossing.Up to now,the genetic characteristics,major agronomic traits,photosynthetic pigments content,chloroplast ultrastructure and gene mapping of Ygm have been studied well.However,little is known about the molecular mechanisms leading to the chlorophyll deficiency of Ygm,candidate genes closely related to leaf color mutation have yet to be tapped and utilized.To further understand the molecular mechanism underlying chlorophyll metabolism and photosynthesis and search for candidate genes associated with above biological process.Herein,the half-developed leaves come from Y(yellow leaf color)and corresponding G(normal green color)of wheat Ygm mutant progeny were subjected to transcriptome sequencing combined with determination of chlorophyll biosynthesis precursors and carotenoid composition.The main findings are as follows:1.Using the RNA-seq technology,we obtained approximately 389.20 million paired-end clean reads.Totally,1227 differentially expressed genes(DEGs)were identified in Y compared to G,of which,689 were up-regulated and 538 were down-regulated.GO enrichment analysis revealed that a large number of DEGs involved in metabolism,binding,and catalytic function.KEGG pathway analysis indicated that the DEGs were involved in chlorophyll biosynthesis,carotenoid biosynthesis,photosynthesis,carbon fixation and carbon metabolism in photosynthetic organisms are closely related to the yellow leaf phenotype in Y.Moreover,early light-induced proteins(ELIPs)and a large number of heat-shock proteins(HSPs)encoding genes associated with chlorophyll biosynthesis and chloroplast development were identified.Of the 44 identified transcription factors,heat shock transcription factors(Hsfs)accounted for the highest proportion.Seven Hsfs target genes prediction showed that genes encoding heat shock proteins(HSPs)comprised the major group that interacted with those Hsfs.The down-regulated genes encoding HSPs and Hsfs may contribute to the yellow leaf color formation in Y.The expression of fifteen selected genes that involved with the photosynthetic biosynthesis and chloroplast development were analyzed by qRT-PCR to validate the reliability of RNA-seq data,further indicating the data from the RNA-Seq were credible.2.Analysis of seven intermediates of Chl synthesis pathway in Y and G indicated that Y plants accumulated higher levels of ALA,PBG,Urogen III,Coprogen III,and Proto IX especially,the accumulation of Proto IX(the substrate of Mg-chelatase).However,the contents of Mg-proto IX(the product of Mg-chelatase)and Pchlide dramatically decreased in Y plants,indicating that the inhibition of chlorophyll biosynthesis in Y occurred from Proto IX to Mg-proto IX.Combining with the transcriptome sequencing data,it was concluded that the down-regulated CHLH,which encodes the Mg-chelatase H subunit,may reduced the Mg-chelatase activity,thereby resulting in decreased Chl content in Y.3.The contents of five carotenoid compounds in Y and G were determined by high performance liquid chromatography(HPLC).The results showed that β-carotene and lutein were the major carotenoids found in G plants,while zeaxanthin,luein and β-carotene were the main carotenoids of Y.Compared to G,the zeaxanthin level was significantly increased in Y,whereas the β-carotene and lutein levels were significantly lower.β-carotene hydroxylase(BCH)to catalyze the production of zeaxanthin from β-carotene,further demonstrating that the up-regulated expression of the BCH encoding genes is closely related to yellow leaf phenothpe of Y plants.This study provides further insights into the molecular mechanisms underlying the phenotype of chlorophyll deficiency of Ygm and identified the key genes may be responsible for the metabolite biosynthesis differences among the different leaf color of Y and G plant.