| Iris japonica var.variegata,with green-white striped leaves,which is a evergreen plant,belonging to Iridaceae.Due to its shade-tolerant and cold-tolerant trait,it can be conducive to widely planted.At present,the research of Iris L mainly focuses on floral characteristics such as flower color and flower type,but few researches about the leaf color variation were reported.In this study,we used the green leaf tissues and white leaf tissues as the materials.To explore the leaf structural changes and physiological mechanism on the formation of variegated leaves,the analysis on chlorophyll,chlorophyll precursors,chloroplast ultrastructure of I.japonica var.variegata were carried out.Then,through the RNA-seq analysis,the differentially expressed genes(DEGs)of the green leaf tissue and white leaf tissue were compared.The candidate genes closely related to leaf variegation have be identified,which revealed the molecular regulation mechanism of leaf color variation.The main results are as follows:1.The physiological determination of chlorophyll results showed that the content of Chl a and Chl b in the WL is significantly lower than that of GL.On the other hand,the relative contents of seven chlorophyll synthesis precursors were analyzed.Further detailed analysis showed thatδ-aminolevulinic acid(ALA),porphobilinogen(PBG),uroporphyrin Ⅲ(Urogen Ⅲ)and coproporphyrin Ⅲ(Coprogen Ⅲ)contents decreased,by about 10-60%in the WL leaves,the concentration of protoporphyrin Ⅸ(Proto Ⅸ),magnesium protoporphyrin Ⅸ(Mg-Proto Ⅸ)and protochlorophyllide(Pchlide)contents decreased markedly by about 97-99% in the WL leaves.Observation of chloroplast ultrastructure revealed that the chloroplasts of white leaf tissue did not have a complete thylakoid membrane structure.The chloroplasts were filled with a large number of vesicles and a very small amount of osmophilic granules,without starch granules.These results indicating that the white-green striped phenotype of I.japonica var.variegata might result from deficient Chl contents and abnormal chloroplast morphology.2.Six cDNA libraries were created and sequenced using the Illumina platform,which yielded 23.4G raw data.Using Trinity,the high-quality clean reads of GL and WL leaves were assembled into 159,677 unigenes with mean lengths of 730.19 bp.A total of 29738 unigenes were divided into 25 KOG categories.Besides,when comparing the two types of the library concerning the RPKM calculation,we identified 1327 genes that were expressed specifically between the GL and WL samples,including 342 up-regulated genes and 985down-regulated genes.GO annotions showed that DEGs were classified into three categories(cellular component,molecular function,and biological process).In the cellular component category,genes associated with “cell part”,“organelle”,“membrane”,“membrane part”,and“organelle part ”,which include Chloroplast thylakoid membrane related genes,were highly enriched among the down-regulated.The down-regulated genes associated with “cellular process”,“metabolic process”,“biological regulation”,and “binding”,“catalytic activity”under GO molecular functions.3.Genes associated with porphyrin and chlorophyll metabolism,chloroplast development,thylakoid membrane,and photosynthesis were identified.In the porphyrin and chlorophyll biosynthetic pathway,DEGs encoding enzymes such as siroheme synthase(CYSG)and protochlorophyllide reductase(POR),was up-regulated in WL.The downregulated genes encoding CYSG may contribute to the decrease in uroporphyrin Ⅲ(Urogen Ⅲ),which is the substrate either for siroheme synthesis or for porphyrin synthesis.PORA catalyzes the photoreduction of protochlorophyllide to chlorophyllide in Chl biosynthesis.Chloroplast development also affected the expression of genes encoding proteins.The expression of GLK gene was down-regulated,GLK gene is closely related to chloroplast development,which showed a lower expression in WL compared to GL.Meanwhile,we found that the genes expression involved in photosynthesis-light harvesting,light reaction,photosynthetic electron transport chain were significantly down-regulated in WL.Moreover,DEGs involved in chloroplast thylakoid membrane formation and genes encoding Ribulose-1,5-bisphosphate carboxylase oxygenase(Rubisco)were significantly repressed.4.We further selected 15 DEGs related to chlorophyll metabolism,chloroplast development,and photosynthesis and then examined by Real-time RT-PCR techniques.In our study,the q RT-PCR results showed that the expression patterns of 14 unigenes were largely keeping the same levels with the transcriptome data,where only one gene encoding transcription factor PIF3 had a different expression pattern.These observations indicated that the gene expression changes as detected by RNA-seq reflect the transcriptome profile differences between GL and WL. |
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