Studies On Photosynthetic Characteristics Of Citrus Etiolated Seedlings,transcriptome Analysis And CitXET Gene Identification

Citrus is a perennial plant of Rutaceae and Subfamily aurantioideae,has high dietary property and economic value,but also is one of the most important fruit trees in southern China.Over the past few decades,there has been a lot of research on etiolation of plant leaves,but most of them focus on explaining the differences of metabolic pathways,protein translation and genetic improvement resources during the process of de-etiolation.A large number of proteins and genes related to de-etiolation or greening have been found and supplied,few studies have been carried out on the transcriptome sequence of etiolation of Citrus.Studies on genes related to chloroplast development and photosynthesis pathway during seedling etiolation have not been carried out.Therefore,it is of great significance to promote the normal growth and the healthy development of citrus industry to understand the influence of etiolation on photosynthetic parameters,to develop transcriptome analysis of Citrus seedlings with different degrees of etiolation,to explore the differential expression of genes related to chloroplast development and photosynthesis,and to understand the role of XET gene in the growth of etiolated seedlings.1.In order to study the effect of different etiolation degree on Citrus seedlings growth and photosynthesis,Citrus cultivars Huangguogan and Shiranuhiseedlings were used as the materials,to analyse the changes of seedling biomass,chlorophyll content,photosynthetic parameters and chlorophyll fluorescence characteristics of etiolated,multicoloured and green seedlings.The results showed that the normal growth of Citrus cultivars Huangguogan and Shiranuhi seedlings was inhibited by etiolation,and the growth of etiolated seedlings was significantly lower than that of green seedlings.The contents of free proline?Pro?,malondialdehyde?MDA?,soluble sugar?SS?and soluble protein?SP?were significantly increased in the leaves of etiolated seedlings.Seedlings etiolation could significantly reduce the content of chlorophyll a,chlorophyll b,carotenoid and photosynthetic gas exchange parameters?stomatal conductance,intercellular CO₂ concentration,transpiration rate?,and change the composition ratio of the chlorophyll.Maximal photochemical efficiency?F_v/F_m?,photochemicalquenching?qP?,and non-photochemical quenching?NPQ?of etiolated seedlings were significantly lower than those of green seedlings,but the F_v/F_m of multicoloured seedlings was decreased slightly.This study indicatied that seedlings etiolation inhibited the normal growth and photosynthetic capacity.2.Using transcriptome sequencing technique,modification of global gene expression in two hybrid Citrus cultivars—Huangguogan and Shiranuhi,respectively—were investigated.More than 834.16 million clean reads and 125.12 Gb of RNA-seq data were obtained,more than 91.37%reads had a quality score of Q30.124,952 unigenes were finally generated with a mean length of 1,189 bp.79.15%,84.35%,33.62%,63.12%,57.67%,57.99%and 37.06%of these unigenes had been annotated in NR,NT,KO,SwissProt,PFAM,GO and KOG databases,respectively.Further,we identified 604 differentially expressed genes in multicoloured and etiolated seedlings of Shiranuhi,including 180 up-regulated genes and 424 down-regulated genes.While in Huangguogan,we found 1,035 DEGs,271 of which were increasing and the others were decreasing.No de-etiolated process?GO:0009704?was found in seedlings of two Citrus Cultivars with different degrees of etiolation.7 DEGs were commonly up-regulated,and 59 DEGs down-regulated in multicoloured and etiolated seedlings of these two cultivars,suggesting that some genes play fundamental roles in two hybrid citrus seedlings during etiolation.Our study is the first to provide the transcriptome sequence resource for seedlings etiolation ofShiranuhi and Huangguogan.3.According to the transcriptome sequencing data,we analyzed the mechanisms controlling the expression and function of genes associated with photosynthesis and chlorophyll biosynthesis in multicolored and greenseedlings of Shiranuhi and Huangguogan.We obtained 567.07 million clean reads and 85.05 Gb of RNA-sequencing data,with more than 94.19%of the reads having a quality score of Q30?sequencing error rate=0.1%?.Furthermore,we detected 4,786 and 7,007 differentially expressed genes?DEGs?between multicolored and green Shiranuhi and Huangguogan seedlings.Thirty common pathways were differentially regulated,including pathways related to photosynthesis?GO:0015979?and the chloroplast?GO:0009507?.The photosynthesis?44 and 63 DEGs?,photosynthesis-antenna proteins?14 and 29 DEGs?,and flavonoid biosynthesis?16 and 29 DEGs?pathways were the most common KEGG pathways detected in two analyzed libraries.Differences in the expression patterns of PsbQ,PetF,PetB,PsaA,PsaN,PsbP,PsaF,and XET might be responsible for the etiolation in citrusseedlings.4.Considering the known effects of xyloglucan endotransglycosylase?XET?on plant growth and development,we aimed to determine whether XETs help regulate the growth and elongation of Huangguogan shoots and roots.We confirmed a possible role for XET during seedling etiolation.We also observed positive and negative effects on the xyloglucan-degrading activity of XET in the etiolated Huangguogan seedling root sub-apical region and shoots,respectively.There was a significant down-regulation in CitXET expression in the etiolated shoots at 15 days after germination.On the contrary,it was significantly increased in the root sub-apical region of etiolated and multicolored seedlings at 15 days after germination.Analyses involving the NCBI Conserved Domains Database?NCBI-CDD?,InterPro,and ScanProsite revealed that CitXET is a member of the glycosyl hydrolase family 16?GH16?,and includes the DEIDFEFLG motif.Our results indicate that the extent of etiolation influences the CitXETexpression pattern and XET activity in Huangguogan seedlings.The differential changes in XET activity and CitXET expression levels in Huangguogan seedlings may influence the regulation of root and shoot development,and may be important for seedling etiolation.5.In order to elucidate the genetic and evolutionary patterns of XET genes,we analyzed the codon usage bias using 4,500 codons from 20 XET genes to elucidate the genetic and evolutionary patterns.Phylogenetic and hierarchical cluster analyses of coding sequences and relative synonymous codon usage?RSCU?values,respectively,revealed the 20 XET genes belonged to two groups.The top four codons in the 20 XET genes were AGA,AGG,AUC,and GUG.Additionally,we identified eight optimal codonsof CitXET?i.e.,AGA,AUU,UCU,CUU,CCA,GCU,GUU,and AAA?.Base composition was observed to influence codon usage bias,possibly because of the specific XET gene functions.The two main factors affecting codon usage bias?i.e.,Axes 1 and 2?represented 54.8%and 17.6%of the total variation,respectively.PgXET,ZmXET,VlXET,VrXET,and PcXET were biased toward codons ending with G/C.In contrast CitXET,DpXET,and BrpXET werestrongly biased toward codons ending with A/U.Different XET genes generally exhibited diverse codon usage patterns,although there were also some codon usage biases.The closer the genetic distance,the more similar their codon usage preference.