Albino Mechanism Of Tea Plant Based On Multi-omics Analysis

Albino of tea plants is a kind of mutant phenotype with important economic value.Tea made by albino tea shoots shows strong umami taste and slight bitter taste because of high amino acids and low phenols/amino acids ratio.The albino mutant tea germplasms have been therefore concerned by researchers.Now,albino tea plants have been widely cultivated in many new tea gardens,producing good social and economic benefits,eg.'Baiye 1'.At present,the quality and yield of tea are severely limited because of the single albino variety and short production period.Therefore,it is particularly important to explore new high-quality albino tea resources.Although there have been many reports on the research of albino tea varieties,the molecular mechanism of the albino phenotype is still unclear.In this study,the newly discovered albino tea resources 'Huangjinya' and 'Huabai 1'were used as materials,transcriptome and metabolome technologies were used to screen albino related differentially expressed genes and metabolites and to explore the relationship between these genes and related metabolites to reveal the molecular mechanism of albino tea plants.Meanwhile,the chloroplast genome structure of 'Huabai 1' and 'Huangjinya'was analyzed.The main research results are as follows:1.As a light-sensitive albino tea variety,phenotype changes of 'Huangjinya' were observed by shading treatment.The results showed that chlorophyll and carotene increased significantly after shading.However,due to the slight albino phenotype of 'Huangjinya',chlorophyll a/b value was not significantly different from that of green leaves after shading,and it still had good photosynthetic performance.The high-throughput sequencing method was used to sequence the transcriptome of new shoots for 'Huangjinya' exposed to light and shading treatment,and a total of 127,253 unigenes were obtained.The sequence annotation and expression data have been uploaded to the GEO database with accession of GSE97659.A total of 81,128 gene sequences were annotated by the databases of NR,NT,swiss-prot,KEGG,GO and COG.Compared to 'Huangjinya' under normal light conditions,207 differentially expressed genes were obtained after shading treatment,including 110 up-regulated genes and 97 down-regulated genes.GO biological process annotation analysis showed that most of the differentially expressed genes were mainly involved in cell process,metabolic process and stress response process.Meanwhile,the GO molecular functional classification indicated that these differentially expressed genes mainly acted on binding and oxidoreductase activity.KEGG enrichment analysis showed that the differentially expressed genes were mainly involved in flavonoid biosynthesis,linolenic acid metabolism and other metabolic pathways.QRT-PCR was used to analyze the gene expression in the flavonoids and carotenoids biosynthesis.It was found that most of the genes in flavonoids biosynthesis showed a downregulated expression trend in shaded 'Huangjinya' shoots,while the genes related to the carotenoids biosynthesis showed an opposite expression trend.Therefore,it was speculated that light could inhibit the accumulation of carotenoids and flavonoids by regulating the expression of flavonoids and carotenoid biosynthesis related genes in 'Huangjinya',thus leading to the yellowing of the new shoots.At the same time,shading can reverse the anabolism of flavonoids and carotenoids and make the leaves turn green.Therefore,the color change of 'Huangjinya' depends on the combination of flavonoid and carotenoid biosynthesis pathways.2.Baed on the newly discovered albino resource 'Huabai 1',the transcriptomes of albino and green shoots were determined(SRA accession number:SRP126084).A total of 483 differentially expressed genes were identified by comparative analysis,471 of which were annotated.The KEGG pathway enrichment analysis showed that most of the differentially expressed genes were mainly concentrated in the metabolic pathways,especially the phenylpropanoid metabolism.Differential expression analysis showed that expression of phenylpropanoid metabolism related genes was higher in albino shoots than in green shoots of 'Huabai 1',especially genes act on deoxidization,hydrogen reduction reaction,such as cytochrome P450 subtype ferulic acid-5 hydroxylation enzyme gene(F5H),cinnamyl alcohol dehydrogenase(CAD)and peroxidase(POD)genes,etc.In addition to the phenylpropanoid biosynthesis pathway,other metabolic pathways such as tyrosine metabolism,tryptophan metabolism and flavonoid synthesis also enriched many differentially expressed genes,indicating that phenylpropanoid biosynthesis and related metabolic pathways jointly participated in the leaf color change of 'Huabai 1'.In the albino shoots of 'Huabai 1',the expression of LHCII which is a key gene in chlorophyll synthesis was significantly lower than that of in the green shoots,while the expression of the chlorophyll degradation related gene SGR in albino shoots of 'Huabai 1' was significantly higher than that of the green shoots.Therefore,the expression of these two genes is also an important factor for the color change of 'Huabai 1'.In addition,protein interaction analysis revealed that NEDD8,a protein associated with ubiquitination,interacted with proteins in many metabolic pathways such as phenylpropanoid biosynthesis.To sum up,the phenylpropanoid biosynthesis pathway plays important roles in the color change of the new shoots of 'Huabai 1' Chlorophyll biosynthesis related genes(LHCII and SGR)and protein interaction network with the hub protein of NEDD8 may also be important factors for the color change of‘Huabai 1'leaves.3.In order to study the albino metabolism mechanism of tea plants,LC-MS was used to determine the metabolome before and after the leaf color changes of 'Huangjinya' and'Huabai 1'.The results showed that 991 metabolites were identified in the shoots of'Huangjinya' and 'Huabai 1',and the classification showed that flavonoids,amino acids and organic acids were the main metabolic components in tea shoots,and they were also the key components controlling the quality and flavor of tea.The KEGG enrichment analysis revealed that a total of 235 metabolites could be mapped to the metabolic pathways,among which amino acid metabolism and secondary metabolic pathway enriched the most metabolites.Compared the albino with the green shoots of 'Huangjinya',242 differential metabolites were identified,which mainly metabolites related to the metabolism of flavonoids and pyrimides.In 'Huabai 1',a total of 359 differential metabolites were identified,mainly related to the phenylpropanoid biosynthesis.This indicated that there were significant differences in the albino mechanism between different abino tea germplasms.4.In order to understand the variation of chloroplast genomes of albino tea germplasms'Huangjinya' and 'Huabai 1',the chloroplast genomes of both varieties were obtained by sequencing.The chloroplast genome of 'Huangjinya' and 'Huabai 1' was 158,196 bp and 157,028 bp,respectively.The chloroplast genome structure of both varieties was similar,including LSC,SSC and two IR regions.The IR region of 'Huabai 1' chloroplast genome was 1,819 bp less than that of 'Huangjinya'.The GC content of chloroplast genome of'Huabai 1' was 37.3%,slightly higher than that of 'Huangjinya'.Codon usage analysis showed that 'Huabai 1' chloroplast genome had 29 high-frequency codons,and the RSCU value was very close to that of 'Huangjinya',indicating that the codon usage bias between the two varieties was relatively close and the chloroplast genomes of tea plants were generally conservative.Based on the nucleotide sequences of rbcL,matK,trnH-psbA and ycfl,it is found that ycfl is most suitable for developing as a DNA barcode to identify tea tree varieties and analyze the evolutionary relationship between different tea varieties.Through cluster analysis,the relationship between 'Huangjinya' and 'Longjing43' were classed in the same branch,while 'Huabai 1' and 'Fudingdabai' were in the same branch,indicating that the two albino varieties differentiated long before,or they may come from different ancestors.