Gene Differential Analysis And Physicochemical Characteristics Of Camellia Sinensis Cv. Baijiguan In Response To Light

The tea plant [Camellia sinensis (L.) O. Kuntze] exhibits albino shoots is a rare germplasm, the mechanisms underlying leaf color and specific chemical composition have been the hot research. As a stably heritable albino tea,’Baijiguan’ is a good material for breeding. To conduct a study on its physiology, biochemistry and gene expression regulation, which may help to understand the metabolic changes and genetic variation in tea plant and possess a high research value and practical significance. In this study, the light-sensitive albino tea strain’Baijiguan’was used as the experimental material to study the formation mechanism of albino leaf at the physiological, biochemical and transcriptional level. Firstly, light and temperature tests were performed; the results showed that’Baijiguan’belongs to light-sensitive albino type. To understand the molecular mechanisms underlying light-responsive albinism, we performed de novo transcriptome sequencing combined with digital gene expression profiling (DGE) using Illumina sequencing technology. Based on the differential expressed genes (DEGs), GO (Gene Ontology) functional assignment and KEGG pathway analyses were conducted to enrich the genes involved in light regulatory and albino traits. Furthermore, using quantitative real time PCR (qRT-PCR) technology to validate DGE data and detect gene expression involved in chlorophyll biosynthesis. In order to investigate the relationship of phenotype with chloroplast development and antioxidant defense enzymes in the plant, the chloroplast structure observation and determinenation of peroxidases activity enzymes were conducted by transmission electron microscope and enzyme activity assay kits, respectively. By spectrophotometry, high performance liquid chromatography (HPLC) and gas chromatograph-mass spectrometer (GC-MS) technologies, the total catechins, polyphenols, total amino acids and aroma components were measured in order to investigate the relationship of phenotype with pigments, the main flavor substances and aroma. The main findings were as follows.1. Different light intensity and temperature tests showed that’Baijiguan’ belonged to the light-sensitive albino tea plant. The formation of yellow leave color is mediated by the induction of light.2. The leaves of ’Baijiguan’ shaded three days (pale green leaf half and the control, T3d_Z vs. T3d_W) and six days (green leaf half and the control, T6d_Z vs. T6d_W) by half leaf shading method were used as materials, transcriptome sequencing was performed using the second generation of sequencing technology to construct a new transcriptome of albino leaf in tea plant. By de novo assembly,16.1 G clean reads and 88788 Unigene were obtained. After searching against the public database, a total of 35703 Unigene were identified, accounting for 40.2% of the total Unigene. Among them,24846 Unigene were assigned to 47 categories by GO classification. In the sub-level of biological processes, cellular processes and metabolic processes were the most enriched. In the sub-level of cellular component, the most classified categories were cell, cell part and organelles. In the sub-level of molecular function, the representative classifications were binding, catalytic activity and transport activity.10213 Unigene were classified into 26 gene clusters by KOG database, including general function prediction that represented the largest groups, followed by post-translational modification/protein turnover/chaperon, and then signal transduction, translation and intracellular transport. In addition, lipid metabolism, cell wal’l/membrane/envelop biogenesis that closely related to cell structure were also assigned.8561 Unigene were classified into 31 categories of the second level by KO database. Metabolic pathways including carbohydrate metabolism, energy metabolism, lipid metabolism, amino acid metabolism and other secondary metabolites biosynthesis; genetic information processing were annotated to folding, sorting and degradation, translation, transcription, replication and repair. Signal transduction pathways and membrane transport in information processing environment that were worthy to further study also enriched.1652 Unigene were classified into 25 transcription factor families by Blast2GO annotation software, the most categories were MYB transcription factor, AP2 domain, homeobox, zinc finger protein, bZIP family and bHLH family.3. Analysis of differential gene expression profilingThe results showed that:A total of 6382 differentially expressed genes set were obtained by combination of all comparisons.4072 significantly DGEs (1597 up-regulated,2475 down-regulated) were identified in three-day shading group.4863 DEGs were identified in six-day shading group, in which 32 up-regulated DEGs and 759 down-regulated DEGs were greater than three-day shading group. The down-regulated genes were greater in number than the up-regulated genes following this treatment.Down-regulated DEGs of the green half leaf in ’Baijiguan’ compared to the albino half leaf, were assigned to GO database. The top 20 classification including cytosolic part, chloroplasts, plastids, ribosomes, ribosome biogenesis, biosynthesis ribonucleoprotein complex, the structural components of the ribosome, translation, ribonucleoprotein complex, plastid part, chloroplast part, biosynthesis of cellular components, thylakoid, structural molecular activity, cell part, intracellular, organization or biosynthesis of cellular components, organelles, chloroplast envelope and chloroplast development, the light-harvesting chlorophyll protein genes. Annotation of up-regulated DEGs showed that oxidation-reduction process accounted for the highest one of biological processes. Cellular component assigned the cell periphery and cell wall categories. Oxidoreductase activity, nucleic acid-binding transcription factor activity, sequence-specific DNA-binding transcription factor activity were the largest categories in the molecule functions.KEGG pathway enrichment analysis showed that, photosynthesis, porphyrin and chlorophyll metabolism, metabolism of carotenoids, flavonoids and other synthetic pathways significantly enriched in both of 3-day shading group and 6-day shading groups. Those pathways are closely related to leaf color. In addition, fatty acid metabolism and synthesis, unsaturated fatty acid metabolism (involved in membrane fluidity), nitrogen metabolism and secondary metabolic pathways were significantly enriched, which were consistent with the found of degradation of chloroplast membrane structure and the significant changes of polyphenols and amino acid composition.4. DAG enrichment analysis of the shading groups and the two treatments based on GO classificationA total of 2779 genes (928 up-regulated,1851 down-regulated) were differentially co-expressed between the two shading groups. Up-regulated differentially co-expressed genes were assigned to plant-type cell wall and xyloglucan transferase activity that were closely related to a plant cell wall and chloroplast envelope gene function. Based on the differentially co-expressed genes,81 transcription factors were identified. Among them, the most enriched categories were 11 for HLH/bHLH transcription factor and 8 for MYB transcription factor. In addition, 3 for bZIP, ethylene-responsive factor, homeobox and WRKY transcription factors, respectively; 2 for NAC and 1 for Trihelix transcription factors. Most of these transcription factor families were reported in respond to environmental changes. Among them, the transcription factor HY5 (belong to bZIP family) plays an important role in response to light.131 DEGs (42 up-regulated,89 down-regulated) were unique between the two treatments, most DEGs involved in pigments, secondary metabolites biosynthesis, photosynthetic system Ⅱ and peroxisome-related pathways. There were 72 DEGs in the 3 day-shading group,6 day-shading groupand between treatments (T6d_Z vs. T3d_Z). Compared with the control, chlorophyll biosynthesis related genes glutamyl-tRNA reductase (hemA), magnesium chelatase subunit H(CHLH), methyl magnesium protoporphyrin IX ester cyclase (CRD1), chlorophyll enzyme (E3.1.1.14) were down-regulated. Terpene biosynthesis related genes 1-deoxy-D-xylulose 5-phosphate synthase (DXS), terpene synthases were up regulated, and the DXS increased significantly under shading conditions. Flavonoids biosynthesis related genes anthocyanin reductase (E1.3.1.77), leucoanthocyanidin reductase (LAR), flavonoid 3’, 5’-hydroxylase (F3’5’H) were down-regulated. Chloroplast development related genes ferredoxin protein (petF), Rubisco large subunit binding protein (rbcL) were down-regulated; photosynthetic system Ⅱ 10kDa protein (psbR), light harvesting complex Ⅱ chlorophyll a/b-binding protein 1 (LHCB1) were up-regulated, psb27 protein (psb27), reaction center X protein (psbX) were down-regulated. Peroxisomes related gene superoxide dismutase (SOD], Cu-Zn family), acyl-activating enzyme were down-regulated. Five genes involved in photosystem Ⅱ stabilization, thirteen genes involved in chloroplast thylakoid membrane and seven genes involved in photosystem Ⅱ by DAG enrichment analysis of down-regulated DEGs.5. qRT-PCR validated genes related to enriched pathwaysDGE data were validated using qRT-PCR techniques, the results showed that:In the three-day shading group, the expression patterns of 19 genes detected via qRT-PCR were similar to those observed in the DGE data, whereas the JAZ gene showed a different expression pattern. In the six-day shading group, three genes (COP1, CHLD and CHL1) showed different expression patterns compared with the DGE data (Figure 11B). In general, the qRT-PCR data were consistent with the Illumina sequencing results, suggesting that the RNA-Seq data are reliable.The leaves of ’Baijiguan’ and ’Rougui’ under natural comditions were used as research material to detect the relative expression of genes involved in chlorophyll metabolism, the results showed that POR (protochlorophyllide reductase) plays an important role in responsive to light regulation and the formation of leaf color.6. The impact of light on ultrastructure and defense enzymes in’Baijiguan’tea plantTranscriptome and gene expression profiling analysis showed that GO functional annotation revealed many genes related to chloroplast development, photosynthesis and peroxisome relevant, therefore, to conduct ultrastructure observation and determination of antioxidant defense enzyme activity under natural and shading conditions. Chloroplast displayed abnormal ultrastructure with photosynthetic membrane structure degradation and few chloroplasts under bright light conditions. After shading, chloroplast exhibited stacked thylakoids and normal morphology, the thylakoid membrane rebuilt and the grana lamellae developed compact structure, with a significant increase of defense enzyme activity in vivo.7. The impact of light on the main pigment, flavor and aroma substances in ’Baijiguan’ tea plantThe results showed that:Porphyrins and chlorophyll metabolism, carotenoid biosynthesis, biosynthesis of flavonoids and terpene biosynthesis pathway were enriched by KEGG pathway enrichment analysis. Therefore, we investigated the impact of shading on these substances. The pigments, flavor substances and aroma in ’Baijiguan’ significantly changed after shading. After shading, the leaf of ’Baijiguan’ turned green, and Chl a, Chl b, total chlorophyll, β- carotene increased significantly. The main changes of the flavor substances were as follows:6 days of shading compared to the control, the content of non-galloylated including EGC, C and EC were significantly reduced. The total amino acids were significantly lower than those without shading. Caffeine increased slightly, water extracts decreased slightly, but they did not show significant changes. Aroma determination showed that six days of shading compared with three days of shading, linalool is not detected, but nerolidol as the important aroma of oolong tea were determined, while methyl salicylate, geranylacetone increased.