Tea plant (Camellia sinensis) leaf is an important non-alcoholic beverage resource. The application of quantitative real time polymerase chain reaction (qRT-PCR) has a profound significance for the gene expression studies of tea plant, especially when applied to tea leaf development and metabolism. In this study, nine candidate reference genes (i.e.,CsACT7,CsEF-1α,CseIF-4α, CsGAPDH,CsPP2A,CsSAND,CsTBP,CsTIP41andCsTUB) ofC. sinensiswere cloned. The quantitative expression data of these genes were investigated in five tea leaf developmental stages (i.e., 1st, 2nd, 3rd, 4th and older leaves) and normal growth tea leaves subjected to five hormonal stimuli (i.e., ABA, GA, IAA, MeJA and SA) and gene expression stability was calculated using three common statistical algorithms, namely, geNorm, NormFinder and Bestkeeper. Results indicated thatCsTBPandCsTIP41were the most stable genes in tea leaf development andCsTBPwas the best gene under hormonal stimuli; by contrast,CsGAPDHandCsTUBgenes showed the least stability. The gene expression profile ofCsNAMgene was analyzed to confirm the validity of the reference genes in this study. Our data provide basis for the selection of reference genes for future biological research in the leaf development and hormonal stimuli ofC. sinensis.

Tea is a popular natural non-alcoholic beverage consumed worldwide due to its bioactive ingredients, particularly catechins (flavan-3-ols). Catechins not only contribute to tea quality but also serve important functions in the anti-stress regulation of secondary metabolic pathways. However, the percentages of various catechins are different among tea plant [Camellia sinensis (L.) O. Kuntze] cultivars. This study aimed to elucidate the biosynthetic mechanism of catechins. Transcriptomes from leaf tissues of four tea plant cultivars, ‘Yunnanshilixiang’, ‘Chawansanhao’, ‘Ruchengmaoyecha’, and ‘Anjibaicha’, were sequenced using the high-throughput sequencing platform Illumina HiSeq™ 2000. De novo assemble were also performed. Catechins contents were measured through reversed-phase high-performance liquid chromatography (RP-HPLC), and the biosynthetic pathway was also surveyed. We constructed a unified unigene database. A total of 146,342 pairs of putative orthologs from the four tea plant cultivars, ‘Yunnanshilixiang’, ‘Chawansanhao’, ‘Ruchengmaoyecha’, and ‘Anjibaicha’ were generated. Approximately 68,890 unigenes (47.1%) were aligned to the sequences of seven public databases with a cut-off E-value of 1E-5. A total of 217 differentially expressed genes were found through RPKM values, and 150 unigenes were assigned to the flavonoid biosynthetic pathway using the integrated function annotation. The (−)-EGC and (−)-EC contents were significantly lower and the (+)-GC and (+)-C contents were abnormally higher in ‘Ruchengmaoyecha’ than in ‘Yunnanshilixiang’, ‘Chawansanhao’, and ‘Anjibaicha’. The proportion of catechins was confirmed by selecting critical genes (ANS, ANR, and LAR) for qRT-PCR analysis. This study provided a global survey of transcriptomes from four tea plant cultivars and serves as an available resource of genetic diversity. The analyses of transcriptome profiles and physiological indicators not only identified the putative genes involved in the flavonoid biosynthetic pathway but also provided some novel insights for the mechanisms of catechins biosynthesis.

GRAS proteins are important transcription factors that play multifarious roles in regulating the growth and development as well as stress responses of plants. Tea plant is an economically important leaf -type beverage crop. Information concerning GRAS family transcription factors in tea plant is insufficient. In this study, 52 CsGRAS genes encoding GRAS proteins were identified from tea plant genome database. Phylogenetic analysis of the identified GRAS proteins from tea plant, Arabidopsis, and rice divided these proteins into at least 13 subgroups. Conserved motif analysis revealed that the gene structure and motif compositions of the proteins were considerably conserved among the same subgroup. Functional divergence analysis indicated that the shifted evolutionary rate might act as a major evolutionary force driving subfamily-specific functional diversification. Transcriptome analysis showed that the transcriptional levels of CsGRAS genes under non-stress conditions varied among different tea plant cultivars. qRT-PCR analysis revealed tissue and development stage-specific expression patterns of CsGRAS genes in tea plant. The expression patterns of CsGRAS genes in response to abiotic stresses and gibberellin treatment suggested the possible multiple functions of these genes. This study provides insights into the potential functions of GRAS genes.