BackgroundTea is the most popular non-alcoholic health beverage in the world. The tea plant (Camellia sinensis (L.) O. Kuntze) needs to undergo a cold acclimation process to enhance its freezing tolerance in winter. Changes that occur at the molecular level in response to low temperatures are poorly understood in tea plants. To elucidate the molecular mechanisms of cold acclimation, we employed RNA-Seq and digital gene expression (DGE) technologies to the study of genome-wide expression profiles during cold acclimation in tea plants.ResultsUsing the Illumina sequencing platform, we obtained approximately 57.35 million RNA-Seq reads. These reads were assembled into 216,831 transcripts, with an average length of 356 bp and an N50 of 529 bp. In total, 1,770 differentially expressed transcripts were identified, of which 1,168 were up-regulated and 602 down-regulated. These include a group of cold sensor or signal transduction genes, cold-responsive transcription factor genes, plasma membrane stabilization related genes, osmosensing-responsive genes, and detoxification enzyme genes. DGE and quantitative RT-PCR analysis further confirmed the results from RNA-Seq analysis. Pathway analysis indicated that the “carbohydrate metabolism pathway” and the “calcium signaling pathway” might play a vital role in tea plants’ responses to cold stress.ConclusionsOur study presents a global survey of transcriptome profiles of tea plants in response to low, non-freezing temperatures and yields insights into the molecular mechanisms of tea plants during the cold acclimation process. It could also serve as a valuable resource for relevant research on cold-tolerance and help to explore the cold-related genes in improving the understanding of low-temperature tolerance and plant-environment interactions.

Anthracnose caused by Colletotrichum is one of the most severe diseases that can afflict Camellia sinensis. However, research on the diversity and geographical distribution of Colletotrichum in China remain limited. In this study, 106 Colletotrichum isolates were collected from diseased leaves of Ca. sinensis cultivated in the 15 main tea production provinces in China. Multi-locus phylogenetic analysis coupled with morphological identification showed that the collected isolates belonged to 11 species, including 6 known species (C. camelliae, C. cliviae, C. fioriniae, C. fructicola, C. karstii, and C. siamense), 3 new record species (C. aenigma, C. endophytica, and C. truncatum), 1 novel species (C. wuxiense), and 1 indistinguishable strain, herein described as Colletotrichum sp. Of these species, C. camelliae and C. fructicola were the dominant species causing anthracnose in Ca. sinensis. In addition, our study provided further evidence that phylogenetic analysis using a combination of ApMat and GS sequences can be used to effectively resolve the taxonomic relationships within the C. gloeosporioides species complex. Finally, pathogenicity tests suggested that C. camelliae, C. aenigma, and C. endophytica are more invasive than other species after the inoculation of the leaves of Ca. sinensis.

Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions.

Sugar plays an essential role in plant cold acclimation (CA), but the interaction between CA and sugar remains unclear in tea plants. In this study, during the whole winter season, we investigated the variations of sugar contents and the expression of a large number of sugar-related genes in tea leaves. Results indicated that cold tolerance of tea plant was improved with the development of CA during early winter season. At this stage, starch was dramatically degraded, whereas the content of total sugars and several specific sugars including sucrose, glucose and fructose were constantly elevated. Beyond the CA stage, the content of starch was maintained at a low level during winter hardiness (WH) period and then was elevated during de-acclimation (DC) period. Conversely, the content of sugar reached a peak at WH stage followed by a decrease during DC stage. Moreover, gene expression results showed that, during CA period, sugar metabolism-related genes exhibited different expression pattern, in which beta-amylase gene (CsBAM), invertase gene (CsINV5) and raffinose synthase gene (CsRS2) engaged in starch, sucrose and raffinose metabolism respectively were solidly up-regulated; the expressions of sugar transporters were stimulated in general except the down-regulations of CsSWEET2, 3, 16, CsERD6.7 and CsINT2; interestingly, the sugar-signaling related CsHXK3 and CsHXK2 had opposite expression patterns at the early stage of CA. These provided comprehensive insight into the effects of CA on carbohydrates indicating that sugar accumulation contributes to tea plant cold tolerance during winter season, and a simply model of sugar regulation in response to cold stimuli is proposed.

Tea is an economically important plant characterized by a large genome, high heterozygosity, and high species diversity. In this study, we assemble a 3.26-Gb high-quality chromosome-scale genome for the ‘Longjing 43’ cultivar of Camellia sinensis var. sinensis. Genomic resequencing of 139 tea accessions from around the world is used to investigate the evolution and phylogenetic relationships of tea accessions. We find that hybridization has increased the heterozygosity and wide-ranging gene flow among tea populations with the spread of tea cultivation. Population genetic and transcriptomic analyses reveal that during domestication, selection for disease resistance and flavor in C. sinensis var. sinensis populations has been stronger than that in C. sinensis var. assamica populations. This study provides resources for marker-assisted breeding of tea and sets the foundation for further research on tea genetics and evolution.

Tea plant (Camellia sinensis) is one of the most economically valuable crops in the world. Anthracnose can affect the growth of leaves and cause serious yield losses of tea. Tea plants are rich in secondary metabolites; however, their roles in resistance to anthracnose are unclear. Herein we compared the contents of total phenolics, catechins, and caffeine in two cultivars with different resistances to anthracnose during Colletotrichum fructicola infection. (-)-Epigallocatechin-3-gallate (EGCG), (+)-catechin (C), caffeine, and critical regulatory genes were induced in C. fructicola-resistant tissues. In vitro antifungal tests showed that caffeine more strongly inhibited mycelial growth than tea polyphenols and catechins. Both electron microscopy and bioactivity analysis results showed that caffeine can affect mycelial cell walls and plasma membranes. Through promoter sequences analysis, a number of stress response-related cis-acting elements were identified in S-adenosylmethionine synthetase and tea caffeine synthase. These results demonstrated that (-)-EGCG, (+)-C, and caffeine may be involved in the resistance of tea plants to anthracnose.

Anthracnose causes severe losses of tea production in China. Although genes and biological processes involved in anthracnose resistance have been reported in other plants, the molecular response to anthracnose in tea plant is unknown. We used the susceptible tea cultivar Longjing 43 and the resistant cultivar Zhongcha 108 as materials and compared transcriptome changes in the leaves of both cultivars following Colletotrichum fructicola inoculation. In all, 9015 and 8624 genes were differentially expressed between the resistant and susceptible cultivars and their controls (0 h), respectively. In both cultivars, the differentially expressed genes (DEGs) were enriched in 215 pathways, including responses to sugar metabolism, phytohormones, reactive oxygen species (ROS), biotic stimuli and signalling, transmembrane transporter activity, protease activity and signalling receptor activity, but DEG expression levels were higher in Zhongcha 108 than in Longjing 43. Moreover, functional enrichment analysis of the DEGs showed that hydrogen peroxide (H2O2) metabolism, cell death, secondary metabolism, and carbohydrate metabolism are involved in the defence of Zhongcha 108, and 88 key genes were identified. Protein–protein interaction (PPI) network demonstrated that putative mitogen-activated protein kinase (MAPK) cascades are activated by resistance (R) genes and mediate downstream defence responses. Histochemical analysis subsequently validated the strong hypersensitive response (HR) and H2O2 accumulation that occurred around the hyphal infection sites in Zhongcha 108. Overall, our results indicate that the HR and H2O2 are critical mechanisms in tea plant defence against anthracnose and may be activated by R genes via MAPK cascades.

Winter dormancy is an important biological feature for tea plant to survive cold winters, and it also affects the economic output of tea plant, one of the few woody plants in the world whose leaves are harvested and one of the few non-conifer evergreen species with characterized dormancies. To discover the bud dormancy regulation mechanism of tea plant in winter, we analyzed the global gene expression profiles of axillary buds at the paradormancy, endodormancy, ecodormancy, and bud flush stages by RNA-Seq analysis. In total, 16,125 differentially expressed genes (DEGs) were identified among the different measured conditions. Gene set enrichment analysis was performed on the DEGs identified from each dormancy transition. Enriched gene ontology terms, gene sets and transcription factors were mainly associated with epigenetic mechanisms, phytohormone signaling pathways, and callose-related cellular communication regulation. Furthermore, differentially expressed transcription factors as well as chromatin- and phytohormone-associated genes were identified. GI-, CAL-, SVP-, PHYB-, SFR6-, LHY-, ZTL-, PIF4/6-, ABI4-, EIN3-, ETR1-, CCA1-, PIN3-, CDK-, and CO-related gene sets were enriched. Based on sequence homology analysis, we summarized the key genes with significant expression differences in poplar and tea plant. The major molecular pathways involved in tea plant dormancy regulation are consistent with those of poplar to a certain extent; however, the gene expression patterns varied. This study provides the global transcriptome profiles of overwintering buds at different dormancy stages and is meaningful for improving the understanding of bud dormancy in tea plant.