Xinyuan Hao

Global transcriptome profiles of Camellia sinensis during cold acclimation

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.

Effects of cold acclimation on sugar metabolism and sugar-related gene expression in tea plant during the winter season

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.

Molecular cloning and expression analysis of tea plant aquaporin (AQP) gene family.

The role of aquaporin proteins (AQPs) has been extensively studied in plants. However, the information of AQPs in the tea plant (Camellia sinensis) is unclear. In this manuscript, we isolated 20 full-length AQP cDNAs from the tea plant, and these sequences were classified into five subfamilies. The genes in these subfamilies displayed differential expression profiles in the studied tissues. The CsAQP expression patterns correlated with flower development and opening (FDO) and bud endodormancy (BED). To better understand the short-term expression patterns of CsAQPs in response to abiotic stress, tea plants were treated with abscisic acid (ABA), cold, salt or drought. ABA treatment down-regulated the expression of various CsAQPs. Salt up-regulated the transcription of most CsAQP genes. Cold treatment resulted in a complicated transcriptional regulation pattern for various CsAQPs. The expression of CsAQPs, especially plasma membrane intrinsic proteins (CsPIPs) and tonoplast intrinsic proteins (CsTIPs), was induced by drought and remained relatively high after rehydration in leaves, whereas almost all the CsAQPs were repressed in roots. Our results highlighted the diversity of CsAQPs in the tea plant and demonstrated that the CsPIP and CsTIP genes play a vital role in the stress response as well as in FDO and BED. Furthermore, certain CsSIPs (small basic intrinsic proteins), CsNIPs (NOD26-like intrinsic proteins) and CsXIPs (X intrinsic proteins) may regulate BED and FDO.

Isolation and expression analysis of 18 CsbZIP genes implicated in abiotic stress responses in the tea plant (Camellia sinensis).

Basic leucine zipper (bZIP) transcription factors (TFs) play essential roles in regulating stress processes in plants. Despite the economic importance of this woody crop, there is little information about bZIP TFs in tea plants. In this study, 18 bZIP genes were isolated from the tea plant (Camellia sinensis) and named sequentially from CsbZIP1 to CsbZIP18. According to the phylogenetic classification as in Arabidopsis, the CsbZIP genes spanned ten subgroups (Group A, B, C, D, E, F, H, I, S and K) of bZIP TFs. When analyzed for organ specific expression, all CsbZIP genes were found to be ubiquitously expressed in roots, stems, leaves and flowers. Expression analysis of CsbZIP genes in response to four abiotic stresses showed that in leaves, 9, 9, 15 and 11 CsbZIPs have 2-fold greater variation in transcript abundance under cold, exogenous ABA, high salinity and dehydration conditions, respectively. In roots, 5, 12, 14 and 11 CsbZIPs were differentially expressed under conditions of cold, exogenous ABA, high salinity and dehydration stresses. Moreover, CsbZIP genes in Groups F, H, S and K exhibited several folds up-and/or down-regulation against the above four stresses. Notably, CsbZIP18 of group K showed significant up-regulation in response to these same stresses, suggesting a vital functional role in stress response. Together, these findings increase our knowledge of bZIP TFs in the tea plant and suggest the significance of CsbZIP genes in plant abiotic responses.

Identification of differential gene expression profiles between winter dormant and sprouting axillary buds in tea plant (Camellia sinensis) by suppression subtractive hybridization

Tea plant (Camellia sinensis (L.) O. Kuntze) is an important cash crop. In temperate regions, bud dormancy in winter and budbreak in spring are important biological phenomena for the tea plant life cycle. To understand the molecular mechanism of dormancy maintenance and release in tea plant, the differentially expressed genes in dormant and sprouting axillary buds were investigated in two cultivars (special early-sprouting tea cultivar ‘Camellia sinensis cv. Longjing 43’ (‘LJ’) and late-sprouting tea cultivar ‘C. sinensis cv. Zhenghe Dabaicha’ (‘ZD’)), using suppression subtractive hybridization (SSH) approach. Four high performance complementary DNA (cDNA)-SSH libraries (‘LJ’ sprouting bud library, ‘LJ’ dormant bud library, ‘ZD’ sprouting bud library and ‘ZD’ dormant bud library) were constructed, and 1,736 valid ESTs were obtained, in which 1,242 ESTs were unique to the sprouting bud libraries and 494 ESTs were unique to the dormant bud libraries. Based on sequence matching and gene ontology analysis, 1,287 unigenes consisting of 208 contigs and 1,079 singletons were identified, in which 995 had Blast hits. The putative functions of differentially regulated sequences were involved in most aspects of plant biological processes. The quality and expression patterns of partial ESTs from these four libraries were validated by qRT-PCR. We identified numerous differentially expressed genes mainly involved in stress response, water metabolism, cell division regulation, energy metabolism and hormone regulation from dormant and sprouting bud libraries. This study provides general information on tea plant axillary bud dormancy and release at the transcriptional level and provides some hypotheses for further exploration on the mechanism of bud dormancy and budbreak in tea plant.

Isolation and expression features of hexose kinase genes under various abiotic stresses in the tea plant (Camellia sinensis)

Hexokinases (HXKs, EC 2.7.1.1) and fructokinases (FRKs, EC 2.7.1.4) play important roles in carbohydrate metabolism and sugar signaling during the growth and development of plants. However, the HXKs and FRKs in the tea plant (Camellia sinensis) remain largely unknown. In this manuscript, we present the molecular characterization, phylogenetic relationships, conserved domains and expression profiles of four HXK and seven FRK genes of the tea plant. The 11 deduced CsHXK and CsFRK proteins were grouped into six main classes. All of the deduced proteins, except for CsFKR7, possessed putative ATP-binding motifs and a sugar recognition region. These genes exhibited tissue-specific expression patterns, which suggests that they play different roles in the metabolism and development of source and sink tissues in the tea plant. There were variations in CsHXKs and CsFRKs transcript abundance in response to four abiotic stresses: cold, salt, drought and exogenous abscisic acid (ABA). Remarkably, CsHXK3 and CsHXK4 were significantly induced in the leaves and roots under cold conditions, CsHXK1 was apparently up-regulated in the leaves and roots under salt and drought stresses, and CsHXK3 was obviously stimulated in the leaves and roots under short-term treatment with exogenous ABA. These findings demonstrate that CsHXKs play critical roles in response to abiotic stresses in the tea plant. Our research provides a fundamental understanding of the CsHXK and CsFRK genes of the tea plant and important information for the breeding of stress-tolerant tea cultivars.

Identification of the invertase gene family (INVs) in tea plant and their expression analysis under abiotic stress

Key messageFourteen invertase genes were identified in the tea plant, all of which were shown to participate in regulating growth and development, as well as in responding to various abiotic stresses.AbstractInvertase (INV) can hydrolyze sucrose into glucose and fructose, which plays a principal role in regulating plant growth and development as well as the plants response to various abiotic and biotic stresses. However, currently, there is a lack of reported information, regarding the roles of INVs in either tea plant development or in the tea plants response to various stresses. In this study, 14 INV genes were identified from the transcriptome data of the tea plant (Camellia sinensis (L.) O. Kuntze), and named CsINV1-5 and CsINV7-15. Based on the results of a Blastx search and phylogenetic analysis, the CsINV genes could be clustered into 6 acid invertase (AI) genes and 8 alkaline/neutral invertase (A/N-Inv) genes. The results of tissue-specific expression analysis showed that the transcripts of all the identified CsINV genes are detectable in various tissues. Under various abiotic stress conditions, the expression patterns of the 14 CsINV genes were diverse in both the leaves and roots, and some of them were shown to be significantly expressed. Overall, we hypothesize that the identified CsINV genes all participate in regulating growth and development in the tea plant, and most likely through different signaling pathways that regulate the carbohydrate allocation and the ratio of hexose and sucrose for improving the resistance of the leaves and the roots of the tea plant to various abiotic stresses.

Transcriptomic analysis of the effects of three different light treatments on the biosynthesis of characteristic compounds in the tea plant by RNA-Seq

The tea plant (Camellia sinensis) is considered an environment-sensitive plant, because the biosynthesis of tea’s characteristic compounds is easily influenced by light quality, ambient temperature, and humidity. The characteristic compounds in tea leaves, especially polyphenols, theanine, and caffeine, are important components that contribute to tea’s flavor. To investigate the effects of light quality on secondary metabolism in young shoots of tea plants at the messenger RNA (mRNA) transcript level, RNA-Seq was performed using one leaf and a bud from tea cultivar 108, which was reared in three different colored light conditions. Plastic film (blue, purple, and yellow) was used to create different monochromatic light conditions, which were compared to natural (white) light treatment as a control. Our results showed that, compared to the control, the 2667, 1938, and 2326 genes were differentially expressed and the 1518, 1032, and 1316 genes were upregulated in blue, purple, and yellow light conditions, respectively. Gene Ontology (GO) analysis indicated that oxidation-reduction, regulation of transcription, and protein phosphorylation were the main enriched biological process terms in all three treatments. Conversely, the top enriched terms in the cellular component and molecular function categories were plasma membrane and ATP binding. Furthermore, KEGG analysis showed that the genes which were downregulated in blue light were enriched for genes in the amino acid biosynthesis, flavonoid biosynthesis, and phenylpropanoid biosynthesis pathways. Similarly, phenylpropanoid biosynthesis and phenylalanine metabolism pathway genes were enriched among the transcripts that were downregulated in purple light and yellow light. Particularly, a few genes encoding enzymes involved in the biosynthesis of catechins, theanine, and caffeine exhibited significantly different expression levels in the different monochromatic light conditions. Compared to purple and yellow light treatments, more genes were differentially expressed in blue light relative to white light. These results demonstrated that light quality affects secondary metabolism in young shoots of the tea plant, and that light color can influence the biosynthesis of characteristic compounds in tea leaves.

Differential expression of gibberellin- and abscisic acid-related genes implies their roles in the bud activity-dormancy transition of tea plants

Key messageThirty genes involved in GA and ABA metabolism and signalling were identified, and the expression profiles indicated that they play crucial roles in the bud activity-dormancy transition in tea plants.AbstractGibberellin (GA) and abscisic acid (ABA) are fundamental phytohormones that extensively regulate plant growth and development, especially bud dormancy and sprouting transition in perennial plants. However, there is little information on GA- and ABA-related genes and their expression profiles during the activity-dormancy transition in tea plants. In the present study, 30 genes involved in the metabolism and signalling pathways of GA and ABA were first identified, and their expression patterns in different tissues were assessed. Further evaluation of the expression patterns of selected genes in response to GA3 and ABA application showed that CsGA3ox, CsGA20ox, CsGA2ox, CsZEP and CsNCED transcripts were differentially expressed after exogenous treatment. The expression profiles of the studied genes during winter dormancy and spring sprouting were investigated, and somewhat diverse expression patterns were found for GA- and ABA-related genes. This diversity was associated with the bud activity-dormancy cycle of tea plants. These results indicate that the genes involved in the metabolism and signalling of GA and ABA are important for regulating the bud activity-dormancy transition in tea plants.

Transcriptome sequencing dissection of the mechanisms underlying differential cold sensitivity in young and mature leaves of the tea plant (Camellia sinensis)

The tea plant originated in tropical and subtropical regions and experiences considerable challenges during cold winters and late spring frosts. After short-term chilling stress, young leaves of tea plants exhibit browning, a significant increase in electrolyte leakage and a marked decrease in the maximal photochemical efficiency of photosystem II (Fv/Fm) compared with mature leaves. To identify the mechanisms underlying the different chilling tolerance between young and mature leaves of the tea plant, we used Illumina RNA-Seq technology to analyse the transcript expression profiles of young and mature leaves exposed to temperatures of 20 °C, 4 °C, and 0 °C for 4 h. A total of 45.70-72.93 million RNA-Seq raw reads were obtained and then de novo assembled into 228,864 unigenes with an average length of 601 bp and an N50 of 867 bp. In addition, the differentially expressed unigenes were identified via Venn diagram analyses for paired comparisons of young and mature leaves. Functional classifications based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that the up-regulated differentially expressed genes were predominantly related to the cellular component terms of chloroplasts and cell membranes, the biological process term of oxidation-reduction process as well as the pathway terms of glutathione metabolism and photosynthesis, suggesting that these components and pathways may contribute to the cold hardiness of mature leaves. Conversely, the inhibited expression of genes related to cell membranes, carotenoid metabolism, photosynthesis, and ROS detoxification in young leaves under cold conditions might lead to the disintegration of cell membranes and oxidative damage to the photosynthetic apparatus. Further quantitative real-time PCR testing validated the reliability of our RNA-Seq results. This work provides valuable information for understanding the mechanisms underlying the cold susceptibility of young tea plant leaves and for breeding tea cultivars with superior frost resistance via the genetic manipulation of antioxidant enzymes.