Project description:Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the cold treatment of tea leaves. Subsequently, aligning these sequencing date with plant known miRNAs, we characterized 112 C. sinensis conserved miRNAs. In addition, 215 potential candidate miRNAs were found; among them, 131 candidates with star sequence were chosen as novel miRNAs. There are both congruously and differently regulated miRNAs, and line-specific miRNAs were identified by microarray-based hybridization in response to cold stress. The miRNA chip included 3228 miRNA probes corresponding to miRNA transcripts listed in Sanger miRBase release 19.0 and 283 novel miRNAs probes founding in tea plant. In the study presented here, two tea plant cultivars, ‘Yingshuang’ (YS, a cold-tolerant tea plant cultivar) and ‘Baiye 1’ (BY, a cold-sensitive tea plant cultivar), were kept at 4°C for 4,12, 24 h, respectively, and 28°C for as control. These samples were used to acquire expression profiles of a total of 3,511 unique genes, leading to the successful construction of supervised
Project description:BACKGROUND: Tea 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. RESULTS: Using 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. CONCLUSIONS: Our 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.
Project description:C-repeat (CRT)/dehydration responsive element (DRE)-binding factor CBFs, a small family of genes encoding transcriptional activators, play important roles in plant cold tolerance. In this study, a comprehensive genome-wide analysis was carried out to identify and characterize the functional dynamics of CsCBFs in tea plant (Camellia sinensis). A total of 6 CBF genes were obtained from the tea plant genome and named CBF1-6. All of the CsCBFs had an AP2/ERF DNA-binding domain and nuclear localization signal (NLS) sequence. CsCBF-eGFP fusion and DAPI staining analysis confirmed the nuclear localization of the CsCBFs. Transactivation assays showed that the CsCBFs, except CsCBF1, had transcriptional activity. CsCBF expression was differentially induced by cold, heat, PEG, salinity, ABA, GA, MeJA, and SA stresses. In particular, the CsCBF genes were significantly induced by cold treatments. To further characterize the functions of CsCBF genes, we overexpressed the CsCBF3 gene in Arabidopsis thaliana plants. The resulting transgenic plants showed increased cold tolerance compared with the wild-type Arabidopsis plant. The enhanced cold tolerance of the transgenic plants was potentially achieved through an ABA-independent pathway. This study will help to increase our understanding of CsCBF genes and their contributions to stress tolerance in tea plants.
Project description:Background:Cold and frost are two serious factors limiting the yield of many crops worldwide, including the tea plant (Camellia sinensis (L.) Kuntze). The acclimatization of tea plant from tropical to temperate climate regions resulted in unique germplasm in the North-Western Caucasus with extremely frost-tolerant genotypes. Methods:The aim of the current research was to evaluate the physiological, biochemical and genetic responses of tolerant and sensitive tea cultivars exposed to cold (0 to +2 °C for 7 days) and frost (-6 to -8 °C for 5 days). Relative water content, cell membranes integrity, pH of the cell sap, water soluble protein, cations, sugars, amino acids were measured under cold and frost. Comparative expression of the following genes ICE1, CBF1, WRKY2, DHN1, DHN2, DHN3, NAC17, NAC26, NAC30, SnRK1.1, SnRK1.2, SnRK1.3, bHLH7, bHLH43, P5CS, LOX1, LOX6, LOX7 were analyzed. Results:We found elevated protein (by 3-4 times) and cations (potassium, calcium and magnesium) contents in the leaves of both cultivars under cold and frost treatments. Meanwhile, Leu, Met, Val, Thr, Ser were increased under cold and frost, however tolerant cv. Gruzinskii7 showed earlier accumulation of these amino acids. Out of 18 studied genes, 11 were expressed at greater level in the frost- tolerant cultivar comparing with frost-sensitive one: ICE1, CBF1, WRKY2, DHN2, NAC17, NAC26, SnRK1.1, SnRK1.3, bHLH43, P5CS and LOX6. Positive correlations between certain amino acids namely, Met, Thr, Leu and Ser and studied genes were found. Taken together, the revealed cold responses in Caucasian tea cultivars help better understanding of tea tolerance to low temperature stress and role of revealed metabolites need to be further evaluated in different tea genotypes.
Project description:Recently, intensive global climate change has become a major factor impacting plant survival during the winter. Freezing cold temperatures during the winter and abnormal temperature fluctuations during the winter and early spring are the most harmful ambient factors threatening tea plant winter survival and currently cause marked economic losses in tea production. In this study, by simulating natural climate change, we established cold acclimation (CA) and rapid cold stress (after CA) conditions to comprehensively investigate the transcriptome changes involved in CA and rapid cold stress. Electrolyte leakage (EL) rate and expression profile clustering analyses confirmed that the experimental design was valid. Comparative transcription analysis identified many differentially expressed genes (DEGs) involved in both processes. Time course and pathway enrichment analyses further revealed the physiological changes that occur during the initial period of CA and the cell wall changes that occur throughout the entire CA process; these changes play crucial roles in increasing freezing tolerance during this process. Compared with CA, different cold response mechanisms were rapidly activated under cold stress; however, the subsequent accumulation of reactive oxygen species, which affect multiple aspects, caused by freezing cold could be the harshest factor impairing tea leaves. Moreover, we investigated 60 DEGs shared by both processes and highlighted the importance of KCSs, HXXXD-type acyl-transferase family proteins, NAC080, SWEETs and ENOs in the responses to various cold conditions. These results greatly improve our knowledge of cold response mechanisms in tea plants and provide meaningful information for functional studies investigating cold tolerance-related genes. Overall design: Total of 8 biosamples including cold acclimation treatment and freezing cold treatment, triplication were applied. Therefore 24 microarray were used in this study.
Project description:Tea [Camellia sinensis (L) O. Kuntze, Theaceae] is one of the most popular non-alcoholic beverages worldwide. Cold stress is one of the most severe abiotic stresses that limit tea plants' growth, survival and geographical distribution. However, the genetic regulatory network and signaling pathways involved in cold stress responses in tea plants remain unearthed. Using RNA-Seq, DGE and sRNA-Seq technologies, we performed an integrative analysis of miRNA and mRNA expression profiling and their regulatory network of tea plants under chilling (4?) and freezing (-5?) stress. Differentially expressed (DE) miRNA and mRNA profiles were obtained based on fold change analysis, miRNAs and target mRNAs were found to show both coherent and incoherent relationships in the regulatory network. Furthermore, we compared several key pathways (e.g., 'Photosynthesis'), GO terms (e.g., 'response to karrikin') and transcriptional factors (TFs, e.g., DREB1b/CBF1) which were identified as involved in the early chilling and/or freezing response of tea plants. Intriguingly, we found that karrikins, a new group of plant growth regulators, and ?-primeverosidase (BPR), a key enzyme functionally relevant with the formation of tea aroma might play an important role in both early chilling and freezing response of tea plants. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-Seq and sRNA-Seq analysis. This is the first study to simultaneously profile the expression patterns of both miRNAs and mRNAs on a genome-wide scale to elucidate the molecular mechanisms of early responses of tea plants to cold stress. In addition to gaining a deeper insight into the cold resistant characteristics of tea plants, we provide a good case study to analyse mRNA/miRNA expression and profiling of non-model plant species using next-generation sequencing technology.
Project description:BACKGROUND:Alternative splicing (AS) may generate multiple mRNA splicing isoforms from a single mRNA precursor using different splicing sites, leading to enhanced diversity of transcripts and proteins. AS has been implicated in cold acclimation by affecting gene expression in various ways, yet little information is known about how AS influences cold responses in tea plant (Camellia sinensis). RESULTS:In this study, the AS transcriptional landscape was characterized in the tea plant genome using high-throughput RNA-seq during cold acclimation. We found that more than 41% (14,103) of genes underwent AS events. We summarize the possible existence of 11 types of AS events, including the four common types of intron retention (IR), exon skipping (ES), alternative 5' splice site (A5SS), and alternative 3' splice site (A3SS); of these, IR was the major type in all samples. The number of AS events increased rapidly during cold treatment, but decreased significantly following de-acclimation (DA). It is notable that the number of differential AS genes gradually increased during cold acclimation, and these genes were enriched in pathways relating to oxidoreductase activity and sugar metabolism during acclimation and de-acclimation. Remarkably, the AS isoforms of bHLH transcription factors showed higher expression levels than their full-length ones during cold acclimation. Interestingly, the expression pattern of some AS transcripts of raffinose and sucrose synthase genes were significantly correlated with sugar contents. CONCLUSION:Our findings demonstrated that changes in AS numbers and transcript expression may contribute to rapid changes in gene expression and metabolite profile during cold acclimation, suggesting that AS events play an important regulatory role in response to cold acclimation in tea plant.
Project description:In field conditions, especially in arid and semi-arid areas, tea plants are often simultaneously exposed to various abiotic stresses such as cold and drought, which have profound effects on leaf senescence process and tea quality. However, most studies of gene expression in stress responses focus on a single inciting agent, and the confounding effect of multiple stresses on crop quality and leaf senescence remain unearthed. Here, global transcriptome profiles of tea leaves under separately cold and drought stress were compared with their combination using RNA-Seq technology. This revealed that tea plants shared a large overlap in unigenes displayed "similar" (26%) expression pattern and avoid antagonistic responses (lowest level of "prioritized" mode: 0%) to exhibit very congruent responses to co-occurring cold and drought stress; 31.5% differential expressed genes and 38% of the transcriptome changes in response to combined stresses were unpredictable from cold or drought single-case studies. We also identified 319 candidate genes for enhancing plant resistance to combined stress. We then investigated the combined effect of cold and drought on tea quality and leaf senescence. Our results showed that drought-induced leaf senescence were severely delayed by (i) modulation of a number of senescence-associated genes and cold responsive genes, (ii) enhancement of antioxidant capacity, (iii) attenuation of lipid degradation, (iv) maintenance of cell wall and photosynthetic system, (v) alteration of senescence-induced sugar effect/sensitivity, as well as (vi) regulation of secondary metabolism pathways that significantly influence the quality of tea during combined stress. Therefore, care should be taken when utilizing a set of stresses to try and maximize leaf longevity and tea quality.
Project description:C-repeat binding factors (CBFs) are key signaling genes that can be rapidly induced by cold and bind to the C-repeat/dehydration-responsive motif (CRT/DRE) in the promoter region of the downstream cold-responsive (COR) genes, which play a vital role in the plant response to low temperature. However, the CBF family in tea plants has not yet been elucidated, and the possible target genes regulated by this family under low temperature are still unclear. In this study, we identified five CsCBF family genes in the tea plant genome and analyzed their phylogenetic tree, conserved domains and motifs, and cis-elements. These results indicate that CsCBF3 may be unique in the CsCBF family. This is further supported by our findings from the low-temperature treatment: all the CsCBF genes except CsCBF3 were significantly induced after treatment at 4 °C. The expression profiles of eight tea plant tissues showed that CsCBFs were mainly expressed in winter mature leaves, roots and fruits. Furthermore, 685 potential target genes were identified by transcriptome data and CRT/DRE element information. These target genes play a functional role under the low temperatures of winter through multiple pathways, including carbohydrate metabolism, lipid metabolism, cell wall modification, circadian rhythm, calcium signaling, transcriptional cascade, and hormone signaling pathways. Our findings will further the understanding of the stress regulatory network of CsCBFs in tea plants.