Two galactinol synthases contribute to the drought response of Camellia sinensis.

MAIN CONCLUSION: CsGolS2-1 and CsGolS2-2 are involved in the transcriptional mechanism and play an important role in the drought response of tea plants. GolS is critical for the biosynthesis of galactinol and has been suggested to contribute to drought tolerance in various plants. However, whether GolS plays a role in drought response and the underlying transcriptional mechanism of GolS genes in response to drought stress in tea plants is still unclear. In this study, we found that drought stress promotes the accumulation of galactinol in tea leaves and that the expression of CsGolS2-1 and CsGolS2-2, which encode proteins capable of catalyzing galactinol biosynthesis, is continuously and dramatically induced by drought stress. Moreover, transgenic Arabidopsis plants expressing CsGolS2-1 and CsGolS2-2 were more drought-tolerant than WT plants, as evidenced by increased cell membrane stability. In addition, the drought-responsive transcription factor CsWRKY2 has been shown to positively regulate the expression of CsGolS2-1 and CsGolS2-2 by directly binding to their promoters. Furthermore, CsVQ9 was found to interact with CsWRKY2 and promote its transcriptional function to activate CsGolS2-1 and CsGolS2-2 expression. Taken together, our findings provide insights not only into the positive role played by CsGolS2-1 and CsGolS2-2 in the drought response of tea plants but also into the transcriptional mechanisms involved.

Accumulation of Galactinol and ABA Is Involved in Exogenous EBR-Induced Drought Tolerance in Tea Plants.

Drought stress severely limits growth and causes losses in the yield of tea plants. Exogenous application of 24-epibrassinolide (EBR) positively regulates drought responses in various plants. However, whether EBR could contribute to drought resistance in tea plants and the underlying mechanisms has not been investigated. Here, we found that EBR application is beneficial for the drought tolerance of tea plants. The transcriptome results revealed that EBR could contribute to tea plant drought resistance by promoting galactinol and abscisic acid (ABA) biosynthesis gene expression. The content of galactinol was elevated by EBR and EBR-responsive CsDof1.1 positively regulated the expression of the galactinol synthase genes CsGolS2-1 and CsGolS2-2 to contribute to the accumulation of galactinol by directly binding to their promoters. Moreover, exogenous EBR was found to elevate the expression of genes related to ABA signal transduction and stomatal closure regulation, which resulted in the promotion of stomatal closure. In addition, EBR-responsive CsMYC2-2 is involved in ABA accumulation by binding to the promoters CsNCED1 and CsNCED2 to activate their expression. In summary, findings in this study provide knowledge into the transcriptional regulatory mechanism of EBR-induced drought resistance in tea plants.

CsGSTU8, a Glutathione S-Transferase From Camellia sinensis, Is Regulated by CsWRKY48 and Plays a Positive Role in Drought Tolerance.

Glutathione S-transferases (GSTs) constitute a large family of enzymes with a wide range of cellular functions. Recently, plant GSTs have gained a great deal of attention due to their involvement in the detoxification of electrophilic xenobiotics and peroxides under adverse environmental conditions, such as salt, cold, UV-B and drought stress. A previous study reported that a GST gene (CsGSTU8) in tea plant was distinctly induced in response to drought, suggesting this gene plays a critical role in the drought stress response. In this study, by using quantitative real-time PCR (qRT-PCR) and ß-glucuronidase (GUS) reporter lines, we further demonstrated that CsGSTU8 was upregulated in response to drought stress and exogenous abscisic acid (ABA) treatments. Overexpression of CsGSTU8 in Arabidopsis resulted in enhanced drought tolerance as indicated by the improved scavenging of excess amounts of reactive oxygen species (ROS) under drought conditions. Furthermore, we found that CsWRKY48 acts as a transcriptional activator and that its expression is induced in response to drought stress and ABA treatment. Electrophoretic mobility shift assays (EMSAs), dual-luciferase (LUC) assays and transient expression assays in tea plant leaves revealed that CsWRKY48 directly binds to the W-box elements in the promoter of CsGSTU8 and activates its expression. Taken together, our results provide additional knowledge of drought stress responses in tea plant.

Integrated Analysis of Long Non-coding RNAs (lncRNAs) and mRNAs Reveals the Regulatory Role of lncRNAs Associated With Salt Resistance in Camellia sinensis.

Tea plant (Camellia sinensis), an important economic crop, is seriously affected by various abiotic stresses, including salt stress, which severely diminishes its widespread planting. However, little is known about the roles of long non-coding RNAs (lncRNAs) in transcriptional regulation under salt stress. In this study, high-throughput sequencing of tea shoots under salt-stress and control conditions was performed. Through sequencing analysis, 16,452 unique lncRNAs were identified, including 172 differentially expressed lncRNAs (DE-lncRNAs). The results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of their cis- and trans-target genes showed that these DE-lncRNAs play important roles in many pathways such as the galactinol synthase (GOLS), calcium signaling pathway, and interact with transcription factors (TFs) under salt stress. The data from the gene-specific antisense oligodeoxynucleotide-mediated reduction in the lncRNA MSTRG.139242.1 and its predicted interacting gene, TEA027212.1 (Ca2+-ATPase 13), in tea leaves revealed that MSTRG.139242.1 may function in the response of tea plants to high salinity. In addition, 12 lncRNAs were predicted to be target mimics of 17 known mature miRNAs, such as miR156, that are related to the salt-stress response in C. sinensis. Our results provide new insights into lncRNAs as ubiquitous regulators in response to salt stress in tea plants.

Genome-wide identification and characterization of the CsSnRK2 family in Camellia sinensis.

The sucrose nonfermenting 1 (SNF1)-related protein kinase 2 (SnRK2) genes play central roles in plant stress signal transduction. In this study, 8 SnRK2 genes were identified from the tea plant genome database and named CsSnRK2.1-8. Phylogenetic analysis showed that the CsSnRK2 genes were classifiable into three groups, similar to those of Arabidopsis thaliana, Oryza sativa and maize. The coding sequences (CDSs) of all CsSnRK2s were separated by eight introns, and their exon-intron organizations exhibited high similarity to those of other plants. The fluorescence of GFP fused with CsSnRK2.3 was detected in only the cytoplasm, while the rest of the proteins showed GFP signal in both the nucleus and the cytoplasm. The results of the expression patterns of the CsSnRK2 genes showed that CsSnRK2s were differentially induced by salt, polyethylene glycol (PEG) and abscisic acid (ABA) stress. Interestingly, The expression of CsSnRK2.3 was inhibited by ABA, suggesting the complicated roles of CsSnRK2s in the ABA signal transduction pathway. Some CsSnRK2 gene pairs showed significant expression change correlations under stresses, indicating that CsSnRK2s might exhibit synergistic effects of signal regulation in response to various stresses. In summary, this comprehensive analysis will facilitate further studies of the SnRK2 family of Camellia sinensis and provide useful information for the functional validation of CsSnRK2s.

Genome-Wide Identification, Classification and Expression Analysis of the HSP Gene Superfamily in Tea Plant (Camellia sinensis).

Heat shock proteins (HSPs) function as molecular chaperones. These proteins are encoded by a multigene family whose members play crucial roles in plant growth, development and stress response. However, little is known about the HSP gene superfamily in tea plant. In this study, a total of 47 CsHSP genes were identified, including 7 CsHSP90, 18 CsHSP70, and 22 CssHSP genes. Phylogenetic and composition analyses showed that CsHSP proteins in the same subfamily have similar gene structures and conserved motifs, but significant differences exist in the different subfamilies. In addition, expression analysis revealed that almost all CsHSP genes were specifically expressed in one or more tissues, and significantly induced under heat and drought stress, implying that CsHSP genes play important roles in tea plant growth, development, and response to heat and drought stress. Furthermore, a potential interaction network dominated by CsHSPs, including HSP70/HSP90 organizing protein (HOP) and heat shock transcription factor (HSF), is closely related to the abovementioned processes. These results increase our understanding of CsHSP genes and their roles in tea plant, and thus, this study could contribute to the cloning and functional analysis of CsHSP genes and their encoded proteins in the future.