Comprehensive analysis of the laccase gene family in tea plant highlights its roles in development and stress responses.

BACKGROUND: Laccase (LAC) is the pivotal enzyme responsible for the polymerization of monolignols and stress responses in plants. However, the roles of LAC genes in plant development and tolerance to diverse stresses are still largely unknown, especially in tea plant (Camellia sinensis), one of the most economically important crops worldwide. RESULTS: In total, 51 CsLAC genes were identified, they were unevenly distributed on different chromosomes and classified into six groups based on phylogenetic analysis. The CsLAC gene family had diverse intron-exon patterns and a highly conserved motif distribution. Cis-acting elements in the promoter demonstrated that promoter regions of CsLACs encode various elements associated with light, phytohormones, development and stresses. Collinearity analysis identified some orthologous gene pairs in C. sinensis and many paralogous gene pairs among C. sinensis, Arabidopsis and Populus. Tissue-specific expression profiles revealed that the majority of CsLACs had high expression in roots and stems and some members had specific expression patterns in other tissues, and the expression patterns of six genes by qRT‒PCR were highly consistent with the transcriptome data. Most CsLACs showed significant variation in their expression level under abiotic (cold and drought) and biotic (insect and fungus) stresses via transcriptome data. Among them, CsLAC3 was localized in the plasma membrane and its expression level increased significantly at 13 d under gray blight treatment. We found that 12 CsLACs were predicted to be targets of cs-miR397a, and most CsLACs showed opposite expression patterns compared to cs-miR397a under gray blight infection. Additionally, 18 highly polymorphic SSR markers were developed, these markers can be widely used for diverse genetic studies of tea plants. CONCLUSIONS: This study provides a comprehensive understanding of the classification, evolution, structure, tissue-specific profiles, and (a)biotic stress responses of CsLAC genes. It also provides valuable genetic resources for functional characterization towards enhancing tea plant tolerance to multiple (a)biotic stresses.

A monoterpene synthase gene cluster of tea plant (Camellia sinensis) potentially involved in constitutive and herbivore-induced terpene formation.

Monoterpenes and sesquiterpenes are the most abundant volatiles in tea plants and have dual functions in aroma quality formation and defense responses in tea plants. Terpene synthases (TPS) are the key enzymes for the synthesis of terpenes in plants; however, the functions of most of them in tea plants are still unknown. In this study, six putative terpene biosynthesis gene clusters were identified from the tea plant genome. Then we cloned three new TPS-b subfamily genes, CsTPS08, CsTPS10 and CsTPS58. In vitro enzyme assays showed that CsTPS08 and CsTPS58 are two multiple-product terpene synthases, with the former synthesizing linalool as the main product, and ß-myrcene, α-phellandrene, α-terpinolene, D-limonene, cis-ß-ocimene, trans-ß-ocimene and (4E,6Z)-allo-ocimene as minor products are also detected, while the latter catalyzing the formation of α-pinene and D-limonene using GPP as the substrate. No product of CsTPS10 was detected in the prokaryotic expression system, but geraniol production was detected when transiently expressed in tobacco leaves. CsTPS08 and CsTPS10 are two functional members of a monoterpene synthase gene cluster, which were significantly induced during both Ectropis oblique feeding and fresh leaf spreading treatments, suggesting that they have dual functions involved in tea plant pest defense and tea aroma quality regulation. In addition, the differences in their expression levels in different tea plant cultivars provide a possibility for the subsequent screening of tea plant resources with a specific aroma flavor. Our results deepen the understanding of terpenoid synthesis in tea plants.

miR319a targeting of CsTCP10 plays an important role in defense against gray blight disease in tea plant (Camellia sinensis).

Gray blight disease occurs widely in major tea-producing areas and harms the leaves of tea trees, which affects the quality and yield of processed tea. According to an analysis of previous sequencing data, miR319a may be important in the resistance of tea plants to gray blight disease. In this study, based on 5'RLM-RACE, qRT-PCR, sODN, CIN and transient transformation experiments in tobacco, CsTCP10 and CsTCP4 were found to be cleaved by miR319a. qRT-PCR and northern blotting also revealed that the expression pattern of CsTCP10 in tea leaves was opposite to that of miR319a, while that of CsTCP4 displayed no similar change. Furthermore, a large amount of reactive oxygen species was found to accumulate in tea leaves in the antisense oligodeoxynucleotide experiment, while the expression of CsTCP10 was inhibited. These results suggest that CsTCP10 is a positive regulator of the resistance of tea plants to gray blight disease. Compared with the wild-type, the expression of AtTCP10 in transgenic Arabidopsis plants was downregulated. After infection with the pathogen, the transgenic plants were more severely damaged. Our results suggest that miR319a facilitates Pestalotiopsis infection by suppressing the expression of CsTCP10 in tea plants.

Integrated metabolic phenotypes and gene expression profiles revealed the effect of spreading on aroma volatiles formation in postharvest leaves of green tea.

Spreading is an indispensable process in the aroma formation of premium green tea. In this study, volatile metabolomics and transcriptomics were performed for three tea plant cultivars to investigate the mechanism of changes occurring in volatile compounds during green tea spreading. The content of primary aroma compounds significantly increased after spreading, the Wickremasinghe-Yamanishi ratio decreased and the Owuor's flavor index increased with the extension of spreading time, and the degree of aroma production was genotype-dependent. Volatile terpenes and fatty acid-derived volatiles were the principal aroma volatiles that accumulated during the spreading of green tea, and the trends of their changes were consistent with the expression pattern of related synthesis pathway genes, indicating that they were primarily derived from de novo synthesis rather than glycoside hydrolysis. Two co-expression networks that were highly correlated with variations in the volatile component contents during the spreading process were identified via WGCNA. Our results provide insights into spreading that can be considered to improve the quality of green tea.