Characterization of Cuticular Wax in Tea Plant and Its Modification in Response to Low Temperature.

Cuticular wax ubiquitously covers the outer layer of plants and protects them against various abiotic and biotic stresses. Nevertheless, the characteristics of cuticular wax and its role in cold resistance in tea plants remain unclear. In our study, cuticular wax from different tissues, cultivars, and leaves during different spatio-temporal growth stages were characterized and compared in tea plants. The composition, distribution pattern, and structural profile of cuticular wax showed considerable tissue specificity, particularly in petals and seeds. During the spatial development of tea leaves, total wax content increased from the first to fifth leaf in June, while a decreasing pattern was observed in September. Additionally, the total wax content and number of wax compounds were enhanced, and the wax composition significantly varied with leaf growth from June to September. Ten cultivars showed considerable differences in total wax content and composition, such as the predominance of saturated fatty acids and primary alcohols in SYH and HJY cultivars, respectively. Correlation analysis suggested that n-hexadecanoic acid is positively related to cold resistance in tea plants. Further transcriptome analysis from cold-sensitive AJBC, cold-tolerant CYQ, and EC 12 cultivars indicated that the inducible expression of wax-related genes was associated with the cold tolerance of different cultivars in response to cold stress. Our results revealed the characterization of cuticular wax in tea plants and provided new insights into its modification in cold tolerance.

Duplication and transcriptional divergence of three Kunitz protease inhibitor genes that modulate insect and pathogen defenses in tea plant (Camellia sinensis).

Kunitz protease inhibitors (KPIs) are ubiquitous in plants and act as crucial compounds in defense responses against insect attack and pathogen infection. However, the influence of gene duplication on the postdivergence of the CsKPI genes involved in biotic stresses in tea plant is not well known. Here, we identified three CsKPI genes from tea plant (Camellia sinensis) and characterized their expression and evolutionary patterns among plant species. We found that CsKPI1, CsKPI2, and CsKPI3 diverged from their common ancestor 72.94 million years ago (MYA), and the tandem duplication of CsKPI2 and CsKPI3 occurred 26.78 MYA. An in vitro protein assay showed that the three CsKPI proteins were functional and inhibited the production of p-nitroanilide (PNA) from an artificial substrate. The three CsKPI-GFP fusion proteins localized to the cytoplasm. We showed that salicylic acid (SA) and transcripts of CsKPI2 and CsKPI3 significantly accumulated after infection with Glomerella cingulata. The application of exogenous SA stimulated the high expression of both CsKPI2 and CsKPI3 by activating cis-elements within their promoters. Under Ectropis oblique attack, CsKPI1 expression and jasmonic acid (JA) levels were more abundant in both insect-damaged leaf tissues and undamaged neighboring leaves. The application of jasmonic acid methyl ester elicited high expression levels of CsKPI1, suggesting that CsKPI1 accumulation requires JA production in tea plant. The overall findings suggest that the transcriptional divergence of KPI genes after duplication led to the specialized role of CsKPI1 in the physiological response to insect stress; the functional conservation between CsKPI2 and CsKPI3 confers resistance to pathogen infection in tea plant.