Light induces an increasing release of benzyl nitrile against diurnal herbivore Ectropis grisescens Warren attack in tea (Camellia sinensis) plants.

Herbivore-induced plant volatiles (HIPVs) are critical compounds that directly or indirectly regulate the tritrophic interactions among herbivores, natural enemies and plants. The synthesis and release of HIPVs are regulated by many biotic and abiotic factors. However, the mechanism by which multiple factors synergistically affect HIPVs release remains unclear. Tea plant (Camellia sinensis) is the object of this study because of its rich and varied volatile metabolites. In this study, benzyl nitrile was released from herbivore-attacked tea plants more in the daytime than at night, which was consistent with the feeding behaviour of tea geometrid (Ectropis grisescens Warren) larvae. The Y-tube olfactometer assay and insect resistance analysis revealed that benzyl nitrile can repel tea geometrid larvae and inhibit their growth. On the basis of enzyme activities in transiently transformed Nicotiana benthamiana plants, CsCYP79 was identified as a crucial regulator in the benzyl nitrile biosynthetic pathway. Light signalling-related transcription factor CsPIF1-like and the jasmonic acid (JA) signalling-related transcription factor CsMYC2 serve as the activator of CsCYP79 under light and damage conditions. Our study revealed that light (abiotic factor) and herbivore-induced damage (biotic stress) synergistically regulate the synthesis and release of benzyl nitrile to protect plants from diurnal herbivorous tea geometrid larvae.

Histone deacetylase CsHDA6 mediates the regulated formation of the anti-insect metabolite α-farnesene in tea (Camellia sinensis).

α-Farnesene accumulated in tea plants following infestations by most insects, and mechanical wounding is the common factor. However, the specific mechanism underlying the wounding-regulated accumulation of α-farnesene in tea plants remains unclear. In this study, we observed that histone deacetylase inhibitor treatment induced the accumulation of α-farnesene. The histone deacetylase CsHDA6 interacted directly with CsMYC2, which was an important transcription factor in the jasmonic acid (JA) pathway, and co-regulated the expression of the key α-farnesene synthesis gene CsAFS. Wounding caused by insect infestation affected CsHDA6 production at the transcript and protein levels, while also inhibited the binding of CsHDA6 to the CsAFS promoter. The resulting increased acetylation of histones H3/H4 in CsAFS enhanced the expression of CsAFS and the accumulation of α-farnesene. In conclusion, our study demonstrated the effect of histone acetylation on the production of tea plant HIPVs and revealed the importance of the CsHDA6-CsMYC2 transcriptional regulatory module.

Involvement of DNA methylation in regulating the accumulation of the aroma compound indole in tea (Camellia sinensis) leaves during postharvest processing.

The manufacturing process of tea (Camellia sinensis), especially oolong tea, involves multiple postharvest stresses. These stresses can induce the formation and accumulation of many important aroma compounds, such as indole-a key floral aroma contributor of oolong tea. However, little is known about the regulation mechanisms of aroma compound formation, especially epigenetic regulation. DNA methylation is an important epigenetic modification. Changes in the DNA methylation levels of promoter sequences can regulate gene expression under stress conditions. In this study, the differences in DNA methylation levels and histone 3 lysine 9 dimethylation levels of indole key biosynthetic gene (tryptophan synthase ß-subunit 2, CsTSB2) were detected between untreated and continuous wounding treatment tea leaves. The results show that the DNA methylation levels affect the ability of the basic helix-loop-helix family transcription factor CsMYC2a to bind to the promoter of CsTSB2. Analyses of the transcript levels of DNA methyltransferases during oolong tea processing screened out candidate genes involved in the regulation of secondary metabolite product biosynthesis/accumulation. The results suggest that the domains rearranged methyltransferase 3, a DNA methyltransferase, is involved in the DNA methylation regulation of indole formation during the oolong tea manufacturing process. This is the first report on the involvement of DNA methylation in the regulation of aroma compound formation in tea leaves exposed to postharvest stresses.

Epigenetic Regulation of the Phytohormone Abscisic Acid Accumulation under Dehydration Stress during Postharvest Processing of Tea (Camellia sinensis).

The plant hormone abscisic acid (ABA) accumulates in tea leaves under dehydration stress during the withering process. However, the mechanism underlying ABA biosynthesis regulation remains largely unclear. In the present study, we found increased expression of ABA biosynthesis genes under dehydration stress during postharvest processing of tea. Furthermore, dehydration stress promoted ABA accumulation by increasing histone acetylation of ABA anabolism genes but by decreasing the levels of histone H3 lysine 9 dimethylation and DNA methylation of ABA biosynthesis genes. We screened candidate regulators of histone deacetylation and DNA methylation under dehydration stress. Taken together, our results indicate a role for epigenetic modifications during postharvest processing of tea.

Elucidation of ( Z)-3-Hexenyl-ß-glucopyranoside Enhancement Mechanism under Stresses from the Oolong Tea Manufacturing Process.

The enzymatic hydrolysis of glycosidically bound volatiles (GBVs) plays an important role in tea aroma formation during the tea manufacturing process. However, during the enzyme-active manufacturing process of oolong tea, most GBVs showed no reduction, while ( Z)-3-hexenyl-ß-glucopyranoside significantly enhanced at the turnover stage. This study aimed to determine the reason for this increase in ( Z)-3-hexenyl-ß-glucopyranoside. Continuous wounding stress in the turnover stage did not enhance the expression level of glycosyltransferase 1 ( CsGT1), while it induced a significant increase in the ( Z)-3-hexenol content ( p ≤ 0.05). Furthermore, observing the cell structures of tea leaves exposed to continuous wounding and subcellular localizations of CsGTs suggested that the interaction of ( Z)-3-hexenol (substrate) and CsGT1 (enzyme) was available. In conclusion, both continuous wounding and subcellular localizations led to a ( Z)-3-hexenyl-ß-glucopyranoside enhancement mechanism during the oolong tea's turnover stage. These results advance our understanding of GBV formation during the tea manufacturing process and their relationship with the stress from the tea manufacturing process. In addition, the information will help us further evaluate contribution of GBVs to enzymatic formation of oolong tea aroma compounds.

Influence of Chloroplast Defects on Formation of Jasmonic Acid and Characteristic Aroma Compounds in Tea (Camellia sinensis) Leaves Exposed to Postharvest Stresses.

Characteristic aroma formation in tea (Camellia sinensis) leaves during the oolong tea manufacturing process might result from the defense responses of tea leaves against these various stresses, which involves upregulation of the upstream signal phytohormones related to leaf chloroplasts, such as jasmonic acid (JA). Whether chloroplast changes affect the formation of JA and characteristic aroma compounds in tea leaves exposed to stresses is unknown. In tea germplasms, albino-induced yellow tea leaves have defects in chloroplast ultrastructure and composition. Herein, we have compared the differential responses of phytohormone and characteristic aroma compound formation in normal green and albino-induced yellow tea leaves exposed to continuous wounding stress, which is the main stress in oolong tea manufacture. In contrast to single wounding stress (from picking, as a control), continuous wounding stress can upregulate the expression of CsMYC2, a key transcription factor of JA signaling, and activate the synthesis of JA and characteristic aroma compounds in both normal tea leaves (normal chloroplasts) and albino tea leaves (chloroplast defects). Chloroplast defects had no significant effect on the expression levels of CsMYC2 and JA synthesis-related genes in response to continuous wounding stress, but reduced the increase in JA content in response to continuous wounding stress. Furthermore, chloroplast defects reduced the increase in volatile fatty acid derivatives, including jasmine lactone and green leaf volatile contents, in response to continuous wounding stress. Overall, the formation of metabolites derived from fatty acids, such as JA, jasmine lactone, and green leaf volatiles in tea leaves, in response to continuous wounding stress, was affected by chloroplast defects. This information will improve understanding of the relationship of the stress responses of JA and aroma compound formation with chloroplast changes in tea.