Wound- and pathogen-activated de novo JA synthesis using different ACX isozymes in tea plant (Camellia sinensis).

Acyl-CoA oxidase (ACX; EC 1.3.3.6) plays a vital role in the biosynthesis of jasmonic acid (JA) in plant peroxisomes. We previously identified an herbivore-induced gene CsACX1 in tea plant (Camellia sinensis) and showed CsACX1 was involved in the wound-induced synthesis of jasmonic acid (JA). Here, another ACX gene CsACX3 was isolated from tea plant. CsACX3 was predicted to consist of 684 amino acid residues. CsACX3 can be induced by mechanical wounding, JA application, and infestation by the tea geometrid Ectropis obliqua Prout and the tea green leafhopper Empoasca (Matsumurasca) onukii Matsuda. These expression patterns are consistent with the previously reported expression pattern of CsACX1 under such treatments. Recombinant CsACX3 showed preference for medium-chain acyl-coA oxidase substrates (C8- to C14-CoA). CsACX3 expression could also be induced by the infection of a pathogen Colletotrichum gloeosporioides (Cgl), and the increased ACX activities in tea plants were correlated with the Cgl-induced CsACX3 expression. Cgl could not induce the expression of CsACX1, which showed preference for C12- to C16-CoA substrates. The constitutive expression of CsACX3 rescued wound-induced JA biosynthesis and enhanced the Cgl-induced JA biosynthesis in Arabidopsis mutant atacx1. However, constitutive expression of CsACX1 could not enhance the Cgl-induced JA biosynthesis in atacx1 plant. These results indicate that CsACX1 and CsACX3 functions overlap and have distinct roles in the wound- and pathogen-activated de novo JA synthesis via enzymatic routes that utilize different ACX isozymes in tea plant.

Enhanced transcriptome responses in herbivore-infested tea plants by the green leaf volatile (Z)-3-hexenol.

Green leaf volatiles (GLVs) play a vital role in enhancing herbivore-associated defense responses, but the mechanism by which they precisely regulate such responses is not well understood. (Z)-3-Hexenol (z3HOL), an important component of GLVs, effectively activates the defense of tea plants (Camellia sinensis) against a tea geometrid (TG) Ectropis obliqua Prout. To elucidate the molecular mechanisms of defense activation by z3HOL, RNA-Sequencing was employed to investigate the effect of z3HOL on transcriptome responses to TG in tea plants. A total of 318 upregulated genes were identified, and expression of 10 unigenes was validated by quantitative real-time PCR. Among these 318 upregulated genes, 56 were defense-related, including 6 key enzyme genes in jasmonic acid, and ethylene biosynthesis, 24 signal transduction genes, and 12 insect-responsive transcription factors. Most of the defense-related genes are induced by JA, TG, or wounding treatments, suggesting that JA signaling plays a vital role in z3HOL-induced tea defense against TG.

A Disease Resistance Elicitor Laminarin Enhances Tea Defense against a Piercing Herbivore Empoasca (Matsumurasca) onukii Matsuda.

The tea plant (Camellia sinensis) suffers heavily from a harmful piercing pest, the tea green leafhopper (TLH) Empoasca (Matsumurasca) onukii Matsuda. In the present study, we studied the effect of an efficient elicitor of plant disease resistance, the ß-1,3-glucan laminarin, on the induced defense against TLH in tea plants. Defense responses elicited by laminarin in tea include the activation of mitogen-activated protein kinases and WRKY, the burst of H2O2, salicylic acid, and abscisic acid, and the accumulation of direct-defense chemicals (including chitinase, phenylalanine ammonia lyase, callose, polyphenol oxidase, and flavonol synthase), as well as the production of volatile compounds. The laminarin-treated tea plants reduced the performance of TLH and enhanced the attractiveness to the egg parasitoid wasp of TLH, Stethynium empoascae Subba Rao. In the field experiment, laminarin application effectively reduced the number of TLH by attracting parasitoids. These results suggest that laminarin can induce protection against TLH by regulating signaling pathways in tea plant. Our study also proposes an environment friendly strategy for the integrated management of an economically important piercing pest.

The involvement of a herbivore-induced acyl-CoA oxidase gene, CsACX1, in the synthesis of jasmonic acid and its expression in flower opening in tea plant (Camellia sinensis).

The biosynthesis of jasmonic acid (JA) in plant peroxisomes requires the action of acyl-CoA oxidase (ACX; EC 1.3.3.6). Multiple isoforms of ACXs have been identified in various annual herbaceous plants, but the genes encoding these enzymes in perennial woody plants are yet to be fully investigated. In this study, an ACX gene named CsACX1 (GeneBank accession: KX650077.1) was isolated from tea plant (Camellia sinensis L.). CsACX1 was predicted to consist of 664 amino acid residues. Transcriptional analysis revealed that CsACX1 can be induced by mechanical wounding, JA application, and infestation by the tea geometrid Ectropis obliqua Prout and the tea green leafhopper Empoasca (Matsumurasca) onukii Matsuda. To further elucidate the function of CsACX1, it was heterologously expressed in a bacterial system and characterized. Recombinant CsACX1 showed preference for C12 ∼ C16-CoA substrates. The constitutive expression of CsACX1 can rescue wound-related JA biosynthesis in Arabidopsis mutant acx1. CsACX1 was expressed in different organs, predominantly in flowers. Notably, CsACX1 transcripts were detected up-regulated during flower opening, and the JA levels were correlated with CsACX1 expression. All these results enrich our knowledge of the regulatory pathway involved in the JA biosynthesis in tea, and helps further understand the defense mechanism of tea plant against insects.

A putative 12-oxophytodienoate reductase gene CsOPR3 from Camellia sinensis, is involved in wound and herbivore infestation responses.

12-Oxophytodienoate reductase (OPR) is a key enzyme in the biosynthesis of jasmonic acid (JA), which plays an important role in plant defense responses. Although multiple isoforms of OPRs have been identified in various annual herbaceous plants, genes encoding these enzymes in perennial woody plants have yet to be fully investigated. In the tea plant, Camellia sinensis (L.), no OPR genes have been isolated, and their possible roles in tea plant development and defense mechanism remain unknown. In this study, a putative OPR gene, designated as CsOPR3, was isolated from tea plants for the first time through the rapid amplification of cDNA ends. The open reading frame of CsOPR3 is 1197bp in length, and encodes a protein of 398 amino acids. Real-time qPCR analysis revealed that CsOPR3 was expressed in different organs. In particular, CsOPR3 was highly expressed in flowers, leaves and stems but was weakly expressed in roots and seeds. CsOPR3 expression could be rapidly induced by mechanical wounding, and increased JA levels were correlated with the wound-induced CsOPR3 expression. The infestation of the tea geometrid (TG) Ectropis obliqua Prout, regurgitant derived from TG and exogenous JA application could enhance the CsOPR3 expression. Our study is the first to report that CsOPR3 plays an important role in JA biosynthesis and tea plant defense against herbivorous insects.