Two New Polyphenol Oxidase Genes of Tea Plant (Camellia sinensis) Respond Differentially to the Regurgitant of Tea Geometrid, Ectropis obliqua

Huang, Chen; Jin, Zhang; Zhang, Xin; Yu, Yongchen; Bian, Wenbo; Zeng, Zhongping; Sun, Xiaoling; Li, Xinghui

doi:10.3390/ijms19082414

Cited by 16 publications

(9 citation statements)

References 61 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The expression vector carrying the jrPPO1-wt gene was transformed into E. coli BL21 (DE3) and was expressed similar to previous studies 21,39 , resulting in a final yield of 38 mg pure protein per liter of culture. However, a suitable protocol is required in order to avoid production of insoluble and/ or inactive PPO [40][41][42][43] . Different expression protocols have been reported, including the variation of affinity-tags, incubation temperatures and expression hosts (Table S2).…”

Section: Resultsmentioning

confidence: 99%

Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis

Panis

Kampatsikas

Bijelic

et al. 2020

Sci Rep

View full text Add to dashboard Cite

polyphenol oxidases (ppos) comprise tyrosinases (tYRs) and catechol oxidases (cos), which catalyse the initial reactions in the biosynthesis of melanin. tYRs hydroxylate monophenolic (monophenolase activity) and oxidize diphenolic (diphenolase activity) substrates, whereas cos react only with diphenols. In order to elucidate the biochemical basis for the different reactions in PPOs, cDNA from walnut leaves was synthesized, the target gene encoding the latent walnut tyrosinase (jrPPO1) was cloned, and the enzyme was heterologously expressed in Escherichia coli. Mutations targeting the two activity controller residues (Asn240 and Leu244) as well as the gatekeeper residue (Phe260) were designed to impair monophenolase activity of jrPPO1. For the first time, monophenolase activity of jrPPO1 towards L-tyrosine was blocked in two double mutants (Asn240Lys/Leu244Arg and Asn240Thr/ Leu244Arg) while its diphenolase activity was partially preserved, thereby converting jrPPO1 into a CO. Kinetic data show that recombinant jrPPO1 resembles the natural enzyme, and spectrophotometric investigations proved that the copper content remains unaffected by the mutations. The results presented herein provide experimental evidence that a precisely tuned interplay between the amino acids located around the active center controls the substrate specificity and therewith the mono-versus diphenolase activity in the type-iii copper enzyme jrPPO1.Tyrosinases (TYRs), catechol oxidases (COs) and aurone synthases (AUSs) represent the polyphenol oxidase (PPO) family, which is an umbrella term for copper metalloenzymes 1-3 containing one type-III copper center. TYRs catalyse the hydroxylation of monophenols to o-diphenols (EC 1.14.18.1, monophenolase activity) as well as the subsequent oxidation of o-diphenols to their corresponding o-quinones (EC 1.10.3.1, diphenolase activity) 1,4 , whereas COs catalyse only the latter reaction, unable to react with monophenolic substrates (Fig. 1). AUSs participate in the formation of aurones from chalcone precursors and are involved in plant secondary metabolism 5,6 . Quinones produced by PPOs usually undergo non-enzymatic reactions, polymerize 7 and finally form melanin products 8,9 . PPOs occur in a broad spectrum of organisms, including archaea 10 , bacteria 11 , fungi 2 , plants 8 and animals 12,13 . In plants, they are believed to be involved in defence mechanisms associated with the formation of browning substances, which is triggered by mechanical damage or wounding 14 , while in animals their reaction products are responsible for coloring of skin, hair and eyes 13 .TYR from Juglans regia (walnut, jrPPO1) is expressed in vivo as a latent 66.8 kDa pro-enzyme consisting of three domains 15 : an N-terminal chloroplast transit peptide (~12 kDa) 15 , the catalytically active domain (~39 kDa) and the C-terminal domain (~16 kDa) that shields the entrance to the catalytic pocket and keeps the enzyme in a latent state. In vivo enzymatic activity is triggered by the removal of the C-terminal domain 16 . Alternati...

show abstract

Section: Resultsmentioning

confidence: 99%

Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis

Panis

Kampatsikas

Bijelic

et al. 2020

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Polyphenol oxidase exhibits multiple important physiological effects such as resistance to stress, pests and diseases during growth and development ( Huang et al, 2018 ). During the manufacturing processes of black tea, the flavonoids present in the leaves are oxidized to o-quinones by PPO that are associated with the formation of pigments such as thearubigins, theaflavins, and theabrownin.…”

Section: Discussionmentioning

confidence: 99%

“… Zhang et al (2020) find that methyl jasmonate enhances PPO activity by activating the expression of CsPPO2 and CsPPO4 gene to improve defense against tea geometrid larvae ( Ectropis grisescens ) in tea plant. Additionally, mechanical damage or regurgitant of tea geometrid significantly upregulates the transcriptional expression of CsPPO1 and CsPPO2 ( Huang et al, 2018 ). These findings indicate that increasing the expression of PPO genes in tea plants is of interest; however, the molecular mechanism of the regulation of PPO activity in tea plants remains unclear.…”

Section: Introductionmentioning

confidence: 99%

The R2R3 Transcription Factor CsMYB59 Regulates Polyphenol Oxidase Gene CsPPO1 in Tea Plants (Camellia sinensis)

Huang

Qin

et al. 2021

Front. Plant Sci.

View full text Add to dashboard Cite

Polyphenol oxidase (PPO) plays a role in stress response, secondary metabolism, and other physiological processes during plant growth and development, and is also a critical enzyme in black tea production. However, the regulatory mechanisms of PPO genes and their activity in tea plants are still unclear. In this study, we measured PPO activity in two different tea cultivars, Taoyuandaye (TYDY) and Bixiangzao (BXZ), which are commonly used to produce black tea and green tea, respectively. The expression pattern of CsPPO1 was assessed and validated via transcriptomics and quantitative polymerase chain reaction in both tea varieties. In addition, we isolated and identified an R2R3-MYB transcription factor CsMYB59 that may regulate CsPPO1 expression. CsMYB59 was found to be a nuclear protein, and its expression in tea leaves was positively correlated with CsPPO1 expression and PPO activity. Transcriptional activity analysis showed that CsMYB59 was a transcriptional activator, and the dual-luciferase assay indicated that CsMYB59 could activate the expression of CsPPO1 in tobacco leaves. In summary, our study demonstrates that CsMYB59 represents a transcriptional activator in tea plants and may mediate the regulation of PPO activity by activating CsPPO1 expression. These findings provide novel insights into the regulatory mechanism of PPO gene in Camellia sinensis, which might help to breed tea cultivars with high PPO activity.

show abstract

“…The second tea leaf was damaged mechanically by using a pattern wheel as described by Huang et al (). Both sides of the major vein of the leaf were rolled thrice (W).…”

Section: Plant Treatmentsmentioning

confidence: 99%

Feeding on tea GH19 chitinase enhances tea defense responses induced by regurgitant derived from Ectropis grisescens

Wang

Cai

et al. 2020

Physiologia Plantarum

View full text Add to dashboard Cite

Multiple isoforms of chitinases participate in plant defense against outside invaders. However, the functions of hydrolase family 19 (GH19) chitinases on pest control remain largely unknown. Here we reported the isolation and functional analysis of a gene CsChi19, which encodes a GH19 endochitinase protein of 332 amino acid residues from tea plant (Camellia sinensis). CsChi19 expression levels were upregulated in response to mechanical wounding, infestation by two important pests: the tea geometrid Ectropis grisescens and the tea green leafhopper Empoasca (Matsumurasca) onukii, a fungal pathogen Colletotrichum fructicola, and treatment with two phytohormones: jasmonic acid (JA) and salicylic acid. CsChi19 was heterologously expressed in Escherichia coli, and its catalytic function was further elucidated. The protein could hydrolyze colloidal chitin, and the optimum temperature and pH for its activity was 40°C and pH 5.0. CsChi19 were found to be toxic to tea pests when they were fed on artificial diets containing this protein. Interestingly, the regurgitant derived from E. grisescens fed with artificial diets containing CsChi19 protein induced stronger expression of CsMPK3, more JA burst, more accumulation of defense‐related secondary metabolites, and more emission of volatiles than the regurgitant derived from E. grisescens fed only with artificial diets. Our results provide first evidence that CsChi19 is involved in mediating a novel defense mechanism of tea plant through altering the composition of the regurgitant.

show abstract

Two New Polyphenol Oxidase Genes of Tea Plant (Camellia sinensis) Respond Differentially to the Regurgitant of Tea Geometrid, Ectropis obliqua

Cited by 16 publications

References 61 publications

Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis

Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis

The R2R3 Transcription Factor CsMYB59 Regulates Polyphenol Oxidase Gene CsPPO1 in Tea Plants (Camellia sinensis)

Feeding on tea GH19 chitinase enhances tea defense responses induced by regurgitant derived from Ectropis grisescens

Contact Info

Product

Resources

About