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Tree Physiology Advance Access originally published online on April 20, 2009
Tree Physiology 2009 29(6):837-846; doi:10.1093/treephys/tpp022
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Differential display mediated cloning of anthocyanidin reductase gene from tea (Camellia sinensis) and its relationship with the concentration of epicatechins

Kashmir Singh1,*, Arti Rani1,**, Asosii Paul1, Som Dutt1, Robin Joshi3, Ashu Gulati3, Paramvir Singh Ahuja1 and Sanjay Kumar1,2

1 Biotechnology Division, Institute of Himalayan Bioresource Technology (CSIR), P.O. Box No. 6, Palampur – 176061, India
2 Corresponding author (sanjayplp{at}rediffmail.com, sanjaykumar{at}ihbt.res.in)
3 Hill Area Tea Science Division, Institute of Himalayan Bioresource Technology (CSIR), P.O. Box No. 6, Palampur – 176061, India


  Abstract

Tea [Camellia sinensis (L.) O. Kuntze] leaves are a major source of epicatechin (EC) and its gallolyl derivatives epicatechin gallate, epigallocatechin and epigallocatechin gallate, collectively known as epicatechins (ECs). Epicatechins are important factors determining tea quality, and they also possess many medicinal properties. To gain further information about the regulation of the biosynthesis of ECs, we cloned the gene encoding anthocyanidin reductase from tea (CsANR) by first quantifying changes in the concentrations of ECs in response to drought, gibberellic acid (GA3), abscisic acid (ABA) and wounding treatments, followed by differential display of mRNAs and analysis of those bands exhibiting a change in expression paralleling the treatment-induced changes observed in the EC data. Analysis of 133 bands yielded a partial cDNA of CsANR that was later cloned to the full length by rapid amplification of the cDNA ends. The full-length CsANR (Accession No. AY641729) comprised 1233 bp with an ORF of 1014 bp (from 79 to 1092 bp) encoding a polypeptide of 337 amino acids. Expression of CsANR in an Escherichia coli expression vector yielded a functional protein that catalyzed the conversion of cyanidin to EC in the presence of NADPH. Analysis of ECs and gene expression in leaves at different developmental stages and across five tea clones exhibiting variable concentrations of ECs revealed a positive correlation between concentration of ECs and CsANR expression. Expression of CsANR was down-regulated in response to drought, ABA and GA3 treatments and up-regulated in response to wounding.

Keywords: catechin, flavonoid

Received November 27, 2008; Accepted March 16, 2009

* Present address: Department of Biotechnology, Punjab University, Chandigarh – 160014, India.

** Present address: Vittal Mallya Scientific Research Foundation, Bangalore, India.


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