Poster: Seed Biology

Abs # 557: Isolation and characterization of thioredoxin h and NADP-thioredoxin reductase genes from barley (Hordeum vulgare L.).

Presenter:	Cho, Myeong-Je , mjcho@nature.berkeley.edu
Authors	Cho, Myeong-Je  (A)   Jung, Hye Rim  (A)   Kim, Hyun-Kyung  (A)   Choi, Hae-Woon  (A)   Caillau, Maxime  (A)   del Val, Greg  (A)   Yoo, Hyun Sook  (A)   Kim, Yong-Bum  (A)   Hwang, Keum Hee  (A)   Buchanan, Bob B (A)   Lemaux, Peggy G (A)
Affiliations:	(A): Univ. of California

As in photosynthesis, thioredoxin plays a primary role in regulating heterotrophic processes in plants. In this capacity, the disulfide group of a thioredoxin of the h-type (TRXh) is reduced by NADPH via the flavin enzyme, NADP-thioredoxin reductase (NTR). In the present study, genes encoding barley thioredoxin h (BTRXh) and NADP-thioredoxin reductase (BNTR) were identified in a barley (Hordeum vulgare L.) cDNA library and expressed in both Escherichia coli and barley grain. The deduced proteins of BTRXh and BNTR showed a high similarity to counterparts from other sources. BTRXh has a molecular mass of 13-kDa while BNTR is a 35-kDa protein. Both recombinant BTRXh and BNTR proteins produced in E. coli proved to be biochemically active with the 2’,5’-dithiobis-(2-nitrobenzoic acid) reduction assay. Monobromobimane (mBBr) assays showed that the addition of these 2 proteins together with NADPH significantly reduced target disulfide proteins extracted from barley grain. For in vivo experiments, DNA constructs containing either the btrxh or bntr gene driven by a barley endosperm-specific B₁-hordein promoter with a signal peptide sequence were introduced into barley. The level of overexpression of BTRXh and BNTR in transgenic barley grain was assessed and confirmed by immunoblots. Overexpressed BTRXh and BNTR in transgenic barley grain showed appreciable biochemical activity based on the mBBr assay.