The NADPH-Dependent Thioredoxin Reductase/Thioredoxin System in Germinating Barley Seeds: Gene Expression, Protein Profiles, and Interactions between Isoforms of Thioredoxin h and Thioredoxin Reductase

Abstract

The NADPH-dependent thioredoxin reductase (NTR)/thioredoxin (Trx) system catalyzes disulfide bond reduction in the cytoplasm and mitochondrion. Trx h is suggested to play an important role in seed development, germination, and seedling growth. Plants have multiple isoforms of Trx h and NTR; however, little is known about the roles of the individual isoforms. Trx h isoforms from barley (Hordeum vulgare) seeds (HvTrxh1 and HvTrxh2) were characterized previously. In this study, two NTR isoforms (HvNTR1 and HvNTR2) were identified, enabling comparison of gene expression, protein appearance, and interaction between individual NTR and Trx h isoforms in barley embryo and aleurone layers. Although mRNA encoding both Trx h isoforms is present in embryo and aleurone layers, the corresponding proteins differed in spatiotemporal appearance. HvNTR2, but not HvNTR1, gene expression seems to be regulated by gibberellic acid. Recombinant HvNTR1 and HvNTR2 exhibited virtually the same affinity toward HvTrxh1 and HvTrxh2, whereas HvNTR2 has slightly higher catalytic activity than HvNTR1 with both Trx h isoforms, and HvNTR1 has slightly higher catalytic activity toward HvTrxh1 than HvTrxh2. Notably, both NTRs reduced Trx h at the acidic conditions residing in the starchy endosperm during germination. Interspecies reactions between the barley proteins and Escherichia coli Trx or Arabidopsis thaliana NTR, respectively, occurred with 20- to 90-fold weaker affinity. This first investigation of regulation and interactions between members of the NTR/Trx system in barley seed tissues suggests that different isoforms are differentially regulated but may have overlapping roles, with HvNTR2 and HvTrxh1 being the predominant isoforms in the aleurone layer.

An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

“…A clear distinction of the roles of the two thioredoxin as well as of the two thioredoxin reductase isoforms has not been made. In vitro, one specific pair is up to three times as efficient as other pairs and this pair is moreover enriched in the aleurone layer during germination (Shahpiri et al 2008). Maybe different spatio-temporal occurrence of isoforms is an important factor in efficient recycling of oxidised thioredoxin.…”

“…Barley contains two thioredoxin isoforms as well as two isoforms of an NADPH-dependent thioredoxin reductase that reduce the disulfide formed in the thioredoxin active site motif CXXC after it has reduced a target protein disulfide bond (Maeda et al 2003;Shahpiri et al 2008). We have developed a proteomics-based procedure that allows identification of target disulfides in protein mixtures (Maeda et al 2005; P. Hägglund et al, manuscript in preparation).…”

Dissecting Molecular Interactions Involved in Recognition of Target Disulfides by the Barley Thioredoxin System

“…Wild-type (wt) HvTrxh2 and mutants, BASI, and HvNTR2 were produced as described, purified (His-Trap HP column, GE Healthcare), dialyzed against 30 mM Tris-HCl (pH 8.0), concentrated (Amicon Ultra Centrifugal Filter Device, Millipore), and further purified by gel filtration (HiLoad 26/60 Superdex 75 column, GE Healthcare) in 30 mM Tris-HCl (pH 8.0) and 0.2 M NaCl. 7,23,24 Eluted proteins were dialyzed against 30 mM Tris-HCl (pH 8.0), concentrated as described above, and stored at −80°C. All proteins migrated as single bands via sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE).…”

A cold‐induced thioredoxin h of rice, OsTrx23, negatively regulates kinase activities of OsMPK3 and OsMPK6 in vitro

“…Trxs regulate a variety of physiological processes in plants [2,[7][8][9]. There are two action modes by which Trxs regulate the functions of target proteins.…”

“…Plant Trx h proteins are implicated in many cellular processes, including protein folding, protein repair after damage by oxidation, sulfur metabolism, and regulation of transcription factors [2]. Trx h proteins are also involved in plant development and stress responses, such as seed germination and early seedling development [7], self-incompatibility reactions [8] and oxidative stress [9]. In rice, thirty potential thioredoxin-encoding genes (OsTrxs) have been identified; nine genes of which were predicted to encode Trx h [10].…”