Identification and Differential Expression of Two Thioredoxin h Isoforms in Germinating Seeds from Pea

Abstract

The NADPH/NADP-thioredoxin (Trx) reductase (NTR)/Trx system (NTS) is a redox system that plays a posttranslational regulatory role by reducing protein targets involved in crucial cellular processes in microorganisms and animals. In plants, the system includes several h type Trx isoforms and has been shown to intervene in reserve mobilization during early seedling growth of cereals. To determine whether NTS was operational during germination of legume seeds and which Trx h isoforms could be implicated, Trx h isoforms expression was monitored in germinating pea (Pisum sativum cv Baccara) seeds, together with the amount of NTR and NADPH. Two new isoforms were identified: Trx h3, similar to the two isoforms already described in pea but not expressed in seeds; and the more divergent isoform, Trx h4. Active recombinant proteins were produced in Escherichia coli and used to raise specific antibodies. The expression of new isoforms was analyzed at both mRNA and protein levels. The lack of correlation between mRNA and protein abundances suggests the occurrence of posttranscriptional regulation. Trx h3 protein amount remained constant in both axes and cotyledons of dry and imbibed seeds but then decreased 2 d after radicle protrusion. In contrast, Trx h4 was only expressed in axes of dry and imbibed seeds but not in germinated seeds or in seedlings, therefore appearing as closely linked to germination. The presence of NTR and NADPH in seeds suggests that NTS could be functional during germination. The possible role of Trx h3 and h4 in this context is discussed.

PsTRXh1 and PsTRXh2 Are Both Pea h-Type Thioredoxins with Antagonistic Behavior in Redox Imbalances

“…In addition, three-dimensional models for both deduced sequences, based on resolved h-type TRXs (Coudevylle et al, 2005), fit well to the classical TRX folding (data not shown). Compared to other TRXs from pea, we found a high similarity of both PsTRXh1 and PsTRXh2 with PsTRXh3 (64% and 72% identity, respectively); however, they both share less than 41% identity with PsTRXh4, the latest cytosolic isoform isolated up to now (Montrichard et al, 2003). Contrary to chloroplastic TRXs, PsTRXh1 and PsTRXh2 do not contain any transit peptide.…”

“…This pattern of protein accumulation was not consistent with PsTRXh2 mRNA accumulation. It is likely that these levels mostly correspond to cross recognition of other abundant isoforms such as PsTRXh3, for which similar patterns were previously reported (Montrichard et al, 2003). Therefore, PsTRXh2 accumulation could not be further investigated.…”

“…Three of them, f, m1, and m2, are chloroplastic (Jaramillo et al, 1997;Pagano et al, 2000), two h-type TRXs being cytosolic. These last cytosolic isoforms, PsTRXh3 and PsTRXh4, have recently been well characterized and related to diverse processes during seed germination (Montrichard et al, 2003). In this work, we address the gene, protein, and functional characterization of two additional h-type TRXs from pea, termed PsTRXh1 and PsTRXh2, with the aim to determine whether their role is specific or redundant with other isoforms.…”

Cadmium affects the NADP-thioredoxin reductase/thioredoxin system in germinating pea seeds

“…The pea cytosolic system consists of Trx h and homodimeric NTR (Montrichard et al 2003), and Trx f and a heterodimeric ferredoxin-dependent Trx reductase for the chloroplastic Trx system (Cain et al 2009). …”

“…Subsequent events, including the mobilization of the major reserves, are associated with the growth of the seedling (Bewley 1997). A potential role of Trxs in redox status of storage proteins and proteolysis during germination has been already suggested (Lozano et al 1996;Montrichard et al 2003;Alkhalfioui et al 2007). They appear to play a role in the reduction of critical disulfide bonds of proteins (Kobrehel et al 1992;Besse et al 1996;Lozano et al 1996;Schu¨rmann and Jacquot 2000).…”