Thioredoxin-linked proteins are reduced during germination of Medicago truncatula seeds

Abstract

Germination of cereals is accompanied by extensive change in the redox state of seed proteins. Proteins present in oxidized form in dry seeds are converted to the reduced state following imbibition. Thioredoxin (Trx) appears to play a role in this transition in cereals. It is not known, however, whether Trx-linked redox changes are restricted to cereals or whether they take place more broadly in germinating seeds. To gain information on this point, we have investigated a model legume, Medicago truncatula. Two complementary gel-based proteomic approaches were followed to identify Trx targets in seeds: Proteins were (1) labeled with a thiol-specific probe, monobromobimane (mBBr), following in vitro reduction by an NADP/Trx system, or (2) isolated on a mutant Trx affinity column. Altogether, 111 Trx-linked proteins were identified with few differences between axes and cotyledons. Fifty nine were new, 34 found previously in cereal or peanut seeds, and 18 in other plants or photosynthetic organisms. In parallel, the redox state of proteins assessed in germinating seeds using mBBr revealed that a substantial number of proteins that are oxidized or partly reduced in dry seeds became more reduced upon germination. The patterns were similar for proteins reduced in vivo during germination or in vitro by Trx. In contrast, glutathione and glutaredoxin were less effective as reductants in vitro. Overall, more than half of the potential targets identified with the mBBr labeling procedure were reduced during germination. The results provide evidence that Trx functions in the germination of seeds of dicotyledons as well as monocotyledons.

Figure 1.

Procedures for the identification of potential Trx targets and the determination of the redox state of proteins in germinating seeds of M. truncatula. The targets were identified by proteomic procedures.

Figure 2.

2D gel patterns of potential Trx targets of M. truncatula. Proteins were extracted from embryo axes or cotyledons from seeds imbibed for 14 h. Potential targets were labeled with mBBr (top sections) or isolated by affinity chromatography (bottom sections) then resolved in 2D gels as shown in Figure 1. Fluorescent proteins were detected under UV light at 365 nm and isolated proteins by Coomassie Blue staining. The potential targets identified are numbered and their names and properties appear in Table I. MtNTRA (NTR) and PsTrxh3 (Trx), components of the mBBr labeling procedure, are also indicated.

Figure 3.

Analysis of the redox state of the proteins in whole seeds during germination. Proteins were extracted from dry (0 h), 14, or 22 h-imbibed seeds in the presence of mBBr and resolved in 2D gels. Fluorescent proteins were detected under UV light at 365 nm (left sections) and total proteins were stained with Coomassie Blue (right sections).

Figure 4.

Specific fluorescence of Trx targets during germination. The ratio of fluorescence to normalized density/spot was calculated for the targets recognized in Figure 3 at three germination times (0, 14, and 22 h).