Thioredoxin-Linked Proteins Are Reduced during Germination ofMedicago truncatulaSeeds

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.

Plastidial Thioredoxin z Interacts with Two Fructokinase-Like Proteins in a Thiol-Dependent Manner: Evidence for an Essential Role in Chloroplast Development in Arabidopsis and Nicotiana benthamiana

“…Recent proteomic studies, such as thioredoxin-trapping chromatography or labeled gel electrophoresis, both in combination with protein identification by mass spectrometry, have identified >180 potential TRX target proteins in plants (Motohashi et al, 2001;Balmer et al, 2003Balmer et al, , 2004bBalmer et al, , 2006Wong et al, 2004;Marchand et al, 2006;Alkhalfioui et al, 2007). However, the vast majority of these have yet not been experimentally verified, and in vitro methods appear to suffer from an inherent lack of specificity as chloroplast targets have been identified with cytosolic TRX as bait and vice versa (Meyer et al, 2008).…”

Integrated Proteomic and Cytological Study of Rice Endosperms at the Storage Phase

“…13 The proteins sensitive to redox potential, termed potential thioredoxin targets (PTTs), have been intensively investigated. 13 Here, we screened possible PTTs from the differentially expressed proteins (DEPs) by use of a known PTT database 13,[28][29][30][31][32][33] (Supplemental Table S3). About two-thirds of the DEPs matched known PTTs.…”

Disulfide Proteomics: Current Status in Thioredoxin Biochemistry and Industrial Research

“…Comparative studies of the two procedures were reported in some studies 1,32,49 . In each case, the two procedures were found to be complementary.…”

“…It is therefore possible to confirm whether the putative targets of Trx identified by the in vitro reaction are reduced in vivo. In a study with the legume, Medicago truncatula, more than half the 43 potential Trx targets identified by the in vitro mBBr-labeling reaction were also labeled in vivo during germination 1 . In addition, Yano & Kuroda 55 reported that rice aleurone ESP2, identified as putative targets of Trx, were degraded both in vivo and in vitro by a cysteine protease also activated by Trx.…”