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.

Seeds are in a natural oxidative context leading to protein oxidation. Although inevitable for proper progression from maturation to germination, protein oxidation at high levels is detrimental and associated with seed aging. Oxidation of methionine to methionine sulfoxide is a common form of damage observed during aging in all organisms. This damage is reversible through the action of methionine sulfoxide reductases (MSRs), which play key roles in lifespan control in yeast and animal cells. To investigate the relationship between MSR capacity and longevity in plant seeds, we first used two Medicago truncatula genotypes with contrasting seed quality. After characterizing the MSR family in this species, we analyzed gene expression and enzymatic activity in immature and mature seeds exhibiting distinct quality levels. We found a very strong correlation between the initial MSR capacities in different lots of mature seeds of the two genotypes and the time to a drop in viability to 50% after controlled deterioration. We then analyzed seed longevity in Arabidopsis thaliana lines, in which MSR gene expression has been genetically altered, and observed a positive correlation between MSR capacity and longevity in these seeds as well. Based on our data, we propose that the MSR repair system plays a decisive role in the establishment and preservation of longevity in plant seeds.

NADP-thioredoxin reductases (NTRs) reduce thioredoxins (Trxs), using NADPH as a reductant, together constituting complete redox systems (NTS). Beside NTRA and NTRB targeted to both cytosol and mitochondria of plant cells, there is in chloroplasts an unusual NTR (NTRC) harbouring a Trx domain in a C-terminal extension, as recently reported in Oryza sativa. Although NTRC may constitute a complete NTS, it was described as a bifunctional enzyme. Because the gene is only present in photosynthetic organisms and the protein in green tissues, NTRC was thought to have a role restricted to photosynthetic cells. To determine whether NTRC from dicot plants is a bifunctional enzyme or a complete NTS, as well as to identify its putative target, NTRC from Medicago truncatula was cloned and NTRA was cloned for comparison. Here evidence is presented that MtNTRC (i) acts as an NTS and reduces dithiobisnitrobenzoate (DTNB) with a turnover (0.62 s⁻¹) similar to that measured with MtNTRA in the presence of a Trxh (0.81 s⁻¹); (ii) is able to use both NADPH (k_M=2.4 μM) and NADH (k_M=11 μM) as cofactors; (iii) efficiently reduces BAS1, a plastidial peroxiredoxin; and (iv) is expressed in both leaves and stems but unexpectedly is even more abundant in cotyledons from dry and germinating seeds. Because BAS1 is also present in both green tissues and seeds, NTRC/BAS1 may be involved in the scavenging of peroxides produced in green tissues during the day or the night and in seeds during germination. These results suggest different roles for NTRC in monocot and dicot plants.

Thioredoxins (Trxs) constitute a family of small proteins in plants. This family has been extensively characterized in Arabidopsis (Arabidopsis thaliana), which contains six different Trx types: f, m, x, and y in chloroplasts, o in mitochondria, and h mainly in cytosol. A detailed study of this family in the model legume Medicago truncatula, realized here, has established the existence of two isoforms that do not belong to any of the types previously described. As no possible orthologs were further found in either rice (Oryza sativa) or poplar (Populus spp.), these novel isoforms may be specific for legumes. Nevertheless, on the basis of protein sequence and gene structure, they are both related to Trxs m and probably have evolved from Trxs m after the divergence of the higher plant families. They have redox potential values similar to those of the classical Trxs, and one of them can act as a substrate for the M. truncatula NADP-Trx reductase A. However, they differ from classical Trxs in that they possess an atypical putative catalytic site and lack disulfide reductase activity with insulin. Another important feature is the presence in both proteins of an N-terminal extension containing a putative signal peptide that targets them to the endoplasmic reticulum, as demonstrated by their transient expression in fusion with the green fluorescent protein in M. truncatula or Nicotiana benthamiana leaves. According to their pattern of expression, these novel isoforms function specifically in symbiotic interactions in legumes. They were therefore given the name of Trxs s, s for symbiosis.

Yield losses due to nutrient deficiency are estimated as the primary cause of the yield gap worldwide. Understanding how plant roots perceive external nutrient status and elaborate morphological adaptations in response to it is necessary to develop reliable strategies to increase crop yield. In the last decade, reactive oxygen species (ROS) were shown to be key players of the mechanisms underlying root responses to nutrient limitation. ROS contribute in multiple ways to shape the root system in response to nutritional cues, both as direct effectors acting on cell wall architecture and as second messengers in signalling pathways. Here, we review the mutual interconnections existing between perception and signalling of the most common forms of the major macronutrients (nitrogen, phosphorus and potassium), and ROS in shaping plant root system architecture. We discuss recent advances in dissecting the integration of these elements and their impact on morphological traits of the root system, highlighting the functional ductility of ROS and enzymes implied in ROS metabolism, such as class III peroxidases.