The presence of nitrogen inhibits legume nodule formation, but the mechanism of this inhibition is poorly understood. We found that 2.5 mM nitrate and above significantly inhibited nodule initiation but not root hair curling in Medicago trunatula. We analyzed protein abundance in M. truncatula roots after treatment with either 0 or 2.5 mM nitrate in the presence or absence of its symbiont Sinorhizobium meliloti after 1, 2 and 5 days following inoculation. Two-dimensional gel electrophoresis combined with mass spectrometry was used to identify 106 differentially accumulated proteins responding to nitrate addition, inoculation or time point. While flavonoid-related proteins were less abundant in the presence of nitrate, addition of Nod gene-inducing flavonoids to the Sinorhizobium culture did not rescue nodulation. Accumulation of auxin in response to rhizobia, which is also controlled by flavonoids, still occurred in the presence of nitrate, but did not localize to a nodule initiation site. Several of the changes included defense- and redox-related proteins, and visualization of reactive oxygen species indicated that their induction in root hairs following Sinorhizobium inoculation was inhibited by nitrate. In summary, the presence of nitrate appears to inhibit nodulation via multiple pathways, including changes to flavonoid metabolism, defense responses and redox changes.

Plant growth, development, and yield of current tomato cultivars are directly affected by low temperatures. Although wild tomato species have been suggested as a potential source for low temperature tolerance, very little is known about their behavior during the reproductive phase. Here, we investigated the impact of suboptimal temperatures (SOT, 16/14°C), as compared to control temperatures (CT, 22/20°C), on plant growth, photosynthetic capacity, and carbohydrate metabolism. Under these conditions, two genotypes were analyzed: a Solanum lycopersicum cultivar Moneymaker and a high-altitude wild species Solanum arcanum LA385, from flowering onset until a later stage of fruit development. Total dry matter production in cv. Moneymaker was reduced up to 30% at SOT, whereas it was hardly affected in wild accession LA385. Specific leaf area, total leaf area, and number of fruits were also decreased at SOT in cv. Moneymaker. In contrast, wild accession LA385 showed an acclimation to SOT, in which ΦPSII and net CO2 assimilation rates were less affected; a similar specific leaf area; higher total leaf area; and higher number of fruits compared to those at CT. In addition, LA385 appeared to have a more distinct sucrose metabolism than cv. Moneymaker at both temperatures, in which it had higher contents of sucrose-6-phosphate, sucrose, and ratio of sucrose: starch in leaves and higher ratio of sucrose: hexose in fruits. Overall, our findings indicate that wild accession LA385 is able to acclimate well to SOT during the reproductive phase, whereas growth and development of cv. Moneymaker is reduced at SOT.

Sucrose synthase (SuSy) is one key enzyme directly hydrolyzing sucrose to supply substrates for plant metabolism, and is considered to be a biomarker for plant sink strength. Improvement in plant sink strength could lead to enhanced plant growth and yield. Cultivated tomatoes are known to have a narrow genetic diversity, which hampers further breeding for novel and improved traits in new cultivars. In this study, we observed limited genetic variation in SuSy1, SuSy3 and SuSy4 in 53 accessions of cultivated tomato and landraces, but identified a wealth of genetic diversity in 32 accessions of related wild species. The variation in the deduced amino acid sequences was grouped into 23, 22, and 17 distinct haplotypes for SuSy1/3/4, respectively. Strikingly, all known substrate binding sites were highly conserved, as well as most of the phosphorylation sites except in SuSy1. Two SuSy1 and three SuSy3 protein variants were heterologously expressed to study the effect of the amino acid changes on enzyme kinetic properties, i.e. maximal sucrose hydrolyzing capacity (Vmax), affinity for sucrose (Km), and catalytic efficiency (Vmax/Km) at 25°C and 16°C. SuSy1-haplotype#3 containing phosphorylation site Ser-16 did not have an improvement in the kinetic properties compared to the reference SuSy1-haplotype#1 containing Arg-16. Meanwhile SuSy3-haplotype#9 from a wild accession, containing four amino acid changes S53A, S106I, E727D and K741E, showed an increase in Vmax/Km at 16°C compared to the reference SuSy3-haplotype#1. This study demonstrates that SuSy kinetic properties can be enhanced by exploiting natural variation, and the potential of this enzyme to improve sucrose metabolism and eventually sink strength in planta.

Plant growth, development and yield of current tomato cultivars are directly affected by low temperatures. Although wild tomato species have been suggested as a potential source for low temperature tolerance, very little is known about their behaviour during the reproductive phase. Here we investigated the impact of suboptimal temperatures (SOT, 16/14˚C), as compared to control temperatures (CT, 22/20˚C), on plant growth, photosynthetic capacity, and carbohydrate metabolism. Under these conditions, two genotypes were analysed: a Solanum lycopersicum cultivar Moneymaker and a high altitude wild species Solanum arcanum LA385, from flowering onset until a later stage of fruit development. Total dry matter production in cv Moneymaker was reduced up to 30% at SOT, whereas it was hardly affected in wild accession LA385. Specific leaf area, total leaf area, and number of fruits were also decreased at SOT in cv Moneymaker. In contrast wild accession LA385 showed an acclimation to SOT, in which Φ PSII and net CO 2 assimilation rates were less affected, a similar specific leaf area, higher total leaf area, and higher number of fruits compared to those at CT. In addition, LA385 appeared to have a more distinct sucrose metabolism than cv Monemaker at both temperatures, in which it had higher contents of sucrose-6-phosphate, sucrose, and ratio of sucrose: starch in leaves and higher ratio of sucrose: hexose in fruits. Overall our findings indicate that wild accession LA385 is able to acclimate well to SOT during the reproductive phase, whereas growth and development of cv Moneymaker is reduced at SOT.