A genome-scale metabolic reconstruction of soybean and Bradyrhizobium diazoefficiens reveals the cost-benefit of nitrogen fixation.

Nitrogen-fixing symbioses allow legumes to thrive in nitrogen-poor soils at the cost of diverting some photoassimilate to their microsymbionts. Effort is being made to bioengineer nitrogen fixation into nonleguminous crops. This requires a quantitative understanding of its energetic costs and the links between metabolic variations and symbiotic efficiency. A whole-plant metabolic model for soybean (Glycine max) with its associated microsymbiont Bradyrhizobium diazoefficiens was developed and applied to predict the cost-benefit of nitrogen fixation with varying soil nitrogen availability. The model predicted a nitrogen-fixation cost of c. 4.13 g C g-1 N, which when implemented into a crop scale model, translated to a grain yield reduction of 27% compared with a non-nodulating plant receiving its nitrogen from the soil. Considering the lower nitrogen content of cereals, the yield cost to a hypothetical N-fixing cereal is predicted to be less than half that of soybean. Soybean growth was predicted to be c. 5% greater when the nodule nitrogen export products were amides versus ureides. This is the first metabolic reconstruction in a tropical crop species that simulates the entire plant and nodule metabolism. Going forward, this model will serve as a tool to investigate carbon use efficiency and key mechanisms within N-fixing symbiosis in a tropical species forming determinate nodules.

RpoS contributes in a host-dependent manner to Salmonella colonization of the leaf apoplast during plant disease.

Contaminated fresh produce has been routinely linked to outbreaks of Salmonellosis. Multiple studies have identified Salmonella enterica factors associated with successful colonization of diverse plant niches and tissues. It has also been well documented that S. enterica can benefit from the conditions generated during plant disease by host-compatible plant pathogens. In this study, we compared the capacity of two common S. enterica research strains, 14028s and LT2 (strain DM10000) to opportunistically colonize the leaf apoplast of two model plant hosts Arabidopsis thaliana and Nicotiana benthamiana during disease. While S. enterica 14028s benefited from co-colonization with plant-pathogenic Pseudomonas syringae in both plant hosts, S. enterica LT2 was unable to benefit from Pto co-colonization in N. benthamiana. Counterintuitively, LT2 grew more rapidly in ex planta N. benthamiana apoplastic wash fluid with a distinctly pronounced biphasic growth curve in comparison with 14028s. Using allelic exchange, we demonstrated that both the N. benthamiana infection-depedent colonization and apoplastic wash fluid growth phenotypes of LT2 were associated with mutations in the S. enterica rpoS stress-response sigma factor gene. Mutations of S. enterica rpoS have been previously shown to decrease tolerance to oxidative stress and alter metabolic regulation. We identified rpoS-dependent alterations in the utilization of L-malic acid, an abundant carbon source in N. benthamiana apoplastic wash fluid. We also present data consistent with higher relative basal reactive oxygen species (ROS) in N. benthamiana leaves than in A. thaliana leaves. The differences in basal ROS may explain the host-dependent disease co-colonization defect of the rpoS-mutated LT2 strain. Our results indicate that the conducive environment generated by pathogen modulation of the apoplast niche can vary from hosts to host even with a common disease-compatible pathogen.

Tryptophan Levels as a Marker of Auxins and Nitric Oxide Signaling.

The aromatic amino acid tryptophan is the main precursor for indole-3-acetic acid (IAA), which involves various parallel routes in plants, with indole-3-acetaldoxime (IAOx) being one of the most common intermediates. Auxin signaling is well known to interact with free radical nitric oxide (NO) to perform a more complex effect, including the regulation of root organogenesis and nitrogen nutrition. To fathom the link between IAA and NO, we use a metabolomic approach to analyze the contents of low-molecular-mass molecules in cultured cells of Arabidopsis thaliana after the application of S-nitrosoglutathione (GSNO), an NO donor or IAOx. We separated the crude extracts of the plant cells through ion-exchange columns, and subsequent fractions were analyzed by gas chromatography-mass spectrometry (GC-MS), thus identifying 26 compounds. A principal component analysis (PCA) was performed on N-metabolism-related compounds, as classified by the Kyoto Encyclopedia of Genes and Genomes (KEGG). The differences observed between controls and treatments are mainly explained by the differences in Trp contents, which are much higher in controls. Thus, the Trp is a shared response in both auxin- and NO-mediated signaling, evidencing some common signaling mechanism to both GSNO and IAOx. The differences in the low-molecular-mass-identified compounds between GSNO- and IAOx-treated cells are mainly explained by their concentrations in benzenepropanoic acid, which is highly associated with IAA levels, and salicylic acid, which is related to glutathione. These results show that the contents in Trp can be a marker for the study of auxin and NO signaling.

A hybrid kinetic and constraint-based model of leaf metabolism allows predictions of metabolic fluxes in different environments.

While flux balance analysis (FBA) provides a framework for predicting steady-state leaf metabolic network fluxes, it does not readily capture the response to environmental variables without being coupled to other modelling formulations. To address this, we coupled an FBA model of 903 reactions of soybean (Glycine max) leaf metabolism with e-photosynthesis, a dynamic model that captures the kinetics of 126 reactions of photosynthesis and associated chloroplast carbon metabolism. Successful coupling was achieved in an iterative formulation in which fluxes from e-photosynthesis were used to constrain the FBA model and then, in turn, fluxes computed from the FBA model used to update parameters in e-photosynthesis. This process was repeated until common fluxes in the two models converged. Coupling did not hamper the ability of the kinetic module to accurately predict the carbon assimilation rate, photosystem II electron flux, and starch accumulation of field-grown soybean at two CO2 concentrations. The coupled model also allowed accurate predictions of additional parameters such as nocturnal respiration, as well as analysis of the effect of light intensity and elevated CO2 on leaf metabolism. Predictions included an unexpected decrease in the rate of export of sucrose from the leaf at high light, due to altered starch-sucrose partitioning, and altered daytime flux modes in the tricarboxylic acid cycle at elevated CO2 . Mitochondrial fluxes were notably different between growing and mature leaves, with greater anaplerotic, tricarboxylic acid cycle and mitochondrial ATP synthase fluxes predicted in the former, primarily to provide carbon skeletons and energy for protein synthesis.

An Arabidopsis stomatin-like protein affects mitochondrial respiratory supercomplex organization.

Stomatins belong to the band-7 protein family, a diverse group of conserved eukaryotic and prokaryotic membrane proteins involved in the formation of large protein complexes as protein-lipid scaffolds. The Arabidopsis (Arabidopsis thaliana) genome contains two paralogous genes encoding stomatin-like proteins (SLPs; AtSLP1 and AtSLP2) that are phylogenetically related to human SLP2, a protein involved in mitochondrial fusion and protein complex formation in the mitochondrial inner membrane. We used reverse genetics in combination with biochemical methods to investigate the function of AtSLPs. We demonstrate that both SLPs localize to mitochondrial membranes. SLP1 migrates as a large (approximately 3 MDa) complex in blue-native gel electrophoresis. Remarkably, slp1 knockout mutants have reduced protein and activity levels of complex I and supercomplexes, indicating that SLP affects the assembly and/or stability of these complexes. These findings point to a role for SLP1 in the organization of respiratory supercomplexes in Arabidopsis.