Phenylpropanoid metabolism represents an important metabolic pathway from which originates a wide number of secondary metabolites derived from phenylalanine or tyrosine, such as flavonoids and isoflavonoids, crucial molecules in plants implicated in a large number of biological processes. Therefore, various types of interconnection exist between different aspects of nitrogen metabolism and the biosynthesis of these compounds. For legumes, flavonoids and isoflavonoids are postulated to play pivotal roles in adaptation to their biological environments, both as defensive compounds (phytoalexins) and as chemical signals in symbiotic nitrogen fixation with rhizobia. In this paper, we summarize the recent progress made in the characterization of flavonoid and isoflavonoid biosynthetic pathways in the model legume Lotus japonicus (Regel) Larsen under different abiotic stress situations, such as drought, the impairment of photorespiration and UV-B irradiation. Emphasis is placed on results obtained using photorespiratory mutants deficient in glutamine synthetase. The results provide different types of evidence showing that an enhancement of isoflavonoid compared to standard flavonol metabolism frequently occurs in Lotus under abiotic stress conditions. The advance produced in the analysis of isoflavonoid regulatory proteins by the use of co-expression networks, particularly MYB transcription factors, is also described. The results obtained in Lotus japonicus plants can be also extrapolated to other cultivated legume species, such as soybean, of extraordinary agronomic importance with a high impact in feeding, oil production and human health.

Glutamine synthetase (GS) is the key enzyme involved in the assimilation of ammonia derived either from nitrate reduction, N₂ fixation, photorespiration or asparagine breakdown. A small gene family is encoding for different cytosolic (GS1) or plastidic (GS2) isoforms in legumes. We summarize here the recent advances carried out concerning the quaternary structure of GS, as well as the functional relationship existing between GS2 and processes such as nodulation, photorespiration and water stress, in this latter case by means of proline production. Functional genomic analysis using GS2-minus mutant reveals the key role of GS2 in the metabolic control of the plants and, more particularly, in carbon metabolism.

This paper was aimed to investigate the possible implications of the lack of plastidic glutamine synthetase (GS2) in phenolic metabolism during stress responses in the model legume Lotus japonicus. Important changes in the transcriptome were detected in a GS2 mutant called Ljgln2-2, compared to the wild type, in response to two separate stress conditions, such as drought or the result of the impairment of the photorespiratory cycle. Detailed transcriptomic analysis showed that the biosynthesis of phenolic compounds was affected in the mutant plants in these two different types of stress situations. For this reason, the genes and metabolites related to this metabolic route were further investigated using a combined approach of gene expression analysis and metabolite profiling. A high induction of the expression of several genes for the biosynthesis of different branches of the phenolic biosynthetic pathway was detected by qRT-PCR. The extent of induction was always higher in Ljgln2-2, probably reflecting the higher stress levels present in this genotype. This was paralleled by accumulation of several kaempferol and quercetine glycosides, some of them described for the first time in L. japonicus, and of high levels of the isoflavonoid vestitol. The results obtained indicate that the absence of GS2 affects different aspects of phenolic metabolism in L .japonicus plants in response to stress.

This paper summarizes some recent advances in the understanding of nitrate assimilation in the model legume Lotus japonicus. First, different types of experimental evidence are presented that emphasize the importance of the root in the nitrate-reducing assimilatory processes in this plant. Secondly, the main results from an ethyl methanesulphonate mutagenesis programme are presented. In this programme, chlorate-resistant and photorespiratory mutants were produced and characterized. The phenotype of one particular chlorate-resistant mutant suggested the importance of a low-affinity nitrate transport system for growth of L. japonicus plants under nitrate nutrition. The phenotype of photorespiratory mutants, affected in all forms of plastid glutamine synthetase in leaves, roots, and nodules, indicated that plastid glutamine synthetase was not required for primary nitrate assimilation nor for the symbiotic associations of the plant (nodulation, mycorrhization), provided photorespiration was suppressed. However, the phenotype of these mutants confirmed that plastid glutamine synthetase was required for the reassimilation of ammonium released by photorespiration. Finally, different aspects of the relationship between nitrate assimilation and osmotic stress in L. japonicus are also discussed, with specific reference to the biosynthesis of proline as an osmolyte.

The physiological role of K⁺-dependent and K⁺-independent asparaginases in plants remains unclear, and the contribution from individual isoforms during development is poorly understood. We have used reverse genetics to assess the phenotypes produced by the deficiency of K⁺-dependent NSE1 asparaginase in the model legume Lotus japonicus. For this purpose, four different mutants were identified by TILLING and characterized, two of which affected the structure and function of the asparaginase molecule and caused asparagine accumulation. Plant growth and total seed weight of mature mutant seeds as well as the level of both legumin and convicilin seed storage proteins were affected in the mutants. The mutants isolated in the present work are the first of their type in legumes and have enabled us to demonstrate the importance of asparagine and K⁺-dependent NSE1 asparaginase for nitrogen remobilization and seed production in L. japonicus plants.

Summary: Hydrogen sulfide is a signaling molecule in plants that regulates essential biological processes through protein persulfidation. However, little is known about sulfide‐mediated regulation in relation to photorespiration. Here, we performed label‐free quantitative proteomic analysis and observed a high impact on protein persulfidation levels when plants grown under nonphotorespiratory conditions were transferred to air, with 98.7% of the identified proteins being more persulfidated under suppressed photorespiration. Interestingly, a higher level of reactive oxygen species (ROS) was detected under nonphotorespiratory conditions. Analysis of the effect of sulfide on aspects associated with non‐ or photorespiratory growth conditions has demonstrated that it protects plants grown under suppressed photorespiration. Thus, sulfide amends the imbalance of carbon/nitrogen and restores ATP levels to concentrations like those of air‐grown plants; balances the high level of ROS in plants under nonphotorespiratory conditions to reach a cellular redox state similar to that in air‐grown plants; and regulates stomatal closure, to decrease the high guard cell ROS levels and induce stomatal aperture. In this way, sulfide signals the CO₂‐dependent stomata movement, in the opposite direction of the established abscisic acid‐dependent movement. Our findings suggest that the high persulfidation level under suppressed photorespiration reveals an essential role of sulfide signaling under these conditions.