Summary• The role of plastidic glutamine synthetase (GS2) in proline biosynthesis and drought stress responses in Lotus japonicus was investigated using the GS2 mutant, Ljgln2-2.• Wild-type (WT) and mutant plants were submitted to different lengths of time of water and nutrient solution deprivation. Several biochemical markers were measured and the transcriptional response to drought was determined by both quantitative real-time polymerase chain reaction and transcriptomics.• The Ljgln2-2 mutant exhibited normal sensitivity to mild water deprivation, but physiological, biochemical and massive transcriptional differences were detected in the mutant, which compromised recovery (rehydration) following re-watering after severe drought stress. Proline accumulation during drought was substantially lower in mutant than in WT plants, and significant differences in the pattern of expression of the genes involved in proline metabolism were observed. Transcriptomic analysis revealed that about three times as many genes were regulated in response to drought in Ljgln2-2 plants compared with WT.• The transcriptomic and accompanying biochemical data indicate that the Ljgln2-2 mutant is subject to more intense cellular stress than WT during drought. The results presented here implicate plastidic GS2 in proline production during stress and provide interesting insights into the function of proline in response to drought.

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The accumulation of proline (Pro) and overproduction of reactive oxygen species (ROS) by plants exposed to stress is well-documented. In vitro assays show that enzyme inactivation by hydroxyl radicals ((•)OH) can be avoided in the presence of Pro, suggesting this amino acid might act as a (•)OH scavenger. Although production of hydroxyproline (Hyp) has been hypothesized in connection with such antioxidant activity, no evidence on the detailed mechanism of scavenging has been reported. To elucidate whether and how Hyp might be produced, we used density functional theory calculations coupled to a polarizable continuum model to explore 27 reaction channels including H-abstraction by (•)OH and (•)OH/H2O addition. The structure and energetics of stable species and transition states for each reaction channel were characterized at the PCM-(U)M06/6-31G(d,p) level in aqueous solution. Evidence is found for a main pathway in which Pro scavenges (•)OH by successive H-abstractions (ΔG(‡,298) = 4.1 and 7.5 kcal mol(-1)) to yield 3,4-Δ-Pro. A companion pathway with low barriers yielding Δ(1)-pyrroline-5-carboxylate (P5C) is also supported, linking with 5-Hyp through hydration. However, this connection remains unlikely in stressed plants because P5C would be efficiently recycled to Pro (contributing to its accumulation) by P5C reductase, hypothesis coined here as the "Pro-Pro cycle".

The accumulation of proline (Pro) in plants exposed to biotic/abiotic stress is a well-documented and conserved response in most vegetal species. Stress conditions induce the overproduction of reactive oxygen species which can lead to cellular damage. In vitro assays have shown that enzyme inactivation by hydroxyl radicals (·OH) can be avoided in presence of Pro, suggesting that this amino acid could act as an ·OH scavenger. We applied Density Functional Theory coupled with a polarizable continuum model to elucidate how Pro reacts with ·OH. In this work we suggest that Pro reacts favourably with ·OH by H–abstraction on the amine group. This reaction produces the spontaneous decarboxylation of Pro leading to the formation of pyrrolidin-1-yl. In turn, pyrrolidin-1-yl can easily be converted to Δ1-pyrroline, the substrate of the enzyme Δ1-pyrroline dehydrogenase, which produces γ-aminobutyric acid (GABA). GABA and Pro are frequently accumulated in stressed plants and several protective roles have been assigned to these molecules. Thereby we present an alternative non-enzymatic way to synthetize GABA under oxidative stress. Finally this work sheds light on a new beneficial role of Pro accumulation in the maintenance of photosynthetic activity.

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Plants are commonly subjected to several environmental stresses that lead to an overproduction of reactive oxygen species (ROS). As plants accumulate proline in response to stress conditions, some authors have proposed that proline could act as a non-enzymatic antioxidant against ROS. One type of ROS aimed to be quenched by proline is singlet oxygen (1O2)-molecular oxygen in its lowest energy electronically excited state-constitutively generated in oxygenic, photosynthetic organisms. In this study we clearly prove that proline cannot quench 1O2 in aqueous buffer, giving rise to a rethinking about the antioxidant role of proline against 1O2. Highlights Pro was proposed to be a quencher of 1 O 2 , but a direct prove is still remaining  19 Abstract 20Plants are commonly subjected to several environmental stresses that lead to an overproduction of reactive 21 oxygen species (ROS). As plants accumulate proline in response to stress conditions, some authors have 22proposed that proline could act as a non-enzymatic antioxidant against ROS. One type of ROS aimed to be 23 quenched by proline is singlet oxygen ( 1 O 2 )-molecular oxygen in its lowest energy electronically excited 24 state-constitutively generated in oxygenic, photosynthetic organisms. In this study we clearly prove that proline 25 cannot quench 1 O 2 in aqueous buffer, giving rise to a rethinking about the antioxidant role of proline against 1 O 2 .

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.