Jorge Monza

Water stress induces a differential and spatially distributed nitro-oxidative stress response in roots and leaves of Lotus japonicus

Water stress is one of the most severe problems for plant growth and productivity. Using the legume Lotus japonicus exposed to water stress, a comparative analysis of key components in metabolism of reactive nitrogen and oxygen species (RNS and ROS, respectively) were made. After water stress treatment plants accumulated proline 23 and 10-fold in roots and leaves respectively, compared with well-watered plants. Significant changes in metabolism of RNS and ROS were observed, with an increase in both protein tyrosine nitration and lipid peroxidation, which indicate that water stress induces a nitro-oxidative stress. In roots, ·NO content was increased and S-nitrosoglutathione reductase activity was reduced by 23%, wherein a specific protein nitration pattern was observed. As part of this response, activity of NADPH-generating dehydrogenases was also affected in roots resulting in an increase of the NADPH/NADP(+) ratio. Our results suggest that in comparison with leaves, roots are significantly affected by water stress inducing an increase in proline and NO content which could highlight multiple functions for these metabolites in water stress adaptation, recovery and signaling. Thus, it is proposed that water stress generates a spatial distribution of nitro-oxidative stress with the oxidative stress component being higher in leaves whereas the nitrosative stress component is higher in roots.

Water stress generates an oxidative stress through the induction of a specific Cu/Zn superoxide dismutase in Lotus corniculatus leaves

Drought brings about different biochemical responses in plants in order to minimize its deleterious effects. Drought induces an oxidative stress in Lotus corniculatus leaves, measured as an increment in lipid membrane peroxidation and in situ detected superoxide. As a result, total superoxide dismutase (SOD: E.C. 1.15.1.1) activity increases after a 4-h drought, when the hydric potential has decreased to −0.77 MPa. Assays with specific inhibitors suggest the presence of MnSOD, Cu/ZnSOD and FeSOD activity in L. corniculatus plants, contribute 60, 30 and 10% of total SOD activity, respectively. The Cu/ZnSOD isoform proved to be the most responsive to drought showing a remarkable increase in its activity as that corresponds to the induction of three different isoenzymes. Expression analysis of SOD isoforms revealed an increase in Cu/ZnSOD transcripts with a maximum accumulation 4 h after drought imposition. The possible role of SOD enzymes as an antioxidant protector system under water stress conditions in L. corniculatus is discussed.

Deficiency in plastidic glutamine synthetase alters proline metabolism and transcriptomic response in Lotus japonicus under drought stress

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.

Response to photoxidative stress induced by cold in japonica rice is genotype dependent.

Two japonica rice genotypes, INIA Tacuarí and L2825CA, were analyzed for tolerance to low temperature during early vegetative growth. Effect on photosynthesis, energy dissipation, pigment content, xanthophyll-cycle pool conversion, hydrogen peroxide accumulation, oxidative damage and antioxidant enzyme activities were determined to better understand potential mechanisms for cold tolerance. Photoinhibition was measured using chlorophyll fluorescence and oxidative damage by lipid peroxidation and electrolyte leakage. Both genotypes were demonstrated to be cold tolerant which was consistent with their reduced levels of photoinhibition and oxidative damage compared with a cold-sensitive genotype during chilling stress. The strategy for cold tolerance differed between the two genotypes, and involved different mechanisms for disposal of excess energy. The presence of high lutein concentrations and the existence of active non-harmful energy dissipation processes through the xanthophyll cycle appeared to be responsible for chilling tolerance in INIA Tacuarí. On the other hand, increased cold tolerance of L2825CA relative to INIA Tacuarí was related to the higher constitutive superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11) and catalase (CAT, EC 1.11.1.6).

Lotus-related species and their agronomic importance

More than 180 species within the genus Lotus occur worldwide. Four have been domesticated and improved through selection and plant breeding: Lotus corniculatus, L. uliginosus, L. glaber and L. subbiflorus. Since the model legume L. japonicus is related taxonomically to these species, knowledge can be transferred to the agronomical arena. The slow progress observed in Lotus cultivar improvements to date could be explained by the polyploid nature of some of these species, a feature not present in L. japonicus. This chapter reviews briefly the taxonomical relationships among these species. Secondly, it illustrates how Lotus species are currently used to improve pastures for which other forage legume species are not suitable. Finally, it touches on beneficial microorganism-plant interactions and the benefits of using Lotus species as animal fodder.

Heat stress results in loss of chloroplast Cu/Zn superoxide dismutase and increased damage to Photosystem II in combined drought-heat stressed Lotus japonicus

Drought and heat stress have been studied extensively in plants, but most reports involve analysis of response to only one of these stresses. Studies in which both stresses were studied in combination have less commonly been reported. We report the combined effect of drought and heat stress on Photosystem II (PSII) of Lotus japonicus cv. Gifu plants. Photochemistry of PSII was not affected by drought or heat stress alone, but the two stresses together decreased PSII activity as determined by fluorescence emission. Heat stress alone resulted in degradation of D1 and CP47 proteins, and D2 protein was also degraded by combined drought-heat stress. None of these proteins were degraded by drought stress alone. Drought alone induced accumulation of hydrogen peroxide but the drought-heat combination led to an increase in superoxide levels and a decrease in hydrogen peroxide levels. Furthermore, combined drought-heat stress was correlated with an increase in oxidative damage as determined by increased levels of thiobarbituric acid reactive substances. Heat also induced degradation of chloroplast Cu/Zn superoxide dismutase (SOD: EC 1.15.1.1) as shown by reduced protein levels and isozyme-specific SOD activity. Loss of Cu/Zn SOD and induction of catalase (CAT: EC 1.11.1.6) activity would explain the altered balance between hydrogen peroxide and superoxide in response to drought vs combined drought-heat stress. Degradation of PSII could thus be caused by the loss of components of chloroplast antioxidant defence systems and subsequent decreased function of PSII. A possible explanation for energy dissipation by L. japonicus under stress conditions is discussed.

Proline does not quench singlet oxygen: evidence to reconsider its protective role in plants.

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 ((1)O(2))-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 (1)O(2) in aqueous buffer, giving rise to a rethinking about the antioxidant role of proline against (1)O(2).

Proline is involved in water stress responses of Lotus corniculatus nitrogen fixing and nitrate fed plants

Summary Water stress induced proline accumulation in Lotus corniculatus regardless of the nitrogen source provided to plants, although this accumulation was higher in nitrogen fixing plants. Enzymes related to synthesis and oxidation of proline were analysed. Fd-GOGAT activity and Fd-GOGAT protein content increased in response to water stress in contrast to PDH activity, which decreased under such condition. However, rehydration for 6 h induced an increment in PDH activity. The differential effect of water deficit on enzymes involved in the proline pathway in L. corniculatus plants growing under different nitrogen regimes is discussed. The possible role of nitrate as an osmoprotectant in the place of proline is also considered.

Connecting Proline and γ-Aminobutyric Acid in Stressed Plants through Non-Enzymatic Reactions

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.

Antioxidant and photosystem II responses contribute to explain the drought–heat contrasting tolerance of two forage legumes

Identification of metabolic targets of environmental stress factors is critical to improve the stress tolerance of plants. Studying the biochemical and physiological responses of plants with different capacities to deal with stress is a valid approach to reach this objective. Lotus corniculatus (lotus) and Trifolium pratense (clover) are legumes with contrasting summer stress tolerances. In stress conditions, which are defined as drought, heat or a combination of both, we found that differential biochemical responses of leaves explain these behaviours. Lotus and clover showed differences in water loss control, proline accumulation and antioxidant enzymatic capacity. Drought and/or heat stress induced a large accumulation of proline in the tolerant species (lotus), whereas heat stress did not cause proline accumulation in the sensitive species (clover). In lotus, Mn-SOD and Fe-SOD were induced by drought, but in clover, the SOD-isoform profile was not affected by stress. Moreover, lotus has more SOD-isoforms and a higher total SOD activity than clover. The functionality and electrophoretic profile of photosystem II (PSII) proteins under stress also exhibited differences between the two species. In lotus, PSII activity was drastically affected by combined stress and, interestingly, was correlated with D2 protein degradation. Possible implications of this event as an adaption mechanism in tolerant species are discussed. We conclude that the stress-tolerant capability of lotus is related to its ability to respond to oxidative damage and adaption of the photosynthetic machinery. This reveals that these two aspects should be included in the evaluation of the tolerance of species to stress conditions.