Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots.

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through nanoindentations with an atomic force microscope (AFM) coupled with an optical inverted fluorescence microscope. The method consists of applying controlled forces to the sample while measuring its deformation, allowing quantifying parameters such as the apparent Young's modulus of cell walls at subcellular resolutions. It requires a careful mechanical immobilization of the sample and correct selection of indenters and indentation depths. Although it can be used only in external tissues, this method allows characterizing mechanical changes in plant cell walls during development and enables the correlation of these microscopic changes with the growth of an entire organ.

The Role of Nitric Oxide in Nitrogen Fixation by Legumes.

The legume-rhizobia symbiosis is an important process in agriculture because it allows the biological nitrogen fixation (BNF) which contributes to increasing the levels of nitrogen in the soil. Nitric oxide (⋅NO) is a small free radical molecule having diverse signaling roles in plants. Here we present and discuss evidence showing the role of ⋅NO during different stages of the legume-rhizobia interaction such as recognition, infection, nodule development, and nodule senescence. Although the mechanisms by which ⋅NO modulates this interaction are not fully understood, we discuss potential mechanisms including its interaction with cytokinin, auxin, and abscisic acid signaling pathways. In matures nodules, a more active metabolism of ⋅NO has been reported and both the plant and rhizobia participate in ⋅NO production and scavenging. Although ⋅NO has been shown to induce the expression of genes coding for NITROGENASE, controlling the levels of ⋅NO in mature nodules seems to be crucial as ⋅NO was shown to be a potent inhibitor of NITROGENASE activity, to induce nodule senescence, and reduce nitrogen assimilation. In this sense, LEGHEMOGLOBINS (Lbs) were shown to play an important role in the scavenging of ⋅NO and reactive nitrogen species (RNS), potentially more relevant in senescent nodules. Even though ⋅NO can reduce NITROGENASE activity, most reports have linked ⋅NO to positive effects on BNF. This can relate mainly to the regulation of the spatiotemporal distribution of ⋅NO which favors some effects over others. Another plausible explanation for this observation is that the negative effect of ⋅NO requires its direct interaction with NITROGENASE, whereas the positive effect of ⋅NO is related to its signaling function, which results in an amplifier effect. In the near future, it would be interesting to explore the role of environmental stress-induced ⋅NO in BNF.

Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide-dependent mechanism.

Protein tyrosine (Tyr) nitration is a post-translational modification yielding 3-nitrotyrosine (NO2 -Tyr). Formation of NO2 -Tyr is generally considered as a marker of nitro-oxidative stress and is involved in some human pathophysiological disorders, but has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as an O2 transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO2 -Tyr in Lb. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO2 -Tyr25 and NO2 -Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving NO3- and were found to decrease during senescence. This demonstrates formation of nitric oxide (˙NO) and NO2- by alternative means to nitrate reductase, probably via a ˙NO synthase-like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lb revealed that Tyr nitration requires NO2- + H2 O2 and that peroxynitrite is not an efficient inducer of nitration, probably because Lb isomerizes it to NO3-. Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO2 -Tyr in Lb is a consequence of active metabolism in functional nodules, where Lb may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.

Plant hemoglobins may be maintained in functional form by reduced flavins in the nuclei, and confer differential tolerance to nitro-oxidative stress.

The heme of bacteria, plant and animal hemoglobins (Hbs) must be in the ferrous state to bind O(2) and other physiological ligands. Here we have characterized the full set of non-symbiotic (class 1 and 2) and 'truncated' (class 3) Hbs of Lotus japonicus. Class 1 Hbs are hexacoordinate, but class 2 and 3 Hbs are pentacoordinate. Three of the globins, Glb1-1, Glb2 and Glb3-1, are nodule-enhanced proteins. The O(2) affinity of Glb1-1 (50 pm) was the highest known for any Hb, and the protein may function as an O(2) scavenger. The five globins were reduced by free flavins, which transfer electrons from NAD(P)H to the heme iron under aerobic and anaerobic conditions. Class 1 Hbs were reduced at very fast rates by FAD, class 2 Hbs at slower rates by both FMN and FAD, and class 3 Hbs at intermediate rates by FMN. The members of the three globin classes were immunolocalized predominantly in the nuclei. Flavins were quantified in legume nodules and nuclei, and their concentrations were sufficient to maintain Hbs in their functional state. All Hbs, except Glb1-1, were expressed in a flavohemoglobin-deficient yeast mutant and found to confer tolerance to oxidative stress induced by methyl viologen, copper or low temperature, indicating an anti-oxidative role for the hemes. However, only Glb1-2 and Glb2 afforded protection against nitrosative stress induced by S-nitrosoglutathione. Because this compound is specifically involved in transnitrosylation reactions with thiol groups, our results suggest a contribution of the single cysteine residues of both proteins in the stress response.

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.

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.