Respuesta fisiológica, bioquímica y molecular de las leguminosas a estreses abióticos

Tesis financiada con beca del Programa de Formación de Personal Investigador (BES-2003-1462) y proyectos del Plan Nacional (AGL2002-02876 y AGL2005-01404) del Ministerio de Educación y Ciencia, además de Fondos Europeos de Desarrollo Regional (FEDER).Peer reviewe

Functional Characterization of an Unusual Phytochelatin Synthase, LjPCS3, of Lotus japonicus1[W][OA]

In plants and many other organisms, phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins from glutathione in the presence of certain metals and metalloids. We have used budding yeast (Saccharomyces cerevisiae) as a heterologous system to characterize two PCS proteins, LjPCS1 and LjPCS3, of the model legume Lotus japonicus. Initial experiments revealed that the metal tolerance of yeast cells in vivo depends on the concentrations of divalent cations in the growth medium. Detailed in vivo (intact cells) and in vitro (broken cells) assays of PCS activity were performed with yeast expressing the plant enzymes, and values of phytochelatin production for each metal tested were normalized with respect to those of cadmium to correct for the lower expression level of LjPCS3. Our results showed that lead was the best activator of LjPCS1 in the in vitro assay, whereas, for both assays, arsenic, iron, and aluminum were better activators of LjPCS3 and mercury was similarly active with the two enzymes. Most interestingly, zinc was a powerful activator, especially of LjPCS3, when assayed in vivo, whereas copper and silver were the strongest activators in the in vitro assay. We conclude that the in vivo and in vitro assays are useful and complementary to assess the response of LjPCS1 and LjPCS3 to a wide range of metals and that the differences in the C-terminal domains of the two proteins are responsible for their distinct expression levels or stabilities in heterologous systems and patterns of metal activation

Ammonium stress increases microautophagic activity while impairing macroautophagic flux in Arabidopsis roots

Plant responses to NH4 + stress are complex, and multiple mechanisms underlying NH4 + sensitivity and tolerance in plants may be involved. Here, we demonstrate that macro‐ and microautophagic activities are oppositely affected in plants grown under NH4 + toxicity conditions. When grown under NH4 + stress conditions, macroautophagic activity was impaired in roots. Root cells accumulated autophagosomes in the cytoplasm, but showed less autophagic flux, indicating that late steps of the macroautophagy process are affected under NH4 + stress conditions. Under this scenario, we also found that the CCZ1‐MON1 complex, a critical factor for vacuole delivery pathways, functions in the late step of the macroautophagic pathway in Arabidopsis. In contrast, an accumulation of tonoplast‐derived vesicles was observed in vacuolar lumens of root cells of NH4 +‐stressed plants, suggesting the induction of a microautophagy‐like process. In this sense, some SYP22‐, but mainly VAMP711‐positive vesicles were observed inside vacuole in roots of NH4 +‐stressed plants. Consistent with the increased tonoplast degradation and the reduced membrane flow to the vacuole due to the impaired macroautophagic flux, the vacuoles of root cells of NH4 +‐stressed plants showed a simplified structure and lower tonoplast content. Taken together, this study presents evidence that postulates late steps of the macroautophagic process as a relevant physiological mechanism underlying the NH4 + sensitivity response in Arabidopsis, and additionally provides insights into the molecular tools for studying microautophagy in plants.Instituto de Fisiología y Recursos Genéticos VegetalesFil: Robert, German. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Unidad de Estudios Agropecuarios (UDEA); Argentina. Université Paris‐Saclay. Institut Jean‐Pierre Bourgin, INRAE, AgroParisTech; FranceFil: Yagyu, Mako. Meiji University. School of Agriculture. Department of Life Sciences; JapónFil: Koizumi, Takaya. Meiji University. School of Agriculture. Department of Life Sciences; JapónFil: Naya, Loreto. Université Paris‐Saclay. Institut Jean‐Pierre Bourgin, INRAE, AgroParisTech; FranciaFil: Masclaux‐Daubresse, Céline. Université Paris‐Saclay. Institut Jean‐Pierre Bourgin, INRAE, AgroParisTech; FranciaFil: Yoshimoto, Kohki. Université Paris‐Saclay. Institut Jean‐Pierre Bourgin, INRAE, AgroParisTech; Francia. Meiji University. School of Agriculture. Department of Life Sciences; Japó

A Reassessment of Substrate Specificity and Activation of Phytochelatin Synthases from Model Plants by Physiologically Relevant Metals

Phytochelatin synthases (PCS) catalyze phytochelatin (PC) synthesis from glutathione (GSH) in the presence of certain metals. The resulting PC-metal complexes are transported into the vacuole, avoiding toxic effects on metabolism. Legumes have the unique capacity to partially or completely replace GSH by homoglutathione (hGSH) and PCs by homophytochelatins (hPCs). However, the synthesis of hPCs has received little attention. A search for PCS genes in the model legume Lotus (Lotus japonicus) resulted in the isolation of a cDNA clone encoding a protein (LjPCS1) highly homologous to a previously reported homophytochelatin synthase (hPCS) of Glycine max (GmhPCS1). Recombinant LjPCS1 and Arabidopsis (Arabidopsis thaliana) PCS1 (AtPCS1) were affinity purified and their polyhistidine-tags removed. AtPCS1 catalyzed hPC synthesis from hGSH alone at even higher rates than did LjPCS1, indicating that GmhPCS1 is not a genuine hPCS and that a low ratio of hPC to PC synthesis is an inherent feature of PCS1 enzymes. For both enzymes, hGSH is a good acceptor, but a poor donor, of γ-glutamylcysteine units. Purified AtPCS1 and LjPCS1 were activated (in decreasing order) by Cd(2+), Zn(2+), Cu(2+), and Fe(3+), but not by Co(2+) or Ni(2+), in the presence of 5 mm GSH and 50 μm metal ions. Activation of both enzymes by Fe(3+) was proven by the complete inhibition of PC synthesis by the iron-specific chelator desferrioxamine. Plants of Arabidopsis and Lotus accumulated (h)PCs only in response to a large excess of Cu(2+) and Zn(2+), but to a much lower extent than did with Cd(2+), indicating that (h)PC synthesis does not significantly contribute in vivo to copper, zinc, and iron detoxification

The Response of Carbon Metabolism and Antioxidant Defenses of Alfalfa Nodules to Drought Stress and to the Subsequent Recovery of Plants12[W][OA]

Alfalfa (Medicago sativa) plants were exposed to drought to examine the involvement of carbon metabolism and oxidative stress in the decline of nitrogenase (N2ase) activity. Exposure of plants to a moderate drought (leaf water potential of −1.3 MPa) had no effect on sucrose (Suc) synthase (SS) activity, but caused inhibition of N2ase activity (−43%), accumulation of succinate (+36%) and Suc (+58%), and up-regulation of genes encoding cytosolic CuZn-superoxide dismutase (SOD), plastid FeSOD, cytosolic glutathione reductase, and bacterial MnSOD and catalases B and C. Intensification of stress (−2.1 MPa) decreased N2ase (−82%) and SS (−30%) activities and increased malate (+40%), succinate (+68%), and Suc (+435%). There was also up-regulation (mRNA) of cytosolic ascorbate peroxidase and down-regulation (mRNA) of SS, homoglutathione synthetase, and bacterial catalase A. Drought stress did not affect nifH mRNA level or leghemoglobin expression, but decreased MoFe- and Fe-proteins. Rewatering of plants led to a partial recovery of the activity (75%) and proteins (>64%) of N2ase, a complete recovery of Suc, and a decrease of malate (−48%) relative to control. The increase in O2 diffusion resistance, the decrease in N2ase-linked respiration and N2ase proteins, the accumulation of respiratory substrates and oxidized lipids and proteins, and the up-regulation of antioxidant genes reveal that bacteroids have their respiratory activity impaired and that oxidative stress occurs in nodules under drought conditions prior to any detectable effect on SS or leghemoglobin. We conclude that a limitation in metabolic capacity of bacteroids and oxidative damage of cellular components are contributing factors to the inhibition of N2ase activity in alfalfa nodules

Phytochelatin Synthases of the Model Legume Lotus japonicus. A Small Multigene Family with Differential Response to Cadmium and Alternatively Spliced Variants

The biosynthesis of phytochelatins and homophytochelatins has been studied in nodulated plants of the model legume Lotus (Lotus japonicus). In the first 6 to 24 h of treatment with cadmium (Cd), roots started to synthesize elevated amounts of both polypeptides, with a concomitant increase of glutathione and a decrease of homoglutathione, indicating the presence of active phytochelatin synthase (PCS) genes. Screening of transformation-competent artificial chromosome libraries allowed identification of a cluster of three genes, LjPCS1, LjPCS2, and LjPCS3, which were mapped at 69.0 cM on chromosome 1. The genes differ in exon-intron composition and responsiveness to Cd. Gene structures and phylogenetic analysis of the three protein products, LjPCS1-8R, LjPCS2-7N, and LjPCS3-7N, are consistent with two sequential gene duplication events during evolution of vascular plants. Two sites for alternative splicing in the primary transcripts were identified. One of them, involving intron 2 of the LjPCS2 gene, was confirmed by the finding of the two predicted mRNAs, encoding LjPCS2-7R in roots and LjPCS2-7N in nodules. The amino acid sequences of LjPCS2-7R (or LjPCS2-7N) and LjPCS3-7N share 90% identity, but have only 43% to 59% identity with respect to the typical PCS1 enzymes of Lotus and other plants. The unusual LjPCS2-7N and LjPCS3-7N proteins conferred Cd tolerance when expressed in yeast (Saccharomyces cerevisiae) cells, whereas the alternatively spliced form, LjPCS2-7R, differing only in a five-amino acid motif (GRKWK) did not. These results unveil complex regulatory mechanisms of PCS expression in legume tissues in response to heavy metals and probably to other developmental and environmental factors

Genome-Wide Medicago truncatula Small RNA Analysis Revealed Novel MicroRNAs and Isoforms Differentially Regulated in Roots and Nodules[W]

Posttranscriptional regulation of a variety of mRNAs by small 21- to 24-nucleotide RNAs, notably the microRNAs (miRNAs), is emerging as a novel developmental mechanism. In legumes like the model Medicago truncatula, roots are able to develop a de novo meristem through the symbiotic interaction with nitrogen-fixing rhizobia. We used deep sequencing of small RNAs from root apexes and nodules of M. truncatula to identify 100 novel candidate miRNAs encoded by 265 hairpin precursors. New atypical precursor classes producing only specific 21- and 24-nucleotide small RNAs were found. Statistical analysis on sequencing reads abundance revealed specific miRNA isoforms in a same family showing contrasting expression patterns between nodules and root apexes. The differentially expressed conserved and nonconserved miRNAs may target a large variety of mRNAs. In root nodules, which show diverse cell types ranging from a persistent meristem to a fully differentiated central region, we discovered miRNAs spatially enriched in nodule meristematic tissues, vascular bundles, and bacterial infection zones using in situ hybridization. Spatial regulation of miRNAs may determine specialization of regulatory RNA networks in plant differentiation processes, such as root nodule formation

Autophagy Increases Zinc Bioavailability to Avoid Light-Mediated Reactive Oxygen Species Production under Zinc Deficiency

International audienceesupply of free zinc ions via autophagic degradation suppresses photosynthesis-related Fenton-like reaction-induced chlorosis under zinc starvation in Arabidopsis thaliana.Zinc (Zn) is an essential micronutrient for plant growth. Accordingly, Zn deficiency (-Zn) in agricultural fields is a serious problem, especially in developing regions. Autophagy, a major intracellular degradation system in eukaryotes, plays important roles in nutrient recycling under nitrogen and carbon starvation. However, the relationship between autophagy and deficiencies of other essential elements remains poorly understood, especially in plants. In this study, we focused on Zn due to the property that within cells most Zn is tightly bound to proteins, which can be targets of autophagy. We found that autophagy plays a critical role during -Zn in Arabidopsis (Arabidopsis thaliana). Autophagy-defective plants (atg mutants) failed to grow and developed accelerated chlorosis under -Zn. As expected, -Zn induced autophagy in wild-type plants, whereas in atg mutants, various organelle proteins accumulated to high levels. Additionally, the amount of free Zn2+ was lower in atg mutants than in control plants. Interestingly, -Zn symptoms in atg mutants recovered under low-light, iron-limited conditions. The levels of hydroxyl radicals in chloroplasts were elevated, and the levels of superoxide were reduced in -Zn atg mutants. These results imply that the photosynthesis-mediated Fenton-like reaction, which is responsible for the chlorotic symptom of -Zn, is accelerated in atg mutants. Together, our data indicate that autophagic degradation plays important functions in maintaining Zn pools to increase Zn bioavailability and maintain reactive oxygen species homeostasis under -Zn in plants