Maria C. Rubio

Biochemistry and Molecular Biology of Antioxidants in the Rhizobia-Legume Symbiosis

The complete reduction of molecular oxygen to water requires four electrons and is catalyzed by cytochrome oxidase in aerobic bacteria and mitochondria. However, 1% to 3% of all oxygen consumed by respiration is inevitably reduced to superoxide radicals and hydrogen peroxide (H2O2). These and other

Nodulation and nitrogen fixation by Mimosa spp. in the Cerrado and Caatinga biomes of Brazil

*An extensive survey of nodulation in the legume genus Mimosa was undertaken in two major biomes in Brazil, the Cerrado and the Caatinga, in both of which there are high degrees of endemicity of the genus. *Nodules were collected from 67 of the 70 Mimosa spp. found. Thirteen of the species were newly reported as nodulating. Nodules were examined by light and electron microscopy, and all except for M. gatesiae had a structure typical of effective Mimosa nodules. The endosymbiotic bacteria in nodules from all of the Mimosa spp. were identified as Burkholderia via immunolabelling with an antibody against Burkholderia phymatum STM815. *Twenty of the 23 Mimosa nodules tested were shown to contain nitrogenase by immunolabelling with an antibody to the nitrogenase Fe- (nifH) protein, and using the delta(15)N ((15)N natural abundance) technique, contributions by biological N(2) fixation of up to 60% of total plant N were calculated for Caatinga Mimosa spp. *It is concluded that nodulation in Mimosa is a generic character, and that the preferred symbionts of Brazilian species are Burkholderia. This is the first study to demonstrate N(2) fixation by beta-rhizobial symbioses in the field.

Localization of superoxide dismutases and hydrogen peroxide in legume root nodules.

Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals to O2 and H2O2 and thus represent a primary line of antioxidant defense in all aerobic organisms. H2O2 is a signal molecule involved in the plants response to pathogen attack and other stress conditions as well as in nodulation. In this work, we have tested the hypothesis that SODs are a source of H2O2 in indeterminate alfalfa (Medicago sativa) and pea (Pisum sativum) nodules. The transcripts and proteins of the major SODs of nodules were localized by in situ RNA hybridization and immunogold electron microscopy, respectively, whereas H2O2 was localized cytochemically by electron microscopy of cerium-perfused nodule tissue. The transcript and protein of cytosolic CuZnSOD are most abundant in the meristem (I) and invasion (II) zones, interzone II-III, and distal part of the N2-fixing zone (III), and those of MnSOD in zone III, especially in the infected cells. At the subcellular level, CuZnSOD was found in the infection threads, cytosol adjacent to cell walls, and apoplast, whereas MnSOD was in the bacteroids, bacteria within infection threads, and mitochondria. The distinct expression pattern of CuZnSOD and MnSOD suggests specific roles of the enzymes in nodules. Large amounts of H2O2 were found at the same three nodule sites as CuZnSOD but not in association with MnSOD. This colocalization led us to postulate that cytosolic CuZnSOD is a source of H2O2 in nodules. Furthermore, the absence or large reduction of H2O2 in nodule tissue preincubated with enzyme inhibitors (cyanide, azide, diphenyleneiodonium, diethyldithiocarbamate) provides strong support to the hypothesis that at least some of the H2O2 originates by the sequential operation of an NADPH oxidase-like enzyme and CuZnSOD. Results also show that there is abundant H2O2 associated with degrading bacteroids in the senescent zone (IV), which reflects the oxidative stress ensued during nodule senescence.

Functional Characterization and Expression of a Cytosolic Iron-Superoxide Dismutase from Cowpea Root Nodules

An iron-superoxide dismutase (FeSOD) with an unusual subcellular localization, VuFeSOD, has been purified from cowpea (Vigna unguiculata) nodules and leaves. The enzyme has two identical subunits of 27 kD that are not covalently bound. Comparison of its N-terminal sequence (NVAGINLL) with the cDNA-derived amino acid sequence showed that VuFeSOD is synthesized as a precursor with seven additional amino acids. The mature protein was overexpressed in Escherichia coli, and the recombinant enzyme was used to generate a polyclonal monospecific antibody. Phylogenetic and immunological data demonstrate that there are at least two types of FeSODs in plants. An enzyme homologous to VuFeSOD is present in soybean (Glycine max) and common bean (Phaseolus vulgaris) nodules but not in alfalfa (Medicago sativa) and pea (Pisum sativum) nodules. The latter two species also contain FeSODs in the leaves and nodules, but the enzymes are presumably localized to the chloroplasts and plastids. In contrast, immunoblots of the soluble nodule fraction and immunoelectron microscopy of cryo-processed nodule sections demonstrate that VuFeSOD is localized to the cytosol. Immunoblot analysis showed that the content of VuFeSOD protein increases in senescent nodules with active leghemoglobin degradation, suggesting a direct or indirect (free radical-mediated) role of the released Fe in enzyme induction. Therefore, contrary to the widely held view, FeSODs in plants are not restricted to the chloroplasts and may become an important defensive mechanism against the oxidative stress associated with senescence.

Effects of salt stress on the expression of antioxidant genes and proteins in the model legume Lotus japonicus.

Salt stress negatively affects many physiological processes in plants. Some of these effects may involve the oxidative damage of cellular components, which can be promoted by reactive oxygen species and prevented by antioxidants. The protective role of antioxidants was investigated in Lotus japonicus exposed to two salinization protocols: S1 (150 mM NaCl for 7 d) and S2 (50, 100 and 150 mM NaCl, each concentration for 6 d). Several markers of salt stress were measured and the expression of antioxidant genes was analyzed using quantitative reverse transcription–polymerase chain reaction and, in some cases, immunoblots and enzyme activity assays. Leaves of S1 plants suffered from mild osmotic stress, accumulated proline but noNa+, and showed induction of many superoxide dismutase and glutathione peroxidase genes. Leaves of S2 plants showed increases in Na+ and Ca2+, decreases in K+, and accumulation of proline and malondialdehyde. In leaves and roots of S1 and S2 plants, the mRNA, protein and activity levels of the ascorbate-glutathione enzymes remained constant, with a few exceptions. Notably, there was consistent up-regulation of the gene encoding cytosolic dehydroascorbate reductase, and this was possibly related to its role in ascorbate recycling in the apoplast. The overall results indicate that L. japonicus is more tolerant to salt stress than other legumes, which can be attributed to the capacity of the plant to prevent Na+reaching the shoot and to activate antioxidant defenses.

The Antioxidants of Legume Nodule Mitochondria

The mitochondria of legume root nodules are critical to sustain the energy-intensive process of nitrogen fixation. They also generate reactive oxygen species at high rates and thus require the protection of antioxidant enzymes and metabolites. We show here that highly purified mitochondria from bean nodules (Phaseolus vulgaris L. cv. Contender x Rhizobium leguminosarum bv. phaseoli strain 3622) contain ascorbate peroxidase primarily in the inner membrane (with lesser amounts detected occasionally in the matrix), guaiacol peroxidases in the outer membrane and matrix, and manganese superoxide dismutase (MnSOD) and an ascorbate-regenerating system in the matrix. This regenerating system relies on homoglutathione (instead of glutathione) and pyridine nucleotides as electron donors and involves the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and homoglutathione reductase. Homoglutathione is synthesized in the cytosol and taken up by the mitochondria and bacteroids. Although bacteroids synthesize glutathione, it is not exported to the plant in significant amounts. We propose a model for the detoxification of peroxides in nodule mitochondria in which membrane-bound ascorbate peroxidase scavenges the peroxide formed by the electron transport chain using ascorbate provided by L-galactono-1,4-lactone dehydrogenase in the inner membrane. The resulting monodehydroascorbate and dehydroascorbate can be recycled in the matrix or cytosol. In the matrix, the peroxides formed by oxidative reactions and by MnSOD may be scavenged by specific isozymes of guaiacol peroxidase, ascorbate peroxidase, and catalase.

Characterization of genomic clones and expression analysis of the three types of superoxide dismutases during nodule development in Lotus japonicus

Superoxide dismutases (SODs) are metalloenzymes that play a primary role in the protection against oxidative stress in plants and other organisms. We have characterized four SOD genes in Lotus japonicus and have analyzed their expression in roots and four developmental stages of nodules. The expression of cytosolic CuZnSOD, at the mRNA, protein, and enzyme activity levels, decreases with nodule age, and the protein is localized in the dividing cells and infection threads of emergent nodules and in the infected cells of young nodules. The mitochondrial MnSOD was downregulated, whereas the bacteroidal MnSOD displayed maximal protein and enzyme activity levels in older nodules. Two additional genes, encoding plastidic (FeSOD1) and cytosolic (FeSOD2) FeSOD isoforms, were identified and mapped. The genes are located in different chromosomes and show differential expression. The FeSOD1 mRNA level did not change during nodule development, whereas FeSOD2 was upregulated. The distinct expression patterns of the SOD genes may reflect different regulatory mechanisms of the enzyme activities during nodule ontogeny. In particular, at the mRNA and activity levels, the virtual loss of cytosolic CuZnSOD in mature and old nodules, concomitant with the induction of FeSOD2, suggests that the two enzymes may functionally compensate each other in the cytosol at the late stages of nodule development.

Expression studies of superoxide dismutases in nodules and leaves of transgenic alfalfa reveal abundance of iron-containing isozymes, posttranslational regulation, and compensation of isozyme activities.

The composition of antioxidant enzymes, especially superoxide dismutase (SOD), was studied in one nontransgenic and three transgenic lines of nodulated alfalfa plants. Transgenic lines overproduced MnSOD in the mitochondria of nodules and leaves (line 1-10), MnSOD in the chloroplasts (line 4-6), and FeSOD in the chloroplasts (line 10-7). In nodules of line 10-7, the absence of transgene-encoded FeSOD activity was due to a lack of mRNA, whereas in nodules of line 4-6 the absence of transgene-encoded MnSOD activity was due to enzyme inactivation or degradation. Transgenic alfalfa showed a novel compensatory effect in the activities of MnSOD (mitochondrial) and FeSOD (plastidic) in the leaves, which was not caused by changes in the mRNA levels. These findings imply that SOD activity in plant tissues and organelles is regulated, at least partially, at the posttranslational level. All four lines had low CuZnSOD activities and an abundant FeSOD isozyme, especially in nodules, indicating that FeSOD performs important antioxidant functions other than the scavenging of superoxide radicals generated in photosynthesis. This was confirmed by the detection of FeSOD cDNAs and proteins in nodules of other legumes such as cowpea, pea, and soybean. The cDNA encoding alfalfa nodule FeSOD was characterized and the deduced protein found to contain a plastid transit peptide. A comparison of sequences and other properties reveals that there are two types of FeSODs in nodules.

Hemoglobin LjGlb1-1 is involved in nodulation and regulates the level of nitric oxide in the Lotus japonicus–Mesorhizobium loti symbiosis

Highlight Missense and null Lotus japonicus mutants allowed us to demonstrate that hemoglobin LjGlb1-1 is required for infection thread elongation and nodule formation, probably by regulating nitric oxide production in the roots.

Redefining nitric oxide production in legume nodules through complementary insights from electron paramagnetic resonance spectroscopy and specific fluorescent probes

The combined use of EPR spectroscopy and the specific fluorescent dye 4,5-diaminofluorescein diacetate uncovers sites of nitric oxide production in legume nodules and warns of potential artifacts.