Márcia Margis-Pinheiro

Plant responses to stresses: role of ascorbate peroxidase in the antioxidant protection

When plants are exposed to stressful environmental conditions, the production of Reactive Oxygen Species (ROS) increases and can cause significant damage to the cells. Antioxidant defenses, which can detoxify ROS, are present in plants. A major hydrogen peroxide detoxifying system in plant cells is the ascorbate-glutathione cycle, in which, ascorbate peroxidase (APX) enzymes play a key role catalyzing the conversion of H2O2 into H2O, using ascorbate as a specific electron donor. Different APX isoforms are present in distinct subcellular compartments, such as chloroplasts, mitochondria, peroxisome, and cytosol. The expression of APX genes is regulated in response to biotic and abiotic stresses as well as during plant development. The APX responses are directly involved in the protection of plant cells against adverse environmental conditions. Furthermore, mutant plants APX genes showed alterations in growth, physiology and antioxidant metabolism revealing those enzymes involvement in the normal plant development.

Rice ascorbate peroxidase gene family encodes functionally diverse isoforms localized in different subcellular compartments.

Aerobic organisms evolved a complex antioxidant system, which protect the cells against oxidative damage caused by partially reduced oxygen intermediates, also known as reactive oxygen species. In plants, ascorbate peroxidases (EC, 1.11.1.11) catalyze the conversion of H2O2 to H2O, using ascorbate as the specific electron donor in this enzymatic reaction. Previously, eight APx genes were identified in the rice (Oryza sativa L.) genome through in silico analysis: two cytosolic isoforms, two putative peroxisomal isoforms, and four putative chloroplastic ones. Using gene-specific probes, we confirmed the presence of the eight APx genes in the rice genome by Southern blot hybridization. Transcript accumulation analysis showed specific expression patterns for each member of the APx family according to developmental stage and in response to salt stress, revealing the complexity of the antioxidant system in plants. Finally, the subcellular localization of rice APx isoforms was determined using GFP-fusion proteins in BY-2 tobacco cells. In agreement with the initial prediction, OSAPX3 was localized in the peroxisomes. On the other hand, the OSAPX6-GFP fusion protein was found in mitochondria of the BY-2 cells, in contrast to the chloroplastic location predicted by sequence analysis. Our findings reveal the functional diversity of the rice APx genes and suggest complementation and coordination of the antioxidant defenses in different cellular compartments during development and abiotic stress.

Analysis of the Molecular Evolutionary History of the Ascorbate Peroxidase Gene Family: Inferences from the Rice Genome

Ascorbate peroxidase (APx) is a class I peroxidase that catalyzes the conversion of H2O2 to H2O and O2 using ascorbate as the specific electron donor. This enzyme has a key function in scavenging reactive oxygen species (ROS) and the protection against toxic effects of ROS in higher plants, algae, and Euglena. Here we report the identification of an APx multigene family in rice and propose a molecular evolutionary relationship between the diverse APx isoforms. In rice, the APx gene family has eight members, which encode two cytosolic, two putative peroxisomal, and four chloroplastic isoforms, respectively. Phylogenetic analyses were conducted using all APx protein sequences available in the NCBI databases. The results indicate that the different APx isoforms arose by a complex evolutionary process involving several gene duplications. The structural organization of APx genes also reflects this process and provides evidence for a close relationship among proteins located in the same subcellular compartment. A molecular evolutionary pathway, in which cytosolic and peroxisomal isoforms diverged early from chloroplastic ones, is proposed.

Arabidopsis thaliana class IV chitinase is early induced during the interaction with Xanthomonas campestris

Endochitinases are widely distributed among higher plants, including a number of important crop species. They are generally considered to be involved in plant defence against potential pathogens. We have cloned a class IV chitinase gene (AtchitIV) from Arabidopsis thaliana. Southern blot analysis allowed the detection of two cross‐hybridising genes in the A. thaliana genome. AtchitIV transcripts are detected in seedpods, but not in roots, inflorescence stems, leaves and flowers of healthy plants. The transcripts accumulated very rapidly in leaves after inoculation with Xanthomonas campestris. Maximum mRNA accumulation was reached one hour after infection and decreased to very low levels 72 hours after induction. This result suggests an involvement of AtchitIV in the initial events of the hypersensitive reaction. Nevertheless, A. thaliana plants transformed with the gus gene under the control of a class IV chitinase bean promoter, showed GUS activity in seed embryos. These data, together with the constitutive expression of the endogenous gene in the seedpods, points to additional physiological roles for this protein.

Small heat shock proteins genes are differentially expressed in distinct varieties of common bean

Plants respond to temperature stress by synthesizing a set of heat shock proteins (HSPs), which may be responsible for the acquisition of thermotolerance. In this study, the induction of small HSPs (sHSPs) in eight common bean varieties was evaluated by Northern blot analysis using the W HSP 16.9 cDNA as heterologous probe. Cowpea was used, as a positive control since this plant, as opposed to common bean, is known to grow well under high temperature regimes such as that found in the Brazilian semi-arid region. After the growth period, the plants were submitted to two h of heat shock at 40 oC. All varieties tested were able to induce sHSP mRNAs that hybridized with W HSP 16.9 probe. However, significant kinetic differences were found when comparing different varieties. SHSP mRNA levels induced after heat shock in cowpea was higher than the levels observed on the bean varieties displaying the highest expression of these proteins. Besides, the sHSP expression was also assessed at the protein accumulation level by Western-blot analysis for cowpea and both IPA 7 and Negro Argel varieties of bean plants. The revealed protein pattern confirmed that sHSPs are differentially expressed in distinct varieties of common bean according their heat stress tolerance.

Identification of differentially expressed genes by cDNA‐AFLP technique during heat stress in cowpea nodules

Legume nodules formed by diazotrophic microorganisms are active sites for biological nitrogen fixation (BNF). In tropical regions, a significant part of N supply for soybean, peanut and bean crops is derived from BNF, which is nevertheless often limited by high temperature stress. In contrast, cowpea nodules are very resistant to high temperatures. To understand the molecular bases of thermotolerance during BNF under heat stress, we have used cDNA‐amplified fragment length polymorphism experiments to identify differentially expressed transcripts from cowpea nodules subjected to heat shock treatment. The expression profiles obtained showed approximately 600 bands, 55 up‐regulated and nine corresponding to genes repressed by heat stress. Twenty transcript‐derived fragments were isolated, cloned and sequenced. The Vigna unguiculata nodule and stress response transcripts present similarities to those that encode low molecular weight heat shock proteins, wound‐induced proteins, disease resistance protein, and xylan endohydrolase isoenzyme, as well as different housekeeping genes. The differential expression of 15 genes was confirmed by using Northern blot or reverse Northern hybridization experiments.

Salt-induced antioxidant metabolism defenses in maize (Zea mays L.) seedlings

Abstract Salinity alters general metabolic processes and enzymatic activities, causing increased production of reactive oxygen species (ROS). Expression of antioxidant defense genes would, in turn, be triggered to defend the cell against oxidative damage. We report that salt disturbed antioxidant metabolism in maize seedlings, causing detrimental effects on the growth and development of maize plantlets, increased hydrogen peroxide production and altered antioxidant activities and transcripts profiles. Excessive ROS levels were accompanied by increased catalase (CAT) activity in photosynthesizing shoots, along with induction of mRNA accumulation. Increased accumulation of superoxide dismutase (SOD) transcripts was also observed although no significant changes in total SOD enzymatic activity and isozyme profiles were detected. Higher salt concentrations (above 0.25 M NaCl) were highly detrimental to the plants, causing arrested growth and severe wilting, among other effects. Histochemical detection of H2O2 by 3,3-diaminobenzidine (DAB) staining indicated a collapse of the leaf veins, with hydrogen peroxide leaking to neighboring cells. In agreement to these observations, Sod1, Sod2, Sod4, Sod4A, as well as all Cat transcripts were severely inhibited in plants exposed to high salt concentrations.

Phytocalpains: orthologous calcium-dependent cysteine proteinases

A single calcium-dependent cysteine protease (calpain) gene, essential for aleurone cell development, has been identified recently in maize, although this activity had been described previously in Arabidopsis and maize roots associated with anoxia-induced root-tip death. Calpain genes are ubiquitous in animals and there are up to 12 paralogous genes in humans that exhibit molecular diversity outside of their catalytic domain. Calpain orthologous genes have been identified in 11 plant species. Like their animal counterparts, phytocalpains have significant homology within the catalytic domain, but lack the conserved calcium-binding domain IV, and some members have an N-terminal transmembrane receptor-like domain.

Bean class IV chitinase gene: structure, developmental expression and induction by heat stress

Abstract A P4-chitinase genomic sequence was isolated from a bean (Phaseolus vulgaris) genomic library using a P4-ch cDNA. The complete sequence of the P4-ch gene was determined. Primer extension analysis allowed the identification of the transcriptional start site located 79 bp upstream of the translational initiation codon. The gene is interrupted by an intervening sequence. In the 5′ upstream region, a TATT box occurs in place of a TATA at position −33, while a typical CAAT box is found at position −33 with respect to the transcription initiation site. Gibberellic acid, heat shock and salicylic acid regulatory responsive sequences were also identified. Transient expression of chimeric genes in tobacco protoplasts indicated that all the elements required for expression of the coding sequences are present within the first 600 bp of P4-ch 5′ flanking DNA. Various stress conditions such as wounding, salicylic acid and NaCl treatments, heat and cold stress have been applied to the plants. Whereas wounding, NaCl treatment and cold stress are ineffective, transcription of P4-mRNAs is induced upon salicylic acid treatment and, suprisingly, in response to heat stress. P4-chitinase is induced during germination and seems to be constitutively expressed in roots of mature plants.

Somatic embryo formation in Arabidopsis and eggplant is associated with expression of a glycine-rich protein gene (Atgrp-5)

The isolation of embryogenesis-associated genes and the characterization of their roles during embryo development are important steps towards the elucidation of the molecular mechanisms controlling embryo morphogenesis. Somatic embryogenesis continues to be an effective model for studying gene expression in embryo development. We report the analysis of the transcriptional expression of a glycine-rich gene (Atgrp-5) during somatic embryo morphogenesis. Arabidopsis thaliana transgenic lines carrying chimeric constructs containing the β-glucuronidase (GUS) reporter gene under the control of the Atgrp-5 promoter were used to analyze its expression pattern during somatic embryogenesis. To evaluate whether Atgrp-5 expression observed in Arabidopsis reflects a general pattern during somatic embryogenesis, transgenic eggplant (Solanum melongena L.) was used as non-homologous embryogenic system. High promoter activity was detected in all cells of pro-embryogenic cell clusters and somatic embryos from globular to torpedo stages. During the transition from torpedo to cotyledonar stage the Atgrp-5 gene was gradually turned off and, in mature embryos, its promoter activity was restricted to the protoderm. mRNA in situ hybridization on Arabidopsis somatic embryo cultures have confirmed the expression pattern observed by GUS histochemical assays. These results indicate that Atgrp-5 is expressed in cells that undergo the first anatomical modifications leading to somatic embryo development.