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Section 8: Stresses and Factors Limiting Nitrogen Fixation


I. Iturbe-Ormaetxe, M.A. Matamoros, J.F. Moran, J. Ramos, M.C. Rubio, M.R. Clemente, B. Heras, M. Becana

Estación Experimental de Aula Dei, CSIC, Apdo. 202, 50080 Zaragoza, Spain

1. Introduction

Legume nodules contain an impressive array of antioxidants to cope with reactive oxygen species (ROS), such as the superoxide radical and H2O2, that are generated in different subcellular compartments. ROS are involved in all stages of legume nodule development, from initiation to senescence (Becana et al. 2000; Santos et al. 2001). In the nodule cytosol, ROS are mainly formed in oxidative reactions involving leghemoglobin and are scavenged through the concerted action of CuZn-superoxide dismutase (SOD) and of the four enzymes of the Halliwell-Asada pathway (Dalton 1995). In this pathway, ascorbate peroxidase (APX) catalyzes the reduction of H2O2 to water by ascorbate, and the resulting monodehydroascorbate and dehydroascorbate are reduced back to ascorbate, respectively, by monodehydroascorbate reductase (MR) at the expense of NADH and by dehydroascorbate reductase (DR) plus glutathione reductase (GR) at the expense of NADPH. A key metabolite of the pathway for the detoxification of ROS in nodules is glutathione (GSH; yGlu-Cys-Gly), which is synthesized in both the bacteroids and plant fraction by two ATP-dependent enzymes, y-glutamylcysteine synthetase and glutathione synthetase (GSHS), acting sequentially (Moran et al. 2000). However, some legume species and tissues can synthesize another thiol tripeptide, homoglutathione (hGSH; yGlu-Cys-pAla), in addition to or instead of GSH. It is generally assumed that hGSH is synthesized by a specific hGSH synthetase (hGSHS) and that it performs similar roles to GSH (Klapheck 1988; Matamoros et al. 1999).

The mitochondria are also a main site for generation of ROS in the nodules because of the abundance of these organelles in the cortex and infected region (Millar et al. 1995), their high rates of respiration required for active N2 fixation (Dalton 1995; Millar et al. 1995), and their high content of heme and nonheme Fe, which can catalyze the formation of hydroxyl radicals from H2O2 (Becana et al. 1998). However, very little is known about the antioxidant composition of nodule mitochondria.

In this work we present data on three aspects on the antioxidant protection of nodules. First, we report on changes in transcript abundance for antioxidant enzymes during nodule senescence. Second, we have overexpressed in an heterologous system and characterized for the first time a functional hGSHS from nodules. Third, we analyze the antioxidant composition of nodule mitochondria and present a model for peroxide detoxification in these organelles.

2. Materials and Methods

2.1. Northern analysis. Total RNA was extracted from pea nodules by a phenol-LiCl procedure (Verwoerd et al. 1989), separated on agarose denaturing (formaldehyde) gels, and capillary transferred to Hybond-N+ nylon filters (Amersham). Blot hybridization with DNA probes prepared by random-primed 32P-labeling and autoradiography were performed following standard protocols (Sambrook et al. 1989).

2.2. Overexpression, purification, and characterization of recombinant GSHS2. The open reading frame of GSHS2 was PCR-amplified using gene-specific primers (Moran et al. 2000). The resulting 1.7-kb fragment was gel purified, subcloned into pCRII TOPO (Invitrogen), and transformed into DH5a competent cells. The inserted open reading frame of GSHS2 was digested out with Ncol and

Notl, gel purified, and ligated into pFastBac HTb. Positive colonies of transformed DH5a cells were identified by PCR using pFastBac specific primers. Recombinant bacmid DNA was grown overnight in E. coli, isolated and used to transfect Sf21 cells. Baculoviruses were harvested 72 h post-transfection and amplified by infecting monolayer cultures of insect cells. After optimizing infection conditions, protein production was scaled up by culturing insect cells at 27°C in 50 ml of TC-100 medium (Sigma) supplemented with 10% fetal calf serum (Sigma) and antibiotics. Cells were collected 48 h after virus infection and lysed by osmotic shock. Cell free extracts were loaded onto a cobalt affinity column (Clontech). Fractions were eluted with imidazole and subjected to Western analysis and protein staining to confirm the presence and purity of GSHS2.

2.3. Assay of antioxidant enzymes and metabolites. GPX activity was assayed with pyrogallol as substrate, including 0.5 mM p-chloromercuriphenylsulfonic acid (pCMS) in the reaction mixture (Amako et al. 1994). All the other enzyme activities were determined as described earlier (Gogorcena et al. 1997). Thiol tripeptides were quantified by HPLC with fluorescence detection (Matamoros et al. 1999).

2.4. Localization of enzymes in nodule mitochondria. Mitochondria purification was performed using modifications of published protocols and was monitored using organelle protein markers (Sandalio et al. 1987; Struglics et al. 1996). Solubilization and latency studies were carried out essentially as described by Jiménez et al. (1997).

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