Glutathione and Homoglutathione Synthesis in Legume Root Nodules<sup><a href="#FN1" rid="FN1" class=" fn">1</a></sup>
Abstract
High-performance liquid chromatography (HPLC) with fluorescence detection was used to study thiol metabolism in legume nodules. Glutathione (GSH) was the major non-protein thiol in all indeterminate nodules examined, as well as in the determinate nodules of cowpea (Vigna unguiculata), whereas homoglutathione (hGSH) predominated in soybean (Glycine max), bean (Phaseolus vulgaris), and mungbean (Vigna radiata) nodules. All nodules had greater thiol concentrations than the leaves and roots of the same plants because of active thiol synthesis in nodule tissue. The correlation between thiol tripeptides and the activities of glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) in the nodules of eight legumes, and the contrasting thiol contents and activities in alfalfa (Medicago sativa) leaves (98% hGSH, 100% hGSHS) and nodules (72% GSH, 80% GSHS) indicated that the distribution of GSH and hGSH is determined by specific synthetases. Thiol contents and synthesis decreased with both natural and induced nodule senescence, and were also reduced in the senescent zone of indeterminate nodules. Thiols and GSHS were especially abundant in the meristematic and infected zones of pea (Pisum sativum) nodules. Thiols and γ-glutamylcysteinyl synthetase were also more abundant in the infected zone of bean nodules, but hGSHS was predominant in the cortex. Isolation of full-length cDNA sequences coding for γ-glutamylcysteinyl synthetase from legume nodules revealed that they are highly homologous to those from other higher plants.
The tripeptide glutathione (GSH; γGlu-Cys-Gly) is the major non-protein thiol in most animals, plants, and prokaryotes (Meister and Anderson, 1983; Hausladen and Alscher, 1993; Rennenberg, 1997). In plants, GSH is a versatile antioxidant that can directly scavenge activated oxygen species and participate in the ascorbate-GSH cycle for peroxide removal in the chloroplasts. It is also involved in many other vital functions of plants, including the transport and storage of sulfur, the synthesis of proteins and DNA, tolerance to abiotic and biotic stress, and the detoxification of xenobiotics, air pollutants, and heavy metals (Hausladen and Alscher, 1993; Rennenberg, 1997; May et al., 1998).
The pathway for GSH synthesis is probably shared by all organisms and involves two ATP-dependent steps. In the first reaction, γ-glutamylcysteine (γEC) is formed from Glu and Cys by γ-glutamylcysteinyl synthetase (γECS; EC 6.3.2.2), and in the second reaction Gly is added to the C-terminal site of γEC by GSH synthetase (GSHS; EC 6.3.2.3). In plants γECS and GSHS are present in the chloroplasts and cytosol of leaves (Law and Halliwell, 1986; Klapheck et al., 1987; Hell and Bergmann, 1988, 1990). More recently, the two enzymes have also been found in the roots of maize (Rüegsegger and Brunold, 1993) and of the heavy-metal accumulator Brassica juncea (Schäfer et al., 1998).
Legumes are an interesting plant material with which to study thiol metabolism for various reasons. First, there is an active ascorbate-GSH cycle in the root nodules, which requires a continuous supply of GSH to protect nitrogen fixation against toxic oxygen species (Dalton et al., 1986). Second, the leaves, roots, and seeds of some legumes contain a thiol tripeptide homolog, homoglutathione (hGSH; γGlu-Cys-βAla), instead of or in addition to GSH. The synthesis of hGSH is thought to proceed through γECS and a specific hGSH synthetase (hGSHS; Klapheck, 1988; Macnicol, 1987). Third, GSH is believed to be involved in plant morphogenesis, cell division, control of redox status, and signaling of stress and pathogen attack (Wingate et al., 1988; May et al., 1998). All of these processes, with some modifications (Vasse et al., 1990; Hirsch, 1992; Baron and Zambryski, 1995), are important in nodule formation and functioning, and therefore GSH is likely to be a critical molecule of nodules.
There is scant information about thiol compounds of legume nodules. Thiol tripeptides are known to be at high concentrations in nodules (Dalton et al., 1991; Gogorcena et al., 1995, 1997; Escuredo et al., 1996), but this information is based on an enzymatic assay that does not distinguish between GSH and hGSH (Griffith, 1980). Very recently, Evans et al. (1999) reported that hGSH is more abundant than GSH in soybean nodules. However, they employed an HPLC technique based on the formation and UV detection of dinitrophenyl derivatives from the reaction of 1-fluoro-2,4-dinitrobenzene with the amino groups (Farris and Reed, 1987). The technique is slow since it requires overnight derivatization and lacks the necessary sensitivity and specificity to quantify thiols in small nodule samples or dissected nodule fractions. This is especially true for Cys and γEC, which are present in plant tissues at low concentrations and are also essential for the study of thiol metabolism. Evans et al. (1999) also concluded that natural senescence in soybean nodules is an oxidative stress process. They reported, for example, a decrease in thiol content and increases in catalytic iron, thiol oxidation, and oxidative damage. A few years earlier we reached the same conclusions about stress-induced nodule senescence (Gogorcena et al., 1995, 1997; Escuredo et al., 1996).
The latest paper within this extensive study on stress-induced nodule senescence (Matamoros et al., 1999) reported that thiol contents and thiol synthetase activities of nodules could be conveniently assayed using HPLC with fluorescence detection. In the present study, we have improved this methodology and examined in detail thiol metabolism in legume nodules. Our results show that nodules are a main site of GSH and hGSH synthesis within the plant and provide indirect evidence that thiol compounds play a crucial role in the process of nitrogen fixation.
Data are means ± se of three to six samples.
Data are means ± se of three to six samples.
Data are means ± se of four to eight samples. N, Nodules; R, roots; L, leaves.
Data are means ± se of four to six samples.
ACKNOWLEDGMENTS
We are most grateful to Carroll Vance for the gift of cDNA libraries, Gautam Sarath for the synthesis of hGSH, and Frank Minchin for critically reading the manuscript. We also thank Gloria Rodríguez for growing the plants.
Footnotes
This work was supported by the Dirección General de Enseñanza Superior e Investigación Científica (Ministry of Education and Culture, Spain; grant nos. PB98–0522, 2FD97–1101, and HB98–163). M.A.M., J.F.M., I.I.-O., and M.C.R. were the recipients, respectively, of a predoctoral fellowship from the Gobierno Vasco, a postdoctoral contract from the Ministry of Education and Culture, a postdoctoral fellowship from the European Union (Training and Mobility Program), and a predoctoral fellowship from the Ministry of Education and Culture.