Increased Levels of Peroxisomal Active Oxygen-Related Enzymes in Copper-Tolerant Pea Plants

Abstract

The effect in vivo of high nutrient levels of copper (240 micromolar) on the activity of different metalloenzymes conaining Cu, Mn, Fe, and Zn, distributed in chloroplasts, peroxisomes, and mitochondria, was studied in leaves of two varieties ofPisum satiRvum L. plants with different sensitivity to copper. The metalloenzymes studied were: cytochrome c oxidase, Mn-superoxide dismutase (Mn-SOD) and Cu,Zn-superoxide dismutase I (Cu,Zn-SOD I1), for mitochondria; catalase and Mn-SOD, for peroxisomes; and isozyme C,Zn-SOD II for chloroplasts. The activity of mitochondrial SOD isozymes (Mn-SOD and Cu,Zn-SOD I) was very similar in Cu-tolerant and Cu-sensitive plants, whereas cytochrome c oxidase was lower in Cu-sensitive plants. Chloroplastid Cu,Zn-SOD activity was the same in the two plant varieties. In contrast, the peroxi-somal Mn-SOD activity was considerably higher in Cu-tolerant than in Cu-sensitive plants, and the activity of catalase was also increased in peroxisomes of Cu-tolerant plants. The higher activities of these perox-isomal active oxygen-related enzymes in Cu-tolerant plants suggest the involvement of reactive oxygen intermediates (O2, OH-) in the mechanism of Cu lethality, and also imply a function for peroxisomal Mn-SOD in the molecular mechanisms of plant tolerance to Cu in Pisum sativum L. Different molecular mechanisms have been proposed to explain the tolerance of certain higher-plant species to high concentrations of heavy metals which are toxic for most plants. These mechanisms mainly imply modifications of membrane and cell wall properties, compartmentation ofmetals in vacuoles, immobilization of metals in cell walls, and adaptive mechanisms of metabolic and enzymic nature (12, 30). In addition, inducible metallothionein or metallothionein-like complexes have also been postulated to be involved in the mechanism of tolerance of certain plants to copper (12, 18) and cadmium (28). Although the primary toxic action of copper ions on plant growth apparently takes place in roots, by means of alterations in cell membrane properties (30), high levels of this metal can also affect the metabolism of plant leaves. The effect of excess Cu on the photosynthetic apparatus ofspinach plants was studied by Baszyfiski et al. (3), and Lolkema and Vooijs (13) have investigated the effect of high copper concentrations on plasto-cyanin synthesis in Silene cucubalus leaves. Moreover, in vitro experiments on the phytotoxic action ofcopper on the photosyn-thetic electron transport system of isolated chloroplasts have also been conducted (20, 26). 'Supported by grant 603/275 from the CAICYT-CSIC (Spain). 2 Recipient of a research fellowship from the Caja General de Ahorros y Monte de Piedad de Granada and CSIC. However, there is a paucity of information on the activity response of metalloenzymes present in different cell compartments of plant leaves to an enhanced pool of nutrient copper. Comparative studies of the activity of metalloenzymes in different cell organelles of copper tolerant and nontolerant plants grown in high Cu nutrient levels would allow deeper insights into the molecular mechanisms ofintracellular responses to plant toxicity and metal tolerance. These types of studies may also give information on possible alterations of metalloenzyme characteristics in metal-tolerant plants and on the adaptive nature of enzymes to metal stress in certain plants. The induction of a Mn-SOD3 in leaves of pea plants by high nutrient levels of zinc and manganese has been recently demonstrated (7), as well as the induction of Fe-superoxide dismu-tases in lemon leaves by iron (25). This suggested a possible involvement of the metalloenzyme family of SODs in the mechanism of plant tolerance to metal toxicity. SODs (EC 1.15.1.1) are a group of metalloenzymes that catalyze the disproportiona-tion of superoxide free radicals (02O), produced in certain cellular loci, and appear to play an important role in protecting cells against the indirect lethal effect of O2 radicals (17). There are essentially three types of SODs containing either Mn, Fe, or Cu plus Zn as metal prosthetic groups (17). Leaves of pea plants contain three electrophoretically distinct SODs, a Mn-containing and two Cu,Zn-containing isozymes, named I and II in order of increasing mobility (7). The isozyme Mn-SOD has been fully characterized (24), as well as the two pea leaf Cu,Zn-SODs (9). By studies of subcellular distribution of SODs in pea leaves the presence of Mn-SOD has been demonstrated both in mitochondria and peroxisomes (6, 19) whereas isozyme Cu,Zn-SOD II was located in chloroplasts (15), and Cu,Zn-SOD I appeared to be distributed between mitochondria and the cytosol (19). This location of SOD isozymes in different cell compartments makes this metalloenzyme system a valuable tool for probing plant metal tolerance at the subcellular level, and this is particularly interesting in the case of copper considering that high concentrations of this metal have been reported to induce the generation of O-radicals, the substrate of SODs, in isolated chloroplasts (20). In this work, using two varieties of pea plants with different sensitivity to copper, the effect in vivo of high nutrient levels of copper on the activity of metalloenzymes from chloroplasts (Cu,Zn-SOD II), mitochondria (Mn-SOD, Cu,Zn-SOD I, and Cyt c oxidase) and peroxisomes (catalase and Mn-SOD) is studied. 3 Abbreviations: Mn-SOD, manganese-containing superoxide dismu-tase; NBT, nitroblue tetrazolium; SOD, superoxide dismutase; Cu,Zn-SOD, copper-zinc-containing superoxide dismutase. 570