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23 Pags.- 2 Tabls. The definitive version, with Figs., is available at: http://link.springer.com/journal/425, Pea (Pisum sativum L. cv. Frilene) plants subjected to drought (leaf water potential of ≈-1.3 MPa) showed major reductions in photosynthesis (78‰), transpiration (83‰), and glycolate oxidase (EC 1.1.3.1) activity (44‰), and minor reductions (≈18‰) in the contents of chlorophyll a, carotenoids, and soluble protein. Water stress also led to pronounced decreases (72–85‰) in the activities of catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2), but resulted in the increase (32–42‰) of non-specific peroxidase (EC 1.11.1.7) and superoxide dismutase (EC 1.15.1.1). Ascorbate peroxidase (EC 1.11.1.11) and monodehydroascorbate reductase (EC 1.6.5.4) activities decreased only by 15‰ and the two enzymes acted in a cyclic manner to remove H2O2, which did not accumulate in stressed leaves. Drought had no effect on the levels of ascorbate and oxidized glutathione in leaves, but caused a 25‰ decrease in the content of reduced glutathione and a 67‰ increase in that of vitamin E. In leaves, average concentrations of catalytic Fe, i.e. Fe capable of catalyzing free-radical generation by redox cycling, were estimated as 0.7 to 7 μM (well-watered plants, depending on age) and 16 μM (water-stressed plants); those of catalytic Cu were ≈4.5 μM and 18 μM, respectively. Oxidation of lipids and proteins from leaves was enhanced two- to threefold under stress conditions and both processes were highly correlated. Fenton systems composed of the purported concentrations of ascorbate, H2O2, and catalytic metal ions in leaves produced hydroxyl radicals, peroxidized membrane lipids, and oxidized leaf proteins. It is proposed that augmented levels and decompartmentation of catalytic metals occurring during water stress are responsible for the oxidative damage observed in vivo., J.F.M., I. I., and S.F. were the recipients of predoctoral fellowships from the Comunidades Autónomas de Aragón, País Vasco, and Navarra, respectively. R.V.K. thanks the U.S. Department of Agriculture (grant 91-37305-6705) for travel support. This work was financed by grants from the Comisión Interministerial de Ciencia y Tecnología (AGR-91-0857-C02 to P.A. and M.B.) and Dirección General de Investigación Científica y Técnica (PB92-0058 to M.B).

Although nonsymbiotic hemoglobins (Hbs) are found in different tissues of dicots and monocots, very little is known about hb genes in monocots and the function of Hbs in nonsymbiotic tissues. We report the cloning and analysis of two rice (Oryza sativa L.) hb genes, hb1 and hb2, that code for plant Hbs. Rice hb1 and hb2 genes contain four exons and three introns, as with all of the known plant hb genes. At least three copies of the hb gene were detected in rice DNA, and analysis of gene expression shows that hb1 and hb2 are expressed in leaves but only hb1 is expressed in roots. A cDNA for rice Hb1 was expressed in Escherichia coli, and the recombinant Hb (rHb1) shows an unusually high affinity for O2 because of a very low dissociation constant. The absorbance spectra of the ferric and deoxyferrous rHb1 indicate that, in contrast to symbiotic Hbs, a distal ligand is coordinated to the ligand-binding site. Mutation of the distal His demonstrates that this residue coordinates the heme Fe of ferric and deoxyferrous rHb1 and stabilizes O2 in oxy-rHb1. The biochemical properties of rice rHb1 suggest that this protein probably does not function to facilitate the diffusion of O2.