Oxidative Damage in Pea Plants Exposed to Water Deficit or Paraquat1

The application of a moderate water deficit (water potential of −1.3 MPa) to pea (Pisum sativum L. cv Lincoln) leaves led to a 75% inhibition of photosynthesis and to increases in zeaxanthin, malondialdehyde, oxidized proteins, and mitochondrial, cytosolic, and chloroplastic superoxide dismutase activities. Severe water deficit (−1.9 MPa) almost completely inhibited photosynthesis, decreased chlorophylls, β-carotene, neoxanthin, and lutein, and caused further conversion of violaxanthin to zeaxanthin, suggesting damage to the photosynthetic apparatus. There were consistent decreases in antioxidants and pyridine nucleotides, and accumulation of catalytic Fe, malondialdehyde, and oxidized proteins. Paraquat (PQ) treatment led to similar major decreases in photosynthesis, water content, proteins, and most antioxidants, and induced the accumulation of zeaxanthin and damaged proteins. PQ decreased markedly ascorbate, NADPH, ascorbate peroxidase, and chloroplastic Fe-superoxide dismutase activity, and caused major increases in oxidized glutathione, NAD+, NADH, and catalytic Fe. It is concluded that, in cv Lincoln, the increase in catalytic Fe and the lowering of antioxidant protection may be involved in the oxidative damage caused by severe water deficit and PQ, but not necessarily in the incipient stress induced by moderate water deficit. Results also indicate that the tolerance to water deficit in terms of oxidative damage largely depends on the legume cultivar.

Hydroperoxide lyase depletion in transgenic potato plants leads to an increase in aphid performance

Hydroperoxide lyases (HPLs) catalyze the cleavage of fatty acid hydroperoxides to aldehydes and oxoacids. These volatile aldehydes play a major role in forming the aroma of many plant fruits and flowers. In addition, they have antimicrobial activity in vitro and thus are thought to be involved in the plant defense response against pest and pathogen attack. An HPL activity present in potato leaves has been characterized and shown to cleave specifically 13-hydroperoxides of both linoleic and linolenic acids to yield hexanal and 3-hexenal, respectively, and 12-oxo-dodecenoic acid. A cDNA encoding this HPL has been isolated and used to monitor gene expression in healthy and mechanically damaged potato plants. HPL gene expression is subject to developmental control, being high in young leaves and attenuated in older ones, and it is induced weakly by wounding. HPL enzymatic activity, nevertheless, remains constant in leaves of different ages and also after wounding, suggesting that posttranscriptional mechanisms may regulate its activity levels. Antisense-mediated HPL depletion in transgenic potato plants has identified this enzyme as a major route of 13-fatty acid hydroperoxide degradation in the leaves. Although these transgenic plants have highly reduced levels of both hexanal and 3-hexenal, they show no phenotypic differences compared with wild-type ones, particularly in regard to the expression of wound-induced genes. However, aphids feeding on the HPL-depleted plants display approximately a two-fold increase in fecundity above those feeding on nontransformed plants, consistent with the hypothesis that HPL-derived products have a negative impact on aphid performance. Thus, HPL-catalyzed production of C6 aldehydes may be a key step of a built-in resistance mechanism of plants against some sucking insect pests.

Pea formaldehyde-active class III alcohol dehydrogenase: common derivation of the plant and animal forms but not of the corresponding ethanol-active forms (classes I and P).

A plant class III alcohol dehydrogenase (or glutathione-dependent formaldehyde dehydrogenase) has been characterized. The enzyme is a typical class III member with enzymatic parameters and substrate specificity closely related to those of already established animal forms. Km values with the pea enzyme are 6.5 microM for NAD+, 2 microM for S-hydroxymethylglutathione, and 840 microM for octanol versus 9, 4, and 1200 microM, respectively, with the human enzyme. Structurally, the pea/human class III enzymes are closely related, exhibiting a residue identity of 69% and with only 3 of 23 residues differing among those often considered in substrate and coenzyme binding. In contrast, the corresponding ethanol-active enzymes, the long-known human liver and pea alcohol dehydrogenases, differ more (47% residue identities) and are also in functionally important active site segments, with 12 of the 23 positions exchanged, including no less than 7 at the usually much conserved coenzyme-binding segment. These differences affect functionally important residues that are often class-distinguishing, such as those at positions 48, 51, and 115, where the plant ethanol-active forms resemble class III (Thr, Tyr, and Arg, respectively) rather than the animal ethanol-active class I forms (typically Ser, His, and Asp, respectively). Calculations of phylogenetic trees support the conclusions from functional residues in subgrouping plant ethanol-active dehydrogenases and the animal ethanol-active enzymes (class I) as separate descendants from the class III line. It appears that the classical plant alcohol dehydrogenases (now called class P) have a duplicatory origin separate from that of the animal class I enzymes and therefore a paralogous relationship with functional convergence of their alcohol substrate specificity. Combined, the results establish the conserved nature of class III also in plants, and contribute to the molecular and functional understanding of alcohol dehydrogenases by defining two branches of plant enzymes into the system.

Location of light-repressible, small GTP-binding protein of the YPT/rab family in the growing zone of etiolated pea stems.

YPT/rab proteins are ras-like small GTP-binding proteins that serve as key regulators of vesicular transport. The mRNA levels of two YPT/rab genes in pea plants are repressed by light, with the process mediated by phytochrome. Here, we examined the mRNA expression and the location of the two proteins, pra2- and pra3-encoded proteins, using monoclonal antibodies. The pra2 and pra3 mRNA levels were highest in the stems of dark-grown seedlings. The corresponding proteins were found in the cytosol and the membranes of the stems. Most of the pra2 protein was in the growing internodes, especially in the growing region, but the pra3 protein was widespread. These results suggest that the pra2 protein is important for vesicular transport in stems, possibly contributing to stem growth in the dark, and that the pra3 protein is important for general vesicular transport. The amounts of pra2 and pra3 proteins decreased with illumination. The decrease in these proteins may be related to the phytochrome-dependent inhibition of stem growth that occurs in etiolated pea seedlings.