RESUMO
A full-length cDNA encoding light-activated chloroplast NADP-malate dehydrogenase (NADP-MDH) (EC 1.1.1.82) from pea (Pisum sativum L.) was introduced in the sense and antisense orientation into tobacco (Nicotiana tabacum L.). Transgenic plants with decreased or increased expression levels were obtained. Because of substantial age-dependent differences in individual leaves of a single plant, standardization of NADP-MDH levels was required first. Then, extent and stability of over- or under-expression of Nmdh, the gene encoding NADP-MDH, was characterized in the various transformants. Frequently, cosuppression effects were observed, indicating sufficient homology between the endogenous tobacco and the heterologous pea gene. Analysis of the T1 and T2 progeny of a series of independent transgenic lines revealed that NADP-MDH capacity ranged between 10% and [greater than or equal to]10-fold compared with the wild type. Under ambient conditions whole-plant development, growth period, and fertility were unaffected by NADP-MDH reduction to 20% of the wild-type level; below this threshold plant growth was retarded. A positive growth effect was registered in young plants with stably enhanced NADP-MDH levels within a defined developmental window.
RESUMO
Three light/dark-modulated chloroplast enzymes, namely NADP-dependent malate dehydrogenase (EC 1.1.1.82), D-fructose 1,6-bisphosphatase (EC 3.1.3.11), and phosphoribulokinase (EC 2.7.1.19) were purified to apparent homogeneity from spinach leaves. Equilibrium constants for the covalent modification of the regulatory disulfide bonds of these enzymes in dithiothreitol (DTT)-redox buffer were determined according to a previously published method in the literature (Clancey and Gilbert (1987) J. Biol. Chem. 262, 13545-13549). The thiol/disulfide-redox potential (Kox) was defined as the ratio of reduced to oxidized dithiothreitol at which 50% of the maximal enzyme activity was observed after equilibrium had been established. All Kox values were very high, comparable to those of extracellular disulfide containing proteins: 0.23 +/- 0.02 for NADP-malate dehydrogenase, 0.59 +/- 0.17 for phosphoribulokinase, and 0.70 +/- 0.16 for D-fructose 1,6-bisphosphatase. The equilibrium constants for the reactions between these enzymes and the redox buffers were also determined in the presence of various concentrations of specific metabolites known to influence the rates of reduction and oxidation. Increasing concentrations of D-fructose 1,6-bisphosphate in the presence of Ca2+ shift the equilibrium constant between D-fructose 1,6-bisphosphatase and the DTT-redox buffer to much lower values. A decreasing NADPH/(NADP + NADPH) ratio increases the Kox of NADP-malate dehydrogenase in the redox buffer to very high values. For PRK, low concentrations of ATP result in a slight decrease of the Kox that is not further affected by higher ATP concentrations. The differences of the equilibrium constants of NADP-malate dehydrogenase and D-fructose 1,6-bisphosphatase as dependent upon the NADPH/(NADP + NADPH) ratio and the concentration of D-fructose 1,6-bisphosphate, respectively, reflect a mechanism of feed-back and feed-forward regulation by the product NADP and the substrate D-fructose 1,6-bisphosphate, respectively. Thus the actual activation state of these two key enzymes of chloroplast metabolism are determined in an independent manner. The relatively small effect of the ATP concentration upon the redox potential of phosphoribulokinase indicates that fine-regulation at this step might be achieved on another level (e.g., catalysis or aggregation state).
