Sergio M. Cocucci

Stimulating effect of fusicoccin on K-activated ATPase in plasmalemma preparations from higher plant tissues

Abstract Fusicoccin (FC), a diterpene glucoside very active in stimulating proton/K + exchange in higher plant tissues, is shown to increase in vitro the K + Mg 2+ -stimulated ATPase activity of plasmalemma-enriched membrane preparations from maize coleoptiles and from spinach leaves. The concentration/activity curve of the effect of FC on ATPase closely parallels that of the in vivo effect on K + uptake. DAK, a derivative of FC inactive in vivo on K + uptake and associated responses, is also inactive on the ATPase activity in vitro. These results are consistent with the hypothesis that the stimulating effects of FC on proton extrusion and cation uptake are due to its capacity to activate at cell membrane level a H + /K + exchange system depending on ATP as an energy source.

Effect of ABA, GA3 and FC on the development of potassium uptake in germinating radish seeds

Abstract Germination of Rhaphanus sativus seeds is accompanied in its early phase by a consistent loss to the medium of the main cations. During the first ten hours there is a general efflux of K + , Na + , Mg 2+ and Ca 2+ , while in the following period K + is actively taken up so that after eighteen hours almost no K + is detected in the external medium. When seed germination is stimulated by fusicoccin (FC) the development of uptake capacity is markedly anticipated. Moreover, inhibition of seed germination by abscisic acid (ABA) inhibits the development of K + uptake. The ABA-induced inhibition is reversed by FC and also, even if to a lesser extent, by gibberellic acid (GA 3 ). Preliminary data suggest that a K + activated ATPase present in membrane preparation is involved in the mechanism of active K + uptake in the seeds.

Cation-activated ATPase activity of plasmalemma-enriched membrane preparations from maize coleoptiles.

The ATPase activity present in plasmalemma-enriched preparations from maize coleoptiles shows an optimum at pH 6, a strong dependence on Mg2+, and is stimulated by K+ and other monovalent cations, both organic and inorganic. The activation of ATPase by K+ obeys Michaelis Menten kinetics, saturation being reached at 50 mM K+ concentration. K+, Mg2+-stimulated ATPase activity is strongly inhibited by N,N′-dicyclohexylcarbodiimide and by diethylstilbestrol and, to a lesser extent, by octylguanidine.

Efflux and active re-absorption of glucose in roots of cotton plants grown under saline conditions.

The effect of growth under saline condition (100 mol m-3 NaCl in the nutrient solution) on the influx and the efflux of glucose from roots of cotton plants was analysed utilising the non metabolisable glucose analogue [14C]-3-O-methylglucose ([14C]3-OMG). Apical segments (1 cm long) excised from cotton roots took up [14C]3-OMG. At each tested concentration (5–500 mmol m-3), the influx was completely inhibited by the presence of the protonophore carbonylcyanide-m-chlorophenyl hydrazone (CCCP) indicating that it is mediated by a H+-coupled co-transport mechanism. The CCCP-sensitive [14C]3-OMG influx was lower in the root segments excised from plants grown on saline solution than in the controls, and this was particularly evident at lower external concentrations. This difference was not due to a lower H+ apoplastic availability. In fact, the saline condition did not affect the pH of the rhizosphere and indeed the H+–ATPase activity, evaluated in plasma membrane vesicles purified from saline-treated plants, was higher (+23%) than in the controls. The lower uptake of [14C]3-OMG into saline treated root segments was related to an enhanced value of the apparent Km of the carrier for the glucose analogue. This effect is discussed in relation to either the more positive value of the transmembrane electric potential difference (ΔΨ) measured in these root segments, or a competitive inhibition of Na+ on the H+ binding site of the carrier. Growth in saline solution slightly affected the efflux of the [14C]3-OMG preloaded in root segments, changing the membrane permeability to the molecule. The results strongly suggest that the higher (2.5 fold) net exudation of glucose, observed in short-term (4 h) collection experiments, from roots of cotton plants grown in saline condition, is mainly due to an effect of the saline growth condition on the system involved in the reabsorption of the hexose rather than on its efflux.

Effects of Ni2+ during the early phases of radish (Raphanus sativus) seed germination

Abstract The effects of Ni 2+ on Raphanus sativus seeds during the first 48 h of germination were investigated. Increasing concentrations of Ni 2+ (0, 80, 100, 200 and 400 μM) progressively inhibited the growth of radish seedlings. In parallel, Ni 2+ affected the restoration of reabsorption of K + and Mg 2+ and the release of Ca 2+ into the incubation medium. The transport mechanisms appeared to be well operative, as indicated by the negative values of the transmembrane electrical potential. Ni 2+ strongly affected the reactivation of oxygen uptake and the increase in energy charge, the mobilisation of sugar and phospho-organic compounds and inhibited the synthesis of DNA, RNA and proteins. The presence of Ni 2+ retarded the degradative and synthetic changes of protein of the soluble and microsomal fractions, occurring in seeds during germination; moreover, in both the soluble and microsomial fractions, two polypeptides from each were not expressed.

Changes in the potato (Solanum tuberosum L.) tuber at the onset of dormancy and during storage at 23 °C and 3 °C. I. Biochemical and physiological parameters

SummaryIn the last 30 d of potato (Solanum tuberosum L.) tuber growth metabolic activity decreased. Levels of glucose-6-P and sucrose in whole tuber tissues declined and in tuber slices there was a decrease in the uptake from the medium and in the incorporation into macromolecules of [U-14C]sucrose. During storage at 23°C only the uptake of [U-14C]sucrose increased concomitant with tuber sprouting, indicating a possible involvement of the transport mechanisms in dormancy breaking. At 3°C, levels of reducing sugars and sucrose increased in response to the low temperature and increased release of K+ and malondialdehyde levels indicated cell membrane damage. The cell membrane functionality was restored at sprouting. The sprouting potential of the tubers was evaluated using the sprouting ability of single-bud explants (“seedcores”) in response to water, GA3 or ABA dips. This sprouting potential of tubers changed with stage of tuber growth and storage duration and temperature, indicating that the tissue hormonal state changed strongly throughout tuber life, probably in relation with the “sink” to “source” transition.

Effects of ophiobolin A on potassium permeability, transmembrane electrical potential and proton extrusion in maize roots

Abstract In maize seedling roots ophiobolin (OPH) A at higher concentration (5 × 10−5 M) promoted a depolarization of the transmembrane electrical potential (PD) and increased the depolarizing effect of K+; at lower concentration (7.5 × 10−6 M) it promoted a hyperpolarization of PD completely counteracted by K+ and still detectable in the presence of the uncoupler (p-trifluoromethoxy)-carbonyl cyanide-phenyl-hydrazone (FCCP). OPH A also increased both the leakage and the efflux of K+; moreover, all the concentrations of OPH A promoted the alkalinization of the medium; in the absence of K+, the capacity to acidify the incubation medium was restored by adding K+ to the medium. These data suggest that the lack of proton extrusion is not linked to a direct action of OPH A on the proton pump, but is linked to an effect of OPH A on the plasma-membrane permeability to K+. The above data are also discussed in relation to the regulation of proton extrusion by a chemical coupling with K+ or by an electrical coupling with PD.

Chromium ions toxicity on the membrane transport mechanism in segments of maize seedling roots

Abstract Chromium ions affect the proton extrusion and the potassium uptake capacity in segments of maize seedling roots. The hypothesis that these effects can explain the chromium ions toxicity in plants is discussed.

Changes in the potato (Solanum tuberosum L.) tuber at the onset of dormancy and during storage at 23°C and 3°C. II. Evaluation of protein patterns

SummaryThe changes in polypeptide profiles (2D-PAGE) occurring in the soluble and microsomal fractions of parenchymatic tissue of potato (Solanum tuberosum L.) tubers were studied during the last 30 d of maturation and during storage at 23°C and 3°C. The major changes were observed in the last period of tuber maturation, when several polypeptides disappeared and new ones appeared. At both 23°C and 3°C specific polypeptides disappeared in dormant tubers and new polypeptides appeared during storage. At 3°C specific changes in protein composition occurred, particularly in the microsomal fraction. The changes in polypeptide profiles are discussed in relation to the transition from “sink” to “source” of the tuber, the onset of dormancy and of sprouting ability and the activation of cold acclimation responses. The results are also discussed on the basis of the physiological and biochemical changes that occur in the parenchymatic tissue.

Changes in some physiological and biochemical parameters during two subcultures in kiwi (Actinidia deliciosa) callus

Abstract Kiwi ( Actinidia deliciosa ) calli were grown for two successive 45-day subcultures on agarised medium in order to evaluate the metabolic changes occurring during the growth of the callus. At different times (10, 35 and 45 days of each subculture), increase in fresh weight, oxygen uptake rate, levels of inorganic cations (K + , Ca 2+ , Mg 2+ and Na + ) and of some metabolites (sucrose, glucose-6-phosphate, malic acid, amino acids and adenosine phosphates) were measured. After an adaptive growth phase (0–6 days) and a phase of high growth (6–12 days), the callus growth rate decreased sharply and then remained essentially constant up to the 45th day. The parameter listed above changed with time following a similar pattern in the two analysed subcultures. In the first 10 days, most of them increased (in particular, oxygen uptake rate and the energetic status of the cells); the levels of Na + decreased. After the first 10 days, the levels of glucose-6-phosphate (glu-6-P) and of the adenylate pool and the availability of the high-energy phosphate bonds of ATP and ADP decreased and the Na + levels began to increase; in this period, the rate of oxygen uptake increased, but this corresponded to neither increased availability of the high-energy phosphate bonds of ATP and ADP nor increased growth rate. In the last period (35–45 days) the main metabolic parameters dropped, with a large increase in Na + levels. Transfer onto new medium restored, after the adaptive period, the maximum growth and the levels of the parameters listed above. The data show that during the subculture periods of Actinidia deliciosa calli important metabolic changes occur. They are probably linked to a different status of suffering (for reduction of nutrient availability, accumulation of catabolic products, and/or variations in oxygen and CO 2 diffusion) with the activation of metabolic mechanisms of adaptation and repair. These metabolic changes appear not severe enough to inhibit the growth of the calli. The results obtained with Actinidia deliciosa calli are discussed in relation to protocols of selection and micropropagation.