Maria Teresa Marrè

Effects of orthovanadate on H+ secretion, K+ uptake, electric potential difference and membrane ATPase activities of higher plant tissues.

Abstract Orthovanadate at concentrations between 5 and 20 μM inhibited the K + -stimulated, DCCD-sensitive ATPase activity of membrane preparations (fraction sedimenting between 13 000 and 80 000 g ) from radish seedlings while it had no effect on either the oligomycin-sensitive ATPase activity of mitochondrial preparations, or the phosphorylating activity of isolated mitochondria from radish seedlings or from pea internode segments. In whole germinating radish seeds orthovanadate inhibited the increase in fresh weight, net K + uptake and H + extrusion without inducing any detectable change of respiration nor of ATP and ADP levels, and it induced a rapid, partial depolarisation of the transmembrane electric potential difference. These effects in vivo were interpreted as indicating that orthovanadate inhibits an ATP-driven electrogenic proton pump corresponding to a plasmalemma ATPase.

Effects of calmodulin inhibitors in plants. I. Synergism with fusicoccin in the stimulation of growth and H+ secretion and in the hyperpolarization of the transmembrane electric potential

Abstract The effects of some calmodulin-Ca 2+ complex inhibitors (trifluoperazine (TFP), chlorpromazine (CP) and the cis and trans isomers of flupenthixol) on both basal and fusicoccin(FC)-stimulated growth, proton extrusion and transmembrane electrical potential (PD) in different plant materials have been investigated. TFP and CP significantly promoted growth and acid secretion in the materials investigated. The stimulation of acid secretion under basal conditions was small (pea stems and maize coleoptiles) or not significant (maize roots), while it became important when the inhibitors were fed together with FC at suboptimal concentration (namely, concentrations inducing 20–30% of the maximum activation of electrogenic proton extrusion). A similar behaviour was induced by either of the two cis and trans flupenthixol isomers, which resulted equally active. The effect of the calmodulin inhibitors and that of FC at suboptimal concentrations appeared synergistic. The promoting effect of TFP on H + secretion in maize roots was associated with a marked hyperpolarization of the PD. These results provide a first indication that calmodulin might be in some way involved in the regulation of proton secretion and electrogenesis in higher plants.

A mutant of Arabidopsis thaliana with a reduced response to fusicoccin. I

Because fusicoccin (FC) has the the capacity to promote solute uptake, a selective procedure for isolating mutants of Arabidopsis thaliana with a reduced response to the toxin has been developed. The procedure is based on the incubation of A. thaliana seedlings in a solution containing the cation Paraquat (Pq) at a concentration that per se does not produce bleaching of the leaves upon illumination but does in the presence of FC because of the increased uptake of the toxic cation. Using this procedure, we identified, among the progenies of 2010 M1 ethyl methanesulfonate-mutagenized plants, two mutants that stay green after exposure to FC and Pq. Some properties and inheritance of one of the two mutants (5–2) are described. Morphology of mutant plants is almost indistinguishable from that of the wild type. However, 5–2 seeds germinate and produce viable seedlings in the presence of FC plus the aminoglycoside antibiotic hygromycin B: plants of the mutant do not wilt when exposed to FC and stomata do not open or open only partially. In the presence of FC, the mutant appears less responsive than the wild type as far as the increment in fresh weight, the enlargement of leaf disc area, or the stimulation of H+ extrusion is concerned. Inheritance of the trait is monogenic dominant or semidominant, depending on the test used.

Effects of Ni2+ on proton extrusion and related transport processes and on the transmembrane electrical potential in maize roots

Abstract The presence of Ni 2+ in the incubation medium of maize roots induces an early and significant enhancement of the rate of proton extrusion, an inhibition of K + and leucine uptake and a slight stimulation of 3- O -methylglucose (3- O -MG) and phosphate uptake. The uptake of Ni 2+ is accompanied by a rapid depolarization of the trans-membrane electrical potential (PD). At the 1-mM concentration, both the uptake and the depolarizing effect of Ni 2+ are quantitatively similar to those of K + . These data suggest that the primary effect of Ni 2+ is the depolarization of PD, which would activate H + extrusion; the other transport phenomena would thus be differently influenced by the changes of the electrical and H + gradients. Some probable secondary consequences of Ni 2+ accumulation in the tissue are also discussed.

Ca2+-Counteracted Changes of Membrane Functions Induced by Calmodulin Antagonists in Red Beet Storage Tissue, in Pea and Maize Tissues Treated with Optimal Fusicoccin Concentrations

Summary Previous work shows that the two calmodulin antagonists trifluoperazine (TFP) and chlorpromazine (CP), applied in vivo to isolated segments from Pea stems maize coleoptiles and roots, enhance acid secretion and hyperpolarize the transmembrane electric potential difference (PD) (maize roots) in the absence and, more markedly, in the presence of a fusicoccin (FC) concentration inducing half maximal stimulation of electrogenic H + extrusion. The present data show that, when the two drugs are supplied in the presence of optimal FC concentrations, their stimulating effect on acid secretion disappears and in the roots becomes inhibitory and is associated with a depolarization of the PD. A similar inhibition of acid secretion and depolarization of PD is induced by TFP and CP in red beet root disks. TFP and CP induce an increase of the leakage of ninhydrin-reacting substances in Pea stem and maize root segments and of betacyanine in red beet root disks both in the absence and in the presence of FC. These responses to the calmodulin antagonists are partially counteracted by 0.5 mM CaSO 4 . No correlation is observed between the changes in the leakage of amino acids induced by the drugs and the simultaneous effects of these drugs on acid secretion under the different conditions. These results suggest that the two calmodulin antagonists, when applied with PC at optimal concentration, inhibit electrogenic H + extrusion. The question remains open, however, whether this effect depends on the inactivation of the Ca 2+ -calmodulin complex or on consequent changes of free Ca 2+ concentration or on non-specific modifications of the membrane structure.

Effects of Helminthosporium maydis T toxin on the ATPase activity of maize root membrane preparations and its sensitivity to diethylstilbestrol

Abstract The effects of Helminthosporium maydis race T toxin (HMT) on K + -stimulated and DES-sensitive ATPase activity of membrane preparations from susceptible and resistant maize roots were investigated. HMT slightly but significantly inhibited K + -stimulated ATPase activity of the preparations from the susceptible, but not of those from resistant tissue, thus confirming previously reported data. A much clearer effect of HMT was the protection of ATPase activity against the inhibiting action of DES. This effect appeared much greater for the susceptible than for the resistant preparations and was still evident even on deoxycholate-solubilised ATPase preparations. These data suggest a direct interaction between HMT and an ATPase complex presumably located at the plasmalemma.

The induction of the oxidative burst inElodea densa by sulfhydryl reagents is independent of the inhibiting action of these reagents on photosynthesis

The sulfhydryl group-reagents NEM (N-ethylmaleimide), p-CMBS (parachloromercurybenzenesulfonate), Ag+ and Cu2+ inhibit very rapidly and drastically photosynthetic O2 evolution inElodea densa leaves. This effect has been compared with that of the same reagents in inducing in non illuminated leaves a strong increase of O2 consumption (oxygen burst), presumably due to activation of some plasma membrane redox systems. The results suggest that the structures and mechanisms involved in induction of the oxidative burst on one hand and in the inhibition of photosynthesis on the other are substantially independent.RiassuntoReagenti che modificano i sulfidrili (NEM, p-CMBS, Ag+ e Cu+2) risultano inibire precoeemente e drasticamente la fotosintesi in foglie diElodea densa. Questo effetto è stato confrontato con quello indotto da questi reagenti in foglie non illuminate nell’indurre un forte aumento della respirazione, presumibilmente imputabile all’attivazione di sistemi ossidativi del plasmalemma. I risultati indicano una completa indipendenza nei meccanismi dei due ordini di fenomeni considerati.

Ecologia-Photosynthesis-induced H+ extrusion in aquatic plants. A comparative study

Evidence has been presented showing that, inElodea densa Planchon leaves, the strong activation of electrogenic H+ extrusion by light depends on a photosynthesis-mediated stimulation of the plasmamembrane ATP-driven proton pump. In this work we present the results of a comparative investigation on different aquatic plant species, chosen on the basis of a wide taxonomic distribution, with the purpose to ascertain if the stimulating effect of photosynthesis on proton extrusion is a common mechanism among aquatic plants or if it is related to their phenotypic typology or taxonomic distribution. The data show that, in eight of the twelve tested species, illumination of excised leaves induced a photosynthesis-mediated (DCMU-sensitive) increase of the capacity to acidify the incubation medium, this effect being imputable to the activation of the plasmamembrane H + -ATPase. No correlation was found between the light-induced stimulating effect and the taxonomic position of the plant species. Among the morphological characters considered, only the presence of chloroplasts in the cells of epidermis appeared correlated with the positive response to light.RiassuntoGli autori hanno evidenziato in foglie diElodea densa Planchon la presenza di una pompa elettrogenica estrudente H + ATP-dipendente sita sul plasmalemma fortemente stimolata dalla luce (fotosintesi). Nella presenteNota si è voluto indagare se tale risposta alla luce fosse comune a tutte le piante acquatiche o se fosse correlata a determinate tipologie fenotipiche o alla loro posizione tassonomica. Allo scopo sono state considerate 12 specie spontanee nelle acque dolci italiane; in otto di queste è stata evidenziata una forte capacità di acidificare il mezzo di incubazione in presenza di luce. Non è stata evidenziata correlazione tra le piante ehe hanno mostrato risposta positiva e la loro posizione tassonomica. Tra i caratteri morfologici considerati solo la presenza di cloroplasti nelle cellule epidermiche sembra essere correlata positivamente alla risposta alla luce.

Heterotic behaviour of the transmembrane electric potential difference in some maize hybrid-parents combinations

Abstract In three hybrid-parents combinations the transmembrane (vacuole-exterior) electric potential difference (PD) of cortical cells of maize root is significantly (approx. 15%) higher in the hybrids than in their parental inbreds. To investigate the nature of this PD difference the PD of cortical cells of subapical maize root segments from the inbreds Mo17 and B73 and their hybrid was measured under the following conditions: (1) inhibition of glycolytic and mitochondrial ATP synthesis by AsO43−; (2) inhibition of mitochondrial ATP synthesis and permeabilization of the plasmalemma to H+ by carbonylcyanid-p-trifluorometoxyphenylhydrazon (FCCP); (3) activation of active electrogenic H+ extrusion by fusicoccin. The results show that: (I) The inhibition of the active PD component by either AsO43− or FCCP lowers the PD-values more in the hybrid than in the inbreds, so that after 30–60 min of treatment no difference is observed any more between the three lines. This suggests that the higher PD of the hybrid depends on a higher rate of operation of the active electrogenic mechanism. (II) The treatment with fusicoccin (FC) increases the PD-values of the three maize lines, but more so in the inbreds than in their hybrid. This may suggest that the potentiality of the electrogenic H+ extrusion mechanism is more efficiently expressed in the hybrid.