RESUMO
Many stresses are associated with increased accumulation of reactive oxygen species (ROS) and polyamines (PAs). PAs act as ROS scavengers, but export of putrescine and/or PAs to the apoplast and their catabolization by amine oxidases gives rise to H2O2 and other ROS, including hydroxyl radicals ((â¢)OH). PA catabolization-based signalling in apoplast is implemented in plant development and programmed cell death and in plant responses to a variety of biotic and abiotic stresses. Central to ROS signalling is the induction of Ca(2+) influx across the plasma membrane. Different ion conductances may be activated, depending on ROS, plant species, and tissue. Both H2O2 and (â¢)OH can activate hyperpolarization-activated Ca(2+)-permeable channels. (â¢)OH is also able to activate both outward K(+) current and weakly voltage-dependent conductance (ROSIC), with a variable cation-to-anion selectivity and sensitive to a variety of cation and anion channel blockers. Unexpectedly, PAs potentiated (â¢)OH-induced K(+) efflux in vivo, as well as ROSIC in isolated protoplasts. This synergistic effect is restricted to the mature root zone and is more pronounced in salt-sensitive cultivars compared with salt-tolerant ones. ROS and PAs suppress the activity of some constitutively expressed K(+) and non-selective cation channels. In addition, both (â¢)OH and PAs activate plasma membrane Ca(2+)-ATPase and affect H(+) pumping. Overall, (â¢)OH and PAs may provoke a substantial remodelling of cation and anion conductance at the plasma membrane and affect Ca(2+) signalling.
Assuntos
Membrana Celular/metabolismo , Fenômenos Fisiológicos Vegetais , Poliaminas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transporte de Íons , Potenciais da MembranaRESUMO
Generation of high levels of polyamines and reactive oxygen species (ROS) is common under stress conditions. Our recent study on a salt-sensitive pea species revealed an interaction between natural polyamines and hydroxyl radicals in inducing non-selective conductance and stimulating Ca(2+)-ATPase pumps at the root plasma membrane (I. Zepeda-Jazo, A.M. Velarde-Buendía, R. Enríquez-Figueroa, B. Jayakumar, S. Shabala, J. Muñiz, I. Pottosin, Polyamines interact with hydroxyl radicals in activating Ca2+ and K+ transport across the root epidermal plasma membranes, Plant Phys. 157 (2011) 1-14). In this work, we extended that study to see if interaction between polyamines and ROS may determine the extent of genotypic variation in salinity tolerance. This work was conducted using barley genotypes contrasting in salinity tolerance. Similar to our findings in pea, application of hydroxyl radicals-generating Cu(2+)/ascorbate mixture induced transient Ca(2+) and K(+) fluxes in barley roots. Putrescine and spermine alone induced only transient Ca(2+) efflux and negligible K(+) flux. However, both putrescine and spermine strongly potentiated hydroxyl radicals-induced K(+) efflux and respective non-selective current. This synergistic effect was much more pronounced in a salt-sensitive cultivar Franklin as compared to a salt-tolerant TX9425. As retention of K(+) under salt stress is a key determinant of salinity tolerance in barley, we suggest that the alteration of cytosolic K(+) homeostasis, caused by interaction between polyamines and ROS, may have a substantial contribution to genetic variability in salt sensitivity in this species.
Assuntos
Poliaminas Biogênicas/metabolismo , Cálcio/metabolismo , Hordeum/genética , Potássio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tolerância ao Sal/genética , Estresse Fisiológico/genética , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacologia , Cobre/metabolismo , Cobre/farmacologia , Citosol/metabolismo , Variação Genética , Genótipo , Hordeum/metabolismo , Radical Hidroxila/metabolismo , Radical Hidroxila/farmacologia , Raízes de Plantas , Putrescina/metabolismo , Cloreto de Sódio/metabolismo , Cloreto de Sódio/farmacologia , Espermina/metabolismoRESUMO
The patch-clamp technique was designed to measure any electrogenic transport across the whole cell and organelle (vacuolar) membranes and excised membrane patches. Here, we describe preparation of protoplasts and vacuoles, as well as patch-clamp assays, to detect the functional expression of K(+) and cation channels of plasma membrane and tonoplast, as well as plasma membrane anion channels and vacuolar and plasma membrane H(+) pumps. All of these contribute to the intracellular ionic homeostasis under saline conditions.
Assuntos
Transporte de Íons , Proteínas de Membrana Transportadoras/metabolismo , Técnicas de Patch-Clamp/métodos , Células Vegetais/metabolismo , Cloreto de Sódio , Homeostase , Hordeum/citologia , Hordeum/metabolismo , Canais Iônicos/metabolismo , Íons/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Bombas de Próton/metabolismo , Protoplastos/citologia , Protoplastos/metabolismo , Sementes/crescimento & desenvolvimento , Soluções , Vacúolos/metabolismoRESUMO
Reactive oxygen species (ROS) are integral components of the plant adaptive responses to environment. Importantly, ROS affect the intracellular Ca(2+) dynamics by activating a range of nonselective Ca(2+)-permeable channels in plasma membrane (PM). Using patch-clamp and noninvasive microelectrode ion flux measuring techniques, we have characterized ionic currents and net K(+) and Ca(2+) fluxes induced by hydroxyl radicals (OH(â¢)) in pea (Pisum sativum) roots. OH(â¢), but not hydrogen peroxide, activated a rapid Ca(2+) efflux and a more slowly developing net Ca(2+) influx concurrent with a net K(+) efflux. In isolated protoplasts, OH(â¢) evoked a nonselective current, with a time course and a steady-state magnitude similar to those for a K(+) efflux in intact roots. This current displayed a low ionic selectivity and was permeable to Ca(2+). Active OH(â¢)-induced Ca(2+) efflux in roots was suppressed by the PM Ca(2+) pump inhibitors eosine yellow and erythrosine B. The cation channel blockers gadolinium, nifedipine, and verapamil and the anionic channel blockers 5-nitro-2(3-phenylpropylamino)-benzoate and niflumate inhibited OH(â¢)-induced ionic currents in root protoplasts and K(+) efflux and Ca(2+) influx in roots. Contrary to expectations, polyamines (PAs) did not inhibit the OH(â¢)-induced cation fluxes. The net OH(â¢)-induced Ca(2+) efflux was largely prolonged in the presence of spermine, and all PAs tested (spermine, spermidine, and putrescine) accelerated and augmented the OH(â¢)-induced net K(+) efflux from roots. The latter effect was also observed in patch-clamp experiments on root protoplasts. We conclude that PAs interact with ROS to alter intracellular Ca(2+) homeostasis by modulating both Ca(2+) influx and efflux transport systems at the root cell PM.