AtNPF2.5 Modulates Chloride (Cl-) Efflux from Roots of Arabidopsis thaliana.

The accumulation of high concentrations of chloride (Cl-) in leaves can adversely affect plant growth. When comparing different varieties of the same Cl- sensitive plant species those that exclude relatively more Cl- from their shoots tend to perform better under saline conditions; however, the molecular mechanisms involved in maintaining low shoot Cl- remain largely undefined. Recently, it was shown that the NRT1/PTR Family 2.4 protein (NPF2.4) loads Cl- into the root xylem, which affects the accumulation of Cl- in Arabidopsis shoots. Here we characterize NPF2.5, which is the closest homolog to NPF2.4 sharing 83.2% identity at the amino acid level. NPF2.5 is predominantly expressed in root cortical cells and its transcription is induced by salt. Functional characterisation of NPF2.5 via its heterologous expression in yeast (Saccharomyces cerevisiae) and Xenopus laevis oocytes indicated that NPF2.5 is likely to encode a Cl- permeable transporter. Arabidopsis npf2.5 T-DNA knockout mutant plants exhibited a significantly lower Cl- efflux from roots, and a greater Cl- accumulation in shoots compared to salt-treated Col-0 wild-type plants. At the same time, [Formula: see text] content in the shoot remained unaffected. Accumulation of Cl- in the shoot increased following (1) amiRNA-induced knockdown of NPF2.5 transcript abundance in the root, and (2) constitutive over-expression of NPF2.5. We suggest that both these findings are consistent with a role for NPF2.5 in modulating Cl- transport. Based on these results, we propose that NPF2.5 functions as a pathway for Cl- efflux from the root, contributing to exclusion of Cl- from the shoot of Arabidopsis.

The NPR1-dependent salicylic acid signalling pathway is pivotal for enhanced salt and oxidative stress tolerance in Arabidopsis.

The role of endogenous salicylic acid (SA) signalling cascades in plant responses to salt and oxidative stresses is unclear. Arabidopsis SA signalling mutants, namely npr1-5 (non-expresser of pathogenesis related gene1), which lacks NPR1-dependent SA signalling, and nudt7 (nudix hydrolase7), which has both constitutively expressed NPR1-dependent and NPR1-independent SA signalling pathways, were compared with the wild type (Col-0) during salt or oxidative stresses. Growth and viability staining showed that, compared with wild type, the npr1-5 mutant was sensitive to either salt or oxidative stress, whereas the nudt7 mutant was tolerant. Acute salt stress caused the strongest membrane potential depolarization, highest sodium and proton influx, and potassium loss from npr1-5 roots in comparison with the wild type and nudt7 mutant. Though salt stress-induced hydrogen peroxide production was lowest in the npr1-5 mutant, the reactive oxygen species (ROS) stress (induced by 1mM of hydroxyl-radical-generating copper-ascorbate mix, or either 1 or 10mM hydrogen peroxide) caused a higher potassium loss from the roots of the npr1-5 mutant than the wild type and nudt7 mutant. Long-term salt exposure resulted in the highest sodium and the lowest potassium concentration in the shoots of npr1-5 mutant in comparison with the wild type and nudt7 mutant. The above results demonstrate that NPR1-dependent SA signalling is pivotal to (i) controlling Na(+) entry into the root tissue and its subsequent long-distance transport into the shoot, and (ii) preventing a potassium loss through depolarization-activated outward-rectifying potassium and ROS-activated non-selective cation channels. In conclusion, NPR1-dependent SA signalling is central to the salt and oxidative stress tolerance in Arabidopsis.

Salicylic acid improves salinity tolerance in Arabidopsis by restoring membrane potential and preventing salt-induced K+ loss via a GORK channel.

Despite numerous reports implicating salicylic acid (SA) in plant salinity responses, the specific ionic mechanisms of SA-mediated adaptation to salt stress remain elusive. To address this issue, a non-invasive microelectrode ion flux estimation technique was used to study kinetics of NaCl-induced net ion fluxes in Arabidopsis thaliana in response to various SA concentrations and incubation times. NaCl-induced K(+) efflux and H(+) influx from the mature root zone were both significantly decreased in roots pretreated with 10-500 µM SA, with strongest effect being observed in the 10-50 µM SA range. Considering temporal dynamics (0-8-h SA pretreatment), the 1-h pretreatment was most effective in enhancing K(+) retention in the cytosol. The pharmacological, membrane potential, and shoot K(+) and Na(+) accumulation data were all consistent with the model in which the SA pretreatment enhanced activity of H(+)-ATPase, decreased NaCl-induced membrane depolarization, and minimized NaCl-induced K(+) leakage from the cell within the first hour of salt stress. In long-term treatments, SA increased shoot K(+) and decreased shoot Na(+) accumulation. The short-term NaCl-induced K(+) efflux was smallest in the gork1-1 mutant, followed by the rbohD mutant, and was highest in the wild type. Most significantly, the SA pretreatment decreased the NaCl-induced K(+) efflux from rbohD and the wild type to the level of gork1-1, whereas no effect was observed in gork1-1. These data provide the first direct evidence that the SA pretreatment ameliorates salinity stress by counteracting NaCl-induced membrane depolarization and by decreasing K(+) efflux via GORK channels.

Improved measurements of Na+ fluxes in plants using calixarene-based microelectrodes.

Ion-selective microelectrodes are a powerful tool in studying adaptive responses of plant cells and tissues to various abiotic stresses. However, application of this technique in Na(+) flux measurements was limited due to poor selectivity for Na(+) ions of commercially available Na(+) cocktails. Often, these cocktails cannot discriminate between Na(+) and other interfering ions such as K(+) and Ca(2+), leading to inaccurate measurements of Na(+) concentration and, consequently, inaccurate Na(+) flux calculations. To overcome this problem, three Na(+)-selective cocktail mixtures were prepared using tetramethoxyethyl ester derivative of p-t-butyl calix[4]arene. These cocktail mixtures were compared with commercially available ETH 227-based Na(+) cocktail for selectivity for Na(+) ions over other ions (particularly K(+) and Ca(2+)). Among the three calixarene-based Na(+) cocktails tested, cocktail 2 [in % w/w: Na(+) ionophore (4-tert-butylcalix[4]arene-tetra acetic acid tetraethyl ester) 3.5, the plasticizer (2-nitrophenyl octyl ether) 95.9 and lipophilic anion (potassium tetrakis (4-chlorophenyl) borate) 0.6] showed the best selectivity for Na(+) ions over K(+) and Ca(2+) ions and was highly stable over time (up to 10h). Na(+) flux measurements under a wide range of NaCl concentrations (25-150 mM) using Na(+) cocktail 2 established a clear dose-response relationship between severity of salt stress and magnitude of Na(+) influx at the distal elongation and mature zones of Arabidopsis thaliana roots. Furthermore, Na(+) cocktail 2 was compared with commercially available ETH 227-based Na(+) cocktail by measuring Na(+) fluxes at the two Arabidopsis root zones in response to 100mM NaCl treatment. With calixarene-based Na(+) cocktail 2, a large decreasing Na(+) influx (0-15 min) followed by small Na(+) influx (15-45 min) was measured, whereas with ETH-based Na(+) cocktail Na(+) influx was short-lived (1-3 min) and was followed by Na(+) efflux (3-45 min) that might have been due to K(+) and Ca(2+) efflux measured together with Na(+) influx. In conclusion, Na(+)-selective calixarene-based microelectrodes have excellent potential to be used in real-time Na(+) flux measurements in plants.