Silicate Inhibits the Cytosolic Influx of Chloride in Protoplasts of Wheat and Affects the Chloride Transporters, TaCLC1 and TaNPF2.4/2.5.

Chloride is an essential nutrient for plants, but high concentrations can be harmful. Silicon ameliorates both abiotic and biotic stresses in plants, but it is unknown if it can prevent cellular increase of chloride. Therefore, we investigated the influx of Cl- ions in two wheat cultivars different in salt sensitivity, by epifluorescence microscopy and a highly Cl--sensitive dye, MQAE, N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide, in absence and presence of potassium silicate, K2SiO3. The Cl--influx was higher in the salt-sensitive cv. Vinjett, than in the salt-tolerant cv. S-24, and silicate pre-treatment of protoplasts inhibited the Cl--influx in both cultivars, but more in the sensitive cv. Vinjett. To investigate if the Cl--transporters TaCLC1 and TaNPF2.4/2.5 are affected by silicate, expression analyses by RT-qPCR were undertaken of TaCLC1 and TaNPF 2.4/2.5 transcripts in the absence and presence of 100 mM NaCl, with and without the presence of K2SiO3. The results show that both transporter genes were expressed in roots and shoots of wheat seedlings, but their expressions were differently affected by silicate. The TaNPF2.4/2.5 expression in leaves was markedly depressed by silicate. These findings demonstrate that less chloride accumulates in the cytosol of leaf mesophyll by Si treatment and increases salt tolerance.

Potassium Efflux and Cytosol Acidification as Primary Anoxia-Induced Events in Wheat and Rice Seedlings.

Both ion fluxes and changes of cytosolic pH take an active part in the signal transduction of different environmental stimuli. Here we studied the anoxia-induced alteration of cytosolic K+ concentration, [K+]cyt, and cytosolic pH, pHcyt, in rice and wheat, plants with different tolerances to hypoxia. The [K+]cyt and pHcyt were measured by fluorescence microscopy in single leaf mesophyll protoplasts loaded with the fluorescent potassium-binding dye PBFI-AM and the pH-sensitive probe BCECF-AM, respectively. Anoxic treatment caused an efflux of K+ from protoplasts of both plants after a lag-period of 300-450 s. The [K+]cyt decrease was blocked by tetraethylammonium (1 mM, 30 min pre-treatment) suggesting the involvement of plasma membrane voltage-gated K+ channels. The protoplasts of rice (a hypoxia-tolerant plant) reacted upon anoxia with a higher amplitude of the [K+]cyt drop. There was a simultaneous anoxia-dependent cytosolic acidification of protoplasts of both plants. The decrease of pHcyt was slower in wheat (a hypoxia-sensitive plant) while in rice protoplasts it was rapid and partially reversible. Ion fluxes between the roots of intact seedlings and nutrient solutions were monitored by ion-selective electrodes and revealed significant anoxia-induced acidification and potassium leakage that were inhibited by tetraethylammonium. The K+ efflux from rice was more distinct and reversible upon reoxygenation when compared with wheat seedlings.

Indoleacetic Acid Levels in Wheat and Rice Seedlings under Oxygen Deficiency and Subsequent Reoxygenation.

The lack of oxygen and post-anoxic reactions cause significant alterations of plant growth and metabolism. Plant hormones are active participants in these alterations. This study focuses on auxin-a phytohormone with a wide spectrum of effects on plant growth and stress tolerance. The indoleacetic acid (IAA) content in plants was measured by ELISA. The obtained data revealed anoxia-induced accumulation of IAA in wheat and rice seedlings related to their tolerance of oxygen deprivation. The highest IAA accumulation was detected in rice roots. Subsequent reoxygenation was accompanied with a fast auxin reduction to the control level. A major difference was reported for shoots: wheat seedlings contained less than one-third of normoxic level of auxin during post-anoxia, while IAA level in rice seedlings rapidly recovered to normoxic level. It is likely that the mechanisms of auxin dynamics resulted from oxygen-induced shift in auxin degradation and transport. Exogenous IAA treatment enhanced plant survival under anoxia by decreased electrolyte leakage, production of hydrogen peroxide and lipid peroxidation. The positive effect of external IAA application coincided with improvement of tolerance to oxygen deprivation in the 35S:iaaM × 35S:iaaH lines of transgene tobacco due to its IAA overproduction.

Anoxia-induced elevation of cytosolic Ca2+ concentration depends on different Ca2+ sources in rice and wheat protoplasts.

The anoxia-dependent elevation of cytosolic Ca(2+) concentration, [Ca(2+)](cyt), was investigated in plants differing in tolerance to hypoxia. The [Ca(2+)](cyt) was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca(2+)](cyt) in rice leaf protoplasts. A tenfold drop in the external Ca(2+) concentration (to 0.1 mM) resulted in considerable decrease of the [Ca(2+)](cyt) shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La(3+) and EGTA) and intracellular membrane Ca(2+)-channel antagonists (Li(+), ruthenium red and cyclosporine A) reduced the anoxic Ca(2+)-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca(2+)](cyt). In wheat leaf protoplasts, the amplitude of the Ca(2+)-shift little depended on the external level of Ca(2+). Wheat root protoplasts were characterized by a small shift of [Ca(2+)](cyt) under anoxia. Plasmalemma Ca(2+)-channel blockers had little effect on the elevation of cytosolic Ca(2+) in wheat protoplasts. Intact rice seedlings absorbed Ca(2+) from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca(2+). Verapamil abolished the Ca(2+) influx in rice roots and Ca(2+) efflux from wheat roots. Anoxia-induced [Ca(2+)](cyt) elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca(2+) stores are important for anoxic [Ca(2+)](cyt) elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca(2+)](cyt) rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.