Ascorbate is not often considered as a signalling molecule in plants. This study demonstrates that, in Arabidopsis roots, exogenous l-ascorbic acid triggers a transient increase of the cytosolic free calcium activity ([Ca2+]cyt.) that is central to plant signalling. Exogenous copper and iron stimulate the ascorbate-induced [Ca2+]cyt. elevation, while cation channel blockers, free radical scavengers, low extracellular [Ca2+], transition metal chelators, and removal of the cell wall inhibit this reaction. These data show that apoplastic redox-active transition metals are involved in the ascorbate-induced [Ca2+]cyt. elevation. Exogenous ascorbate also induces a moderate increase in programmed cell death symptoms in intact roots, but it does not activate Ca2+ influx currents in patch-clamped root protoplasts. Intriguingly, the replacement of gluconate with ascorbate in the patch-clamp pipette reveals a large ascorbate efflux current, which shows sensitivity to the anion channel blocker, anthracene-9-carboxylic acid (A9C), indicative of the ascorbate release via anion channels. EPR spectroscopy measurements demonstrate that salinity (NaCl) triggers the accumulation of root apoplastic ascorbyl radicals in an A9C-dependent manner, confirming that l-ascorbate leaks through anion channels under depolarization. This mechanism may underlie ascorbate release, signalling phenomena, apoplastic redox reactions, iron acquisition, and control the ionic and electrical equilibrium (together with K+ efflux via GORK channels).

Helianthus annuus L. is an important oilseed crop, which exhibits moderate salt tolerance and can be cultivated in areas affected by salinity. Using patch-clamp electrophysiology, we have characterized Na+ influx and K+ efflux conductances in protoplasts of salt-tolerant H. annuus L. hybrid KBSH-53 under high salinity. This work demonstrates that the plasma membrane of sunflower root cells has a classic set of ionic conductances dominated by K+ outwardly rectifying channels (KORs) and non-selective cation channels (NSCCs). KORs in sunflower show extreme Na+ sensitivity at high extracellular [Ca2+] that can potentially have a positive adaptive effect under salt stress (decreasing K+ loss). Na+ influx currents in sunflower roots demonstrate voltage-independent activation, lack time-dependent component, and are sensitive to Gd3+. Sunflower Na+-permeable NSCCs mediate a much weaker Na+ influx currents on the background of physiological levels of Ca2+ as compared to other species. This suggests that sunflower NSCCs have greater Ca2+ sensitivity. The responses of Na+ influx to Ca2+ correlates well with protection of sunflower growth by external Ca2+ in seedlings treated with NaCl. It can be, thus, hypothesized that NaCl tolerance in sunflower seedling roots is programmed at the ion channel level via their sensitivity to Ca2+ and Na+.

Functions of exogenous L-ascorbic acid in plant roots are poorly understood. Recent study by Makavitskaya et al. (doi.org/10.1093/jxb/ery056) has demonstrated that exogenous ascorbate can be released from roots in response to salt stress, and can trigger elevation in the cytosolic free Ca. Here, we report that exogenous ascorbate significantly modifies root elongation in Arabidopsis thaliana. Using a medium exchange technique, we have shown that 10-100 µM ascorbate induces small but significant increase in root elongation while higher levels cause its dramatic decrease. Root border cells of Pisum sativum have been losing viability twice faster in the presence of ascorbate that under control conditions, as tested by the confocal microscopy and a combined staining with propidium iodide and fluorescein diacetate.

Environmental stresses induce the release of electrolytes from root cells into the extracellular space, including key inorganic and organic ions. This phenomenon is described for salinity, drought, attack of pathogens, exposure to heavy metals, oxidative stress, etc. The efflux of electrolytes is also observed during some processes of normal physiology, being part of the gravitropic response, the formation of polarity and the growth of the cell elongation. Despite the long study of this phenomenon, today its molecular mechanism remains unclear. In the present study, a combined approach based on the labeled atom method (loading isotope 86Rb+ followed by recording the parameters of its efflux from root cells), reverse genetics and the patch-clamp technique has been developed for testing the biophysical and molecular nature of the efflux of electrolytes from the root cells. It has been demonstrated that in the roots of Arabidоpsis thaliana (L.) Heynh., the K+ efflux during salinisation and oxidative stress is catalysed by K+ channels encoded by the GORK gene (guard cell outward-rectifying K+ channel), and the efflux of organic anions – by anion channels encoded by the ALMT1 gene (aluminum-activated malate transporter 1). The characteristics of the combined functioning of these systems under stress have been established.