Abstract:

Soil salinity is a key abiotic stress in crop production worldwide, especially in countries where irrigation is an essential aid to agriculture. Salt stress disturbs intracellular ion homeostasis of plants, which leads to membrane dysfunction, attenuation of metabolic activity, and secondary effects causing growth inhibition and ultimately death. Salt tolerance in plants is the ultimate manifestation of several physiologic processes, including ion uptake and membrane flux, ionic balance and distribution. Previous researches have shown that K⁺ inward rectifying channels, outward rectifying channels, high affinity K⁺ transporter (HKT) and low affinity cation transporter (LCT) may facilitate Na⁺ influx under NaCl stress. Extracellular free Ca²⁺ concentration ([Ca²⁺]ext) enhances salt tolerance and salinity stress elicits a transient increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]cyt). The transient increase in [Ca2+]cyt activates the PP2B phosphatase calcineurin leading to the transcription of ENA1, which encodes the P-type ATPase that is primarily responsible for Na⁺ efflux across the plasma membrane or activates the SOS signal pathway which negatively controls this Na⁺ influx system. At the plasma membrane, high cytosolic Ca²⁺ levels can cause activation of anion channels and reduce the conductivity of inward K⁺ channels , thus reducing water loss. To better understand Ca²⁺ function in K⁺ and Na⁺ uptake, here, we investigated the effect of calcium on stomatal movement and K+ channels of Vicia faba under NaCl stress. The results showed that 100 mmol L^-1 NaCl significantly induced stomatal opening, CaCl₂ facilitated NaCl-induced stomatal opening at concentration of 0.1 mmol L^-1 and significantly inhibited NaCl-induced stomatal opening at concentration of 10 mmol L^-1. To gain further insights into Ca²⁺ function in NaCl-regulated stomatal movement, Vicia faba guard cell protoplasts were patch-clamped in a whole-cell configuration and the results showed that Ca²⁺ significantly inhibited inward rectifying and outward rectifying K⁺ current when NaCl and CaCl₂ were added to the bath solution together, at concentration of 100 mmol L^-1 and 10 mmol L^-1 respectively, which was alleviated by LaCl₃ at concentration of 1 mmol L^-1. In contrast, 10 mmol L^-1 CaCl₂ alone significantly inhibited inward rectifying K⁺ current and activated outward rectifying K⁺ current, which was not alleviated by LaCl₃. A single-cell analysis of cytosolic H₂O₂ using 2’,7’-dichlorofluorescin (H2DCF-DA) revealed that Ca²⁺ can induce the generating of H₂O₂ in the guard cells with 10 mmol L^-1 CaCl₂ under 100 mmol L^-1 NaCl stress, but 10 mmol L^-1 CaCl₂ or 100 mmol L^-1 NaCl had little effect on the accumulation of H₂O₂ in the guard cells, respectively. These results suggest that calcium may alleviate the damage caused by NaCl stress through inducing the production of H₂O₂，in turn the H₂O₂-activated increases in [Ca²⁺]cyt in guard cells decrease Na+ uptake by the regulation of plasma membrane K⁺ channels in guard cells leading to stomatal closure and reduction of water loss. These findings open new perspectives about Ca²⁺-based signaling in responsive to salt stress in plants.

Key words: Salt stress, Calcium, Hydrogen peroxide, Guard cell, Plasma membrane K⁺ channels