Determination of apoplastic Na+ in intact leaves of cotton by in vivo fluorescence ratio-imaging

Salinity may reduce plant growth via Na+-toxicity symptoms in mature leaves after long-term exposure. It has been suggested by other authors that Na+ accumulates in the leaf apoplast and leads to dehydration of leaves, wilting, and finally to death of these leaves. Two methods were employed to determine the Na+ concentration in the leaf apoplast of salt-tolerant cotton plants under salinity. The ratio imaging of sodium-binding benzofuran isophthalate (SBFI) fluorescence was used to detect in vivo concentration changes and gradients of Na+ within the leaf apoplast under salinity stress, and results were compared with the infiltration-centrifugation method. A significant increase in Na+ concentration was found in the leaf apoplast under salinity (75 mM NaCl), but no further significant increase was determined when NaCl supply was increased from 75 to 150 mM. Both methods revealed that Na+ concentrations remained relatively low, and thus could not be responsible for the decline in yield under salinity. The ratio images showed changes in Na+ concentration and gradients within the leaf apoplast under salt stress, and demonstrated the validity of the method. However, SBFI fluorescence was also influenced by pH, proteins and salt-induced compatible osmolytes.