De-regulated expression of the plant glutamate receptor homolog AtGLR3.1 impairs long-term Ca²⁺-programmed stomatal closure

Cytosolic Ca²⁺ ([Ca²⁺]_cyt) mediates diverse cellular responses in both animal and plant cells in response to various stimuli. Calcium oscillation amplitude and frequency control gene expression. In stomatal guard cells, [Ca²⁺]_cyt has been shown to regulate stomatal movements, and a defined window of Ca²⁺ oscillation kinetic parameters encodes necessary information for long-term stomatal movements. However, it remains unknown how the encrypted information in the cytosolic Ca²⁺ signature is decoded to maintain stomatal closure. Here we report that the Arabidopsis glutamate receptor homolog AtGLR3.1 is preferentially expressed in guard cells compared to mesophyll cells. Furthermore, over-expression of AtGLR3.1 using a viral promoter resulted in impaired external Ca²⁺-induced stomatal closure. Cytosolic Ca ²⁺ activation of S-type anion channels, which play a central role in Ca²⁺-reactive stomatal closure, was normal in the AtGLR3.1 over-expressing plants. Interestingly, AtGLR3.1 over-expression did not affect Ca²⁺-induced Ca²⁺ oscillation kinetics, but resulted in a failure to maintain long-term 'Ca²⁺-programmed' stomatal closure when Ca²⁺ oscillations containing information for maintaining stomatal closure were imposed. By contrast, prompt short-term Ca²⁺-reactive closure was not affected in AtGLR3.1 over-expressing plants. In wild-type plants, the translational inhibitor cyclohexamide partially inhibited Ca ²⁺-programmed stomatal closure induced by experimentally imposed Ca²⁺ oscillations without affecting short-term Ca²⁺- reactive closure, mimicking the guard cell behavior of the AtGLR3.1 over-expressing plants. Our results suggest that over-expression of AtGLR3.1 impairs Ca²⁺ oscillation-regulated stomatal movements, and that de novo protein synthesis contributes to the maintenance of long-term Ca ²⁺-programmed stomatal closure.