Low Levels of Nitric Oxide and Reactive Oxygen Species in Guard Cells Are Required for Stomatal Opening by Fusicoccin or Butyrate.

Stomata play a crucial role in controlling the rate of photosynthesis and transpiration. Both stomatal opening and closure depend on intricate mechanisms involving several signaling components. The rise in nitric oxide (NO), reactive oxygen species (ROS), and cytosolic pH is necessary for inducing stomatal closure. However, the role of NO and ROS during stomatal opening has not been critically studied. Fusicoccin (FC) and butyric acid (BA) are known to induce guard cell cytosolic acidification and stomatal opening. We conducted a comprehensive study on NO and ROS patterns during stomatal opening induced by FC or BA. Both FC and BA suppressed NO and ROS levels of the guard cells as indicated by specific fluorescent dyes. The external addition of GSNO (natural NO-generator) or H₂O₂ (source of ROS) significantly suppressed FC- or BA-induced stomatal opening, confirming the requirement of low NO and ROS levels for stomatal opening. In addition, FC and BA lowered the guard cell pH as indicated by the fluorescent indicator, BCECF-AM. The ability of vanadate (PM-ATPase inhibitor) to restrict FC- or BA-induced opening suggested the importance of PM-ATPase-mediated cytosolic acidification, followed by suppression of NO and ROS levels in guard cells during stomatal opening. Further, RT-PCR analysis confirmed the upregulation of PM-ATPase by FC or BA. We propose that the guard cell acidification by FC or BA, due to PM-ATPase, caused the suppression of NO and ROS levels in guard cells and facilitated stomatal opening.