Nitric oxide inhibits blue light-induced stomatal opening by regulating the K⁺ influx in guard cells

Abstract

Blue light (BL)-induced stomatal opening and nitric oxide (NO)-promoted stomatal closure comprise two main aspects of stomatal regulation. Stomatal movement depends on ion fluxion in guard cells, whereas the physiological roles of BL or NO in regulating ion channel activities remain largely unknown. For gaining further insights into NO function in mediating BL-induced stomatal opening, guard cell protoplasts (GCPs) were patch-clamped in a whole-cell configuration. The results showed that twice BL pulses (100 μmol m⁻² s⁻¹ for 30 s) effectively activated inward rectifying K⁺ channels by 67% and 20% in Vicia GCPs, respectively. In contrast, Red light (RL) showed little effect on inward rectifying K⁺ channels. In accord with this, BL also increased inward K⁺ currents by 54% in Arabidopsis thaliana wild type gl1, but not in phot1-5 phot2-1 (BL receptor phototropin deletion mutant). Sodium nitroprusside (SNP, a NO donor), at 100 μM, inhibited BL-dependent K⁺ influx and stomatal opening, which were abolished by c-PTIO (a specific NO scavenger). These results indicated that NO inhibits BL-induced stomatal opening maybe through restricting the K⁺ influx across plasma membrane in guard cells.

Introduction

Stomatal pores, each surrounded by a pair of guard cells, play a major role in controlling gaseous exchange, especially photosynthetic CO₂ uptake, and in water release by transpiration [1]. Considerable research progress has suggested that blue light (BL)-induced stomatal opening and stress signals (abscisic acid, ABA)-promoted stomatal closure comprise two main aspects of stomatal regulation [2], [3], [4], [5]. Thereinto, stomatal opening induced by BL have been extensively reported, while much effort has focused on elucidating the primary mechanisms underlying phototropin receptor activation [6], [7], [8], [9], [10], the downstream signaling processes remain largely elusive, except the activation of plasma membrane H⁺-ATPases [11], [12], [13]. Early reports showed that light effectively activated K⁺-influx and efflux channels in the Samanea saman [14], [15] and K⁺-efflux channels in Arabidopsis mesophyll cells [16]. As we know, the transmembrane flux of K⁺ directly regulates cell turgor to control stomatal aperture. However, we have not encountered any reports explicitly showing activation of inward rectifying K⁺ channels by BL in guard cells, although studies claimed that BL activated the plasma membrane H⁺-ATPase [13] and inhibited S-type anion channels in guard cells [17] to hyperpolarize the plasma membrane, which maybe generate an electrical gradient for passive K⁺ flux.

Given the fact that stomatal opening induced by BL occurs in the daytime, which usually leads to severe transpiration, so it is extremely important for plants to prevent excessive loss of water by inhibiting stomatal opening, especially under drought stresses. Recently, Nitric oxide (NO) as a ubiquitous bioactive molecule is involved in ABA-induced stomatal closure or -inhibited stomatal opening in Vicia or Arabidopsis [18], [19], [20]. Therefore, to clarify the function of NO on BL-dependent opening responses of stomata will provide an ideal means of understanding the crosstalk between ABA and BL in regulating stomatal movement. Our previous studies showed that NO functioned in the downstream of hydrogen peroxide (H₂O₂) and was involved in ABA-inhibited BL-dependent stomatal opening by inducing the dephosphorylation of H⁺-ATPase on the plasma membrane in Vicia guard cell protoplasts [21], [22]. In addition, we found that ABA inhibited the inward-rectifying K⁺ channels [26] and BL-induced inward K⁺ currents [24], but no data is available on the sites of NO inhibition and downstream elements. Garcia-Mata et al. demonstrated that NO regulated K⁺ and Cl⁻ channels in guard cells through a subset of ABA-evoked signaling pathways [23]. Whether the inhibition of BL-induced stomatal opening by NO involves the regulation of ion channels in guard cells or not, is still awaited to answer.

Here, we applied patch-clamp techniques to protoplasts isolated from guard cells of Vicia faba and Arabidopsis thaliana and investigated directly an effect of BL and NO on inward and outward rectifying K⁺ channels of plasma membrane in a whole-cell configuration to clarify relationship between BL and NO in signal transduction leading to stomatal movement. Our results showed that BL efficiently promotes influx of K⁺ into cytoplasm through activating inward rectifying K⁺ channels and inhibiting outward rectifying K⁺ channels in the guard cells to support stomatal opening. However, NO significantly inhibits BL-activated inward rectifying K⁺ channels to block K⁺ influx into guard cells, thus inhibiting stomatal opening excessively and preventing the excessive loss of water in plants.