Blue light activates the plasma membrane H+-ATPase by phosphorylation of the C-terminus in stomatal guard cells

Abstract

The opening of stomata, which is driven by the accumulation of K ⍣ salt in guard cells, is induced by blue light (BL). The BL activates the H ⍣ pump; however, the mechanism by which the perception of BL is transduced into the pump activation remains unknown. We present evidence that the pump is the plasma membrane H ⍣ -ATPase and that BL activates the H ⍣ -ATPase via phosphorylation. A pulse of BL (30 s, 100 μmol/m 2 /s) increased ATP hydrolysis by the plasma membrane H ϩ -ATPase and H ⍣ pumping in Vicia guard cell protoplasts with a similar time course. The H ⍣ -ATPase was phosphorylated reversibly by BL, and the phosphorylation levels paralleled the ATP hydrolytic activity. The phosphorylation occurred exclusively in the C-termini of H ⍣ -ATPases on both serine and threonine residues in two isoproteins of H ⍣ -ATPase in guard cells. An endogenous 14-3-3 protein was coprecipitated with H ⍣ -ATPase, and the recombinant 14-3-3 protein bound to the phosphorylated C-termini of H ⍣ -ATPases. These findings demonstrate that BL activates the plasma membrane H ⍣ -ATPase via phosphorylation of the C-terminus by a serine/threonine protein kinase, and that the 14-3-3 protein has a key role in the activation.

Phosphorylation of BLUS1 kinase by phototropins is a primary step in stomatal opening

“…4d), with a re-phosphorylation of BLUS1 following a second pulse. The time course of BLUS1 phosphorylation revealed a more rapid response than described for H þ -ATPase phosphorylation 12,26 and coincided with the timing of phot1 autophosphorylation 9,26 . Thus, it appeared conceivable that phototropins directly phosphorylate BLUS1.…”

“…To detect phototropins and BLUS1, the phosphorylation reaction was terminated at 2 min after the start of the BL pulse by adding trichloroacetic acid (TCA) to the guard cell protoplast suspension. For the H þ -ATPase, the phosphorylation was terminated at 3.5 min after the pulse as described previously 12 . Immunoblotting was performed according to Kinoshita and Shimazaki 12 , with slight modifications.…”

“…We generated antibodies against the C-terminal region and phosphorylated Ser-348 of BLUS1 by immunizing rabbits with recombinant GST-BLUS1 (K294-G487) and a synthetic phospho-BLUS1 peptide (KNRRIpSGWNF), respectively. Antibodies against phot1, phot2 and H þ -ATPase were described previously 12,18,19 . Phosphorylation levels of phot1 and the H þ -ATPase were determined by protein blotting using 36).…”

“…However, the mechanisms by which phototropin transduces the light signal into downstream responses are largely unknown. In guard cells, the driving force for stomatal opening is generated by plasma membrane H þ -ATPases that are activated via phototropins [11][12][13] , and it has been established that protein phosphatase 1 (PP1) mediates the signalling between these two molecules 14 . However, the initial signalling event that couples photoreception to activation of the H þ -ATPase has yet to be clarified.…”

Evidence for Involvement of Photosynthetic Processes in the Stomatal Response to CO2

“…The BL response saturates at fluences much lower than photosynthetic saturation (around 50 mmol m 22 s 21 ; Zeiger, 2000). This response has been shown to involve the activation of a plasma membrane H 1 -ATPase (Kinoshita and Shimazaki, 1999), although the steps leading up to this activation are not clear. The receptor for this response has yet to be identified unequivocally, and there is evidence supporting both zeaxanthin Talbott et al, 2003) and phototropins Doi et al, 2004) for this role.…”