Biochemical Characterization of Plasma Membrane H+-ATPase Activation in Guard Cell Protoplasts of Arabidopsis thaliana in Response to Blue Light

Abstract

Recent genetic analysis showed that phototropins (phot1 and phot2) function as blue light receptors in stomatal opening of Arabidopsis thaliana, but no biochemical evidence was provided for this. We prepared a large quantity of guard cell protoplasts from Arabidopsis. The immunological method indicated that phot1 was present in guard cell protoplasts from the wild-type plant and the phot2 mutant, that phot2 was present in those from the wild-type plant and the phot1 mutant, and that neither phot1 nor phot2 was present in those from the phot1 phot2 double mutant. However, the same amounts of plasma membrane H+-ATPase were found in all of these plants. H+ pumping was induced by blue light in isolated guard cell protoplasts from the wild type, from the single mutants of phototropins (phot1-5 and phot2-1), and from the zeaxanthin-less mutant (npq1-2), but not from the phot1 phot2 double mutant. Moreover, increased ATP hydrolysis and the binding of 14-3-3 protein to the H+-ATPase were found in response to blue light in guard cell protoplasts from the wild type, but not from the phot1 phot2 double mutant. These results indicate that phot1 and phot2 mediate blue light-dependent activation of the plasma membrane H+-ATPase and illustrate that Arabidopsis guard cell protoplasts can be useful for biochemical analysis of stomatal functions. We determined isogenes of the plasma membrane H+-ATPase and found the expression of all isogenes of functional plasma membrane H+-ATPases (AHA1-11) in guard cell protoplasts.

Roles of ion channels and transporters in guard cell signal transduction

“…Compared to wild-type plants or single phot1 or phot2 mutant plants, guard cell protoplasts from phot1 phot2 double mutant plants show severely reduced blue light-induced proton pumping and ATP hydrolysis, and loss of 14-3-3-protein binding, confirming a sequence of events starting at perception of blue light by PHOT1 PHOT2 receptors leading to 14-3-3 binding, H + -ATPase activation, H + extrusion and stomatal opening [3,12]. In addition, the cryptochrome blue light receptors Cry1 and Cry2 have also been proposed to regulate blue light-induced stomatal opening collectively with PHOT1 PHOT2, however their roles in ion transport regulation have not been demonstrated [13].…”

“…In Abbreviations: Ca 2þ cyt , Cytosolic free Ca 2+ concentration; pH cyt , cytosolic pH; GC, guard cell; PM, plasma membrane; ROS, reactive oxygen species; CNGC, cyclic nucleotide gated channel; GLR, glutamate receptor family protein plants, H + -ATPases are encoded by multi-gene families with 11 and 10 genes present in the fully sequenced genomes of Arabidopsis and rice respectively. All 11 of the Arabidopsis genes are expressed in guard cells [3]. These proteins may have redundant functions or they may achieve functional specificity at the signal transduction level (specific pathways, interaction partners, etc.…”

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

“…Guard cell protoplasts were prepared enzymatically from leaves 19,36 . A typical yield of guard cell protoplasts was 4.3 Â 10 7 cells (1,000 mg proteins) per 5,000 leaves 19 .…”

“…BL-and Fc-dependent H þ pumping were determined with a glass pH electrode as described previously 19 . The reaction mixture (0.8 ml) contained 0.125 mM MES-NaOH (pH 6.0), 1 mM CaCl 2 , 0.4 M mannitol, 10 mM KCl and guard cell protoplasts (50-80 mg proteins).…”

“…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).…”