Toshinori Kinoshita

phot1 and phot2 mediate blue light regulation of stomatal opening

The stomatal pores of higher plants allow for gaseous exchange into and out of leaves. Situated in the epidermis, they are surrounded by a pair of guard cells which control their opening in response to many environmental stimuli, including blue light. Opening of the pores is mediated by K+ accumulation in guard cells through a K+ channel and driven by an inside-negative electrical potential. Blue light causes phosphorylation and activation of the plasma membrane H+-ATPase that creates this potential. Thus far, no blue light receptor mediating stomatal opening has been identified, although the carotenoid, zeaxanthin, has been proposed. Arabidopsis mutants deficient in specific blue-light-mediated responses have identified four blue light receptors, cryptochrome 1 (cry1), cryptochrome 2 (cry2), phot1 and phot2. Here we show that in a double mutant of phot1 and phot2 stomata do not respond to blue light although single mutants are phenotypically normal. These results demonstrate that phot1 and phot2 act redundantly as blue light receptors mediating stomatal opening.

Binding of brassinosteroids to the extracellular domain of plant receptor kinase BRI1

Both animals and plants use steroids as signalling molecules during growth and development. Animal steroids are principally recognized by members of the nuclear receptor superfamily of transcription factors. In plants, BRI1, a leucine-rich repeat (LRR) receptor kinase localized to the plasma membrane, is a critical component of a receptor complex for brassinosteroids. Here, we present the first evidence for direct binding of active brassinosteroids to BRI1 using a biotin-tagged photoaffinity castasterone (BPCS), a biosynthetic precursor of brassinolide (the most active of the brassinosteroids). Binding studies using BPCS, 3H-labelled brassinolide and recombinant BRI1 fragments show that the minimal binding domain for brassinosteroids consists of a 70-amino acid island domain (ID) located between LRR21 and LRR22 in the extracellular domain of BRI1, together with the carboxy-terminal flanking LRR (ID-LRR22). Our results demonstrate that brassinosteroids bind directly to the 94 amino acids comprising ID-LRR22 in the extracellular domain of BRI1, and define a new binding domain for steroid hormones.

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

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/m2/s) increased ATP hydrolysis by the plasma membrane H+‐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 co‐precipitated 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.

Cytosolic Concentration of Ca2+ Regulates the Plasma Membrane H+-ATPase in Guard Cells of Fava Bean

Opening of the stomata is driven by the light-activated plasma membrane proton pumping ATPase, although the activation and inactivation mechanism of the enzyme is not known. In this study, we show that the H+-ATPase in guard cells is reversibly inhibited by Ca2+ at physiological concentrations. Isolated microsomal membranes of guard cell protoplasts from fava bean exhibited vanadate-sensitive, ATP-dependent proton pumping. The activity was inhibited almost completely by 1 [mu]M Ca2+ with a half-inhibitory concentration at 0.3 [mu]M and was restored immediately by the addition of 1,2-bis(2-aminophenoxy)ethane N,N,N[prime],N[prime]-tetraacetic acid, a calcium chelating reagent. Similar reversible inhibition by Ca2+ was shown by the generation of electrical potential in the membranes. Activity of ATP hydrolysis was inhibited similarly by Ca2+ in the same membrane preparations. The addition of 1,2-bis(2-aminophenoxy)ethane N,N,N[prime],N[prime]-tetraacetic acid and EGTA, Ca2+ chelators, to epidermal peels of fava bean induced stomatal opening in the dark, and the opening was suppressed by vanadate. This suggests that the lowered cytosolic Ca2+ activated the proton pump in vivo and that the activated pump elicited stomatal opening. Inhibition of H+-ATPase by Ca2+ may depolarize the membrane potential and could be a key step in the process of stomatal closing through activation of the anion channels. Furthermore, similar inhibition of the proton pumping and ATP hydrolysis by Ca2+ was found in isolated plasma membranes of mesophyll cells of fava bean. These results suggest that Ca2+ regulates the activity of plasma membrane H+-ATPases in higher plant cells, thereby modulating stomatal movement and other cellular processes in plants.

Blue light-induced autophosphorylation of phototropin is a primary step for signaling

Phototropins are autophosphorylating protein kinases of plant-specific blue light receptors. They regulate various blue light responses, including phototropism, chloroplast movements, hypocotyl growth inhibition, leaf flattening, and stomatal opening. However, the physiological role of autophosphorylation remains unknown. Here, we identified phosphorylation sites of Ser or Thr in the N terminus, Hinge1 region, kinase domain, and C terminus in Arabidopsis phototropin1 (phot1) by liquid chromatography–tandem mass spectrometry in vivo. We substituted these Ser or Thr residues with Ala in phot1 and analyzed their functions by inspecting the phot1-mediated responses of stomatal opening, phototropism, chloroplast accumulation, and leaf flattening after the transformation of the phot1 phot2 double mutant. Among these sites, we found that autophosphorylation of Ser-851 in the activation loop of the kinase domain was required for the responses mentioned above, whereas the phosphorylation of the other Ser and Thr, except those in the activation loop, was not. Ser-849 in the loop may have an additional role in the responses. Immunological analysis revealed that Ser-851 was phosphorylated rapidly by blue light in a fluence-dependent manner and dephosphorylated gradually upon darkness. We conclude that autophosphorylation of Ser-851 is a primary step that mediates signaling between photochemical reaction and physiological events.

Modulation of an RNA-binding protein by abscisic-acid-activated protein kinase

Protein kinases are involved in stress signalling in both plant and animal systems. The hormone abscisic acid mediates the responses of plants to stresses such as drought, salinity and cold. Abscisic-acid-activated protein kinase (AAPK)—found in guard cells, which control stomatal pores—has been shown to regulate plasma membrane ion channels. Here we show that AAPK-interacting protein 1 (AKIP1), with sequence homology to heterogeneous nuclear RNA-binding protein A/B, is a substrate of AAPK. AAPK-dependent phosphorylation is required for the interaction of AKIP1 with messenger RNA that encodes dehydrin, a protein implicated in cell protection under stress conditions. AAPK and AKIP1 are present in the guard-cell nucleus, and in vivo treatment of such cells with abscisic acid enhances the partitioning of AKIP1 into subnuclear foci which are reminiscent of nuclear speckles. These results show that phosphorylation-regulated RNA target discrimination by heterogeneous nuclear RNA-binding proteins may be a general phenomenon in eukaryotes, and implicate a plant hormone in the regulation of protein dynamics during rapid subnuclear reorganization.

Phototropins Promote Plant Growth in Response to Blue Light in Low Light Environments

Phototropins (phot1 and phot2) are plant-specific blue light receptors for phototropism, chloroplast movement, leaf expansion, and stomatal opening. All these responses are thought to optimize photosynthesis by helping to capture light energy efficiently, reduce photodamage, and acquire CO2. However, experimental evidence for the promotion of plant growth through phototropins is lacking. Here, we report dramatic phototropin-dependent effects on plant growth. When plants of Arabidopsis thaliana wild type, the phot1 and phot2 mutants, and the phot1 phot2 double mutant were grown under red light, no significant growth differences were observed. However, if a very low intensity of blue light (0.1 μmol m−2 s−1) was superimposed on red light, large increases in fresh weight up to threefold were found in those plants that carried functional PHOT1 genes. When the intensity of blue light was increased to 1 μmol m−2 s−1, the growth enhancement was also found in the phot1 single mutant, but not in the double mutant, indicating that phot2 mediated similar responses as phot1 with a lower sensitivity. The effects occurred under low photosynthetically active radiation in particular. The well-known physiological phototropin-mediated responses, including chloroplast movement, stomatal opening, and leaf expansion, in the different lines tested indicated an involvement of these responses in the blue light–induced growth enhancement. We conclude that phototropins promote plant growth by controlling and integrating a variety of responses that optimize photosynthetic performance under low photosynthetically active radiation in the natural environment.

Inhibition of Blue Light-Dependent H + Pumping by Abscisic Acid through Hydrogen Peroxide-Induced Dephosphorylation of the Plasma Membrane H + -ATPase in Guard Cell Protoplasts

Blue light (BL)-dependent H+ pumping by guard cells, which drives stomatal opening, is inhibited by abscisic acid (ABA). We investigated this response with respect to the activity of plasma membrane H+-ATPase using Vicia guard cell protoplasts. ATP hydrolysis by the plasma membrane H+-ATPase, phosphorylation of the H+-ATPase, and the binding of 14-3-3 protein to the H+-ATPase stimulated by BL were inhibited by ABA at 10 μm. All of these responses were similarly inhibited by hydrogen peroxide (H2O2) at 1 mm. The ABA-induced inhibitions of BL-dependent H+ pumping and phosphorylation of the H+-ATPase were partially restored by ascorbate, an intracellular H2O2 scavenger. A single-cell analysis of the cytosolic H2O2 using 2′,7′-dichlorofluorescin revealed that H2O2 was generated by ABA in guard cell protoplasts. We also indicated that H+ pumping induced by fusicoccin and the binding of 14-3-3 protein to the H+-ATPase were inhibited slightly (approximately 20%) by both ABA and H2O2. By contrast, H2O2 at 1 mm did not affect H+ pumping by the H+-ATPase in microsomal membranes. From these results, we concluded that inhibition of BL-dependent H+ pumping by ABA was due to a decrease in the phosphorylation levels of H+-ATPase and that H2O2 might be involved in this response. Moreover, there are at least two inhibition sites by ABA in the BL signaling pathway of guard cells.

Blue-Light- and Phosphorylation-Dependent Binding of a 14-3-3 Protein to Phototropins in Stomatal Guard Cells of Broad Bean

Phototropins are blue-light (BL) receptor serine (Ser)/threonine kinases, and contain two light, oxygen, and voltage (LOV) domains, and are members of the PAS domain superfamily. They mediate phototropism, chloroplast movement, leaf expansion, and stomatal opening of higher plants in response to BL. In stomatal guard cells, genetic analysis has revealed that phototropins mediate activation of the plasma membrane H+-ATPase by phosphorylation and drive stomatal opening. However, biochemical evidence for the involvement of phototropins in the BL response of stomata is lacking. Using guard cell protoplasts, we showed that broad bean (Vicia faba) phototropins (Vfphots) were phosphorylated by BL, and that this phosphorylation of Vfphots reached to the maximum level earlier than that of the H+-ATPase. Phosphorylation of both Vfphots and H+-ATPase showed similar sensitivity to BL and were similarly suppressed by protein kinase and flavoprotein inhibitors. We found that a 14-3-3 protein was bound to Vfphots upon phosphorylation, and this binding occurred earlier than the H+-ATPase phosphorylation. Vfphots (Vfphot1a and Vfphot1b) were expressed in Escherichia coli, and phosphorylation sites were determined to be Ser-358 for Vfphot1a and Ser-344 for Vfphot1b, which are localized between LOV1 and LOV2. We conclude that Vfphots act as BL receptors in guard cells and that phosphorylation of a Ser residue between LOV1 and LOV2 and subsequent 14-3-3 protein binding are likely to be key steps of BL response in stomata. The binding of a 14-3-3 protein to Vfphot was found in etiolated seedlings and leaves in response to BL, suggesting that this event was common to phototropin-mediated responses.

Auxin Activates the Plasma Membrane H+-ATPase by Phosphorylation during Hypocotyl Elongation in Arabidopsis

The phytohormone auxin is a major regulator of diverse aspects of plant growth and development. The ubiquitin-ligase complex SCFTIR1/AFB (for Skp1-Cul1-F-box protein), which includes the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) auxin receptor family, has recently been demonstrated to be critical for auxin-mediated transcriptional regulation. Early-phase auxin-induced hypocotyl elongation, on the other hand, has long been explained by the acid-growth theory, for which proton extrusion by the plasma membrane H+-ATPase is a functional prerequisite. However, the mechanism by which auxin mediates H+-ATPase activation has yet to be elucidated. Here, we present direct evidence for H+-ATPase activation in etiolated hypocotyls of Arabidopsis (Arabidopsis thaliana) by auxin through phosphorylation of the penultimate threonine during early-phase hypocotyl elongation. Application of the natural auxin indole-3-acetic acid (IAA) to endogenous auxin-depleted hypocotyl sections induced phosphorylation of the penultimate threonine of the H+-ATPase and increased H+-ATPase activity without altering the amount of the enzyme. Changes in both the phosphorylation level of H+-ATPase and IAA-induced elongation were similarly concentration dependent. Furthermore, IAA-induced H+-ATPase phosphorylation occurred in a tir1-1 afb2-3 double mutant, which is severely defective in auxin-mediated transcriptional regulation. In addition, α-(phenylethyl-2-one)-IAA, the auxin antagonist specific for the nuclear auxin receptor TIR1/AFBs, had no effect on IAA-induced H+-ATPase phosphorylation. These results suggest that the TIR1/AFB auxin receptor family is not involved in auxin-induced H+-ATPase phosphorylation. Our results define the activation mechanism of H+-ATPase by auxin during early-phase hypocotyl elongation; this is the long-sought-after mechanism that is central to the acid-growth theory.