Abstract

Plasma membrane H+-ATPase is thought to mediate hypocotyl elongation, which is induced by the phytohormone auxin through the phosphorylation of the penultimate threonine of H+-ATPase. However, regulation of the H+-ATPase during hypocotyl elongation by other signals has not been elucidated. Hypocotyl elongation in etiolated seedlings of Arabidopsis thaliana was suppressed by the H+-ATPase inhibitors vanadate and erythrosine B, and was significantly reduced in aha2-5, which is a knockout mutant of the major H+-ATPase isoform in etiolated seedlings. Application of the phytohormone ABA to etiolated seedlings suppressed hypocotyl elongation within 30 min at the half-inhibitory concentration (4.2 µM), and induced dephosphorylation of the penultimate threonine of H+-ATPase without affecting the amount of H+-ATPase. Interestingly, an ABA-insensitive mutant, abi1-1, did not show ABA inhibition of hypocotyl elongation or ABA-induced dephosphorylation of H+-ATPase. This indicates that ABI1, which is an early ABA signaling component through the ABA receptor PYR/PYL/RCARs (pyrabactin resistance/pyrabactin resistance 1-like/regulatory component of ABA receptor), is involved in these responses. In addition, we found that the fungal toxin fusiccocin (FC), an H+-ATPase activator, induced hypocotyl elongation and phosphorylation of the penultimate threonine of H+-ATPase, and that FC-induced hypocotyl elongation and phosphorylation of H+-ATPase were significantly suppressed by ABA. Taken together, these results indicate that ABA has an antagonistic effect on hypocotyl elongation through, at least in part, dephosphorylation of H+-ATPase in etiolated seedlings.

Introduction

Hypocotyl elongation is strongly influenced by light, temperature and phytohormones, such as auxin, gibberellin and brassinosteroid (Gendreau et al. 1997, Gray et al. 1998, Collett et al. 2000, Depuydt and Hardtke 2011). Among them, auxin has been known to induce hypocotyl elongation rapidly within several minutes and such an early phase of auxin-induced elongation is explained by the acid-growth theory (Rayle and Cleland 1970, Hager et al. 1971, Hager, 2003). According to this theory, the apoplastic acidification that is governed by proton efflux through plasma membrane H+-ATPase causes wall extension by activating wall-loosening proteins. In addition, the hyperpolarization of the plasma membrane evoked by H+-ATPase enhances K+ uptake through the inward-rectifying K+ channels, such as KAT1 (Claussen et al. 1997, Philippar et al. 2006). These processes cooperatively increase water uptake into cells and permit cell expansion (Katou and Okamoto 1992, Hager 2003). Thus, H+-ATPase is considered to play a key role in the early phase of hypocotyl elongation (Hager et al. 1991, Frias et al. 1996, Jahn et al. 1996, Hager 2003). In fact, the hypocotyl length of de-etiolated and etiolated Arabidopsis seedlings was significantly reduced in aha2-4 and aha2-5, which are knockout mutants of AHA2, a major H+-ATPase isoform (Haruta and Sussman 2012). Moreover, our recent investigation revealed that auxin-induced hypocotyl elongation of etiolated Arabidopsis seedlings is mediated by activation of H+-ATPase via phosphorylation of the penultimate threonine in the C-terminus of H+-ATPase, and that this response is most likely to be mediated by an unidentified auxin receptor, not by TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX protein (TIR1/AFB) family auxin receptors (Takahashi et al. 2012). In addition, auxin-induced and TIR1/AFB-mediated expression of KAT1 is thought to be partially involved in auxin-induced hypocotyl elongation (Takahashi et al. 2012).

Plasma membrane H+-ATPase, a member of the superfamily of P-type ATPases, transports protons out of the cell in a process that is coupled to ATP hydrolysis and is important for regulation of the membrane potential and intracellular pH homeostasis. Arabidopsis thaliana possesses 11 functional H+-ATPase isoforms (AHA1–AHA11; Palmgren 2001). The H+-ATPase creates the electrochemical gradient of protons across the plasma membrane which leads to hyperpolarization and generates the proton motive force. As a result, the H+-ATPase energizes transport of numerous substances through channels and through transporters such as the inward-rectifying K+ channels and Suc/H+ symporters; this process leads to a variety of physiological responses, including phloem loading, stomatal opening, solute uptake by the roots and cell expansion. Phosphorylation of the penultimate amino acid threonine in the C-terminus of H+-ATPase and subsequent binding of a 14-3-3 protein to the phosphorylated C-terminus is the major common mechanism by which H+-ATPase is activated in plant cells (Sondergaard et al. 2004, Duby and Boutry 2009, Kinoshita and Hayashi 2011). Many signals, including light, sucrose, NaCl, the fungal toxin fusicoccin (FC) and the phytohormone auxin regulate phosphorylation levels of the penultimate threonine in the C-terminus of H+-ATPase (Fuglsang et al. 1999, Kinoshita and Shimazaki 1999, Svennelid et al. 1999, Kinoshita and Shimazaki 2001, Kerkeb et al. 2002, Inoue et al. 2005, Niittylä et al. 2007, Chen et al. 2010, Okumura et al. 2012a, Okumura et al. 2012b, Takahashi et al. 2012). It should be noted that H+-ATPase is phosphorylated at multiple sites, in addition to the penultimate threonine (Fuglsang et al. 2007, Duby and Boutry 2009, Rudashevskaya et al. 2012).

The phytohormone ABA regulates many physiological processes, such as seed dormancy, drought resistance, cell growth and stomatal closure in plants (Hirayama and Shinozaki 2007, Cutler et al. 2010). In addition, it suppresses hypocotyl elongation in etiolated squash hypocotyl segments (Wakabayashi et al. 1989). However, the mechanism by which ABA mediates suppression of hypocotyl elongation remains unclear. In the case of stomatal guard cells, blue light activates plasma membrane H+-ATPase through phosphorylation of its penultimate threonine, resulting in stomatal opening (Kinoshita and Shimazaki 1999, Kinoshita and Shimazaki 2002), and ABA inhibits blue light-induced phosphorylation of H+-ATPase in guard cells (Zhang et al. 2004, Hayashi et al. 2011). Furthermore, ABA inhibition of blue light-induced H+-ATPase phosphorylation is mediated by ABI1, ABI2 and OST1/SRK2e, which are early ABA signaling components that function through the PYR/PYL/RCARs (pyrabactin resistance/pyrabactin resistance 1-like/regulatory component of ABA receptor) ABA receptor (Ma et al. 2009, Park et al. 2009, Kim et al. 2010, Nishimura et al. 2010, Hayashi et al. 2011, Hayashi and Kinoshita 2011). More recently, the Mg-chelatase H subunit has been shown to be involved in ABA inhibition of blue light-induced H+-ATPase phosphorylation in stomatal guard cells (Tsuzuki et al. 2013). We hypothesized that ABA may mediate dephosphorylation of H+-ATPase during ABA inhibition of hypocotyl elongation in etiolated seedlings of A. thaliana.

In the present study, we examined the molecular mechanism of ABA-mediated suppression of hypocotyl elongation in etiolated seedlings of A. thaliana, and found that hypocotyl elongation is suppressed by ABA with decreasing phosphorylation levels of plasma membrane H+-ATPase. However, ABA had no effect on hypocotyl elongation and phosphorylation levels of H+-ATPase in an ABA-insensitive mutant, abi1-1, suggesting that an early ABA signaling component ABI1 mediates ABA inhibition of hypocotyl elongation and ABA-induced dephosphorylation of H+-ATPase. In addition, ABA also suppressed the function of the fungal toxin FC, an H+-ATPase activator, which induced hypocotyl elongation and phosphorylation of H+-ATPase in etiolated seedlings.

Results

Involvement of plasma membrane H+-ATPase in hypocotyl elongation

A previous study using auxin-depleted hypocotyl sections from etiolated seedlings of A. thaliana indicated that application of auxin to the hypocotyl sections induces hypocotyl elongation, and auxin-induced hypocotyl elongation was suppressed by an H+-ATPase inhibitor (Takahashi et al. 2012). We performed a detailed analysis of the role of H+-ATPase in hypocotyl elongation using intact etiolated seedlings of A. thaliana. Three-day-old etiolated seedlings had growth rates of approximately 0.20 mm h–1, and the inhibitors of H+-ATPase, erythrosine B and vanadate (Kanczewska et al. 2005, Kinoshita et al. 2011, Koizumi et al. 2011), significantly suppressed hypocotyl elongation. The elongation rates decreased to 55% in the presence of 30 µM erythrosine B and to 75% in the presence of 1 mM vanadate (Fig. 1A). H+-ATPase isoforms AHA1 and AHA2 are the major isoforms expressed in etiolated seedlings of A. thaliana, and the expression level of AHA5 is very low (Hayashi et al. 2010). Therefore, we examined hypocotyl elongation in the AHA1 knockdown mutant aha1-8 (Haruta et al. 2010), the AHA2 knockout mutant aha2-5 (Haruta et al. 2010) and the AHA5 knockout mutant aha5 as a negative control (Supplementary Fig. S1). The elongation rates decreased to 89.8% in aha1-8 and to 89.5% in aha2-5, but did not decrease in aha5 (Fig. 1B). Statistical analysis using the Student’s t-test revealed that the elongation rate in aha2-5 significantly decreased (P < 0.05) compared with a background Columbia (Col-0) plant, but there was no significant change in the elongation rate in aha1-8 (P > 0.05) because the elongation rates varied. These results strongly suggest that plasma membrane H+-ATPase mediates hypocotyl elongation in intact etiolated seedlings of A. thaliana. It should be noted that the H+-ATPase is highly expressed in the upper region of hypocotyls known as the elongation zone (Supplementary Fig. S2), suggesting that the hypocotyl elongation mediated by H+-ATPase mainly occurs in the elongation zone.

Fig. 1

Involvement of H+-ATPase in hypocotyl elongation in etiolated seedlings of Arabidopsis thaliana. (A) Effects of H+-ATPase inhibitors on hypocotyl elongation of etiolated seedlings. Three-day-old etiolated seedlings were placed on agar-solidified medium with 30 µM erythrosine B (filled triangles), 1 mM sodium orthovanadate (vanadate; open triangles) or without inhibitor (Mock; open circles), after which hypocotyl elongation was measured. The upper graph shows time course changes in hypocotyl elongation. The lower graph shows the elongation rate of hypocotyls between 2 and 4 h after inhibitor treatment when the elongation rates reached a constant level. Values represent the mean of 15 seedlings with the SEs. The experiments were repeated on three occasions with similar results. Each letter indicates a significant difference relative to mock treatment based on the Student’s t-test (a, erythrosine B, P < 0.05; b, vanadate, P < 0.05). (B) Comparison of the elongation rate of hypocotyls in Col-0 with three single mutants of Arabidopsis H+-ATPases (aha1-8, aha2-5 and aha5). The elongation rate was calculated from the hypocotyl elongation of 3-day-old etiolated seedlings for a 3 h period. Values are means of three independent experiments with the SEs. The asterisk indicates a significant difference relative to Col-0 based on the Student’s t-test (P < 0.05).

Fig. 1

Involvement of H+-ATPase in hypocotyl elongation in etiolated seedlings of Arabidopsis thaliana. (A) Effects of H+-ATPase inhibitors on hypocotyl elongation of etiolated seedlings. Three-day-old etiolated seedlings were placed on agar-solidified medium with 30 µM erythrosine B (filled triangles), 1 mM sodium orthovanadate (vanadate; open triangles) or without inhibitor (Mock; open circles), after which hypocotyl elongation was measured. The upper graph shows time course changes in hypocotyl elongation. The lower graph shows the elongation rate of hypocotyls between 2 and 4 h after inhibitor treatment when the elongation rates reached a constant level. Values represent the mean of 15 seedlings with the SEs. The experiments were repeated on three occasions with similar results. Each letter indicates a significant difference relative to mock treatment based on the Student’s t-test (a, erythrosine B, P < 0.05; b, vanadate, P < 0.05). (B) Comparison of the elongation rate of hypocotyls in Col-0 with three single mutants of Arabidopsis H+-ATPases (aha1-8, aha2-5 and aha5). The elongation rate was calculated from the hypocotyl elongation of 3-day-old etiolated seedlings for a 3 h period. Values are means of three independent experiments with the SEs. The asterisk indicates a significant difference relative to Col-0 based on the Student’s t-test (P < 0.05).

ABA suppresses hypocotyl elongation in etiolated seedlings

ABA may mediate the suppression of plant cell growth (Hirayama and Shinozaki 2007, Cutler et al. 2010), and it suppresses hypocotyl elongation in etiolated squash seedlings (Wakabayashi et al. 1989). Therefore, we examined the effect of ABA on hypocotyl elongation of intact etiolated seedlings of A. thaliana. Hypocotyl elongation was suppressed by ABA in a dose-dependent manner (Fig. 2A). ABA-induced suppression was observed at 1 µM and saturated at 20 µM. The rate of hypocotyl elongation in the presence of 20 µM ABA after 30–120 min decreased to 43% compared with that in the absence of ABA (Fig. 2B). The half inhibitory concentration (IC50) was estimated to be approximately 4.2 µM. It should be noted that ABA does not completely suppress hypocotyl elongation (Fig. 2B), suggesting that ABA-independent regulation is involved in hypocotyl elongation.

Fig. 2

Effect of ABA on hypocotyl elongation in etiolated Arabidopsis seedlings. Three-day-old etiolated seedlings were incubated on agar-solidified medium containing ABA at the indicated concentrations. Hypocotyl elongation was measured at 30 min intervals from 0 to 120 min. (A) Time course of hypocotyl elongation of etiolated seedlings on medium containing ABA at 0 (filled circles), 1 (open circles), 5 (filled triangles), 10 (open triangles), 20 (open squares) and 50 µM (filled squares). Each value represents the mean of 15 seedlings with the SE. The experiments were repeated on three occasions with similar results. (B) Dose-dependent response of the elongation rate of hypocotyls to ABA. The elongation rates of hypocotyls were calculated between 30 and 120 min when they reached a constant level. Values represent the means of three independent experiments with the SEs. The asterisk indicates a significant difference between each concentration and 0 µM based on the Student’s t-test (P < 0.05).

Fig. 2

Effect of ABA on hypocotyl elongation in etiolated Arabidopsis seedlings. Three-day-old etiolated seedlings were incubated on agar-solidified medium containing ABA at the indicated concentrations. Hypocotyl elongation was measured at 30 min intervals from 0 to 120 min. (A) Time course of hypocotyl elongation of etiolated seedlings on medium containing ABA at 0 (filled circles), 1 (open circles), 5 (filled triangles), 10 (open triangles), 20 (open squares) and 50 µM (filled squares). Each value represents the mean of 15 seedlings with the SE. The experiments were repeated on three occasions with similar results. (B) Dose-dependent response of the elongation rate of hypocotyls to ABA. The elongation rates of hypocotyls were calculated between 30 and 120 min when they reached a constant level. Values represent the means of three independent experiments with the SEs. The asterisk indicates a significant difference between each concentration and 0 µM based on the Student’s t-test (P < 0.05).

Next, we examined ABA inhibition of hypocotyl elongation in abi1-1 mutants, which are ABA insensitive (Leung et al. 1994, Meyer et al. 1994). As shown in Fig. 3, ABA inhibited hypocotyl elongation of etiolated seedlings in the background Landsberg erecta (Ler), but no significant inhibition was observed in the abi1-1 mutant. The rate of elongation from 30 to 120 min after 20 µM ABA application significantly decreased to 32% in Ler. In contrast, ABA had no effect on the rate of elongation in abi1-1. These results are indicative of the involvement of ABI1 in ABA inhibition of hypocotyl elongation in etiolated seedlings.

Fig. 3

Effect of ABA on hypocotyl elongation in etiolated seedlings of an ABA-insensitive mutant, abi1-1. Time course of hypocotyl elongation of etiolated Ler (A) and abi1-1 seedlings (B) on medium in the absence (open circles) or presence (filled circles) of 20 µM ABA. Hypocotyl elongation was measured at 30 min intervals from 0 to 120 min. Each value represents the mean of 15 seedlings with the SE. The asterisks indicate a significant difference based on the Student’s t-test (*P < 0.05; **P < 0.01). The experiments were repeated on three occasions with similar results. (C) Comparison of the elongation rate of hypocotyls in Ler with abi1-1. The elongation rate of hypocotyls between 30 and 90 min after mock (white bars) or 20 µM ABA treatment (black bars) was calculated. Values represent the means of three independent experiments with the SEs. The asterisks indicate a significant difference based on the Student’s t-test (**P < 0.01). ns, not significant at P > 0.05.

Fig. 3

Effect of ABA on hypocotyl elongation in etiolated seedlings of an ABA-insensitive mutant, abi1-1. Time course of hypocotyl elongation of etiolated Ler (A) and abi1-1 seedlings (B) on medium in the absence (open circles) or presence (filled circles) of 20 µM ABA. Hypocotyl elongation was measured at 30 min intervals from 0 to 120 min. Each value represents the mean of 15 seedlings with the SE. The asterisks indicate a significant difference based on the Student’s t-test (*P < 0.05; **P < 0.01). The experiments were repeated on three occasions with similar results. (C) Comparison of the elongation rate of hypocotyls in Ler with abi1-1. The elongation rate of hypocotyls between 30 and 90 min after mock (white bars) or 20 µM ABA treatment (black bars) was calculated. Values represent the means of three independent experiments with the SEs. The asterisks indicate a significant difference based on the Student’s t-test (**P < 0.01). ns, not significant at P > 0.05.

ABA induces dephosphorylation of H+-ATPase in etiolated seedlings

As shown in Fig. 1, H+-ATPase is involved in hypocotyl elongation of intact etiolated seedlings. In addition, auxin-induced elongation in the hypocotyl sections of A. thaliana etiolated seedlings is mediated by plasma membrane H+-ATPase via the phosphorylation of the penultimate threonine (Takahashi et al. 2012). Next, we examined the effect of ABA on the phosphorylation status of the penultimate threonine of H+-ATPase in intact etiolated seedlings (Fig. 4A). We detected the phosphorylation status using a specific antibody (anti-pThr947). When intact etiolated seedlings were incubated in 20 µM ABA for 60 min (which significantly suppresses hypocotyl elongation) (Fig. 2), we observed significantly decreased phosphorylation of H+-ATPase, but the amount of H+-ATPase was not affected. These results indicate that ABA affects the phosphorylation status of H+-ATPase in etiolated seedlings. In accordance with this, ABA suppressed ATP hydrolysis by the H+-ATPase (Fig. 4B). Notably, ABA had no effect on the phosphorylation status of H+-ATPase in abi1-1 or on hypocotyl elongation (Fig. 4C). These results suggest that ABI1 plays a role in ABA-induced dephosphorylation of H+-ATPase in etiolated seedlings.

Fig. 4

Effect of ABA on the phosphorylation level of the penultimate threonine of H+-ATPase in etiolated seedlings of A. thaliana. (A) Effect of ABA on dephosphorylation of H+-ATPase in etiolated seedlings of Col-0. Three-day-old etiolated seedlings were treated with 20 µM ABA (ABA) or 0.1% dimethylsulfoxide (DMSO; Mock) for 60 min. The amount of H+-ATPase and the phosphorylation status of the penultimate threonine in the C-terminus were determined by immunoblot using anti-H+-ATPase and anti-pThr947 antibodies, respectively. The phosphorylation level of H+-ATPase was quantified as the ratio of the signal intensity from the phosphorylated H+-ATPase to that from the amount of H+-ATPase, and was expressed relative to the phosphorylation level of seedlings that were not treated with ABA (graph at bottom). Values represent means ± SD; n = 3 independent experiments. The asterisks indicate a significant difference based on the Student’s t-test (**P < 0.01). (B) Effect of ABA on vanadate-sensitive ATP hydrolytic activity of H+-ATPase in etiolated seedlings of Col-0. Three-day-old etiolated seedlings were treated with 20 µM ABA (ABA) or 0.1% DMSO (Mock) for 60 min. Vanadate-sensitive ATP hydrolysis was measured by determining the inorganic phosphate released from ATP. Values represent means ± SD; n = 3 independent experiments. The asterisk indicates a significant difference based on the Student’s t-test (*P < 0.05). (C) Effect of ABA on the phosphorylation level of H+-ATPase in etiolated seedlings of an ABA-insensitive mutant, abi1-1. Three-day-old etiolated seedlings of Ler and abi1-1 were incubated for 60 min in the absence (–) or presence (+) of 20 µM ABA. The phosphorylation level of H+-ATPase was determined in the same way as described in A and was expressed relative to the phosphorylation level of the seedlings of Ler that were not treated with ABA (graph at bottom). Values represent means ± SD; n = 3 independent experiments. The asterisks indicate a significant difference based on the Student’s t-test (**P < 0.01). ns, not significant at P > 0.05.

Fig. 4

Effect of ABA on the phosphorylation level of the penultimate threonine of H+-ATPase in etiolated seedlings of A. thaliana. (A) Effect of ABA on dephosphorylation of H+-ATPase in etiolated seedlings of Col-0. Three-day-old etiolated seedlings were treated with 20 µM ABA (ABA) or 0.1% dimethylsulfoxide (DMSO; Mock) for 60 min. The amount of H+-ATPase and the phosphorylation status of the penultimate threonine in the C-terminus were determined by immunoblot using anti-H+-ATPase and anti-pThr947 antibodies, respectively. The phosphorylation level of H+-ATPase was quantified as the ratio of the signal intensity from the phosphorylated H+-ATPase to that from the amount of H+-ATPase, and was expressed relative to the phosphorylation level of seedlings that were not treated with ABA (graph at bottom). Values represent means ± SD; n = 3 independent experiments. The asterisks indicate a significant difference based on the Student’s t-test (**P < 0.01). (B) Effect of ABA on vanadate-sensitive ATP hydrolytic activity of H+-ATPase in etiolated seedlings of Col-0. Three-day-old etiolated seedlings were treated with 20 µM ABA (ABA) or 0.1% DMSO (Mock) for 60 min. Vanadate-sensitive ATP hydrolysis was measured by determining the inorganic phosphate released from ATP. Values represent means ± SD; n = 3 independent experiments. The asterisk indicates a significant difference based on the Student’s t-test (*P < 0.05). (C) Effect of ABA on the phosphorylation level of H+-ATPase in etiolated seedlings of an ABA-insensitive mutant, abi1-1. Three-day-old etiolated seedlings of Ler and abi1-1 were incubated for 60 min in the absence (–) or presence (+) of 20 µM ABA. The phosphorylation level of H+-ATPase was determined in the same way as described in A and was expressed relative to the phosphorylation level of the seedlings of Ler that were not treated with ABA (graph at bottom). Values represent means ± SD; n = 3 independent experiments. The asterisks indicate a significant difference based on the Student’s t-test (**P < 0.01). ns, not significant at P > 0.05.

ABA suppresses FC-induced hypocotyl elongation and phosphorylation of H+-ATPase in etiolated seedlings

FC, an activator of H+-ATPase, induces phosphorylation of H+-ATPase by inhibiting its dephosphorylation (Kinoshita and Shimazaki 2001, Hayashi et al. 2010). Thus, we examined the effect of FC on hypocotyl elongation and the phosphorylation status of the penultimate threonine of H+-ATPase in the presence and absence of ABA. Etiolated seedlings incubated in 10 µM FC showed enhanced hypocotyl elongation (Fig. 5A). The rate of hypocotyl elongation in the presence of FC was almost 3-fold higher than in the absence of FC. FC-induced elongation was also suppressed in the presence of 20 µM ABA. The rate of hypocotyl elongation significantly decreased to 68% in the presence of ABA. Next, we examined the effect of FC on the phosphorylation status of H+-ATPase. Addition of FC at 10 µM to the etiolated seedlings increased the phosphorylation level of the penultimate threonine without affecting the amount of the H+-ATPase (Fig. 5B). The phosphorylation level of H+-ATPase increased to 169% in response to FC for 60 min. FC-induced phosphorylation was inhibited in the presence of 20 µM ABA. The phosphorylation level of H+-ATPase in ABA-treated etiolated seedlings was approximately 80% compared with non-ABA-treated seedlings at 60 min after addition of FC, indicating that ABA suppresses both FC-induced hypocotyl elongation and FC-induced phosphorylation of H+-ATPase in etiolated seedlings.

Fig. 5

Effect of FC on hypocotyl elongation and H+-ATPase phosphorylation in ABA-treated etiolated seedlings of A. thaliana. Three-day-old etiolated seedlings (Col-0) were pre-treated with 20 µM ABA (ABA) or 0.1% DMSO (Mock) for 60 min and then incubated for 60 min in the absence (–) or presence (+) of 10 µM FC. (A) FC-induced hypocotyl elongation for 60 min. Values represent the means ± SD; n = 3 independent experiments. Different letters indicate significant differences based on Tukey’s test (P < 0.05). (B) The amount of H+-ATPase and the phosphorylation status of the penultimate threonine in the C-terminus. Details were the same as described in Fig. 4A. Values represent the means ± SD; n = 3 independent experiments. Different letters indicate significant differences based on Tukey’s test (P < 0.05).

Fig. 5

Effect of FC on hypocotyl elongation and H+-ATPase phosphorylation in ABA-treated etiolated seedlings of A. thaliana. Three-day-old etiolated seedlings (Col-0) were pre-treated with 20 µM ABA (ABA) or 0.1% DMSO (Mock) for 60 min and then incubated for 60 min in the absence (–) or presence (+) of 10 µM FC. (A) FC-induced hypocotyl elongation for 60 min. Values represent the means ± SD; n = 3 independent experiments. Different letters indicate significant differences based on Tukey’s test (P < 0.05). (B) The amount of H+-ATPase and the phosphorylation status of the penultimate threonine in the C-terminus. Details were the same as described in Fig. 4A. Values represent the means ± SD; n = 3 independent experiments. Different letters indicate significant differences based on Tukey’s test (P < 0.05).

ABA affects the expression of an inward-rectifying K+ channel in etiolated seedlings

A previous report suggested that auxin-induced and TIR1/AFB-mediated expression of an inward-rectifying K+ channel, such as KAT1, is partially involved in auxin-induced hypocotyl elongation of etiolated seedlings of A. thaliana (Takahashi et al. 2012). Therefore, we examined the effect of ABA on expression of KAT1. Incubation of wild-type etiolated seedlings with 20 µM ABA for 1 h significantly reduced the expression of KAT1 by 25% (Fig. 6). These conditions increased the expression of typical ABA-responsive genes, RAB18 and RD29B (Leonhardt et al. 2004, Fujii et al. 2007). In contrast, the expression levels of KAT1, RAB18 and RD29B were not affected in abi1-1 mutants, suggesting that ABA negatively regulates the expression of KAT1 via ABI1 in hypocotyls.

Fig. 6

Effect of ABA on the expression level of the inward-rectifying K+ channel KAT1 in etiolated seedlings of A. thaliana. qRT-PCR analysis of KAT1 and the known ABA-inducible genes RD29B and RAB18 is shown. Total RNA was obtained from the etiolated seedlings 60 min after the application of 20 µM ABA (ABA) or 0.1% DMSO (Mock). Values represent the means ± SD; n = 3. The asterisks indicate significant differences based on the Student’s t-test (*P < 0.05; **P < 0.01). ns, not significant at P > 0.05.

Fig. 6

Effect of ABA on the expression level of the inward-rectifying K+ channel KAT1 in etiolated seedlings of A. thaliana. qRT-PCR analysis of KAT1 and the known ABA-inducible genes RD29B and RAB18 is shown. Total RNA was obtained from the etiolated seedlings 60 min after the application of 20 µM ABA (ABA) or 0.1% DMSO (Mock). Values represent the means ± SD; n = 3. The asterisks indicate significant differences based on the Student’s t-test (*P < 0.05; **P < 0.01). ns, not significant at P > 0.05.

Discussion

Direct evidence for the involvement of plasma membrane H+-ATPase in hypocotyl elongation of etiolated seedlings

Hypocotyl elongation in plants is thought to involve plasma membrane H+-ATPase (Hager 2003, Takahashi et al. 2012). Moreover, hypocotyl elongation in de-etiolated and etiolated seedlings of A. thaliana is reduced in the H+-ATPase AHA2 knockout plants aha2-4 and aha2-5 (Haruta and Sussman 2012). In the present study, we found that H+-ATPase inhibitors (vanadate and erythrosine B) suppressed hypocotyl elongation in etiolated seedlings of A. thaliana (Fig. 1A), and that hypocotyl elongation was significantly decreased in the AHA2 knockout plant aha2-5 (Fig. 1B). Furthermore, the fungal toxin FC, an H+-ATPase activator, induced hypocotyl elongation (Fig. 5A). These pharmacological and genetic results demonstrate that plasma membrane H+-ATPase plays a role in hypocotyl elongation in etiolated seedlings of A. thaliana.

Minor differences in the elongation rate between the wild type and aha2-5 are due to the redundant role of AHA1 in etiolated seedlings, because AHA1 and AHA2 are predominantly expressed isoforms in etiolated seedlings of A. thaliana (Hayashi et al. 2010). However, aha1 aha2 double mutants show embryonic lethal phenotypes (Haruta et al. 2010). Therefore, further investigation using conditional double mutants will increase our understanding of plasma membrane H+-ATPase during hypocotyl elongation.

ABA suppresses hypocotyl elongation in etiolated seedlings through dephosphorylation of plasma membrane H+-ATPase

The present results clearly indicate that ABA suppresses hypocotyl elongation in etiolated seedlings of A. thaliana (Fig. 2). Furthermore, ABA induced dephosphorylation of the H+-ATPase, which resulted in inactivation of the H+-ATPase (Fig. 4A, B). This suggests that ABA inhibition of hypocotyl elongation is mediated at least partly by dephosphorylation of H+-ATPase. In support of this hypothesis, the fungal toxin FC, an activator of plasma membrane H+-ATPase, induced hypocotyl elongation and phosphorylation of H+-ATPase, which was significantly suppressed by ABA (Fig. 5). To the best of our knowledge, this is the first study to show that dephosphorylation of plasma membrane H+-ATPase plays a role in ABA inhibition of hypocotyl elongation in etiolated seedlings. In addition, the present study suggested that ABA regulates hypocotyl elongation via cell expansion, which is involved in other plant responses such as root elongation (Baskin 2005). Further analysis using a specific cell type or tissue will be needed to clarify the contribution of ABA-induced dephosphorylation of H+-ATPase to the plant responses.

FC inhibits the dephosphorylation of H+-ATPase through FC-mediated tight binding between phosphorylated H+-ATPase and the 14-3-3 protein (Fuglsang et al. 1999, Svennelid et al. 1999, Kinoshita and Shimazaki 2001). In the present study, we found that ABA suppresses FC-induced phosphorylation of H+-ATPase in etiolated seedlings (Fig. 5B), suggesting that ABA decreases phosphorylation activity for the H+-ATPase, which is mediated by an unidentified protein kinase in the plasma membrane (Svennelid et al. 1999, Hayashi et al. 2010). It should be noted that ABA does not completely inhibit FC-induced phosphorylation of H+-ATPase, suggesting that both ABA-dependent and independent kinases are involved in the phosphorylation of the H+-ATPase during hypocotyl elongation. In addition, recent studies indicated that translocation of the H+-ATPase is mediated by PATROL1, and localization of PATROL1 in stomatal guard cells is regulated by numerous signals including drought stress and ABA (Hashimoto-Sugimoto et al. 2013, Higaki et al. 2014). Therefore, it is possible that localization of H+-ATPase to the plasma membrane is required for proper phosphorylation of H+-ATPase. Further investigation is required to examine the subcellular localization of H+-ATPase in response to ABA in etiolated seedlings.

ABA inhibition of hypocotyl elongation may be mediated by an early ABA signaling pathway, including ABI1

A family of START domain proteins, termed the PYR/PYL/RCARs, have recently been identified as cytosolic ABA receptors, and recognition of ABA by these proteins activates members of the protein kinase SRK2/SnRK2 family, including OST1/SRK2e, by inactivating the centrally acting negative regulators, the type 2C protein phosphatases (PP2Cs), that include ABI1 and ABI2 (Ma et al. 2009, Park et al. 2009, Kim et al. 2010, Nishimura et al. 2010). At present, an early-acting ABA signaling pathway, PYR/PYL/RCARs–PP2Cs–SRK2/SnRK2s, is thought to mediate the majority of ABA-triggered plant responses, including the inhibition of seed germination and root growth, stomatal closure and the expression of particular genes (Cutler et al. 2010). However, it remains unclear whether the early-acting PYR/PYL/RCARs–PP2Cs–SRK2/SnRK2s ABA signaling pathway mediates ABA inhibition of hypocotyl elongation in etiolated seedlings. Therefore, we examined the effect of ABA on hypocotyl elongation and phosphorylation levels of plasma membrane H+-ATPase in an ABA-insensitive mutant, abi1-1. As expected, ABA had no effect on hypocotyl elongation and phosphorylation levels of plasma membrane H+-ATPase in the etiolated seedlings of A. thaliana (Figs. 3, 4C), suggestive of the involvement of the early-acting PYR/PYL/RCARs–PP2Cs–SRK2/SnRK2s ABA signaling pathway in ABA inhibition of hypocotyl elongation and ABA-induced dephosphorylation of plasma membrane H+-ATPase.

A hypothetical model for the regulation of elongation growth by ABA and auxin in etiolated seedlings

Based on the present results, we propose a model for the regulation of elongation by ABA and auxin in etiolated seedlings (Fig. 7). Endogenous auxin flowing from the apical cotyledon to the basal root activates plasma membrane H+-ATPase via phosphorylation of the penultimate threonine in etiolated seedlings and induces hypocotyl elongation (Hager 2003, Vanneste and Friml 2009, Takahashi et al. 2012). When the ABA level increases in response to drought stress, ABA suppresses hypocotyl elongation by inhibiting the phosphorylation of plasma membrane H+-ATPase. Signaling is thought to be mediated by an early-acting ABA signaling pathway, PYR/PYL/RCARs–PP2Cs–SRK2/SnRK2s, which includes ABI1. In addition, ABA signaling may affect expression of the inward-rectifying K+ channels to decrease cell growth. Plant growth requires sufficient water to maintain water potential in the cells. Therefore, ABA inhibition of hypocotyl elongation may be advantageous to plants growing under water-limited conditions, because reduction of plant growth would improve plant water status (Creelman et al. 1990). The present results increase our understanding of the regulation of plant growth in response to the drought-responsive hormone ABA in plants.

Fig. 7

Hypothetical model for the regulation of elongation by ABA and auxin in etiolated seedlings.

Fig. 7

Hypothetical model for the regulation of elongation by ABA and auxin in etiolated seedlings.

Materials and Methods

Plant materials and growth conditions

Plants of A. thaliana Col-0 and Ler were used as wild types. Col-0 is the background ecotype of T-DNA insertion mutants of AHA1 (aha1-8, SALK_118350), AHA2 (aha2-5, SALK_022010) and AHA5 (aha5, SALK_127844C), and Ler is the background ecotype of an ABA-insensitive mutant, abi1-1 (Leung et al., 1994). These mutants were obtained from the Arabidopsis Biological Resource Center (Ohio State University). aha1-8 and aha2-5 with T-DNA insertions within an intron are a knockdown mutant of AHA1 and a knockout mutant of AHA2, respectively (Haruta et al., 2010, Hayashi et al., 2011). In aha5, a T-DNA insertion occurred within an exon (Supplementary Fig. S1). The surface-sterilized Arabidopsis seeds were placed on agar-solidified half-strength Murashige and Skoog medium containing 1% (w/v) sucrose (growth medium), and then treated for 2–4 d at 4°C. After that the seeds were irradiated with white light at 50 µmol m–2 s–1 for 4–8 h to enhance germination, then transferred in darkness for 3 d at 24°C.

Treatment with ABA and other reagents

ABA (Sigma-Aldrich), erythrosine B (Wako) and sodium orthovanadate (Sigma-Aldrich) experiments were performed by transferring 3-day-old etiolated seedlings to growth medium containing each reagent. To examine the effect of FC (Sigma-Aldrich), seedlings pre-treated with 20 µM ABA for 1 h were transferred to medium supplemented with 10 µM FC. We transferred seedlings by lifting them using tweezers, without closing or pinching them, in order to prevent seedlings from being damaged. The seedlings were photographed or collected at the indicated time after reagent treatments. All manipulations were performed under dim red light. The hypocotyl length was measured as the length of the center line drawn on the hypocotyl using ImageJ software according to a previously reported method (Takahashi et al., 2012).

Determination of H+-ATPase phosphorylation levels

Plasma membrane H+-ATPases and the phosphorylation level of its penultimate threonine in H+-ATPases were detected by immunoblot analysis using specific antibodies against the catalytic domain of AHA2 (anti-H+-ATPase antibody) and phosphorylated Thr947 (anti-pThr947 antibody) in AHA2 (Hayashi et al. 2010, Takahashi et al. 2012). The collected seedlings were immediately frozen with liquid N2 and stored at –80°C as necessary. The frozen seedlings were ground with a plastic pestle, followed by solubilization in SDS buffer [3% (w/v) SDS, 30 mM Tris–HCl (pH 8.0), 10 mM EDTA, 10 mM NaF, 30% (w/v) sucrose, 0.012% (w/v) Coomassie Brilliant Blue and 15% (v/v) 2-mercaptoethanol], and the homogenates were centrifuged at room temperature (10,000×g for 5 min). The supernatant was analyzed by immunoblot using anti-H+-ATPase and anti-pThr947 antibodies as primary antibodies. A goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad Laboratories) was used as a secondary antibody, and the chemiluminescence from the horseradish peroxidase reaction with a chemiluminescence substrate (Thermo Scientific) was detected using the Light Capture AE-2150 system (Atto). The chemiluminescent signal was quantified using ImageJ software. The phosphorylation level of H+-ATPase was quantified based on the ratio and expressed relative to the phosphorylation level of a control sample.

Measurement of vanadate-sensitive ATPase activity

ATP hydrolysis by the plasma membrane H+-ATPase was measured in a vanadate-sensitive manner according to a previously reported method (Takahashi et al. 2012).

Supplementary data

Supplementary data are available at PCP online.

Funding

This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology [Grants in Aid for Scientific Research (23370019, 22119005 and 21227001 to T.K.)]; the Japan Society for the Promotion of Science [Grants in Aid for Young Scientist (23-10022 to Y.H.)]; the Japan Science and Technology Agency [Advanced Low Carbon Technology Research and Development Program (643 to T.K.)].

Disclosures

The authors have no conflicts of interest to declare.

Abbreviations

    Abbreviations
  • AHA2

    Arabidopsis plasma membrane H+-ATPase 2

  • DMSO

    dimethylsulfoxide

  • FC

    fusicoccin

  • PP2C

    type 2C protein phosphatase

  • PYR/PYL/RCAR

    pyrabactin resistance/pyrabactin resistance 1-like/regulatory component of ABA receptor

References

Baskin
TI
Anisotropic expansion of the plant cell wall
Annu. Rev. Cell Dev. Biol.
 , 
2005
, vol. 
21
 (pg. 
203
-
222
)
Chen
Y
Hoehenwarter
W
Weckwerth
W
Comparative analysis of phytohormone-responsive phosphoproteins in Arabidopsis thaliana using TiO2-phosphopeptide enrichment and mass accuracy precursor alignment
Plant J.
 , 
2010
, vol. 
63
 (pg. 
1
-
17
)
Claussen
M
Lüthen
H
Blatt
M
Böttger
M
Auxin-induced growth and its linkage to potassium channels
Planta
 , 
1997
, vol. 
201
 (pg. 
227
-
234
)
Collett
CE
Harberd
NP
Leyser
O
Hormonal interactions in the control of Arabidopsis hypocotyl elongation
Plant Physiol.
 , 
2000
, vol. 
124
 (pg. 
553
-
561
)
Creelman
RA
Mason
HS
Bensen
RJ
Boyer
JS
Mullet
JE
Water deficit and abscisic acid cause differential inhibition of shoot versus root growth in soybean seedlings
Plant Physiol.
 , 
1990
, vol. 
92
 (pg. 
205
-
214
)
Cutler
SR
Rodriguez
PL
Finkelstein
RR
Abrams
SR
Abscisic acid: emergence of a core signaling network
Annu. Rev. Plant Biol.
 , 
2010
, vol. 
61
 (pg. 
651
-
679
)
Depuydt
S
Hardtke
CS
Hormone signaling crosstalk in plant growth regulation
Curr. Biol.
 , 
2011
, vol. 
21
 (pg. 
R365
-
R373
)
Duby
G
Boutry
M
The plant plasma membrane proton pump ATPase: a highly regulated P-type ATPase with multiple physiological roles
Pflugers Arch.
 , 
2009
, vol. 
457
 (pg. 
645
-
655
)
Frias
I
Caldeira
MT
Pérez-Castiñeira
JR
Navarro-Aviñó
JP
Culiañez-Maciá
FA
Kuppinger
O
, et al.  . 
A major isoform of the maize plasma membrane H+-ATPase: characterization and induction by auxin in coleoptiles
Plant Cell
 , 
1996
, vol. 
8
 (pg. 
1533
-
1544
)
Fuglsang
AT
Guo
Y
Cuin
TA
Qiu
QS
Song
CP
Kristiansen
KA
, et al.  . 
Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+-ATPase by preventing interaction with 14-3-3 protein
Plant Cell
 , 
2007
, vol. 
19
 (pg. 
1617
-
1634
)
Fuglsang
AT
Visconti
S
Drumm
K
Jahn
T
Stensballe.
A
Mattei
B
, et al.  . 
Binding of 14-3-3 protein to the plasma membrane H+-ATPase AHA2 involves the three C-terminal residues Tyr946-Thr-Val and requires phosphorylation of Thr947
J. Biol. Chem.
 , 
1999
, vol. 
51
 (pg. 
36774
-
36780
)
Fujii
H
Verslues
PE
Zhu
JK
Identification of two protein kinases required for abscisic acid regulation of seed germination, root growth, and gene expression in Arabidopsis
Plant Cell
 , 
2007
, vol. 
19
 (pg. 
485
-
494
)
Gendreau
E
Traas
J
Desnos
T
Grandjean
O
Caboche
M
Höfte
H
Cellular basis of hypocotyl growth in Arabidopsis thaliana
Plant Physiol.
 , 
1997
, vol. 
114
 (pg. 
295
-
305
)
Gray
WM
Östin
A
Sandberg
G
Romano
CP
Estelle
M
High temperature promotes auxin-mediated hypocotyl elongation in Arabidopsis
Proc. Natl Acad. Sci. USA
 , 
1998
, vol. 
95
 (pg. 
7197
-
7202
)
Hager
A
Role of the plasma membrane H+-ATPase in auxin-induced elongation growth: historical and new aspects
J. Plant Res.
 , 
2003
, vol. 
116
 (pg. 
483
-
505
)
Hager
A
Debus
G
Edel
HG
Stransky
H
Serrano
R
Auxin induces exocytosis and the rapid synthesis of a high-turnover pool of plasma-membrane H+-ATPase
Planta
 , 
1991
, vol. 
185
 (pg. 
527
-
537
)
Hager
A
Menzel
H
Krauss
A
Versuche und Hypothese zur Primärwirkung des Auxins beim Streckungswachtum
Planta
 , 
1971
, vol. 
100
 (pg. 
47
-
75
)
Haruta
M
Burch
HL
Nelson
RB
Barrett-Wilt
G
Kline
KG
Mohsin
SB
, et al.  . 
Molecular characterization of mutant Arabidopsis plants with reduced plasma membrane proton pump activity
J. Biol. Chem.
 , 
2010
, vol. 
285
 (pg. 
17918
-
17929
)
Haruta
M
Sussman
MR
The effect of a genetically reduced plasma membrane protonmotive force on vegetative growth of Arabidopsis
Plant Physiol.
 , 
2012
, vol. 
158
 (pg. 
1158
-
1171
)
Hashimoto-Sugimoto
M
Higaki
T
Yaeno
T
Nagami
A
Irie
M
Fujimi
M
, et al.  . 
A Munc13-like protein in Arabidopsis mediates H+-ATPase translocation that is essential for stomatal responses
Nat. Commun.
 , 
2013
, vol. 
4
 pg. 
2215
 
Hayashi
M
Inoue
S
Takahashi
K
Kinoshita
T
Immunohistochemical detection of blue light-induced phosphorylation of the plasma membrane H+-ATPase in stomatal guard cells
Plant Cell Physiol.
 , 
2011
, vol. 
52
 (pg. 
1238
-
1248
)
Hayashi
M
Kinoshita
T
Crosstalk between blue-light- and ABA-signaling pathways in stomatal guard cells
Plant Signal. Behav.
 , 
2011
, vol. 
6
 (pg. 
1662
-
1664
)
Hayashi
Y
Nakamura
S
Takemiya
A
Takahashi
Y
Shimazaki
K
Kinoshita
T
Biochemical characterization of in vitro phosphorylation and dephosphorylation of the plasma membrane H+-ATPase
Plant Cell Physiol.
 , 
2010
, vol. 
51
 (pg. 
1186
-
1196
)
Higaki
T
Hashimoto-Sugimoto
M
Akita
K
Iba
K
Hasezawa
S
Dynamics and environmental responses of PATROL1 in Arabidopsis subsidiary cells
Plant Cell Physiol.
 , 
2014
, vol. 
55
 pg. 
in press
 
Hirayama
T
Shinozaki
K
Perception and transduction of abscisic acid signals: keys to the function of the versatile plant hormone ABA
Trends Plant Sci.
 , 
2007
, vol. 
12
 (pg. 
343
-
351
)
Inoue
S
Kinoshita
T
Shimazaki
K
Possible involvement of phototropins in leaf movement of kidney bean in response to blue light
Plant Physiol.
 , 
2005
, vol. 
138
 (pg. 
1994
-
2004
)
Jahn
T
Johansson
F
Lüthen
H
Volkmann
D
Larsson
C
Reinvestigation of auxin and fusicoccin stimulation of the plasma-membrane H+-ATPase activity
Planta
 , 
1996
, vol. 
199
 (pg. 
359
-
365
)
Kanczewska
J
Marco
S
Vandermeeren
S
Maudoux
O
Rigaud
JL
Boutry
M
Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3 proteins converts a dimer into a hexamer
Proc. Natl Acad. Sci. USA
 , 
2005
, vol. 
102
 (pg. 
11675
-
11680
)
Katou
K
Okamoto
H
Symplast as a functional unit in plant growth
Int. Rev. Cytol.
 , 
1992
, vol. 
142
 (pg. 
263
-
304
)
Kerkeb
L
Venema
K
Donaire
JP
Rodríguez-Rosales
MP
Enhanced H+/ATP coupling ratio of H+-ATPase and increased 14-3-3 protein content in plasma membrane of tomato cells upon osmotic shock
Physiol. Plant.
 , 
2002
, vol. 
116
 (pg. 
37
-
41
)
Kim
TH
Böhmer
M
Hu
H
Nishimura
N
Schroeder
JI
Guard cell signal transduction network: advances in understanding abscisic acid, CO2, and Ca2+ signaling
Annu. Rev. Plant Biol.
 , 
2010
, vol. 
61
 (pg. 
561
-
591
)
Kinoshita
T
Hayashi
Y
New insights into the regulation of stomatal opening by blue light and plasma membrane H+-ATPase
Int. Rev. Cell Mol. Biol.
 , 
2011
, vol. 
289
 (pg. 
89
-
115
)
Kinoshita
T
Ono
N
Hayashi
Y
Morimoto
S
Nakamura
S
Soda
M
, et al.  . 
FLOWERING LOCUS T regulates stomatal opening
Curr. Biol.
 , 
2011
, vol. 
21
 (pg. 
1232
-
1238
)
Kinoshita
T
Shimazaki
K
Blue light activates the plasma membrane H+-ATPase by phosphorylation of the C-terminus in stomatal guard cells
EMBO J.
 , 
1999
, vol. 
18
 (pg. 
5548
-
5558
)
Kinoshita
T
Shimazaki
K
Analysis of the phosphorylation level in guard-cell plasma membrane H+-ATPase in response to fusicoccin
Plant Cell Physiol.
 , 
2001
, vol. 
42
 (pg. 
424
-
432
)
Kinoshita
T
Shimazaki
K
Biochemical evidence for the requirement of 14-3-3 protein binding in activation of the guard-cell plasma membrane H+-ATPase by blue light
Plant Cell Physiol.
 , 
2002
, vol. 
43
 (pg. 
1359
-
1365
)
Koizumi
Y
Hara
Y
Yazaki
Y
Sakano
K
Ishizawa
K
Involvement of plasma membrane H+-ATPase in anoxic elongation of stems in pondweed (Potamogeton distinctus) turions
New Phyt.
 , 
2011
, vol. 
190
 (pg. 
421
-
430
)
Leonhardt
N
Kwak
JM
Robert
N
Waner
D
Leonhardt
G
Schroeder
JI
Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant
Plant Cell
 , 
2004
, vol. 
16
 (pg. 
596
-
615
)
Leung
J
Bouvier-Durand
M
Morris
PC
Guerrier
D
Chefdor
F
Giraudat
J
Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase
Science
 , 
1994
, vol. 
264
 (pg. 
1448
-
1452
)
Ma
Y
Szostkiewicz
I
Korte
A
Moes
D
Yang
Y
Christmann
A
, et al.  . 
Regulators of PP2C phosphatase activity function as abscisic acid sensors
Science
 , 
2009
, vol. 
324
 (pg. 
1064
-
1068
)
Meyer
K
Leube
MP
Grill
E
A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana
Science
 , 
1994
, vol. 
264
 (pg. 
1452
-
1455
)
Niittylä
T
Fuglsang
AT
Palmgren
MG
Frommer
WB
Schulze
WX
Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis
Mol. Cell Proteomics
 , 
2007
, vol. 
6
 (pg. 
1711
-
1726
)
Nishimura
N
Sarkeshik
A
Nito
K
Park
SY
Wang
A
Carvalho
PC
, et al.  . 
PYR/PYL/RCAR family members are major in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis
Plant J.
 , 
2010
, vol. 
61
 (pg. 
290
-
299
)
Okumura
M
Inoue
S
Takahashi
K
Ishizaki
K
Kohchi
T
Kinoshita
T
Characterization of the plasma membrane H+-ATPase in the liverwort Marchantia polymorpha
Plant Physiol.
 , 
2012a
, vol. 
159
 (pg. 
826
-
834
)
Okumura
M
Takahashi
K
Inoue
S
Kinoshita
T
Evolutionary appearance of the plasma membrane H+-ATPase containing a penultimate threonine in the bryophyte
Plant Signal. Behav.
 , 
2012b
, vol. 
7
 (pg. 
979
-
982
)
Palmgren
MG
Plant plasma membrane H+-ATPases: powerhouses for nutrient uptake
Annu. Rev. Plant Physiol. Plant Mol. Biol.
 , 
2001
, vol. 
52
 (pg. 
817
-
845
)
Park
SY
Fung
P
Nishimura
N
Jensen
DR
Fujii
H
Zhao
Y
, et al.  . 
Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins
Science
 , 
2009
, vol. 
324
 (pg. 
1068
-
1071
)
Philippar
K
Büchsenschütz
K
Edwards
D
Löffler
J
Lüthen
H
Kranz
E
, et al.  . 
The auxin-induced K+ channel gene Zmk1 in maize functions in coleoptile growth and is required for embryo development
Plant Mol. Biol.
 , 
2006
, vol. 
61
 (pg. 
757
-
768
)
Rayle
DL
Cleland
R
Enhancement of wall loosening and elongation by acid solutions
Plant Physiol.
 , 
1970
, vol. 
46
 (pg. 
250
-
253
)
Rudashevskaya
EL
Ye
J
Jensen
ON
Fuglsang
AT
Palmgren
MG
Phosphosite mapping of the P-type plasma membrane H+-ATPase in homologous and heterologous environments
J. Biol. Chem.
 , 
2012
, vol. 
287
 (pg. 
4904
-
4913
)
Sondergaard
TE
Schulz
A
Palmgren
MG
Energization of transport processes in plants. Roles of the plasma membrane H+-ATPase
Plant Physiol.
 , 
2004
, vol. 
136
 (pg. 
2475
-
2482
)
Svennelid
F
Olsson
A
Piotrowski
M
Rosenquist
M
Ottman
C
Larsson
C
, et al.  . 
Phosphorylation of Thr-948 at the C terminus of the plasma membrane H+-ATPase creates a binding site for the regulatory 14-3-3 protein
Plant Cell
 , 
1999
, vol. 
11
 (pg. 
2379
-
2391
)
Takahashi
K
Hayashi
K
Kinoshita
T
Auxin activates the plasma membrane H+-ATPase by phosphorylation during hypocotyl elongation in Arabidopsis
Plant Physiol.
 , 
2012
, vol. 
159
 (pg. 
632
-
641
)
Tsuzuki
T
Takahashi
K
Tomiyama
M
Inoue
S
Kinoshita
T
Overexpression of the Mg-chelatase H subunit in guard cells confers drought tolerance via promotion of stomatal closure in Arabidopsis thaliana
Front. Plant Sci.
 , 
2013
, vol. 
4
 pg. 
440
 
Vanneste
F
Friml
J
Auxin: a trigger for change in plant development
Cell
 , 
2009
, vol. 
136
 (pg. 
1005
-
1016
)
Wakabayashi
K
Sakurai
N
Kuraishi
S
Role of the outer tissue in abscisic acid-mediated growth suppression of etiolated squash hypocotyl segments
Physiol. Plant.
 , 
1989
, vol. 
75
 (pg. 
151
-
156
)
Zhang
X
Wang
H
Takemiya
A
Song
CP
Kinoshita
T
Shimazaki
K
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
Plant Physiol.
 , 
2004
, vol. 
136
 (pg. 
4150
-
4158
)