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. 2016 Mar 25;171(1):580–589. doi: 10.1104/pp.16.00355

Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation1,[OPEN]

Masaki Okumura 1, Shin-ichiro Inoue 1, Keiko Kuwata 1, Toshinori Kinoshita 1,*
PMCID: PMC4854722  PMID: 27016447

Photosynthesis activates the plasma membrane H+-ATPase in Arabidopsis mesophyll cells through C-terminal phosphorylation, and this activation is mediated by photosynthetic sugars, including sucrose.

Abstract

Plant plasma membrane H+-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H+-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha. However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H+-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H+-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H+-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H+-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H+-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H+-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H+-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism.

Photosynthesis is the planet’s essential biochemical reaction. It converts the photon flux of sunlight into the chemical energy required by nearly all organisms on Earth. Plants produce carbohydrates and oxygen from carbon dioxide and water through the photosynthetic process. Photosynthetic sugars are used not only as energy sources but also as signaling molecules in plant life cycles (Smeekens, 2000; Rolland et al., 2002, 2006; Lastdrager et al., 2014). Plant sugar levels are influenced by biotic and abiotic stresses (Roitsch, 1999; Roitsch and González, 2004). Plants sense these stresses by monitoring sugar levels and adapt to ever-changing environments by regulating their metabolism, growth, and development. Plant growth and development are also controlled by plant hormones, and sugar signaling is closely coordinated with plant hormone signaling (León and Sheen, 2003; Ljung et al., 2015).

Plasma membrane H+-ATPase, a crucial enzyme for plant life, acts as a primary transporter in fungi and plants. It actively transports H+ to extracellular spaces using the energy provided by ATP hydrolysis. This mechanism regulates pH homeostasis and membrane potential and creates a driving force for a variety of solute transport processes operating via secondary transporters (Palmgren, 2001). H+-ATPase is responsible for diverse physiological processes, including nutrient uptake in roots, stomatal opening, phloem loading, and cell expansion (Palmgren, 2001; Duby and Boutry, 2009). H+-ATPase is kept in a low-activity state by its C-terminal autoinhibitory domain; it is activated through the phosphorylation of a penultimate Thr and subsequent binding of 14-3-3 proteins to its phosphorylated C terminus in response to physiological stimuli, such as blue light in guard cells, the plant hormone auxin, and Suc in elongating tissues (Fuglsang et al., 1999; Kinoshita and Shimazaki, 1999; Svennelid et al., 1999; Maudoux et al., 2000; Niittylä et al., 2007; Takahashi et al., 2012). This process is probably the primary mechanism by which H+-ATPase is activated.

Photosynthesis regulates ion transport across the plasma membrane (Spanswick, 1981; Marten et al., 2010). Light-induced hyperpolarization of the plasma membrane has been particularly well studied in a diversity of plants, including Chara corallina and Vallisneria spiralis (Prins et al., 1980; Mimura and Tazawa, 1986). Photosynthesis-dependent membrane hyperpolarization is believed to result from activation of the plasma membrane H+-ATPase. In Vallisneria gigantia, H+-ATPase is likely involved in photosynthesis-dependent membrane hyperpolarization in the mesophyll cells (Harada et al., 2002). Our recent studies showed that photosynthesis induces the phosphorylation of the penultimate Thr of H+-ATPase in the nonvascular bryophytes Marchantia polymorpha and Physcomitrella patens (Okumura et al., 2012a, 2012b). Although it is supposed that photosynthesis induces the activation of the H+-ATPase in large plant species, it remains unknown whether photosynthesis-dependent phosphorylation of H+-ATPase is conserved in vascular plants, and the signaling mechanism by which photosynthesis controls the phosphorylation status of H+-ATPase is unclear.

In this study, we show that light illumination induces the phosphorylation of H+-ATPase in the mesophyll cells of Arabidopsis (Arabidopsis thaliana) in a photosynthesis-dependent manner. Furthermore, we demonstrate that (1) exogenous and endogenous sugars induce the phosphorylation of H+-ATPase and (2) the defect of carbohydrate production suppresses the light-induced phosphorylation of H+-ATPase. Our investigation shows that photosynthetically produced sugar activates H+-ATPase through phosphorylation.

RESULTS

Photosynthesis Activates Plasma Membrane H+-ATPase in Arabidopsis Leaves via Phosphorylation

To determine whether H+-ATPase is phosphorylated by light in the vascular plant Arabidopsis, we illuminated detached dark-adapted leaves with white light. The phosphorylation status of the penultimate Thr of H+-ATPase was detected by immunoblot analyses using the antibody against the phosphorylated penultimate Thr of H+-ATPase (anti-pThr; Hayashi et al., 2010). Illumination of dark-adapted leaves induced the phosphorylation of H+-ATPase with no changes in the quantity of H+-ATPase (Fig. 1A). We also measured the ATP hydrolytic activity of H+-ATPase in microsomal membranes isolated from light-treated and dark-adapted leaves. ATP hydrolytic activity was 1.5-fold higher in light-treated leaves than in dark-adapted leaves (Fig. 1B).

Figure 1.

Figure 1.

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Light-induced activation of plasma membrane H+-ATPase in Arabidopsis leaves via C-terminal phosphorylation. A, Light-induced phosphorylation of the penultimate Thr of H+-ATPase in leaves. Dark-adapted leaves were kept in darkness (Dk) or illuminated with white light (WL; 50 µmol m–2 s–1) for 30 min and then ground. Protein extracts from the leaves were used for immunoblots of phosphorylated H+-ATPase (top; anti-pThr) and total H+-ATPase (bottom; anti-H+-ATPase). Experiments were repeated on at least three occasions with similar results. B, Increase in ATP hydrolytic activity of H+-ATPase in leaves in response to light. Microsomal membranes were prepared from light- or dark-treated leaves. ATP hydrolysis was measured by determining the inorganic phosphate release from ATP in microsomal proteins. Values are means ± sd (n = 3 independent experiments). Asterisks indicate a significant difference between darkness and white light (**, Student’s t test; P < 0.01). C, Time course of H+-ATPase phosphorylation in response to light. Dark-adapted leaves were illuminated with white light (50 µmol m–2 s–1) for 0, 15, 30, 60, and 120 min. Immunoblotting was performed as detailed in A. The phosphorylation level was determined using the ratio of phosphorylated H+-ATPase band intensity to H+-ATPase band intensity; the relative phosphorylation level was expressed as the phosphorylation level ratio of leaves kept in darkness. Values are means ± sd (n = 3 independent experiments). D, Time course of H+-ATPase dephosphorylation in response to light extinction. Dark-adapted leaves were illuminated with white light (50 µmol m–2 s–1) for 30 min and then kept in darkness for 0, 30, 60, and 120 min. Immunoblotting and the quantification of phosphorylation levels were performed as detailed in C. The relative phosphorylation level was expressed as the phosphorylation level ratio of leaves illuminated for 30 min. Values are means ± sd (n = 3 independent experiments). *, P < 0.05 and **, P < 0.01, significant differences from 0 min by Student’s t test.

We subsequently examined the time courses of H+-ATPase phosphorylation and dephosphorylation. The phosphorylation status of H+-ATPase reached a peak after 30 min of illumination (Fig. 1C). The phosphorylation status of the H+-ATPase decreased gradually after the illumination period ended and returned to the original status after approximately 120 min (Fig. 1D). Thus, the H+-ATPase in Arabidopsis leaves was reversibly phosphorylated and activated by light.

To determine whether photosynthesis regulates the phosphorylation of H+-ATPase, we examined the effect of two inhibitors of photosynthetic electron transport, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), on the light-induced phosphorylation of H+-ATPase. DCMU and DBMIB at a concentration of 10 µm strongly inhibited light-induced phosphorylation of H+-ATPase (Fig. 2A; Supplemental Fig. S1). To genetically corroborate our findings, we examined the light-induced phosphorylation of H+-ATPase in the yellow variegated2 (var2-2) mutant of Arabidopsis, which has variegated leaves with no photosynthetic activity in its white sections (Fig. 2B; Kato et al., 2007; Yoshida et al., 2008). Light-induced phosphorylation of H+-ATPase was strongly suppressed in this mutant (Fig. 2C). These findings were congruent with results showing that the major photoreceptor mutants, including the phytochrome double mutant (phyA-201 phyB-1), the cryptochrome double mutant (cry1-304 cry2-1), and the phototropin double mutant (phot1-5 phot2-1), had normal light-induced phosphorylation of H+-ATPase (Fig. 2D). These results indicate that phosphorylation was not mediated by these photoreceptors in Arabidopsis but by photosynthesis, which also was the case in M. polymorpha (Okumura et al., 2012a).

Figure 2.

Figure 2.

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Light-induced H+-ATPase phosphorylation dependence on photosynthesis. A, Effect of DCMU on the light-induced phosphorylation of H+-ATPase. Dark-adapted leaves were either pretreated or not pretreated with 10 µm DCMU for 1 h in darkness (Dk) and then illuminated with white light (WL; 50 µmol m–2 s–1) for 30 min. Immunoblotting was performed as detailed in Figure 1A. B, Visible phenotypes of Columbia (Col) and var2-2 plants. Plants were grown for 4 weeks. Bars = 1 cm. C, Light-induced H+-ATPase phosphorylation in Col and var2-2 leaves. Experimental details are as in Figure 1A. D, Light-induced H+-ATPase phosphorylation in leaves from the photoreceptor mutants phyA phyB, cry1 cry2, and phot1 phot2. Experiments were performed as detailed in Figure 1A. All immunoblotting was repeated on at least three occasions with similar results.

Photosynthesis-Dependent Phosphorylation of H+-ATPase Occurs in Mesophyll Cells

To identify the tissue in which photosynthesis regulates the phosphorylation status of H+-ATPase, we performed an immunohistochemical procedure for detecting H+-ATPase in transverse sections of rosette leaves; we used an anti-H+-ATPase for this purpose. As shown in Figure 3A, the fluorescence signal from anti-H+-ATPase was detectable in all leaf section tissues and was especially strong in vascular cells. When the preserum was used in place of the primary antibody, we detected no significant signals in leaf sections (Fig. 3B).

Figure 3.

Figure 3.

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Autonomous mesophyll cell H+-ATPase phosphorylation by light. A and B, Immunohistochemical detection of H+-ATPase in transverse sections of rosette leaves. Fluorescence images were obtained using anti-H+-ATPase (A) and preserum (B). Bars = 100 µm. C, Immunohistochemical detection of H+-ATPase phosphorylation by light in transverse leaf sections. Fluorescence images were obtained using anti-pThr. Dark-adapted leaves were kept in darkness (Dk) or illuminated with white light (WL; 50 µmol m–2 s–1) for 30 min. Areas in the white boxes are provided as magnified versions in the bottom images. White arrows indicate fluorescence signals from the peripheral regions of mesophyll cells. Bars = 100 µm. D, Red light-induced phosphorylation of H+-ATPase in MCPs. MCPs were pretreated or not pretreated with 10 µm DCMU for 20 min in darkness and then kept in darkness or illuminated with red light (RL; 600 µmol m–2 s–1) for 30 min. Immunoblotting was performed as detailed in Figure 1A. E, Effect of red light on H+-ATPase phosphorylation in GCPs. GCPs were kept in darkness or illuminated with red light (600 µmol m–2 s–1) for 30 min. Immunoblotting was performed as detailed in Figure 1A. All immunoblotting was repeated on at least three occasions with similar results.

We subsequently performed an immunohistochemical procedure to detect the phosphorylated H+-ATPase; we used anti-pThr for this purpose. In dark-adapted leaves, the fluorescence signal from anti-pThr was strong in vascular tissues but weak in the peripheral regions of mesophyll cells (Fig. 3C, left). After the light treatment, the signal in the peripheral region of mesophyll cells increased in strength (Fig. 3C, right). In contrast, the strength of the signals in vascular tissues was unaffected by the level of illumination (Fig. 3C). Thus, light-induced phosphorylation of H+-ATPase occurred in mesophyll cells. Point signals, probably from chloroplasts, also were detected by anti-pThr but not by anti-H+-ATPase (Fig. 3, A and C), although anti-pThr specifically recognizes the phosphorylated penultimate Thr of H+-ATPase in immunoblot analyses (Supplemental Fig. S2). These results suggest that the anti-pThr-detected point signals likely were due to nonspecific fluorescence made visible by the immunohistochemical procedure.

We also examined the light-induced phosphorylation of H+-ATPase in mesophyll cell protoplasts (MCPs) using an immunoblot analysis. Strong red light illumination of MCPs induced the phosphorylation of H+-ATPase; DCMU inhibited the light-induced phosphorylation of the H+-ATPase in both MCPs and rosette leaves (Fig. 3D). These results are consistent with those of the immunohistochemical analyses and indicate that photosynthesis-dependent phosphorylation of the H+-ATPase occurred autonomously in mesophyll cells. We also investigated the light-induced phosphorylation of H+-ATPase in stomatal guard cells, which contain chloroplasts. The H+-ATPase in guard cell protoplasts (GCPs) was not phosphorylated in response to red light (Fig. 3E).

sucrose proton symporter2 Mutants Show High Levels of H+-ATPase Phosphorylation in Darkness

To determine the mechanism by which photosynthesis controls the phosphorylation status of H+-ATPase, we screened ethyl methanesulfonate-mutagenized Arabidopsis plants for their H+-ATPase phosphorylation status and found a mutant with high phosphorylation levels and activities of H+-ATPase in darkness (Fig. 4, A and B). To identify the responsible gene in this mutant, we performed map-based cloning to map the candidate mutation, which was located on chromosome 1. Sequencing of the candidate genes revealed a point mutation in the SUCROSE PROTON SYMPORTER2 (SUC2) locus, which resulted in the substitution of Leu-282 for Phe in the seventh transmembrane domain (Fig. 4C). Accordingly, we assigned the name suc2-7 to the mutant. suc2-7 mutant plants had dwarf phenotypes and accumulated anthocyanin, Suc, and starch in mature leaves, as reported previously in other T-DNA insertion mutants of the SUC2 gene (Fig. 4D; Supplemental Fig. S3; Gottwald et al., 2000; Lloyd and Zakhleniuk, 2004). It was reported that anthocyanin accumulation is mediated by Suc-specific induction of the anthocyanin biosynthetic pathway (Solfanelli et al., 2006). The T-DNA insertion knockout line of SUC2 (suc2-5) also had high phosphorylation levels of H+-ATPase in darkness (Fig. 4E). Transformation with a wild-type genomic fragment of the SUC2 gene complemented the visible phenotypes of suc2-7 (Fig. 4F) and restored the low phosphorylation level of H+-ATPase in darkness (Fig. 4G). Thus, the suc2 mutation was responsible for a high H+-ATPase phosphorylation status in darkness.

Figure 4.

Figure 4.

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Increase in H+-ATPase phosphorylation and activity in suc2 mutants. A and B, Light-induced phosphorylation (A) and activation (B) of H+-ATPase in Col and suc2-7 leaves. Experiments were performed as detailed in Figure 1, A and B. Values are means ± sd (n = 5 independent experiments). Asterisks indicate significant differences from Col leaves kept in darkness (Dk; Student’s t test; *, P < 0.05). WL, White light. C, Genomic structure of the SUC2 gene. Black boxes indicate exons. The suc2-7 mutant has a single nucleotide substitution (C to T) in the first exon. The substitution caused an amino acid change from Leu-282 to Phe. The transfer DNA (T-DNA) insertion in suc2-5 is shown. D, Visible phenotypes of Col and suc2 plants. Plants were grown for 6 weeks. Bar = 1 cm. E, Light-induced H+-ATPase phosphorylation in Col and suc2-5. Immunoblotting was performed as detailed in Figure 1A. F and G, Complementation of growth (F) and increased H+-ATPase phosphorylation (G) in suc2-7 caused by a wild-type SUC2 gene. A 5.3-kb genomic fragment of the SUC2 gene was prepared from the wild type (Col) and introduced into the suc2-7 mutant. The T3 generation of the transgenic plants (gSUC2/suc2-7 #1, #2, and #3) was used in the experiment. Plants were grown for 4 weeks. Immunoblotting was performed as detailed in Figure 1A. All immunoblotting was repeated on at least three occasions with similar results. Bars = 1 cm.

Sugars Mediate Light-Induced Phosphorylation of H+-ATPase

Previous reports indicate that SUC2 is specifically expressed in companion cells and regulates Suc loading from the apoplast into the phloem (Gottwald et al., 2000). The T-DNA insertion mutants of SUC2 fail to export Suc from source leaves to sink tissues and accumulate sugars in mature leaves (Gottwald et al., 2000; Srivastava et al., 2008). We found that mature leaves of suc2-7 accumulated Suc to higher levels than the wild type; however, the Suc accumulation level in young leaves was comparable to that of the wild type (Fig. 5A). In agreement with our results on Suc accumulation, the H+-ATPase phosphorylation levels in suc2-7 plants were low in young leaves but high in mature leaves kept in darkness (Fig. 5B). Furthermore, Suc induced H+-ATPase phosphorylation in the rosette leaves of the wild type (Fig. 5C), as demonstrated previously in Arabidopsis seedlings and M. polymorpha thalli (Niittylä et al., 2007; Okumura et al., 2012a). In contrast, Suc did not further elevate the H+-ATPase phosphorylation status of suc2-7 (Fig. 5C), since the H+-ATPase phosphorylation level in suc2-7 may be saturated in darkness due to a high level of endogenous Suc. These findings indicate that the accumulation of endogenous Suc occurred in concert with increasing H+-ATPase phosphorylation. Therefore, we postulate that light-induced H+-ATPase phosphorylation is mediated by endogenous sugars present as photosynthetic products.

Figure 5.

Figure 5.

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Light-induced H+-ATPase phosphorylation requirement for photosynthetic sugar accumulation. A and B, Suc accumulation (A) and phosphorylation of H+-ATPase (B) in young and mature leaves of suc2-7. Young and mature leaves were detached from dark-adapted suc2-7 plants and kept in darkness (Dk) for 30 min. The detached wild-type leaves were kept in darkness or illuminated with white light (WL; 50 µmol m–2 s–1) for 30 min. The Suc content in leaves was measured by liquid chromatography-mass spectrometry (A); phosphorylation of H+-ATPase was determined by immunoblots as detailed in Figure 1A (B). Values are means ± sd (n = 3 independent experiments). Asterisks indicate significant differences from Col leaves in the dark (Student’s t test; **, P < 0.01). FW, Fresh weight. C, Phosphorylation of H+-ATPase in response to Suc application to the leaves of Col and suc2-7. Dark-adapted leaves were treated with 30 mm Suc or mannitol (Man) for 30 min in the dark. Immunoblotting was performed as detailed in Figure 1A. Mannitol was used as an osmotic control. D, Time course of Suc, Glc, and Fru contents in leaves in response to light. Dark-adapted leaves were illuminated with white light (50 µmol m–2 s–1) for 5, 15, 30, 60, and 120 min. Sugar content was measured as detailed in A. Values are means ± sd (n = 3 independent experiments). E, Time course of Suc, Glc, and Fru contents in leaves in response to light extinction. Dark-adapted leaves were illuminated with white light (50 µmol m–2 s–1) for 30 min and then kept in darkness for 0, 30, 60, and 120 min. Sugar content was measured as detailed in A. Values are means ± sd (n = 3 independent experiments). Gray and white areas represent darkness and white light illumination, respectively. F, Effect of DCMU on Suc accumulation in leaves. Treatments with DCMU and light, and measurement of Suc, were performed as detailed in Figure 2A and A, respectively. Values are means ± sd (n = 3 independent experiments). Asterisks indicate a significant difference from Col leaves kept in darkness (Student’s t test; **, P < 0.01). G, Light-induced phosphorylation of H+-ATPase in the adg1-1 tpt-2 mutant. Experiments were performed as detailed in Figure 1A. All immunoblotting was repeated on at least three occasions with similar results.

To test this postulate, we examined the time courses of Suc, Glc, Fru, Glc-1-P, and Glc-6-P contents after exposure to light. Suc accumulation began within 15 min of the onset of illumination and saturated after 1 h (Fig. 5D). Glc and Fru accumulated gradually compared with Suc accumulation (Fig. 5D). Glc-1-P and Glc-6-P increased rapidly in response to illumination and reached saturation within 5 min of the onset of illumination (Supplemental Fig. S4A). In addition, we found that exogenous Glc, Fru, Glc-1-P, and Glc-6-P also induced H+-ATPase phosphorylation (Supplemental Fig. S5). We also investigated the time courses of sugar contents after the end of illumination under the same conditions as in Figure 1D. Suc decreased gradually after the end of the illumination period, while Glc and Fru remained largely unchanged because they did not increase under this light condition (Fig. 5E). Glc-1-P and Glc-6-P increased rapidly in response to illumination and decreased to the basal level within 30 min after the end of the illumination period (Supplemental Fig. S4B). These results suggest that the time course of Suc content closely tracked that of H+-ATPase phosphorylation and dephosphorylation (Fig. 1, C and D). Inhibition of photosynthesis by DCMU totally suppressed Suc accumulation in leaves exposed to light (Fig. 5F). Furthermore, we examined light-induced H+-ATPase phosphorylation in a double mutant defective in ADP-Glc pyrophosphorylase and triose phosphate/phosphate translocator (TPT; adg1-1 tpt-2) activities; this mutant has defective carbohydrate production in the leaves (Schmitz et al., 2012). As expected, light-induced H+-ATPase phosphorylation was suppressed markedly in adg1-1 tpt-2 (Fig. 5G). These results indicate that photosynthetic sugars mediate H+-ATPase phosphorylation.

DISCUSSION

Photosynthesis regulates diverse ion transports across the plasma membrane in many plant species (Marten et al., 2010). Plasma membrane H+-ATPase is responsible for photosynthesis-dependent H+ transport, which causes membrane hyperpolarization. However, little is known of the mechanism by which photosynthesis activates H+-ATPase (Harada et al., 2002). In bryophytes, photosynthesis induces the phosphorylation of the penultimate Thr of H+-ATPase, which is required for the activation of H+-ATPase (Okumura et al., 2012a, 2012b). However, whether photosynthesis-dependent phosphorylation of H+-ATPase occurs in vascular plants remains an unresolved issue. In this study, we demonstrated that photosynthesis activates H+-ATPase via phosphorylation in a process mediated by photosynthetic sugars in Arabidopsis leaves. Photosynthesis-dependent phosphorylation and activation of H+-ATPase occurred in Arabidopsis leaves (Figs. 1 and 2), just as in bryophytes (Fig. 6; Okumura et al., 2012a, 2012b). This response also was observed in the leaves of other vascular plants, including rice (Oryza sativa), Nicotiana benthamiana, and fava bean (Vicia faba; Fig. 6). Therefore, photosynthesis-dependent activation of H+-ATPase through the phosphorylation of the penultimate Thr is likely common among land plants. We also showed that photosynthesis-induced phosphorylation occurs in MCPs (Fig. 3D).

Figure 6.

Figure 6.

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Light-induced phosphorylation of H+-ATPase in thalli of M. polymorpha and leaves of rice, N. benthamiana (Tobacco), and fava bean. Dark-adapted thalli and leaves were kept in darkness (Dk) or illuminated with white light (WL; 50 µmol m–2 s–1) for 30 min. Immunoblotting was performed as detailed in Figure 1A. All immunoblotting was repeated on at least three occasions with similar results.

The physiological role of the photosynthesis-dependent activation of H+-ATPase has not been fully elucidated. Previous studies have reported that photosynthesis induces H+ efflux, K+ influx, and cell expansion growth in leaf segments (Stahlberg and Van Volkenburgh, 1999; Zivanović et al., 2005). Accordingly, light-induced H+ and K+ flux are probably involved in leaf expansion growth. Photosynthesis-dependent activation of H+-ATPase generates an electrochemical gradient of H+ across the plasma membrane of mesophyll cells. This gradient may drive the efflux of photosynthetic products and the influx of ions and nutrients (which are required for photosynthesis) across the plasma membranes of mesophyll cells. Moreover, depletion of carbon dioxide levels in the extracellular spaces of mesophyll cells through photosynthetic consumption causes alkalization of the apoplast; thus, H+-ATPase activity may maintain pH homeostasis in the photosynthetic process (Neumann and Levine, 1971; Shabala and Newman, 1999). Further research is required for improved understanding of the physiological roles of the light-induced activation of H+-ATPase in the plasma membranes of land plants.

The H+-ATPase phosphorylation status in leaves was low and high in darkness and light, respectively (Fig. 1). We found that the H+-ATPase phosphorylation status of suc2 mutants increased in darkness, likely due to the accumulation of sugars (Figs. 4 and 5). Illumination and Suc application did not further elevate the H+-ATPase phosphorylation status of suc2 mutants (Figs. 4A and 5C). Furthermore, the adg1-1 tpt-2 double mutant had defective sugar production (Schmitz et al., 2012) and reduced H+-ATPase phosphorylation levels compared with the wild type, even under illumination (Fig. 5G). Therefore, the sugars derived from photosynthesis induced H+-ATPase phosphorylation. However, the H+-ATPase phosphorylation level of the adg1-1 tpt-2 double mutant was elevated slightly by illumination. This mutant has low sugar production (Schmitz et al., 2012), which likely explains the low H+-ATPase phosphorylation we observed. Since ATP and reducing equivalents generated by photosynthesis are utilized for sugar production, we suspect that they are also candidates for signaling components of the photosynthesis-dependent phosphorylation of H+-ATPase. However, it was reported that ATP content is not significantly different in dark versus light conditions (Keifer and Spanswick, 1979; Stitt et al., 1982). In addition, sodium hydrosulfite, an efficient reducing agent, did not induce the phosphorylation of H+-ATPase (Supplemental Fig. S6). Furthermore, sugar application clearly increased the phosphorylation of H+-ATPase (Fig. 5C; Supplemental Fig. S5). From these results, we conclude that it is not ATP and reducing equivalents, but rather photosynthetic sugars, that mediate the photosynthesis-dependent phosphorylation of H+-ATPase.

In guard cells, H+-ATPase is activated through the phosphorylation of the penultimate Thr, not via photosynthesis but via phototropins (Fig. 3E; Kinoshita and Shimazaki, 1999; Ueno et al., 2005). In contrast, the H+-ATPase in mesophyll cells was phosphorylated not via phototropins but via photosynthesis (Fig. 2). Chloroplasts in guard cells often are small and sparse compared with those of mesophyll cells; Rubisco activity also is very low in guard cells (Shimazaki et al., 1989; Lawson, 2009). Interestingly, exogenous Suc induced the phosphorylation of H+-ATPase in GCPs (Supplemental Fig. S7). It is possible, therefore, that sugar production by photosynthesis is insufficient for H+-ATPase phosphorylation in Arabidopsis GCPs. Mesophyll cells have functional phototropins, but not BLUE LIGHT SIGNALING1, which mediates phototropin-dependent H+-ATPase phosphorylation in guard cells (Takemiya et al., 2013). Thus, H+-ATPase may not be phosphorylated via phototropins in mesophyll cells. These results suggest that the signals for H+-ATPase activation differ among various cell types and are involved in diverse responses.

Diverse sugars act as signaling molecules; sugar signaling regulates plant hormone biosynthesis and signaling components to control metabolism, growth, and development (Smeekens, 2000; Rolland et al., 2002, 2006; Lastdrager et al., 2014). Glc sensing and signaling are the best characterized among sugars (Rolland et al., 2006; Lastdrager et al., 2014). In this study, exogenous Suc, Glc, Fru, Glc-1-P, and Glc-6-P similarly induced H+-ATPase phosphorylation (Fig. 5C; Supplemental Fig. S5). However, only the time course of Suc content tracked that of H+-ATPase phosphorylation in response to light illumination and extinction (Figs. 1, C and D, and 5, D and E). Glc and Fru content did not increase within 30 min after light illumination, despite an elevation of the H+-ATPase phosphorylation level (Figs. 1C and 5D). Glc-1-P and Glc-6-P contents increased rapidly earlier than H+-ATPase phosphorylation after the onset of illumination and decreased to the basal level within 30 min, earlier than H+-ATPase dephosphorylation after the end of illumination (Fig. 1, D and E; Supplemental Fig. S4). Therefore, we conclude that light-induced H+-ATPase phosphorylation is probably mediated by Suc in leaves in our light conditions. We propose a novel sugar-signaling pathway between the chloroplasts and plasma membrane that regulates the plasma membrane H+-ATPase in response to light.

H+-ATPase is activated via phosphorylation of the penultimate Thr. A recent study showed that the phosphorylated penultimate Thr of H+-ATPase is dephosphorylated directly by the D-clade of type 2C protein phosphatase (PP2C-D), which is inhibited by SMALL AUXIN UP RNA (SAUR) proteins (Spartz et al., 2014). Many SAUR genes are inducible by plant hormones, including auxin, brassinosteroids, and GAs; sugar signaling regulates plant hormone biosynthesis and signaling (Lastdrager et al., 2014; Ljung et al., 2015; Ren and Gray, 2015). These findings suggest that light or Suc may induce the expression of SAUR genes and that H+-ATPase is phosphorylated by the SAUR-mediated inhibition of PP2C-D.

CONCLUSION

In this study, we showed that photosynthesis activates H+-ATPase in Arabidopsis mesophyll cells through the phosphorylation of the penultimate Thr and that this activation is mediated by photosynthetic sugars, including Suc. Light-induced H+-ATPase phosphorylation occurs in other land plants, suggesting that the regulatory mechanism for H+-ATPase is conserved. Our findings provide new insight into the mechanism by which photosynthesis in chloroplasts regulates H+-ATPase at the plasma membrane in mesophyll cells.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) mutants var2-2 (Chen et al., 2000), suc2-5 (Wippel and Sauer, 2012), adg1-1 tpt-2 (Schmitz et al., 2012), cry1-304 cry2-1 (Mockler et al., 2003), gl1, and gl1 phot1-5 phot2-1 (Kinoshita et al., 2001) had a Col background, and phyA-201 phyB-1 (Mazzella et al., 1997) had a Landsberg erecta background. The gl1 mutant was used as a control for the gl1 phot1-5 phot2-1 (phot1 phot2) mutant. Plants were grown on soil for 4 to 6 weeks at 23°C under a 16-h-light/8-h-dark cycle with a photon fluence of 50 µmol m–2 s–1 light. Plants of adg1-1 tpt-2 were grown for 3 to 4 weeks on one-half-strength Murashige and Skoog medium containing 1% (w/v) Suc. Fava bean (Vicia faba) and Nicotiana benthamiana plants were grown for 3 to 4 weeks under the same conditions as the Arabidopsis plants. Rice (Oryza sativa) plants were grown on soil for 4 weeks at 27°C under a 14-h-light/10-h-dark cycle with 130 to 150 µmol m–2 s–1 illumination. The liverwort Marchantia polymorpha was grown following previously described procedures (Okumura et al., 2012a).

Enzymatic Isolation of Protoplasts

The MCPs and GCPs were enzymatically isolated from rosette leaves following a previously described procedure (Ueno et al., 2005) with minor modification. Macerozyme R-10 (Yakult Pharmaceutical Industry) was used in the enzymatic solution instead of Pectolyase Y-23 (Seishin Pharmaceutical). MCPs and GCPs were suspended in a reaction buffer (0.4 m mannitol, 1 mm CaCl2, and 10 mm HEPES-KOH [pH 7.5]) and kept on ice in darkness until used. For investigation of the effect of Suc on H+-ATPase phosphorylation, GCPs were suspended in a buffer containing 5 mm MES-KOH (pH 5.7), 0.4 m mannitol, and 1 mm CaCl2.

Immunoblot Analysis

Rosette leaves (25–30 mg fresh weight) were detached from dark-adapted plants and illuminated with white light (50 µmol m–2 s–1) or kept in darkness for 30 min. The leaves were homogenized in 120 µL of ice-cold homogenization buffer (50 mm MOPS-KOH [pH 7.5], 100 mm NaCl, 2.5 mm EDTA, 10 mm NaF, 5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride [PMSF], and 20 µm leupeptin). The homogenate was centrifuged at 14,000 rpm for 1 min; the supernatant obtained was solubilized by the addition of half the volume of SDS sample buffer (4.5% [w/v] SDS, 30% [w/v] Suc, 25% [v/v] 2-mercaptoethanol, 0.02% [w/v] Coomassie Brilliant Blue, 4.5 mm EDTA, and 45 mm Tris-HCl [pH 8]). The same amount of solubilized protein (15 µL) was loaded into each lane. MCP and GCP suspensions were illuminated with red light (600 µmol m–2 s–1) or kept in darkness for 30 min at room temperature. The protoplasts (60 µg of protein from MCPs and 40 µg of protein from GCPs) were collected by centrifugation at 10,000g for 15 s and then mixed with 50 µL of extraction buffer (10 mm MOPS-KOH [pH 7.5], 2.5 mm EDTA, 1 mm PMSF, and 20 µm leupeptin). The amount of protein was determined using a protein assay kit (Bio-Rad). The mixtures were solubilized with 25 µL of SDS sample buffer. The solubilized proteins were subjected to SDS-PAGE followed by immunoblotting. We used anti-H+-ATPase and anti-pThr to detect H+-ATPase and the phosphorylation of the penultimate Thr of H+-ATPase, respectively (Hayashi et al., 2010). The secondary antibody was goat anti-rabbit IgG-horseradish peroxidase conjugate (Bio-Rad). The chemiluminescence signal was detected using a LightCapture system (ATTO). The signal intensities were analyzed using ImageJ software. We performed data quantification of all immunoblots, and we present them with statistical analyses in Supplemental Figure S8.

Measurement of the ATP Hydrolytic Activity of H+-ATPase

ATP hydrolytic activity was measured following a previously described procedure (Uemura and Yoshida, 1986) with some modifications. Forty-five microliters of microsomal proteins (15 µg) was mixed with 45 µL of ATPase reaction buffer (6 mm MgSO4, 60 mm Tris-MES [pH 6.5], 100 mm KCl, 200 mm KNO3, 1 mm ammonium molybdate, 10 µg mL–1 oligomycin, 0.1% [w/w] Triton X-100, 0.5 mm PMSF, and 10 µm leupeptin). To measure vanadate-sensitive ATP hydrolysis activity, the mixture was combined with or without 2 µL of 10 mm orthovanadate. The reaction was initiated by adding 10 µL of 2 mm ATP. After incubation at 30°C for 30 min, the reaction was terminated by the addition of 1 mL of 1.3% (w/v) SDS, 0.25% (w/v) sodium molybdate, and 0.3 n H2SO4. The inorganic phosphate release was measured by a previously described procedure (Kinoshita and Shimazaki, 1999).

Immunohistochemical Analysis

Immunohistochemical analyses were performed following previously described procedures (Hayashi et al., 2011; Takahashi et al., 2012) with some modifications. Rosette leaves were detached and illuminated in white light as described above. The leaves were fixed in methanol for 1 h at –25°C. Antigens were retrieved in 1 mm EDTA (pH 8) at 105°C for 1 min. H+-ATPase and the phosphorylated penultimate Thr of H+-ATPase were detected with anti-H+-ATPase and anti-pThr, respectively. As a secondary antibody, we used Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen). Fluorescence was observed with a fluorescence microscope (BX50; Olympus).

Isolation of Mutants with Impaired Light-Dependent H+-ATPase Phosphorylation in Their Rosette Leaves

Ethyl methanesulfonate-mutagenized M2 Col seeds were prepared (Lightner and Caspar, 1998). We screened 5,000 ethyl methanesulfonate mutant plants and isolated a mutant lacking light-induced H+-ATPase phosphorylation. The phosphorylation of H+-ATPase was determined by dot blotting rather than immunoblotting to improve throughput. Detached leaves from the dark-adapted plants were subjected to illumination or darkness, as detailed above, and then were ground in homogenization buffer using a ShakeMaster Auto version 1.5 device (BioMedical Science). The extracts were centrifuged at 2,500g for 5 min, and 5 µL of the supernatant was mixed with 120 µL of homogenization buffer. The mixtures were loaded onto a nitrocellulose membrane with a 96-well Bio-Dot Apparatus (Bio-Rad). H+-ATPase and H+-ATPase phosphorylation were detected with anti-H+-ATPase and anti-pThr, respectively. We confirmed the phosphorylation phenotype by immunoblotting the M3 generation of the mutants obtained.

Transgenic Plant Generation

For complementation analysis, a gene-transfer vector bearing the wild-type SUC2 genomic fragment was constructed. The genomic SUC2 fragment, including the promoter and 3′ untranslated regions, was amplified by PCR from the genomic DNA of Col using the primers 5′-GCAGGATCCTCTGGTTTCATATTAATTTCACACACC-3′ and 5′-GCAGGATCCTGTTTGAAGAACCATGTTCGATTCG-3′. The amplified fragment was cloned into the BamHI site of pCAMBIA1300 (Cambia). The construct was introduced into the suc2-7 mutant through the Agrobacterium tumefaciens-mediated procedure (Clough and Bent, 1998).

Measurement of Sugar Accumulation by Liquid Chromatography-Mass Spectrometry

Sugars were extracted from the plant tissues following a previously described procedure with some modifications (Kusano et al., 2007). Detached leaves that had been kept either in darkness or under light were immediately frozen in liquid nitrogen and homogenized in an extraction medium (methanol:chloroform:water [3:1:1, v/v/v], 50 µm of each deuterated sugar [d-Fru, d-Glc, and d-Suc; CIL]; 25 mg of plant material in 50 µL of extraction medium). In the case of the suc2-7 mutant, each deuterated sugar was used at a concentration of 500 µm. The extracts were centrifuged at 15,000g for 5 min, and the supernatant was subjected to liquid chromatography-mass spectrometry analysis (Dionex Ultimate 3000 HPLC system equipped with an autosampler and an EXACTIVE Plus mass spectrometer [Thermo] fitted with a Unison UK-Amino column [3 µm, 4.6 × 250 mm; Imtakt]) at 60°C using 1 mm aqueous ammonium acetate (pH 7) and acetonitrile (1:9, v/v; 500 µL min–1, isocratic). For the Glc-1-P and Glc-6-P quantitative measurements, a Scherzo SM-C18 column (3 µm, 2.1 × 150 mm; Imtakt) was used at 37°C with 0.1% (v/v) aqueous formic acid (200 µL min–1, isocratic). Samples were detected in the electrospray ionization negative ion mode. Fru, Glc, and Suc were quantified against the corresponding deuterated sugars as internal standards. Glc-1-P and Glc-6-P were quantified using authentic standard Glc-1-P and Glc-6-P (Sigma) as external standards. Data were acquired and analyzed with Xcalibur version 2.2 software (Thermo).

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: VAR2 (At2g30950), PHYA (At1g09570), PHYB (At2g18790), CRY1 (At4g08920), CRY2 (At1g04400), GL1 (At3g27920), PHOT1 (At3g45780), PHOT2 (At5g58140), SUC2 (At1g22710), ADG1 (At5g48300), and TPT (At5g46110).

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_171_1_580__index.html (994B, html)

Acknowledgments

We thank Dr. Anzu Minami for technical advice on the measurement of ATP hydrolytic activity, Dr. Koji Takahashi for technical advice on the immunohistochemical analysis, and Tameo You for technical assistance in mutant screening.

Glossary

DCMU

3-(3,4-dichlorophenyl)-1,1-dimethylurea

DBMIB

2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone

MCP

mesophyll cell protoplast

GCP

guard cell protoplast

T-DNA

transfer DNA

Col

Columbia

PMSF

phenylmethylsulfonyl fluoride

Footnotes

1

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (grant nos. 15H05956 and 15H04386 to T.K.) and by a Grant-in-Aid for Japan Society for the Promotion of Science fellows (grant no. 253307 to M.O.).

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