Characterization of the plasma membrane H + -ATPase in the liverwort Marchantia polymorpha

The plasma membrane H + -ATPase generates an electrochemical gradient of H + across the plasma membrane that provides the driving force for solute transport and regulates pH homeostasis and membrane potential in plant cells. Recent studies have demonstrated that phosphorylation of the penultimate threonine (Thr) in H + -ATPase and subsequent binding of a 14-3-3 protein is the major common activation mechanism for H + -ATPase in vascular plants. However, there is very little information on the plasma membrane H + -ATPase in non-vascular plant bryophyte. Here, we show that the liverwort Marchantia polymorpha , which is the most basal lineage of extant land plants, expresses both the penultimate Thr-containing H + -ATPase (pT H + -ATPase) and non-pT H + -ATPase as in the green algae, and that pT H + -ATPase is regulated by phosphorylation of its penultimate Thr. A search in the EST database of M. polymorpha revealed eight H + -ATPase genes, designated MpHA ( Marchantia polymorpha H + -ATPase). Four isoforms are the pT H + -ATPase; the remaining isoforms are non-pT H + -ATPase. An apparent 95-kD protein was recognized by anti-H + -ATPase antibodies against an Arabidopsis isoform and was phosphorylated on the penultimate Thr in response to fungal toxin fusicoccin in thalli, indicating that the 95-kD protein contains pT H + -ATPase. Furthermore, we found that the pT H + -ATPase in thalli is phosphorylated in response to light, sucrose, and osmotic shock, and that light-induced phosphorylation depends on photosynthesis. Our results define physiological signals for regulation of pT H + -ATPase in the liverwort M. polymorpha , which is one of the earliest plants to acquire pT H + -ATPase. plant plasma membrane H + -ATPase expressed in yeast is activated by phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins Chlamydomonas genome reveals the evolution of key animal and plant functions. and female, Tak-2) were treated with (+) or without (-) 10 µM fusicoccin (FC) in the dark for 30 min. Then thalli were disrupted and the protein extracts subjected to SDS-PAGE. A, FC-induced phosphorylation of the penultimate Thr of H + -ATPase. Phosphorylated H + -ATPase was detected by immunoblot using anti-pThr. B, FC-induced binding of 14-3-3 protein to the H + -ATPase. Protein blots were performed using GST-14-3-3 protein (Arabidopsis GF14phi) as probe. C, Amount of the H + -ATPase. The H + -ATPase was detected by immunoblot using antibodies for


Introduction
The plasma membrane H + -ATPase, a member of the superfamily of P-type ATPases, which are characterized by the formation of phosphorylated intermediates during catalysis, has ten transmembrane segments and an N-and C-terminus in the cytosol (Sussman, 1994;Palmgren, 2001). The H + -ATPase is a ubiquitous enzyme from fungi to vascular plants and is a functional monomer with a molecular mass of about 100 kD that can form a dimer or hexamer (Goormaghtigh et al., 1986;Briskin and Reynolds-Niesman, 1989;Kanczewska et al., 2005). The H + -ATPase actively transports H + out of the cell, coupled with ATP hydrolysis, and creates an electrochemical gradient of H + across the plasma membrane for energizing substance transport, coupled with many secondary transporters, the maintenance of membrane potential, and pH homeostasis (Duby and Boutry, 2009). Indeed, the H + -ATPase has been shown to be an essential enzyme in yeast and Arabidopsis plant (Serrano et al., 1986;Haruta et al., 2010).
The structure of the H + -ATPase is highly conserved from fungi to the vascular plants, apart from the C-terminal region. In vascular plants, including one of the most basal lineage of vascular plant lycophytes, Selaginella moellendorffii, the C-terminal region of the H + -ATPase consisting around 100 amino acids is known as an autoinhibitory domain and contains a penultimate threonine (Thr) (Palmgren et al., 1990(Palmgren et al., , 1991Palmgren, 2001;Banks et al., 2011). On the other hand, the plasma membrane H + -ATPases in yeasts, red algae (Cyanidioschyzon merolae 10D), and green algae (Chlamydomonas reinhardtii, Volvox carteri, and Chlorella variabilis NC64A) lack such a C-terminus, and the length of the C-terminus varies among species (Portillo, 2000;Matsuzaki et al., 2004;Merchant et al., 2007;Blanc et al., 2010;Prochnik et al., 2010). Here we define the H + -ATPase having the C-terminal region containing the penultimate Thr as a pT H + -ATPase and others as the non-pT H + -ATPase. Taken together, the pT H + -ATPases probably did exist in the last common ancestor of liverworts and other land plants. However, when pT H + -ATPase appeared in the evolution of plants remains unknown.
The H + -ATPase is known to be regulated by physiological signals at both transcriptional and posttranscriptional levels (Portillo, 2000). Posttranslational 6 regulation of the pT H + -ATPase has been studied extensively. The C-terminal region keeps the H + -ATPase in a low activity state via an interaction with the catalytic domain under normal conditions, and phosphorylation of the penultimate Thr and subsequent binding of the 14-3-3 protein to the phosphorylated penultimate Thr in response to physiological signals results in activation of the H + -ATPase (Olsson et al., 1998;Fuglsang et al., 1999Fuglsang et al., , 2003Svennelid et al., 1999;Maudoux et al., 2000;Kinoshita and Shimazaki, 2002). As a physiological signal, blue light is known to activate the H + -ATPase via phosphorylation of the penultimate Thr in stomatal guard cells (Kinoshita and Shimazaki, 1999;Kinoshita et al., 2001;Shimazaki et al., 2007).
Moreover, it has been reported that sucrose and phytohormones, such as auxin and gibberellic acid, induce phosphorylation of the penultimate Thr in seedlings and culture cells from Arabidopsis thaliana (Niittylä et al., 2007;Chen et al., 2010). In addition, osmotic shock is most likely to induce phosphorylation of the penultimate Thr of the H + -ATPase in tomato culture cells (Kerkeb et al., 2002). These results indicate that many physiological signals regulate H + -ATPase activity via regulation of the phosphorylation status of the penultimate Thr of H + -ATPase, and that phosphorylation of the penultimate Thr is a major common regulatory mechanism of H + -ATPase in vascular plants (Sze et al., 1999;Kinoshita and Hayashi, 2011). It should be noted that the pT H + -ATPase has been reported to be phosphorylated at multiple sites in addition to the penultimate Thr in vascular plants (Fuglsang et al., 2007;Duby et al., 2009;Rudashevskaya et al., 2012).
The C-terminus of non-pT H + -ATPase is also thought to be important for regulation of its activity. In the yeast Saccharomyces cerevisiae, it has been reported that phosphorylation of two tandemly positioned residues in the C-terminus (Ser-911 and Thr-912 in PMA1) activates the H + -ATPase in response to glucose (Lecchi et al., 2007), indicating that the fungal H + -ATPase is regulated in a different manner from the pT H + -ATPase. Posttranslational regulation of the H + -ATPases in red and green algae remains unresolved.
In the present study, we performed molecular characterization of plasma membrane H + -ATPase in the liverwort Marchantia polymorpha as a non-vascular plant bryophyte, which represents the most basal lineage of extant land plants. We found that M. polymorpha expresses both the pT H + -ATPase and non-pT H + -ATPase. We further clustered with Arabidopsis H + -ATPase, and that MpHA6, MpHA7, and MpHA8 are close to the non-pT H + -ATPase of Chlamydomonas reinhardtii (Crpump), which has no penultimate Thr (Fig. 1B). According to classification of gene families in the pT H + -ATPase, MpHA2, MpHA3, and MpHA4 localize between subfamilies I and IV (Arango et al., 2003). These results suggest that M. polymorpha genome encodes both pT H + -ATPase and non-pT H + -ATPase genes. Note that MpHA5 has high sequence identity with AHA2 as well as MpHA1-MpHA4 but no conserved penultimate Thr, and that MpHA6 has insertions of over 40 residues in the C-terminal region and C-terminal extension of 39 residues (Fig. 1A, Supplemental Table S1).
To examine the expression of MpHAs, reverse transcriptase (RT)-PCR analysis using total RNA from thalli was performed. The results showed that the H + -ATPase isoforms, except for MpHA7, were expressed in thalli (Fig. 1C). All MpHAs showed identical expression properties in both male (Tak-1) and female (Tak-2) thalli (Fig. 1C).

Fusicoccin induces phosphorylation of the penultimate Thr of pT H + -ATPases
We first performed immunoblot analysis using antibodies raised against the conserved catalytic domain of AHA2 (anti-H + -ATPase) (Hayashi et al., 2010), and found that only an apparent 95-kD protein in thalli was recognized (Fig. 2C). This suggests that the 95-kD protein is most likely involved in MpHA1-MpHA5, since these isoforms show high identity with AHA2 (>80%) and have very similar molecular masses to AHA2 (Supplemental Table S1).
To analyze whether the pT H + -ATPase in M. polymorpha (MpHA1-MpHA4) is regulated by phosphorylation of the penultimate Thr, we treated thalli with the fungal toxin fusicoccin (FC), which is an activator of H + -ATPase and accumulates phosphorylated H + -ATPase through inhibition of dephosphorylation of the phosphorylated penultimate Thr in vascular plants (Kinoshita and Shimazaki, 2001;Hayashi et al., 2010). Phosphorylation of the penultimate Thr was detected using antibodies raised against the phosphorylated penultimate Thr 947 of AHA2 (anti-pThr) (Hayashi et al., 2010). The results showed that FC at 10 µM induced phosphorylation of the 95-kD protein in thalli without altering the amount of H + -ATPase present in the cells (Fig. 2, A and C). Moreover, protein blot analysis using 14-3-3 protein (Arabidopsis GF14phi) as a probe revealed that phosphorylated H + -ATPase bound with the 14-3-3 protein. These results indicate that the phosphorylated penultimate Thr creates a binding motif for the 14-3-3 protein, as also seen in vascular plants (Fig. 2B), and that the 95-kD protein contains the pT H + -ATPase in M. polymorpha. Figure 2A, the H + -ATPases in both male (Tak-1) and female (Tak-2) thalli showed an identical response to FC. We performed further experiments using Tak-1.

Mp14-3-3a binds to the phosphorylated H + -ATPase
We detected endogenous 14-3-3 proteins in thalli having molecular masses of 31 and 32 kD using antibodies raised against Arabidopsis GF14phi ( confirmed by RT-PCR (Fig. 3B). Moreover, protein blot analysis using the recombinant Mp14-3-3a as a probe revealed that it bound to phosphorylated H + -ATPase in thalli (Fig.   3C). These results indicate that the pT H + -ATPases in M. polymorpha might be activated via phosphorylation of the penultimate Thr and subsequent binding of the endogenous 14-3-3 protein, as in vascular plants.

M. polymorpha
To clarify physiological signals that regulate phosphorylation status of the pT Interestingly, all treatments induced phosphorylation of the H + -ATPase in thalli without altering the H + -ATPase amount. Sucrose-induced phosphorylation cannot be interpreted as a result of its osmotic pressure, since treatment with the same concentration of mannitol (30 mM for 30 min) had no effect on phosphorylation level (Fig. 4B). Osmotic shock-dependent phosphorylation required over 100 mM mannitol and was concentration-dependent between 100 mM and 200 mM (Fig. 4C).
Notably, light illumination had a drastic effect on the phosphorylation level of the H + -ATPase in thalli (Fig. 4A). Therefore, we examined light-induced phosphorylation of the H + -ATPase in more detail. As shown in Figure 5A, the phosphorylation level of the H + -ATPase reached a maximum within 15 min after the start of illumination.

Analyses of the signaling pathway in light-induced phosphorylation of the H + -ATPase in thalli
Previous studies have shown that a potent protein kinase inhibitor, K-252a, and a type1/2A protein phosphatase inhibitor, calyculin A (CA), inhibit blue light-induced phosphorylation/activation of the plasma membrane H + -ATPase in stomatal guard cells (Kinoshita andShimazaki, 1997, 1999;Hayashi et al., 2011). We tested the effects of K-252a and CA on light-induced phosphorylation of the H + -ATPase in thalli and found that both K-252a at 10 µM and CA at 0.5 µM severely inhibited phosphorylation (Fig. 7, A and C). In contrast, K-252a and CA had no effect on FC-induced phosphorylation of the H + -ATPase ( C-terminus is the most common activation mechanism for the H + -ATPase (Palmgren, 2001;Duby and Boutry, 2009;Kinoshita and Hayashi, 2011). We found that the pT H + -ATPase in thalli of M. polymorpha is phosphorylated in its penultimate Thr and binds to the 14-3-3 protein in response to FC (Fig. 2). The results clearly indicate that the pT H + -ATPase in M. polymorpha might be activated via an identical mechanism to that in vascular plants. Moreover, we showed that the phosphorylation status of the penultimate Thr of the pT H + -ATPase in thalli is regulated by phosphorylation in response to physiological signals such as light, sucrose, and osmotic shock (Fig. 4).
Similarly, sucrose was reported to induce phosphorylation of the plasma membrane H + -ATPase in Arabidopsis seedlings (Niittylä et al., 2007), and osmotic shock likely induced phosphorylation of the plasma membrane H + -ATPase in tomato culture cells (Kerkeb et al., 2002), suggesting that phosphorylation status of the pT H + -ATPase in the liverwort M. polymorpha is also regulated by similar physiological signals to those in vasculr plants. It should be noted that we measured ATP hydrolytic activity of the H + -ATPase according to a previous method for vascular plants (Kinoshita and Shimazaki, 1999), but we could not detect increased ATP hydrolytic activity of the H + -ATPase in response to physiological signals in cell extracts and microsomes from thalli because of high background noise from non-specific ATP hydrolytic activity in these samples (data not shown). Further investigations will be needed to establish measurement methods for plasma membrane H + -ATPase activity in the liverwort M.
polymorpha and to demonstrate that phosphorylation of the penultimate Thr is correlated with the activation status of the H + -ATPase.
In addition, the present results suggest that M. polymorpha possesses the identical/similar protein kinase and protein phosphatase that directly regulate the phosphorylation status of the pT H + -ATPase, and might have obtained these components in parallel with evolution of the pT H + -ATPase. We should note, however, that the protein kinase and phosphatase have not yet been identified in vascular plants, although they have been extensively investigated (Svennelid et al., 1999;Camoni et al., 2000;Hayashi et al., 2010). Identification of protein kinase and phosphatase, including those of M. polymorpha, will provide novel understanding for the regulation of pT H + -ATPase in plants.  sucrose also induced phosphorylation of the pT H + -ATPase in thalli (Fig. 4B), it is possible that light-induced and photosynthesis-dependent phosphorylation of the H + -ATPase is mediated by sucrose as a photosynthetic product. Light-induced phosphorylation, however, began within 5 min (Fig. 5A). Further investigation will be needed to clarify the relationship between time courses of sucrose production and H + -ATPase phosphorylation in thalli.
In vascular plants, the H + -ATPase plays a crucial role in the transport of photosynthetic products into sink tissues, such as fruits, tubers, and roots (Palmgren, 2001;Duby and Boutry, 2009). In the case of phloem loading, sucrose produced by photosynthesis in mesophyll cells is uploaded into the phloem companion cells by the sucrose/H + symporter utilizing the electrochemical gradient generated by H + -ATPase and is transported to sink tissues via the phloem (Stadler et al., 1995;Burkle et al., 1998;Zhao et al., 2000). However, the liverwort M. polymorpha is a non-vascular plant and has no phloem companion cells. Supplemental Figure S4  Further investigation will be needed to elucidate the localization of the H + -ATPase in the thalli, which is phosphorylated in response to light. This will clarify whether H + -ATPase phosphorylation is mediated by an intracellular or an intercellular signaling pathway and will elucidate the physiological role of photosynthetic control of H + -ATPase phosphorylation in thalli.

Expression of transcripts determined by RT-PCR analysis
Total RNA was extracted from 3-week-old thalli using the RNeasy Plant Mini Kit

Preparation of GST-fused Mp14-3-3 protein
The glutathione S-transferase (GST)-fused Mp14-3-3a (GST-Mp14-3-3a) was expressed in E. coli cells, and the recombinant protein was purified and used as probe in protein blots. The full-length Mp14-3-3a cDNA was amplified by RT-PCR with two oligonucleotide primers: 5'-CGGGATCCCTTCGTCGAGGATTAGCGATGG-3' and 5'-CGGGATCCTTAACTGTCCTCGGCATCCTC-3'. The amplified DNA was cloned into the BamHI site of the pGEX-2T vector (GE Healthcare), and the plasmids were transformed into the E. coli BL21 strain. The polypeptide was expressed as a fusion protein with a GST-tag and was purified using glutathione sepharose 4B beads (GE Healthcare) as described previously (Kinoshita and Shimazaki, 1999). The purified protein was frozen and kept at -25°C until use.

Immunoblot and protein blot analyses
Immunoblot and protein blots were performed according to previous methods    Arabidopsis AHA2 (anti-H + -ATPase). D, Amount of 14-3-3 protein. 14-3-3 protein was detected by immunoblot using antibodies for Arabidopsis GF14phi (anti-14-3-3 protein).   Dark-adapted thalli were illuminated with white light at 50 µmol m -2 s -1 and disrupted at 1, 5, 15, and 30 min after the start of illumination. The protein extracts were subjected to SDS-PAGE. Other procedures were the same as in Figure 4A. B, Time course of dephosphorylation of the H + -ATPase at the end of illumination. Dark-adapted thalli (Dk) were illuminated with white light for 30 min at 50 µmol m -2 s -1 and kept in darkness at the end of the illumination. The thalli were disrupted at 0, 30, 60, and 120 min after the end of illumination. The protein extracts were subjected to SDS-PAGE.
Other procedures were the same as in Figure 4A. The graphs represent the phosphorylation level of H + -ATPase, as quantified from the ratio of signal intensity from the phosphorylated H + -ATPase to that from the H + -ATPase, and expressed relative to the phosphorylation level of the dark-adapted thalli. Values represent means of three independent experiments with SD. µmol m -2 s -1 . The protein extracts were subjected to SDS-PAGE. Other procedures were the same as in Figure 4A. B, and C, Effects of DCMU and DBMIB on light-induced phosphorylation of H + -ATPase. Dark-adapted thalli were incubated with (+) or without (-) 10 µM DCMU or 10 µM DBMIB for 30 min in the dark. Thalli were then illuminated with white light (Lt) at 50 µmol m -2 s -1 or kept in the dark (Dk) for 30 min.
Other procedures were the same as in Figure 4A.  . thaliana with ClustalW (Thompson et al., 1994). Black blocks indicate highly conserved residues. Asterisks mean that the proteins were not full-length and N-terminal information was not acquired. Figure S2. Alignment of 14-3-3 proteins from M. polymorpha (Mp14-3-3a) and A. thaliana (GF14phi) with ClustalW (Thompson et al., 1994). Black blocks indicate identical amino acid residues; dashes indicate gaps introduced to allow for optimal alignment of sequences.   (Thompson et al., 1994). Black blocks indicate highly conserved residues. Tenth transmembrane segment (TM10) and Region I (R-I) and Region II (R-II) within the C-terminal region are indicated by lines. The arrow denotes the penultimate Thr of AHA2, which is the phosphorylated site for activity regulation. Asterisks are amino acid numbers of the partial sequences not acquiring N-terminal information for MpHA1 and MpHA5. Dashes indicate gaps introduced to allow for optimal alignment of sequences. B, Phylogenetic tree of the H + -ATPase proteins from M. polymorpha, A. thaliana (AHA1-AHA11), and C. reinhardtii (Crpump: XP_001698580). The alignment for the phylogenetic tree was performed with Clustal W using full-length amino acid sequences (Thompson et al., 1994). The phylogenetic tree was created with the MEGA 5 software (Tamura et al., 2011) and the neighborjoining program with 1,000 bootstrap replications. Bootstrap values at the branches represent the percentage obtained in 1,000 replications. C. reinhardtii pump sequence was used as outgroup. MpHA1 and MpHA5 were not used to construct the phylogenetic tree as full-length sequences were not acquired. Roman numerals designate the subfamilies. The bar represents 0.05 substitutions/site. C, RT-PCR analysis of isogenes of the H + -ATPase in M. polymorpha. RNA was extracted from each thallus of the male (Tak-1) and female (Tak-2) gametophyte. PCRs were performed with 30 cycles using specific primers for MpHA1-8. Figure 2. Phosphorylation of the pT H + -ATPase in M. polymorpha. Dark-adapted thalli (male, Tak-1, and female, Tak-2) were treated with (+) or without (-) 10 µM fusicoccin (FC) in the dark for 30 min. Then thalli were disrupted and the protein extracts subjected to SDS-PAGE. A, FC-induced phosphorylation of the penultimate Thr of H + -ATPase. Phosphorylated H + -ATPase was detected by immunoblot using anti-pThr. B, FC-induced binding of 14-3-3 protein to the H + -ATPase. Protein blots were performed using GST-14-3-3 protein (Arabidopsis GF14phi) as probe. C, Amount of the H + -ATPase. The H + -ATPase was detected by immunoblot using antibodies for Arabidopsis AHA2 (anti-H + -ATPase). D, Amount of 14-3-3 protein. 14-3-3 protein was detected by immunoblot using antibodies for Arabidopsis GF14phi (anti-14-3-3 protein).   (Mp14-3-3a), A. thaliana (GF14chi, GF14omega, GF14psi, GF14phi, GF14upsilon, GF14lambda, GF14nu, GF14kappa, GF14mu, GF14epsilon, GF14omicron, GF14iota, GF14pi), and Dictyostelium discoideum 14-3-3 (X95568). The alignment for the phylogenetic tree was performed with Clustal W using full-length amino acid sequences (Thompson et al., 1994). The phylogenetic tree was created with the MEGA 5 software (Tamura et al., 2011) and the neighbor-joining program with 1,000 bootstrap replications. Bootstrap values at the branches represent the percentage obtained in 1,000 replications. The D. discoideum 14-3-3 sequence was used as outgroup.

Supplemental
The bar represents 0.02 substitutions/site. B, RT-PCR analysis of 14-3-3 protein expression in M. polymorpha thalli. Total RNA was extracted from thalli and RT-PCR was performed.
MpEF was used as a loading control. C, FC-induced binding of 14-3-3 protein of M. polymorpha to the phosphorylated H + -ATPase. Procedures were the same as in Figure 2. Binding of 14-3-3 protein was detected by protein blot using GST-Mp14-3-3a as probe.  Dark-adapted thalli were illuminated with white light for 3 h at 50 µmol m -2 s -1 (Lt) or kept in the dark (Dk). The thalli were then disrupted and the protein extracts subjected to SDS-PAGE. Phosphorylated H + -ATPase and the H + -ATPase were detected by immunoblot using anti-pThr and anti-H + -ATPase, respectively. B, Sucrose-induced phosphorylation of the H + -ATPase in thalli. Dark-adapted thalli were treated with 30 mM sucrose or 30 mM mannitol for 30 min in the dark. Other procedures were the same as in Figure 4A. Mannitol was used for osmotic control. C, Mannitol-induced phosphorylation of the H + -ATPase in thalli. Dark-adapted thalli were treated with 0, 100, or 200 mM mannnitol for 30 min in the dark. Other procedures were the same as in Figure 4A.  Dark-adapted thalli were illuminated with white light at 50 µmol m -2 s -1 and disrupted at 1, 5, 15, and 30 min after the start of illumination. The protein extracts were subjected to SDS-PAGE. Other procedures were the same as in Figure 4A. B, Time course of dephosphorylation of the H + -ATPase at the end of illumination. Dark-adapted thalli (Dk) were illuminated with white light for 30 min at 50 µmol m -2 s -1 and kept in darkness at the end of the illumination. The thalli were disrupted at 0, 30, 60, and 120 min after the end of illumination. The protein extracts were subjected to SDS-PAGE. Other procedures were the same as in Figure 4A. The graphs represent the phosphorylation level of H + -ATPase, as quantified from the ratio of signal intensity from the phosphorylated H + -ATPase to that from the H + -ATPase, and expressed relative to the phosphorylation level of the dark-adapted thalli. Values represent means of three independent experiments with SD.