Evolutionary appearance of the plasma membrane H (+) -ATPase containing a penultimate threonine in the bryophyte.

The plasma membrane H (+) -ATPase provides the driving force for solute transport via an electrochemical gradient of H (+) across the plasma membrane, and regulates pH homeostasis and membrane potential in plant cells. However, the plasma membrane H (+) -ATPase in non-vascular plant bryophyte is largely unknown. Here, we show that the moss Physcomitrella patens, which is known as a model bryophyte, 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 P. patens genome database revealed seven H (+) -ATPase genes, designated PpHA (Physcomitrella patens H (+) -ATPase). Six isoforms are the pT H (+) -ATPase; a remaining isoform is non-pT H (+) -ATPase. An apparent 95-kD protein was recognized by anti-H (+) -ATPase antibodies against an isoform of Arabidopsis thaliana and was phosphorylated on the penultimate Thr in response to a fungal toxin fusicoccin and light in protonemata, indicating that the 95-kD protein contains pT H (+) -ATPase. Furthermore, we could not detect the pT H (+) -ATPase in the charophyte alga Chara braunii, which is the closest relative of the land plants, by immunological methods. These results strongly suggest the pT H (+) -ATPase most likely appeared for the first time in bryophyte.

The Plasma Membrane H+-ATPase in the Moss Physcomitrella patens

The plasma membrane H+-ATPase actively transports H+ out of the cell 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.- The structure of the H+-ATPase is highly conserved, apart from the C-terminal region. In vascular plants, the H+-ATPase contains the C-terminal region consisting around 100 amino acids, which is known as an autoinhibitory domain and contains a penultimate threonine (Thr). It has been demonstrated that phosphorylation of the penultimate Thr and subsequent binding of the 14–3-3 protein to the phosphorylated penultimate Thr in response to physiological signals has shown to be a major common regulatory mechanism of H+-ATPase in vascular plants.- On the other hand, the H+-ATPases in yeasts and green algae, including Chlamydomonas reinhardtii, Chlorella variabilis NC64A and Volvox carteri, lack such a C-terminus,- suggesting that the H+-ATPase containing a penultimate Thr might have appeared after green algae during evolution.

A recent our study has revealed that 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, expresses both the penultimate Thr-containing H+-ATPase (pT H+-ATPase) and non-penultimate Thr-containing H+-ATPase (non-pT H+-ATPase). We further provided the evidence that the pT H+-ATPase in M. polymorpha is regulated by phosphorylation of its penultimate Thr in response to physiological signals, such as light, sucrose, and osmotic shock, and that light-induced phosphorylation of the pT H+-ATPase depends on photosynthesis.

In this study, we examined the plasma membrane H+-ATPase in the moss Physcomitrella patens, which is known as a model bryophyte, and the genome has been sequenced., We searched H+-ATPase genes with similarity to the typical plasma membrane H+-ATPase in Arabidopsis thaliana, AHA2 in the P. patens genome database (http://www.cosmoss.org) and found seven H+-ATPase homologs, designated PpHA1–PpHA7. Of these, six isoforms (PpHA1–PpHA6) possess a penultimate Thr in the C-terminal region. In contrast, the remaining isoform, PpHA7, lack such a penultimate Thr in the C-terminus. Phylogenetic analysis using full-length amino acid sequences indicated that PpHA1–PpHA5 are clustered with Arabidopsis H+-ATPase, and that PpHA7 is close to the non-pT H+-ATPase of Chlamydomonas reinhardtii (Crpump), which has no penultimate Thr (Fig. 1A). Note that PpHA6 is clustered with the non-pT H+-ATPases, although this isoform possesses the penultimate Thr. According to classification of gene families in the pT H+-ATPase, PpHA1–PpHA5 localize between subfamilies I and IV. These results suggest that P. patens genome encodes both pT H+-ATPase and non-pT H+-ATPase genes (Fig. 1A). In addition, we identified 11 typical 14–3-3 protein genes in P. patens (Fig. 1B).

Figure 1. Molecular characterization of the H+-ATPase in P. patens. (A) Phylogenetic tree of the H+-ATPase proteins from P. patens (PpHA1-PpHA7), M. polymorpha (MpHA1 and MpHA8),A. thaliana (AHA1, AHA6, and AHA11), and C. reinhardtii (Crpump: XP_001698580). The tree was constructed with ClustalW using full-length amino acid sequences. Roman numerals designate the subfamilies. The bar represents 0.05 substitutions/site. Bootstrap values at the branches represent the percentage obtained in 1,000 replications. Sequence data can be found in the P. patens genome database (www.cosmoss.org) under the following accession numbers: PpHA1 (Pp1s6_11V6.1), PpHA2 (Pp1s133_22V6.1), PpHA3 (Pp1s321_33V6.1), PpHA4 (Pp1s321_30V6.1), PpHA5 (Pp1s137_291V6.1), PpHA6 (Pp1s302_18V6.1), and PpHA7 (Pp1s404_34V6.1). (B) Alignment of 14–3-3 proteins from P. patens (Pp1s73_90V6.1) and A. thaliana (GF14phi). We found ten 14–3-3 genes (Pp1s46_127V6.1, Pp1s201_25V6.1, Pp1s348_15V6.1, Pp1s67_176V6.1, Pp1s137_194V6.1, Pp1s348_9V6.1, Pp1s73_133V6.1, Pp1s137_232V6.1, Pp1s140_61V6.1, and Pp1s140_63V6.1) in addition to Pp1s73_90V6.1. Pp1s73_90V6.1 has the highest identity with GF14phi. Black blocks indicate identical residues; dashes indicate gaps introduced to allow for optimal alignment of sequences. (C) Phosphorylation of the pT H+-ATPase in response to FC. Dark-adapted P. patens protonemata and M. polymorpha thalli were treated with (+) or without (-) 10 µM FC in the dark for 30 min., Then the samples were disrupted and the protein extracts subjected to SDS-PAGE. Phosphorylated H+-ATPase was detected by immunoblot using anti-pThr, which recognizes specifically phosphorylated penultimate threonine in the pT H+-ATPase. The H+-ATPase was detected by immunoblot using antibodies for Arabidopsis AHA2 (anti-H+-ATPase), which recognizes specifically the pT H+-ATPase. Arrowheads indicate position of the H+-ATPase. (D) Phosphorylation of the pT H+-ATPase in response to light. Dark-adapted P. patens protonemata and M. polymorpha thalli were illuminated with white light for 3 h at 50 µmol m−2 s−1 (Lt) or kept in the dark (Dk). Others were the same as in (C).

We then examined a fungal toxin fusicoccin (FC)- and light-induced phosphorylation of the penultimate Thr in H+-ATPase in protonemata of P. patens. An apparent 95-kD protein as the H+-ATPase was phosphorylated in response to FC and light (50 µmol m−2 s−1 for 30 min) as well as in thalli of M. polymorpha (Fig. 1, C and D), suggesting that the 95-kD protein contains the pT H+-ATPase in P. patens, and that light also acts as a physiological signal regulating phosphorylation status of the pT H+-ATPase in protonemata of P. patens.

The Charophyte Green Alga Chara braunii is Unlikely to Express the pT H+-ATPase

Next, we investigated whether the charophyte alga Chara braunii, which is the closest relative of the land plants, expresses the pT H+-ATPase using immunoblot and protein blot analyses. The results showed that there are no proteins in C. braunii that are recognized by anti-H+-ATPase against Arabidopsis H+-ATPase; furthermore, the signal did not appear in immunoblots using anti-pThr and protein blots using 14–3-3 protein as a probe when characean cells were treated with FC (Fig. 2A), although endogenous 14–3-3 proteins (31.6 kD, 32.5 kD, and 32.9 kD) were recognized by anti-14–3-3 against Arabidopsis GF14phi (Fig. 2B). In contrast, Arabidopsis H+-ATPase having an apparent mass of 95 kD from the etiolated seedlings was recognized by anti-H+-ATPase and was phosphorylated and bound to 14–3-3 protein in response to FC. In addition, we could not find the H+-ATPase containing the penultimate Thr of charophyte algae in the available database such as National Center for Biotechnology Information. Characean cells, however, have plasma membrane H+-ATPase activity, suggesting that characean cells are unlikely to express pT H+-ATPase, which binds with 14–3-3 protein on phosphorylation of the penultimate Thr, and that they express only the non-pT H+-ATPase.

Figure 2. Effect of FC on the phosphorylation level of the H+-ATPase in A. thaliana and C. braunii. (A) Etiolated Arabidopsis seedlings and dark-adapted C. braunii were treated with (+) or without (-) 10 µM FC for 30 min in the dark. Microsomal membranes obtained from A. thaliana and C. braunii were subjected to SDS-PAGE. Ponceau S staining was used as loading control. Protein blot (GST-14–3-3) was performed using GST-14–3-3 protein (Arabidopsis GF14phi) as probe., Others were the same as in Figure 1C. (B) Detection of 14–3-3 protein in A. thaliana and C. braunii. The microsomes obtained from A. thaliana and C. braunii were subjected to SDS-PAGE. 14–3-3 protein was detected by immunoblot using antibodies for Arabidopsis GF14phi (anti-14–3-3 protein).

From these results, we conclude that the pT H+-ATPase most likely appeared for the first time in the bryophyte; in other words, during the transition of plants from water to the terrestrial land. To verify the evolutionary appearance of the pT H+-ATPase in plants, elucidation of whole genome sequence in charophyte algae is required.