Exchangers man the pumps

Abstract

Tonoplast-localised proton-coupled Ca²⁺ transporters encoded by cation/H⁺ exchanger (CAX) genes play a critical role in sequestering Ca²⁺ into the vacuole. These transporters may function in coordination with Ca²⁺ release channels, to shape stimulus-induced cytosolic Ca²⁺ elevations. Recent analysis of Arabidopsis CAX knockout mutants, particularly cax1 and cax3, identified a variety of phenotypes including sensitivity to abiotic stresses, which indicated that these transporters might play a role in mediating the plant's stress response. A common feature of these mutants was the perturbation of H⁺-ATPase activity at both the tonoplast and the plasma membrane, suggesting a tight interplay between the Ca²⁺/H⁺ exchangers and H⁺ pumps. We speculate that indirect regulation of proton flux by the exchangers may be as important as the direct regulation of Ca²⁺ flux. These results suggest cautious interpretation of mutant Ca²⁺/H⁺ exchanger phenotypes that may be due to either perturbed Ca²⁺ or H⁺ transport.

Ca²⁺ plays a fundamental role in the plant cell, functioning as a highly versatile second messenger controlling a multitude of cellular reactions and adaptive responses.^1,2 Ca²⁺ dynamics are maintained by precise interplay among transporters involved in its release from or uptake into Ca²⁺ stores. The vacuole, as the largest internal Ca²⁺ pool, is assumed to play a major role in Ca²⁺ signalling, and has been shown to be the source of Ca²⁺ release following various abiotic stresses such as cold and osmotic stress.^3,4 Rapid, stimulus-induced release of Ca²⁺ from the vacuole is attributable to selectively permeable Ca²⁺ channels, however, the identity of candidate genes encoding this mechanism remains contested.^5,6 Better understood, are the two major vacuolar uptake mechanisms; P-type Ca²⁺ pumps, including ACA4 and ACA11, which mediate high-affinity Ca²⁺ uptake, and a family of cation/H⁺ exchangers (CAX), responsible for lower-affinity but high-capacity Ca²⁺ uptake.^7,8 While Ca²⁺ pumps rely directly on the hydrolysis of ATP to drive Ca²⁺ uptake, Ca²⁺/H⁺ exchangers are energized indirectly by the pH gradient generated by electrogenic H⁺ pumps located on the tonoplast, including the vacuolar-type H⁺-ATPase (V-ATPase).⁹

With the aim of further understanding the role of specific CAX isoforms in Arabidopsis, we and others have recently characterized CAX mutants and overexpression lines and observed a variety of phenotypes, including altered response to abiotic stresses.^10–14 While some phenotypes are identical among different CAX mutants, others are specific to individual lines.¹⁴ Moreover, these analyses have highlighted the interplay of these transporters with H⁺ pumps at both the tonoplast and the plasma membrane. Overexpression of CAX1 in Arabidopsis results in increased activity of the V-ATPase, whereas mutations in CAX1 cause a concomitant decrease in measured V-ATPase activity (Fig. 1).¹¹ Similar reductions in V-ATPase activity are also observed in cax2 and cax3 mutant plants but to a lesser extent,^12,13 and a significant reduction is observed in a cax1 cax3 double knockout line.¹³ At the plasma membrane, P-type H⁺-ATPase (P-ATPase) activity is increased in cax1 but decreased in cax3 (Fig. 1).¹⁴ Indeed cax3 lines appeared more sensitive to changes in the pH of the growth media.¹⁴ This implies that unlike cax1, cax3 is less efficient at cytoplasmic pH adjustment. Another intriguing observation is that activity of the H⁺-pyrophosphatase (H⁺-PPase) at the tonoplast is largely unaltered following CAX gene deletion. While overexpression of the Arabidopsis H⁺-PPase AVP1 leads to increased Ca²⁺/H⁺ exchange activity,^15,16 there is little alteration in H⁺-PPase activity following perturbed expression of CAX1 or CAX2.^11,12 Thus, this feedback interplay appears to exist only between exchangers and H⁺-ATPases.

Figure 1.

Tonoplast H⁺-ATPase (V-ATPase) activity and plasma membrane H⁺-ATPase (P-ATPase) activity in wild type Arabidopsis (ecotype Col-0) and Arabidopsis lines with manipulated tonoplast Ca²⁺/H⁺ exchange activity. 35S::CAX1 and 35S::CAX2 denote lines that overexpress a constitutively active N-terminally truncated CAX1 or CAX2 construct driven by the CaMV 35S promoter in the cax1-1 or cax2-1 mutant background, respectively. V-ATPase H⁺-transport activity was measured by the ATP-dependent quenching of quinacrine fluorescence, and rates of bafilomycin-sensitive, vanadate-resistant hydrolytic activity of the V-ATPase were determined in isolated tonoplast membranes, as described in refs. ¹¹ and ¹³. Rates of vanadate-sensitive, bafilomycin- and azide-resistant hydrolytic activity of the P-ATPase were determined in isolated plasma membranes, as described in ref. 14. Results are shown as % of wild type (Col-0) ATPase activity and are means ± SE of 3–4 independent experiments. Data taken and modified from refs. ¹¹–¹⁴.

The V-ATPase is important not only for maintenance of a pH gradient across the tonoplast, but also in maintenance of Golgi structure, endocytosis and secretory trafficking.^17,18 The V-ATPase is localised at the Golgi, endoplasmic reticulum and endosomes, in addition to the tonoplast.⁹ The det3 mutant, with a mutation in subunit C (VHA-C), has a 40–60% reduction in V-ATPase activity, but numerous severe developmental phenotypes.¹⁹ In contrast, the cax1 and cax1 cax3 mutants have a reduction in V-ATPase activity equivalent to det3 (Fig. 1), but the morphological phenotypes are not as pronounced.¹³ It is therefore likely that reduction of tonoplast Ca²⁺/H⁺ exchange primarily affects tonoplast V-ATPase activity, while V-ATPase activity in the secretory pathway is unperturbed. The V-ATPase is a multi-subunit protein and some of these subunit gene products appear to be either tonoplast-specific or tonoplast-enriched. Mutations in tonoplast subunits may cause defective V-ATPase activity only at the tonoplast.⁹ It will be of interest to see whether such tonoplast-specific V-ATPase mutants phenocopy the cax mutants, and possess perturbed Ca²⁺/H⁺ exchange activity and altered abiotic stress responses.

CAX-mediated transport may alter both cytoplasmic and lumenal pH, as well as intracellular Ca²⁺ gradients. In the case of the V-ATPase, evidence is emerging for a role not only in the generation of a pH gradient across membranes, but also in the direct sensing of pH within the compartment,^20,21 creating a feedback mechanism which regulates pump activity. Thus, in cax1 lines, abnormal acidification of the lumen is detected by the V-ATPase resulting in a dampening of its activity. This would conserve ATP, which we postulate could be utilized to drive the tonoplast Ca²⁺ pump which itself is upregulated in cax1 as a compensatory mechanism to correct perturbations in the Ca²⁺ gradient.¹¹ In the case of cax1, this in turn may signal the P-ATPase to remove surplus H⁺ from the cytoplasm, triggering its upregulation (Fig. 1). However, not all CAX mutants show this complex H⁺ feedback mechanism.

Co-ordinate downregulation of the V-ATPase in the cax1 mutant lines may also be a result of activity of the SOS2 kinase. This Ser/Thr kinase, which specifically interacts with the N-terminus of CAX1 resulting in Ca²⁺/H⁺ exchange activation,²² upregulates V-ATPase activity through interactions with the VHA-B regulatory subunit.²³ Loss of CAX1 may be signalling to the V-ATPase through changes in SOS2 activity resulting in a compensatory downregulation of the pump. It is tempting to speculate that SOS2 may signal the alteration in P-ATPase activity, as it is known to regulate other plasma membrane proteins, notably the Na⁺/H⁺ exchanger SOS1.²⁴ It will be interesting to determine if SOS2 and the P-ATPase interact directly. It is notable, however, that SOS2 does not appear to interact with CAX3,²² while P-ATPase activity is reduced in cax3 plants.¹⁴

Our recent results indicate there are at least two modes by which Ca²⁺/H⁺ exchangers can mediate adaptive responses to stress: direct manipulation of cytosolic Ca²⁺ and indirect feedback of H⁺ flux (Fig. 2). For example, salt stress responses are likely controlled via the generation of a specific cytosolic Ca²⁺ signature, which mediates a downstream signalling pathway. CAX3 appears to be the principle isoform providing tonoplast Ca²⁺/H⁺ exchange in response to salt stress.¹⁴ Disruption of CAX3-mediated tonoplast Ca²⁺ transport and the alteration of cytosolic Ca²⁺ dynamics may therefore alter the plant's normal response to salt stress (Fig. 2). Maintenance of H⁺ gradients at both the vacuole and plasma membrane are also critical for salt tolerance, such that salt treatment upregulates V-ATPase and P-ATPase activity.²⁵ This energizes Na⁺ efflux from the cytosol mediated by Na⁺/H⁺ exchangers at the plasma membrane and the tonoplast.^24,26 Therefore downregulation of H⁺ pumps at both membranes in the cax3 mutant is likely to perturb the ability of the cell to remove Na⁺ (Fig. 2). Further analysis of cax mutants, P-ATPase mutants, and tonoplast-specific V-ATPase mutants will be required to determine whether many of the phenotypes resulting from lack of Ca²⁺/H⁺ exchange activity are due to altered Ca²⁺ transport or H⁺ transport.

Figure 2.

Model of tonoplast Ca²⁺/H⁺ exchanger interaction with H⁺ pumps in response to salt stress. (A) In response to NaCl treatment, an elevation in cytosolic Ca²⁺ will occur, possibly due to vacuolar Ca²⁺ release.³ Increased CAX3-mediated Ca²⁺/H⁺ exchange activity¹⁴ will sequester excess Ca²⁺ into the vacuole. CAX3 may be involved in the generation of a specific Ca²⁺ signature that is recognised by the cell to mediate downstream stress responses. In addition, salt stress will lead to upregulation of H⁺ pumps at both the plasma membrane and the tonoplast (P-ATPase and V-ATPase)²⁵ which will in turn energize Na⁺/H⁺ exchange activity encoded by SOS1 and NHX1, promoting Na⁺ efflux from the cell. Increased V-ATPase activity may also upregulate Ca²⁺/H⁺ exchange. Activity of SOS1 requires activation by the kinase SOS2²⁴ which may also regulate tonoplast Na⁺/H⁺ exchange and V-ATPase activity.²³,²⁴ (B) In a cax3 knockout mutant experiencing salt stress, the cytosolic Ca²⁺ elevation may be sustained due to reduced vacuolar Ca²⁺ sequestration and normal salinity-induced Ca²⁺ signalling pathways may be perturbed. Lack of CAX3 downregulates both P-ATPase and V-ATPase activity¹⁴ thereby reducing energization of the plasma membrane and tonoplast Na⁺/H⁺ exchangers and reducing Na⁺ efflux from the cell. Some energization of H⁺-coupled processes at the vacuole may be maintained by residual H⁺-pyrophosphatase (V-PPase) activity.

The phenomenon observed between tonoplast Ca²⁺/H⁺ exchangers and H⁺ pumps at both the tonoplast and plasma membranes, suggesting a co-ordinate regulation between several transporters, is not solely restricted to this family of transporters. It is a common observation emerging from recent research on the manipulation of tonoplast transporters. Several labs have reported unpredictable phenotypes associated with ectopic expression of tonoplast proteins.^26–28 Until we understand the significance of these types of unexpected interactions, it is naïve to believe that engineering plants will provide predictable results.

Addendum to: Zhao J, Barkla BJ, Marshall J, Pittman JK, Hirschi KD. The Arabidopsis cax3 mutants display altered salt tolerance, pH sensitivity and reduced plasma membrane H+-ATPase activity. Planta. 2008;227:659–669. doi: 10.1007/s00425-007-0648-2.