Tonoplast-localised proton-coupled Ca
2+ transporters encoded by
cation/H
+ exchanger (CAX) genes play a critical role in sequestering Ca
2+ into the vacuole. These transporters may function in coordination with Ca
2+ release channels, to shape stimulus-induced cytosolic Ca
2+ 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 Ca
2+/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 Ca
2+ flux. These results suggest cautious interpretation of mutant Ca
2+/H
+ exchanger phenotypes that may be due to either perturbed Ca
2+ or H
+ transport.Key words:
abiotic stress, Ca2+ transport, Ca2+/H+ exchanger, H+-ATPase, Na+ transport, pH, salt stress, vacuoleCa
2+ 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 Ca
2+ dynamics are maintained by precise interplay among transporters involved in its release from or uptake into Ca
2+ stores. The vacuole, as the largest internal Ca
2+ pool, is assumed to play a major role in Ca
2+ signalling, and has been shown to be the source of Ca
2+ release following various abiotic stresses such as cold and osmotic stress.
3,4 Rapid, stimulus-induced release of Ca
2+ from the vacuole is attributable to selectively permeable Ca
2+ 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 Ca
2+ pumps, including ACA4 and ACA11, which mediate high-affinity Ca
2+ uptake, and a family of cation/H
+ exchangers (CAX), responsible for lower-affinity but high-capacity Ca
2+ uptake.
7,8 While Ca
2+ pumps rely directly on the hydrolysis of ATP to drive Ca
2+ uptake, Ca
2+/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).
9With 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 ().
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 ().
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 Ca
2+/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.
Open in a separate windowTonoplast 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 Ca
2+/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 (), but the morphological phenotypes are not as pronounced.
13 It is therefore likely that reduction of tonoplast Ca
2+/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 Ca
2+/H
+ exchange activity and altered abiotic stress responses.CAX-mediated transport may alter both cytoplasmic and lumenal pH, as well as intracellular Ca
2+ 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 Ca
2+ pump which itself is upregulated in
cax1 as a compensatory mechanism to correct perturbations in the Ca
2+ 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 (). 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 Ca
2+/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.
14Our recent results indicate there are at least two modes by which Ca
2+/H
+ exchangers can mediate adaptive responses to stress: direct manipulation of cytosolic Ca
2+ and indirect feedback of H
+ flux (). For example, salt stress responses are likely controlled via the generation of a specific cytosolic Ca
2+ signature, which mediates a downstream signalling pathway. CAX3 appears to be the principle isoform providing tonoplast Ca
2+/H
+ exchange in response to salt stress.
14 Disruption of CAX3-mediated tonoplast Ca
2+ transport and the alteration of cytosolic Ca
2+ dynamics may therefore alter the plant''s normal response to salt stress (). 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
+ (). 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 Ca
2+/H
+ exchange activity are due to altered Ca
2+ transport or H
+ transport.
Open in a separate windowModel of tonoplast Ca
2+/H
+ exchanger interaction with H
+ pumps in response to salt stress. (A) In response to NaCl treatment, an elevation in cytosolic Ca
2+ will occur, possibly due to vacuolar Ca
2+ release.
3 Increased CAX3-mediated Ca
2+/H
+ exchange activity
14 will sequester excess Ca
2+ into the vacuole. CAX3 may be involved in the generation of a specific Ca
2+ 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 Ca
2+/H
+ exchange. Activity of SOS1 requires activation by the kinase SOS2
24 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 Ca
2+ elevation may be sustained due to reduced vacuolar Ca
2+ sequestration and normal salinity-induced Ca
2+ signalling pathways may be perturbed. Lack of CAX3 downregulates both P-ATPase and V-ATPase activity
14 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 Ca
2+/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.
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