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Inorganic cations play a crucial role in many cellular and physiological processes in plants, and are essential components of plant nutrition (Taiz & Zeiger, 2006). Therefore, the uptake of cations and their redistribution must be precisely controlled to maintain normal physiology and to respond to endogenous and exogenous stimuli in a timely manner. Vacuolar antiporters are important elements in mediating the intracellular sequestration of cations (Busch & Saier, 2002). These antiporters are energized by the proton gradient across the vacuolar membrane and allow the rapid transport of cations into the vacuole. Among such vacuolar antiporters, CAXs (for CAtion eXchanger) have been shown to be involved in a multitude of cellular responses (Shigaki & Hirschi, 2006). However, the role of individual transporters in specific cellular processes has not been completely defined.
The primary cation substrate of CAXs is thought to be Ca2+. Ca2+ acts as a secondary messenger in many cellular processes, including hormone-mediated signaling (Cunningham & Fink, 1994; White & Broadley, 2003). CAX-mediated calcium and metal transport may have an impact on signaling events (Hirschi, 2004; Shigaki & Hirschi, 2006; McAinsh & Pittman, 2009). For example, with regard to auxin, the modulation of cation levels is required for aspects of auxin metabolism and signaling (Gehring et al., 1990; Felle, 1994; Magidin et al., 2003; Rampey et al., 2006), which controls the normal development of roots (Muday & Haworth, 1994; Woodward & Bartel, 2005; Wu et al., 2007). In addition, there is evidence that Ca2+ plays a role in PIN-FORMED (PIN) transporter-mediated auxin efflux activity. The PIN regulator PINOID (PID), a protein kinase, interacts with Ca2+-calmodulin and appears to be negatively regulated by Ca2+-calmodulin (Benjamins et al., 2003). Thus, it appears reasonable to postulate that CAXs, particularly those expressed in roots, are involved in auxin signaling and metabolism.
In Arabidopsis, there are six CAXs: CAX1–CAX6 (Shigaki et al., 2006). Members of the Arabidopsis CAX gene family, such as CAX1, CAX2 and CAX3, have been characterized at both the molecular and whole-plant level (Hirschi et al., 1996; Pittman & Hirschi, 2001; Pittman et al., 2002, 2004; Cheng et al., 2003, 2005; Shigaki et al., 2003). CAX1 was identified by its ability to suppress the Ca2+ sensitivity of a yeast mutant strain lacking vacuolar Ca2+ transport activity (Hirschi et al., 1996). CAX1 is a low-affinity and high-capacity Ca2+/H+antiporter, but CAXs may have a wide substrate range (Hirschi, 1999; Shigaki et al., 2003; Pittman et al., 2004; Korenkov et al., 2007b; Edmond et al., 2009). CAX3 is 77% identical at the amino acid level to CAX1 and, together with CAX4, is the most closely related gene to CAX1 (Shigaki & Hirschi, 2000; Shigaki et al., 2006). In Arabidopsis, CAX1 is highly expressed in leaf tissue, and modestly expressed in roots, stems, and flowers (Cheng et al., 2005). The cax1 knockout lines exhibit a 50% reduction in total vacuolar Ca2+/H+antiport activity, despite the up-regulation of CAX3 and CAX4, and alterations in vacuolar Ca2+-ATPase activity, but the phenotypes of CAX1 deletion on plant growth are subtle (Cheng et al., 2003). However, when deregulated N-terminal truncated CAX1 (sCAX1) was ectopically expressed in tobacco, the plants displayed dramatic Ca2+ deficiency phenotypes, such as tip burning and increased sensitivity to cation imbalances (for a review, see Shigaki & Hirschi, 2006). Meanwhile, CAX3 is expressed mainly in Arabidopsis roots and flowers. The cax3 knockout lines display altered Na+, Li+ and low pH sensitivity (Zhao et al., 2008). However, when CAX3 was expressed in tobacco, no visible phenotypes were observed (Shigaki et al., 2002). CAX4 has been partially biochemically characterized by heterologous expression in yeast and tobacco to determine its cation transport characteristics (Cheng et al., 2002; Korenkov et al., 2007a,b). Expression of full-length CAX4 in tobacco demonstrated that it can mediate proton-coupled Cd2+, Ca2+, Zn2+ and Mn2+ transport, with highest transport activity for Cd2+ (Korenkov et al., 2007b). A yeast-based assay has been informative to elucidate the function of CAX4. For example, when expressed in yeast, mutated variants of CAX4 can transport Ca2+and Cd2+, whereas addition of the hemagglutinin (HA) epitope tag fused at the N-terminus of CAX4 activates this Ca2+/H+ transporter (Cheng et al., 2002; Park et al., 2005). HA-CAX4 is localized on the vacuolar membrane in both yeast and plant cells (Cheng et al., 2002). Thus, we hypothesize that CAX4 is predominantly a vacuolar Ca2+ transporter under physiological conditions, but can efficiently transport Cd2+ when exposed to this metal.
Unlike CAX1 and CAX3, CAX4 is expressed at very low levels in most tissues (Cheng et al., 2002). Given this weak CAX4 expression, we initially postulated that CAX4 phenotypes would be difficult to ascertain. However, CAX4 is moderately expressed in roots (Cheng et al., 2002). With this relatively root-specific expression of CAX4 in mind, we were interested to investigate the role of this gene in Arabidopsis root development to reveal specific functions of vacuolar Ca2+/H+transporters in plant growth and development.
In this study, we dissect the function of CAX4 in planta. We show the expression pattern of CAX4 in Arabidopsis roots and identify stimuli which modulate gene expression. We report the isolation of plants perturbed in CAX4 expression and some of the phenotypes associated with altered expression. Collectively, these findings offer insights into the physiological function of CAX4 in root development and stress responses.
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The Arabidopsis genome contains six CAX transporter genes (Shigaki et al., 2006). To address the functional diversity and physiological role of these transporters, we have further characterized CAX4. In this study, we have demonstrated that CAX4 is required for root growth and development under metal stress conditions. In particular, we have demonstrated that CAX4 is specifically expressed in the primary root apex and lateral root primordia from early stages through post-emergence development under normal growth conditions (Fig. 1). CAX3 is also highly expressed in root tips and the root elongation zone, but its expression is not root specific (Cheng et al., 2005). Expression profiles of CAXs from rice (Oryza sativa) suggest that none of the five OsCAXs is exclusively expressed in roots (Kamiya et al., 2005, 2006). Although the expression of CAX4 is relatively low, it is modulated by environmental conditions. The CAX4::GUS signal is highly induced by Ni2+ and Mn2+ in the root apex and throughout the whole lateral roots (Fig. 1). Contrary to CAX1 and CAX3, whose expression is up-regulated by exogenous Ca2+ (Hirschi, 1999; Shigaki & Hirschi, 2000), the CAX4::GUS signal is only increased under Ca2+-limiting conditions (Fig. 1). The induction of CAX4 expression by Ca2+ depletion conditions is puzzling, considering its putative vacuolar cation/H+ function in excess cation tolerance. This might be an indirect Ca2+ effect, such that, when Ca2+ is depleted, the accumulation of other cations in the roots may be increased, and this imbalance up-regulates CAX4 expression. For example, root Ca2+ channels have good permeability for other cations, such as Mn2+ (White, 1998), and, when Ca2+ is limiting, it might lead to the accumulation of higher concentrations of other cations. Alternatively, CAX4 may be induced as a response to low Ca2+ conditions in order to maintain a pool of Ca2+ in the vacuole. Overall, the expression data suggest that CAX4 functions in root growth and adaptation.
Among the CAXs, the root expression pattern of CAX4 appears to be unique; however, when expressed at high levels in plants, its biochemical properties resemble other CAXs. The expression of 35S::CAX4 partially suppresses the cax1 defect in vacuolar Ca2+/H+ transport (Fig. 4). Previous studies using yeast have found that CAX4 can only transport Ca2+ if the N-terminus is modified (Cheng et al., 2002). These data therefore suggest that, when expressed in Arabidopsis, CAX4 can be activated without requiring artificial deregulation. Previous work in both yeast and plant expression systems suggests that CAX4 may modulate both vacuolar Ca2+ and metal levels, including Cd2+, depending on the environmental conditions (Park et al., 2005; Korenkov et al., 2007a,b). Given the similar ionic radius of Cd2+ and Ca2+, it is not surprising that CAX4 can transport both Cd2+ and Ca2+. Evidence for a role of CAX4 in Ca2+ homeostasis is also suggested by the ion sensitivity phenotypes of tobacco and Arabidopsis plants expressing 35S::CAX4 (Figs 5, 6). These phenotypes are similar to those of sCAX1-expressing tobacco and tomato plants (Hirschi, 1999; Park et al., 2005). Ectopic expression of sCAX1 in tobacco causes sensitivity to Mg2+, K+ and Na+ stresses, which are associated with Ca2+ deficiency (Hirschi, 1999). We speculate that this is caused by Ca2+ deficiencies that arise from the over-accumulation of Ca2+ in the vacuole. Consistent with this explanation, the addition of Ca2+ to the medium restores the normal growth of CAX4-expressing tobacco and Arabidopsis lines (Fig. 5). Although CAX4 expression causes numerous ion sensitivities, CAX4-expressing tobacco plants are also more tolerant to Cd2+ and Mn2+ (Korenkov et al., 2007a). The increased Cd2+/H+antiport activity and Cd2+ tolerance in CAX4-expressing tobacco and yeast (Cheng et al., 2002; Korenkov et al., 2007a) suggest that CAX4 is a cation transporter which has a high affinity for both Cd2+and Ca2+. We postulate that, at the root apex, CAX4 is important for the regulation of Cd2+ and Ca2+ levels. It is probable, however, that the predominant substrate for CAX4 under normal growth conditions is Ca2+.
The CAX4 silencing experiments indicate that CAX4 functions in root growth and development during heavy metal stress conditions. The cax4-1 and CAX4 RNAi lines display altered root architectures only when exposed to Cd2+ or Mn2+ stress (Fig. 3). Such Cd2+ sensitivity is also found in other Cd2+ transport mutants. For example, a knockout of ATM3, which encodes an ATP-binding cassette metal transporter, is more sensitive to Cd2+ than are wild-type plants (Kim et al., 2006). Likewise, knockout of the vacuolar localized P1B-type ATPase AtHMA3 causes Cd2+ sensitivity (Morel et al., 2008). These metal sensitivity root phenotypes of cax4 are distinct from other cax lines. Arabidopsis lines deleted for CAX3, which is also highly expressed in roots, are sensitive to salt stress, low pH and abscisic acid, but not Cd2+ (Zhao et al., 2008). These observations suggest that each CAX transporter may be involved in the response to specific environmental stress conditions. The cax4 mutant root phenotypes are consistent with its biochemical functions as a Ca2+ and Cd2+ transporter, and highlight its importance in root growth and development through the mediation of metal ion sequestration.
The results presented here support the concept that CAX4 cation/H+ antiport activity is required for the fidelity of auxin-mediated root growth and development. The CAX4 mutants exhibit altered responses to exogenous auxin (Fig. 6), but no other hormones perturb cax4 mutant growth. A reduction in IAA content in the root tip in cax4 mutants, as indicated by reduced DR5::GUS expression (Fig. 7a), leads to a loss of primary root meristem function and inhibition of root growth. A similar phenotype has been seen in plants overexpressing an AGC-type kinase PID (Friml et al., 2004). Previous studies have indicated a link between cytosolic Ca2+ signaling and auxin-regulated plant development via PID and other AGC kinases (Benjamins et al., 2003; Robert & Offringa, 2008). Ca2+ regulates these kinases through interaction with Ca2+-binding proteins, and the kinase, in turn, regulates the PIN auxin efflux carrier. Thus, it is possible that increased cytosolic Ca2+ levels, which may occur in some root cells lacking CAX4 (because of reduced transport into the vacuole), could cause altered auxin efflux, impaired root auxin gradients and altered root development. By contrast, the auxin sensitivity phenotype could be a result of altered homeostasis of metals, such as Cd2+ and Mn2+, in cells lacking CAX4. Metal homeostasis plays a role in auxin responses because it is important for some aspects of IAA metabolism (Tam et al., 2000; Walz et al., 2002; Magidin et al., 2003; Rampey et al., 2006). For example, amidohydrolases, which cleave IAA–amino acid conjugates, require metal cofactors for activity, and metal homeostasis regulators are involved in this process (Lasswell et al., 2000; Magidin et al., 2003; Rampey et al., 2006). Our observations suggest a link between Cd2+ homeostasis and auxin responses. Previous studies have indicated links between auxin homeostasis and Cd2+ stress (Hagen & Guilfoyle, 1985; Hagen et al., 1988; Rajkumar et al., 2005; Ganesan, 2008). It is possible that cax4 lines that are unable to tolerate Cd2+ stress also uncouple auxin production and/or metabolism in the roots. Further studies are required to determine the relationship between metal homeostasis, auxin sensitivity and CAX4 function. In addition, the relationship between specific CAX transporters and auxin signaling requires further study. For example, roots of cax1 lines are slightly more tolerant to auxin (Cheng et al., 2003), whereas cax4 lines are more sensitive. This may be caused by differences in expression between CAX1 and CAX4. Ca2+imaging studies and measurements of auxin transport in each CAX mutant will be useful in dissecting the specific roles of each transporter in auxin responses.