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Keywords:

  • Arabidopsis;
  • cation/H+ antiporter;
  • CAX;
  • metal homeostasis;
  • root development

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    The Arabidopsis vacuolar CAtion eXchangers (CAXs) play a key role in mediating cation influx into the vacuole. In Arabidopsis, there are six CAX genes. However, some members are yet to be characterized fully.
  • • 
    In this study, we show that CAX4 is expressed in the root apex and lateral root primordia, and that expression is increased when Ni2+ or Mn2+ levels are elevated or Ca2+ is depleted.
  • • 
    Transgenic plants expressing increased levels of CAX4 display symptoms consistent with increased sequestration of Ca2+ and Cd2+ into the vacuole. When CAX4 is highly expressed in an Arabidopsis cax1 mutant line with weak vacuolar Ca2+/H+ antiport activity, a 29% increase in Ca2+/H+ antiport is measured. A cax4 loss-of-function mutant and CAX4 RNA interference lines display altered root growth in response to Cd2+, Mn2+ and auxin. The DR5::GUS auxin reporter detected reduces auxin responses in the cax4 lines.
  • • 
    These results indicate that CAX4 is a cation/H+ antiporter that plays an important function in root growth under heavy metal stress conditions.

Abbreviations: 
CaMV

cauliflower mosaic virus

CAX

CAtion eXchanger

2,4-D

2,4-dichlorophenoxyacetic acid

GUS

β-glucuronidase

HA

hemagglutinin

IAA

indole-3-acetic acid

PID

PINOID

PIN

PIN-FORMED

RNAi

RNA interference

RT-PCR

reverse transcriptase-polymerase chain reaction

SLAT

Sainsbury Laboratory Arabidopsis thaliana Transposants

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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: CAX1CAX6 (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.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plasmid DNA constructs and plant transformation

To make a transcriptional fusion to the β-glucuronidase (GUS) reporter gene, the CAX4 promoter region, 740 bp upstream of the ATG start codon, was amplified from Arabidopsis genomic DNA by PCR using the following primers: forward primer, 5′-GCCAAGCTTGGGAGGCCTAAAGCAAATCAC-3′; reverse primer, 5′-GCCGGATCCCTTCTTCGTTGACTCTCTTTG-3′. This was cloned into the pBI121 vector to replace the cauliflower mosaic virus (CaMV) 35S promoter.

The CAX4 and triple HA epitope-tagged CAX4 (HA-CAX4) cDNA constructs have been described previously (Cheng et al., 2002). CAX4 and HA-CAX4 were subcloned into the plant expression vector pBI121 by replacing the GUS gene, which is driven by a CaMV 35S promoter.

For modification of CAX4 expression by RNA interference (RNAi), a 1.2-kb cDNA from CAX4 was used. This cDNA fragment was amplified by PCR using the following primer pairs: forward primer, 5′-GCGTTAATTAAGGCGCGCCGGAAGAGATCGGATCTGAAA-3′; reverse primer, 5′GCGGGATCCATTTAAATGCAATGAGCAGCATCACGAAGCTC-3′. The forward primer contained PacI and AscI restriction sites and the reverse primer contained BamHI and SwaI sites. This CAX4 fragment was cloned into the pFGC5941 binary vector (Kerschen et al., 2004) in both sense and antisense orientations, and was used to transform wild-type (Columbia ecotype) plants.

The recombinant plasmids, the CAX4 RNAi construct and empty vector controls were transformed into Agrobacterium tumefaciens GV3101 (Sambrook et al., 1989) or LBA4404 (Invitrogen). These strains were used to transform Arabidopsis Col-0 using the floral dip method (Clough & Bent, 1998). Transgenic tobacco (Nicotiana tabacum cv. KY14) overexpressing CAX4 has been described previously (Korenkov et al., 2007a,b).

Histochemical assay of GUS gene expression

Histochemical assays for GUS activity in the T3 generation of Arabidopsis transgenic plants harboring CAX4::GUS were performed according to the protocol described previously (Cheng et al., 2003).

Isolation of cax4 and creation of CAX4 RNAi lines

To isolate a cax4 null allele, the line SM_3.16922, carrying a dSpm transposon insertion in CAX4 (ecotype Col-0), was obtained from the SLAT collection (Sainsbury Laboratory Arabidopsis thaliana Transposants) (Tissier et al., 1999). Homozygous plants from the T3 generation were obtained by PCR screening using CAX4-specific (P1 and P2) and dSpm-specific (P3) primers; the dSpm-specific primer P3, 5′-TACGAATAAGAGCGTCCATTTTAGAGTGA-3′, and the CAX4 reverse primer P2, 5′-TGATACCCTTAAACATAACTTACCTTTTCGT-3′, were used to screen for the cax4-1 allele. The CAX4 forward primer P1, 5′-AATTGTTGGTAATGCAGCTGAGCAT-3′, and the CAX4 reverse primer (as above) were used to amplify the wild-type CAX4 gene. Two sets of CAX4-specific primers were used in reverse transcriptase-polymerase chain reaction (RT-PCR) to confirm a lack of transcript in the cax4-1 allele: the CAX4 set 1 forward primer CAX4F1, 5′-ATCGGCGTCGTCGTTGATAAGGAA-3′, and the CAX4 set 1 reverse primer CAX4R1, 5′-ACAGCGCCAGATACAACAACATGC-3′; the CAX4 set 2 forward primer CAX4F2, 5′-GCATGTTGTTGTATCTGGCGCTGT-3′, and the CAX4 set 2 reverse primer CAX4R2, 5′-CAGCATGCTCAGCTGCATTACCAA-3′.

Northern blot analysis

For Northern blot analysis, total RNA was extracted from transgenic Arabidopsis and tobacco flowering plants overexpressing CAX4, blotted and hybridized with 32P-labeled CAX4 gene-specific probes, as described previously (Cheng et al., 2003).

Western blot analysis

For Western blot analysis, total microsomal protein was extracted from transgenic Arabidopsis overexpressing HA-CAX4 and wild-type Arabidopsis (Randall & Sze, 1986). Immunoblot analysis was performed and the HA epitope was detected as described previously (Pittman & Hirschi, 2001).

Plant materials and growth conditions

Arabidopsis Col-0 was used in this study. Wild-type, cax4-1, CAX4 RNAi and cax4-1/HA-CAX4 seeds were surface sterilized, germinated and grown on one half-strength Murashige and Skoog medium (Murashige & Skoog, 1962). All plates were sealed with paper surgical tape and incubated at 22°C under continuous cool-fluorescent illumination. For the ion sensitivity assays, 5-d-old Arabidopsis wild-type, cax4-1, CAX4 RNAi and cax4-1/HA-CAX4 seedlings grown under normal conditions were transferred onto half-strength MS medium and half-strength MS medium supplemented with various metal ions. The ion sensitivity assay for the transgenic tobacco plants was performed as described previously (Hirschi, 1999).

Preparation of membrane vesicles and transport measurements

For the measurement of Ca2+ uptake, vacuole-enriched membrane vesicles were prepared from root tissue obtained from 2-wk-old Col-0, cax1-1 and cax1-1/CAX4 plants cultured in Gamborg's B5 medium and pretreated with 100 mM CaCl2 for 18 h before harvest. Membrane vesicle preparation and Ca2+ uptake assay were performed as described previously (Hirschi, 1999; Cheng et al., 2003).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

CAX4 expression in Arabidopsis roots

RT-PCR analysis indicates that CAX4 transcripts can be detected in all Arabidopsis tissues analyzed, including stems, flowers, leaves, siliques and roots (Cheng et al., 2002). However, the expression of CAX4 is the highest in root tissue. This relatively root-specific expression for CAX4 is also observed in publicly available microarray data. To further study the expression pattern of CAX4 in roots, histochemical analysis of transgenic plants harboring the CAX4::GUS reporter was performed. A 740-bp fragment containing the CAX4 promoter was fused to the GUS gene. The CAX4::GUS construct was introduced into wild-type Arabidopsis plants, and GUS activities of T3 plants were analyzed at different root growth and developmental stages. At a very early stage (1 d after germination), the GUS signal was detected in the root tip, as well as hypocotyls and the base of the cotyledon (data not shown). Three days after germination, the GUS signal was visualized in the primary root apex and lateral root primordium (Fig. 1). When seedlings were grown on MS medium, the CAX4::GUS signal was weak (Fig. 1a), which is consistent with previous findings (Cheng et al., 2002). However, when seedlings were exposed to elevated levels of Ni2+ and Mn2+ or depleted levels of Ca2+, the CAX4::GUS signal was enhanced in the primary root apex, lateral root primordium and primary root elongation zone. These same conditions also increased CAX4 expression in the lateral root apex and throughout the lateral roots (Fig. 1). Cd2+ treatment did not enhance CAX4::GUS levels. In addition, exogenous indole-3-acetic acid (IAA) induced CAX4::GUS expression in the primary root apex and lateral root elongation zone (Fig. 1e). RT-PCR analysis confirmed the GUS results. These experiments were performed using RNA extracted from the roots of wild-type plants grown on the same metal and hormone treatments for which the CAX4::GUS-expressing plants were tested (Fig. 1f).

image

Figure 1. (a) CAX4 promoter::GUS expression in transgenic Arabidopsis roots. (a) CAX4::GUS expression in 7-d-old seedlings. Note the preferential GUS staining in the root meristem. (b) GUS staining in the primary root apex, lateral root primordia and lateral root under Ca2+-depleted conditions. (c) GUS staining in the primary root apex, lateral root primordia and lateral root elongation zone after treatment with 2 mM Mn2+. (d) GUS staining in the primary root apex, lateral root primordia and lateral root elongation zone after treatment with 0.1 mM Ni2+. (e) GUS staining in the primary root apex, lateral root primordia and lateral root elongation zone after treatment with 20 nM indole-3-acetic acid (IAA). All stress conditions were administered for 2 d. (f) CAX4 transcripts were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in total RNA from roots of wild-type seedlings treated for 2 d with the following conditions: half-strength Murashige and Skoog medium (1/2MS), 1/2MS + 2 mM Mn2+, 1/2MS + 0.1 mM Ni2+, depletion of Ca2+ from 1/2MS, and 1/2MS + 25 nM IAA. Top panel: a 414-bp CAX4-specific fragment was amplified by RT-PCR. Bottom panel: a 457-bp ACTIN fragment was amplified by RT-PCR as an internal control. The numbers indicate the relative intensities of the PCR bands in percentages relative to the control (grown on 1/2MS as a control).

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Identification of a CAX4 mutant allele and CAX4 RNAi lines

To investigate the physiological function of CAX4 in planta, we used a transposon insertion line, SM_3.16922. The transposon insertion was confirmed by PCR with a combination of gene-specific (P1 and P2) and transposon-specific (P3) primers, as shown in Fig. 2a. A homozygous line was isolated by PCR analysis and was termed cax4-1. The cax4-1 line contains the transposon insertion in the eighth intron, 2226 nucleotides downstream of the start codon of the CAX4 gene (Fig. 2a). RT-PCR was performed to determine the CAX4 transcript levels in cax4-1. As shown in Fig. 2b, no detectable CAX4 expression was observed in the cax4-1 plants, indicating that the expression of CAX4 was completely disrupted by the transposon insertion. Because only one cax4 line was obtained, we generated Arabidopsis lines harboring RNAi constructs for CAX4 to down-regulate its expression. The effect of RNAi on CAX4 transcript accumulation was assessed by RT-PCR. Two RNAi lines with clearly decreased expression of CAX4 were employed for further studies (Fig. 2d). Lines of cax4-1 expressing the deregulated version of CAX4 (HA-CAX4) (Cheng et al., 2002) were also generated, so that any CAX4 loss-of-function phenotype could be rescued. Three transgenic lines (cax4-1/HA-CAX4) harboring HA-CAX4 under the control of the CaMV 35S promoter were generated. The high level of HA-CAX4 expression in cax4-1 was confirmed using both RT-PCR and Western blot analysis employing an antibody against the HA epitope (Fig. 2c).

image

Figure 2. Analysis of a cax4 knockout allele and CAX4 RNA interference (RNAi) lines (CAX, CAtion eXchanger). (a) Diagram of the CAX4 gene depicting the site of the transposon insertion. The c. 3 kb of genomic CAX4 DNA is represented by 11 introns (lines) and 12 exons (boxes). The triangle indicates the sites of the transposon location. (b) Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of CAX4 expression in wild-type and cax4-1. (c) RT-PCR and Western analysis of CAX4 expression in wild-type and cax4-1/HA-CAX4 lines. (d) RT-PCR analysis of CAX4 expression in wild-type, CAX4 RNAi-1 and CAX4 RNAi-2. Total RNA was extracted from the roots of 3-wk-old wild-type, cax4-1, cax4-1/HA-CAX4, CAX4 RNAi-1 and CAX4 RNAi-2 seedlings. (b, d) Top panel: a 520-bp CAX4-specific fragment was amplified by RT-PCR. (b) Middle panel: a 430-bp CAX4-specific fragment was amplified by RT-PCR. (b–d) Bottom panel: a 457-bp ACTIN fragment was amplified by RT-PCR as an internal control. Total microsomal proteins were extracted from 3-wk-old wild-type and cax4-1/HA-CAX4 seedlings. (c) Top panel: a band of c. 49 kDa, which is the expected protein size of CAX4, was detected. (c) Bottom panel: Coomassie Blue G-250 (Invitrogen)-stained protein gel is shown as the loading control.

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Defects in cax4 root development under stress conditions

We examined cax4-1 and CAX4 RNAi transgenic lines under a wide range of conditions, comparing their growth and development with controls. Given that cations modified CAX4 expression, we tested cax4-1 and CAX4 RNAi transgenic lines on Ca2+-depleted and Ni2+-, Cd2+-, Ca2+- and Mn2+-containing media. When cax4-1 and CAX4 RNAi plants were germinated and grown on Cd2+- and Mn2+-containing media, altered root growth and development were observed in the cax4-1 and CAX4 RNAi lines compared with the wild-type (Fig. 3b). Under these conditions, the cax4-1 and CAX4 RNAi seedlings were much smaller than the control seedlings. Both primary root length and lateral root number were reduced significantly (Fig. 3b). The most severe phenotype of cax4-1 was observed on Cd2+-containing medium, where the development of lateral roots was almost completely disrupted (reduced by 70% compared with the wild-type on 2 µM Cd2+) (Fig. 3b). In addition, on Cd2+-containing medium, the cotyledons of the cax4-1 and CAX4 RNAi seedlings became yellow, and a marked reduction in chlorophyll was observed (Fig. 3). On Ca2+-depleted medium and Ni2+- or Ca2+-containing medium, the growth and development of cax4-1 and CAX4 RNAi transgenic lines were similar to those of control plants. Likewise, when grown on soil, no differences from controls were observed (data not shown). In all cases in which cax4-1 growth was perturbed, expression of HA-CAX4 was able to restore the growth changes that were attributed to reduced CAX4 expression (Fig. 3).

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Figure 3. Ion sensitivity of cax4-1 and cax4-1/HA-CAX4 seedlings (CAX, CAtion eXchanger). (a) In each panel, four wild-type plants are shown on the left, four cax4-1 plants are shown in the middle and four cax4-1/HA-CAX4 plants are shown on the right. All photographs are representative of more than 100 plants grown in each condition. Top panel: seeds were germinated on half-strength Murashige and Skoog medium (1/2MS) and grown for 10 d. Middle panel: seeds were germinated on 1/2MS containing 2 µM Cd2+ and grown for 10 d. Bottom panel: seeds were germinated on 1/2MS containing 5 µM Cd2+ and grown for 10 d. (b) Root growth measurements. Primary roots and lateral roots of seedlings grown on 1/2MS containing 2 µM Cd2+, 5 µM Cd2+ or 0.5 mM Mn2+ were measured and counted after 10 d. Error bars represent standard error of the means (n ≥ 12). cax4-1 had significantly shorter primary roots on 0.5 mM Mn2+ (P ≤ 0.0003), 2 µM Cd2+(P ≤ 0.0001) and 5 µM Cd2+ (P ≤ 0.003) and fewer lateral roots than the wild-type on 0.5 mM Mn2+ (P ≤ 0.0001), 2 µM Cd2+(P ≤ 0.005) and 5 µM Cd2+ (P ≤ 0.0001) in two-tailed t-tests, assuming unequal variance. The CAX4 RNA interference (RNAi) line also had significantly shorter primary roots on 0.5 mM Mn2+ (P ≤ 0.0001), 2 µM Cd2+(P ≤ 0.0001) and 5 µM Cd2+ (P ≤ 0.009) and fewer lateral roots than the wild-type on 0.5 mM Mn2+ (P ≤ 0.0001) and 5 µM Cd2+ (P ≤ 0.0006) in two-tailed t-tests, assuming unequal variance.

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CAX4 expression in cax1-1 mutant plants partially restores Ca2+/H+ antiport activity

To determine whether CAX4 functions in Arabidopsis Ca2+ transport, CaMV 35S::CAX4 was expressed in cax1-1 mutant plants. This mutant background is ideal for measuring vacuolar Ca2+/H+ antiport activity, because the mutant lines have 50% less activity than control plants (Cheng et al., 2003). Consistent with our previous findings, the vacuolar Ca2+/H+ antiport activity of cax1 membrane vesicles was decreased by 61% compared with wild-type controls (Fig. 4). By contrast, the cax1-1 lines expressing CaMV 35S::CAX4 displayed only a 33% decrease in vacuolar H+/Ca2+ antiport activity (Fig. 4).

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Figure 4. Ca2+ uptake into vacuole-enriched membrane vesicles from Ca2+-treated wild-type (squares), cax1-1 (circles) and cax1-1/CAX4 (triangles) root tissues. Time courses of Ca2+ uptake by Mg2+-ATP-energized vacuole-enriched membranes were determined in the presence of 0.1 mM NaN3, 10 mM KCl, 1 mM ATP and 10 µM Ca2+. ΔpH-dependent Ca2+/H+ antiport activity was confirmed by adding 5 µM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) in uptake reaction as a negative control, and net H+/Ca2+ antiport activity was shown after the subtraction of the FCCP background values. The Ca2+ ionophore A23187 (5 µM) was added after the 12 min time point and dissipated Ca2+ accumulation mediated by Ca2+/H+ antiport when measured at the 22 min time point. All results are the means ± standard error of experiments from three independent membrane preparations.

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Expression of CAX4 in transgenic Arabidopsis and tobacco induces ion sensitivity

We postulate that CAX4 has similar biochemical functions to CAX1 in plants. To investigate this possibility, we compared the phenotypes of plants expressing CAX4 with those expressing sCAX1 from previous studies (Hirschi, 1999; Cheng et al., 2003). We used independently transformed tobacco and Arabidopsis plants, which expressed high levels of CAX4 under the control of the CaMV 35S promoter. As shown in Fig. 5a,c, expression of CAX4 in five independent transgenic tobacco and four independent Arabidopsis lines was confirmed by Northern blot, and the lines with the highest expression (tobacco T1-35 and Arabidopsis F3-5-8) were used for further studies. As described previously, constitutive expression of deregulated sCAX1 in tobacco plants causes dramatic changes in plant growth and stress responses (Hirschi, 1999). The same conditions that were previously tested for sCAX1-expressing tobacco plants were used in this study to assay CAX4-expressing transgenic tobacco lines. The CAX4-expressing tobacco lines, like sCAX1-expressing lines, showed apical burning and necrotic lesions on their leaves in media containing Mg2+ and Mn2+ or depleted of Ca2+(Fig. 5b). These growth conditions did not affect the controls. Arabidopsis lines expressing CaMV 35S::CAX4 also showed severe necrotic lesions in leaves when grown in the presence of increased levels of Mg2+ (Fig. 5d). Previous studies have demonstrated that the addition of Ca2+ ameliorates the toxicity of Mg2+ in the medium in sCAX1-expressing tobacco lines (Hirschi, 1999). In a similar manner, the increased sensitivity of CAX4-expressing tobacco or Arabidopsis plants was alleviated with the addition of Ca2+ (Fig. 5b,d).

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Figure 5. Expression of CAX4 (CAX, CAtion eXchanger) in transgenic plants and ion sensitivity of CAX4-expressing plants. (a, c) Ten micrograms of total RNA extracted from fully expanded leaves of 6-wk-old T2 tobacco plants or 2-wk-old Arabidopsis plants were analyzed by RNA gel blotting. The blot was hybridized with the CAX4 cDNA probe. Ethidium bromide-stained rRNA before transfer is shown at the bottom. (b) Two vector plants are shown on the left and two CAX4-expressing tobacco plants (35S::CAX4) on the right. 10-d-old tobacco seedlings grown in half-strength Murashige and Skoog medium (1/2MS) were transferred to 1/2MS or 1/2MS containing the following metals, Mg2+, Mn2+, K+, Na+, Ca2+, Cd2+or Ca2+ depletion (concentrations of metals are indicated in the figure), and grown for 10 d. (d) Two vector control plants are shown on the left and CAX4-overexpressing Arabidopsis plants (35S::CAX4) plants on the right. Five-day-old Arabidopsis plants were transferred to 1/2MS or 1/2MS containing different metals (concentrations of metals are indicated in the figure) and grown for 5 d.

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cax4 is more sensitive to exogenous auxin

CAX4 is highly expressed in areas of the root in which auxin may influence growth and development. Furthermore, plants altered in CAX1 expression have altered auxin perception phenotypes (Cheng et al., 2003). We thus sought to address the role of CAX4 in auxin-mediated root growth. Figure 6 shows that the root elongation of cax4-1 plants was inhibited by IAA and by synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), in plants (data not shown). The inhibition of root elongation was more pronounced in cax4-1 lines than in CAX4 RNAi lines. In all cases, expression of HA-CAX4 caused the cax4-1 line to grow in a manner indistinguishable from controls (Fig. 6). These cax4 phenotypes appeared to be auxin specific. We tested the cax4-1 line and CAX4 RNAi lines on media containing cytokinin, gibberellins, abscisic acid or brassinolides, and no visible growth differences were observed between cax4-1 and control plants (data not shown).

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Figure 6. Root elongation inhibition by exogenous auxin. (a) In each panel, three wild-type plants are shown on the left, three cax4-1 plants in the middle and three cax4-1/HA-CAX4 plants on the right (CAX, CAtion eXchanger). All photographs are representative of > 100 plants grown in each condition. (a) Top panel: seeds were germinated on half-strength Murashige and Skoog medium (1/2MS) and grown for 10 d. Bottom panel: seeds were germinated on 1/2MS and grown for 6 d, and then transferred to 1/2MS containing 50 nM indole-3-acetic acid (IAA) and grown for 4 d. (b) Root growth measurements. Primary roots and lateral roots of seedling grown on 1/2MS containing 50 nM IAA were measured and counted after 4 d. Error bars represent standard error of the means (n ≥ 14). cax4-1 had significantly shorter primary roots (P ≤ 0.001) and less lateral roots (P ≤ 0.001) than the wild-type in two-tailed t-tests (assuming unequal variance).

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To determine whether the growth inhibition in cax4 is a result of the altered auxin response, histochemical analysis of the DR5::GUS auxin reporter in wild-type and cax4 roots was performed. DR5 is a synthetic auxin-responsive promoter that consists of a seven times repeat of the ARF site (AuxRE) from the soybean GH3 promoter and a CaMV 35S minimal promoter fused to the GUS-encoding reporter gene (Ulmasov et al., 1997). In Arabidopsis, DR5::GUS is sensitive to auxin in a dosage-dependent manner, and its activity reflects auxin responses in plant parts under the staining conditions typically used. Through genetic crossing, the DR5::GUS cassette was expressed in the cax4-1 line and DR5::GUS expression was monitored. Under normal growth conditions, the DR5::GUS signal was restricted to the root apical meristem, quiescent center and the provascular tissue in both wild-type and cax4-1 plants (Fig. 7). When exposed to exogenous IAA or 2,4-D for 24 or 48 h, DR5::GUS expression in the wild-type was increased (Ulmasov et al., 1997; Sabatini et al., 1999; Fig. 7a), whereas, in the cax4-1 line, DR5::GUS expression was unchanged (Fig. 7a). This result suggests that CAX4 may be required for auxin responses of DR5::GUS expression. However, there is the possibility that the effects of altered CAX4 levels on DR5::GUS expression are indirect through CAX4-mediating metal ion sequestration. To test whether metals are involved in these auxin responses, DR5::GUS expression was examined under different metal treatments. Interestingly, when grown on medium containing Cd2+, DR5::GUS expression in the wild-type was increased after 24 h (data not shown) and 48 h of Cd2+ treatment (Fig. 7b). By contrast, other metals tested, including K+, Na+, Mg2+, Mn2+, Ni2+ or Ca2+, did not affect DR5::GUS expression in the wild-type (data not shown).

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Figure 7. (a) DR5::GUS assay for auxin response in the cax4 allele (CAX, CAtion eXchanger; GUS, β-glucuronidase). Top panel: wild-type DR5::GUS seedlings and cax4-1 DR5::GUS seedlings were grown on half-strength Murashige and Skoog medium (1/2MS) for 12 d before staining for GUS activity. Bottom panel: wild-type DR5::GUS seedlings and cax4-1/DR5::GUS seedlings were grown on 1/2MS for 10 d and then transferred to 1/2MS containing 50 nM indole-3-acetic acid (IAA) for 2 d before staining for GUS activity. (b) DR5::GUS assay for Cd2+ responses in wild-type plants. Top panel: wild-type DR5::GUS seedlings were grown on 1/2MS for 12 d before staining for GUS activity. Bottom panel: wild-type DR5::GUS seedlings were grown on 1/2MS for 10 d and then transferred to 1/2MS containing 5 µM Cd2+ for 2 d before staining for GUS activity.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by the United States Department of Agriculture/Agricultural Research Service under Cooperative Agreement 58-6250-6001, National Science Foundation Grants #90344350 and #0209777, and USDA-CSREES #2005-34402-17121, Designing Foods for Health.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References