JXB Advance Access published online on September 26, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm190
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RESEARCH PAPER |
In planta regulation of the Arabidopsis Ca2+/H+ antiporter CAX1
1Molecular and Environmental Plant Science, Texas A&M University, College Station, Texas 77843, USA
2United States Department of Agriculture–Agricultural Research Service Children's Nutrition Research Center, Baylor College of Medicine, 1100 Bates Street, Houston, Texas 77030, USA
3Faculty of Life Sciences, University of Manchester, 3.614 Stopford Building, Oxford Road, Manchester M13 9PT, UK
4Vegetable and Fruit Improvement Center, Texas A&M University, College Station, Texas 77845, USA
* To whom correspondence should be addressed at the Baylor College of Medicine: E-mail: kendalh{at}bcm.tmc.edu
Received 8 May 2007; Revised 19 June 2007 Accepted 10 July 2007
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Abstract |
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Vacuolar localized Ca2+/H+ exchangers such as Arabidopsis thaliana cation exchanger 1 (CAX1) play important roles in Ca2+ homeostasis. When expressed in yeast, CAX1 is regulated via an N-terminal autoinhibitory domain. In yeast expression assays, a 36 amino acid N-terminal truncation of CAX1, termed sCAX1, and variants with specific mutations in this N-terminus, show CAX1-mediated Ca2+/H+ antiport activity. Furthermore, transgenic plants expressing sCAX1 display increased Ca2+ accumulation and heightened activity of vacuolar Ca2+/H+ antiport. Here the properties of N-terminal CAX1 variants in plants and yeast expression systems are compared and contrasted to determine if autoinhibition of CAX1 is occurring in planta. Initially, using ionome analysis, it has been demonstrated that only yeast cells expressing activated CAX1 transporters have altered total calcium content and fluctuations in zinc and nickel. Tobacco plants expressing activated CAX1 variants displayed hypersensitivity to ion imbalances, increased calcium accumulation, heightened concentrations of other mineral nutrients such as potassium, magnesium and manganese, and increased activity of tonoplast-enriched Ca2+/H+ transport. Despite high in planta gene expression, CAX1 and N-terminal variants of CAX1 which were not active in yeast, displayed none of the aforementioned phenotypes. Although several plant transporters appear to contain N-terminal autoinhibitory domains, this work is the first to document clearly N-terminal-dependent regulation of a Ca2+ transporter in transgenic plants. Engineering the autoinhibitory domain thus provides a strategy to enhance transport function to affect agronomic traits.
Key words: Arabidopsis, autoinhibition, calcium, ionome, tobacco, transport
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Introduction |
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In all biological systems, the intracellular calcium (Ca2+) concentrations fluctuate in response to environmental and internal cues (Sanders et al., 1999; Curran et al., 2000). The cytosolic Ca2+ levels in plant cells are tightly regulated by two opposing fluxes, the influx via Ca2+ channels and the efflux via Ca2+-ATPases and Ca2+/H+ antiporters (Sze et al., 2000). Up to now, the regulation of these transporters has been studied almost exclusively in heterologous systems such as yeast (Saccharomyces cerevisiae).
Ca2+/H+ antiporters are high-capacity, low-affinity transporters that have been physiologically characterized from a variety of plants and appear to locate predominantly on the vacuolar membrane, but also on the plasma membrane and chloroplast thylakoid membrane (Blumwald and Poole, 1986; Kasai and Muto, 1990; Ettinger et al., 1999; Cheng et al., 2001; Luo et al., 2005; Qi et al., 2005). To identify the first two plant Ca2+/H+ antiporter genes, a yeast suppression assay was carried out (Hirschi et al., 1996). Yeast strains lacking the vacuolar P-type Ca2+-ATPase (PMC1) and the vacuolar Ca2+/H+ antiporter (VCX1) grow slowly in media containing high levels of Ca2+(Cunningham and Fink, 1996). Two different Arabidopsis thaliana cDNAs were isolated which allowed pmc1 vcx1 double mutant yeast stains to grow well in the media containing high levels of Ca2+. These genes were termed cation exchanger (CAX) 1 and 2 (Hirschi et al., 1996). Experiments utilizing vacuolar membranes from yeast cells expressing CAX1 or CAX2 demonstrate that CAX1 is a Ca2+/H+ antiporter and CAX2 is a cation/H+ antiporter which transports Ca2+, Cd2+, and Mn2+ into the vacuole (Hirschi et al., 2000; Shigaki et al., 2003). Subsequently, numerous CAX transporters have been identified from various plant species (Kamiya et al., 2005; Shigaki and Hirschi, 2006) and their N-terminal autoinhibitory regions defined using yeast assays (Pittman and Hirschi, 2001).
An N-terminal autoinhibitory domain controls CAX1 activity as well as other CAX transporters including VCAX1 and CAX2 (Pittman and Hirschi, 2001; Pittman et al., 2002a, b, 2004). The originally identified CAX1 and CAX2 transporters are, in fact, products of partial length cDNAs and contain an N-terminal truncation which facilitates activity in yeast (now termed sCAX1 and sCAX2). In planta, CAX1 contains an additional 36 amino acid at the N-terminus which is not present in sCAX1. In yeast, full-length CAX1 acts as a weak vacuolar Ca2+/H+ antiporter, as transport activity is severely reduced when compared with sCAX1 (Cheng et al., 2005). This autoinhibition is caused by the N-terminus physically interacting with a neighbouring N-terminal region (residues 56–62) (Pittman and Hirschi, 2001; Pittman et al., 2002a). Further studies have identified crucial residues in these first 36 amino acids that are involved in N-terminal autoinhibition. For example, expressing a Thr-33 to Ala variant (T33A) of CAX1 effectively suppresses Ca2+ sensitivity in yeast mutants in a manner indistinguishable from sCAX1 expressing cells. Mutation of a Ser residue at position 25 to Asp (S25D) also activated CAX1, suggesting this Ser may be a potential target for phosphorylation as this mutation mimics constitutive phosphorylation, while a CAX1-S25A mutation gave a CAX1 mutant that was indistinguishable from wild-type CAX1 when expressed in the Ca2+-sensitive yeast cells (Pittman et al., 2002a). However, these plant transporter activities were assayed exclusively in yeast cells and it is not known if this activity, or lack thereof, is maintained in transgenic plants expressing these constructs. To date, the only evidence to hint that autoinhibition occurs in the plant and is not purely an artefact of the yeast expression is the ability of a synthetic peptide, which corresponds to the first 36 amino acids of CAX1, to inhibit tonoplast Ca2+/H+ transport activity in Arabidopsis (Pittman et al., 2002b; Cheng et al., 2003); however, a more rigorous confirmation is required.
Transgenic plants expressing CAX transporters may be a component of long-range goals to enhance plant yield and improve human nutrition (Shigaki and Hirschi, 2006). Tobacco, carrots, potatoes, and tomatoes expressing sCAX1 all contain high levels of calcium (Hirschi, 1999; Park et al., 2004, 2005a, b). In some cases, when transgenic plants express sCAX1, the phenotypes are dramatic. Tobacco (Nicotiana tabacum) lines expressing sCAX1 exhibit Ca2+-deficient symptoms such as leaf necrosis, tip burning, and hypersensitivity to ion imbalance as well as increased tonoplast Ca2+/H+ transport activity (Hirschi, 1999). If the aim is to carefully modulate Ca2+/H+ transport activity in various crop plants, it is important to delineate if these phenotypes are caused by the deregulated nature of sCAX1. In addition, insights into the phenotypes associated with expression of various CAX1 variants should facilitate the rational design of transporters which maximize calcium accumulation but minimize deleterious phenotypes.
Recent technologies make it possible to measure how CAX expression modulates the sum total of all the mineral nutrients and trace elements, termed the ionome (Lahner et al., 2003; Cheng et al., 2005). This technology can now be harnessed as a means not only to measure phenotypes but also provide important clues about gene function. For example, yeast ionome profiles have been used to reveal the function of previously uncharacterized yeast genes (Eide et al., 2005). In theory, heterologous expression and ionome measurements could be used to characterize plant transporters.
Here, N-terminal autoinhibition of CAX1 is evaluated in transgenic tobacco plants. Initially, the yeast ionome was used to compare the activity of CAX1 variants in yeast cells. CAX1 variants were then expressd in transgenic tobacco plants in order to assess differences in growth and biochemical phenotypes. These results were compared with our previous findings utilizing the yeast expression assays. Collectively, the results suggest that the yeast assays provide a robust model to assess CAX autoinhibition. To our knowledge, this is the first report to demonstrate clearly the presence of N-terminal autoinhibition of transport function in planta. These findings also offer insights into engineering enhanced transporter activity into agriculturally important crops.
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Yeast strain and plasmids
The yeast (Saccharomyces cerevisiae) strain K667 (cnb1::LEU2 pmc1::TRP1 vcx1
; Cunningham and Fink, 1996) was transformed and grown as described previously (Shigaki et al., 2003). The CAX1, sCAX1, CAX1-T33A, and CAX1-S25A cDNA clones were subcloned into the yeast expression vector piHGpd (Nathan et al., 1999) as described in previous studies (Pittman and Hirschi, 2001; Pittman et al., 2002a).
Yeast culture conditions, sample processing, and ICP-AES analysis
Yeast culture conditions and sample processing were modified from a previous study (Eide et al., 2005). Yeast cultures were inoculated in 5 ml yeast–peptone–dextrose (YPD) and 1/100 vol. of 100x mineral supplement stock (Eide et al., 2005) with additional 10 mM CaCl2. The cells were grown at 30 °C to stationary phase. A 2.5 ml sample of each culture was collected by vacuum filtration using isopore membrane filters (1.2 µm pore size) (Fisher Scientific, PA, USA). Cells were washed three times with 1 ml of 1 µM ethylenediaminetetraacetic acid disodium salt solution, pH 8.0, by vacuum filtration followed by three washes with 1 ml of distilled, deionized H2O. The filters were dried at 70 °C in an oven for 48 h before inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis, performed as previously described (Lahner et al., 2003).
Plant materials, transformation, and growth conditions
The generation of the sCAX1 in the pBin19 construct has been previously described (Hirschi, 1999). The CAX1, CAX1-T33A, and CAX1-S25A cDNAs were subcloned into the plant expression vector pBin19 (Clontech, CA, USA), which contained the cauliflower mosaic virus (CaMV) 35S promoter fragment. The recombinant plasmids or vector controls were transformed into Agrobacterium tumefaciens LBA4404 (Invitrogen, CA, USA). Tobacco (Nicotiana tabacum) plants (cvs KY160 and KY14) were transformed along with the empty vector as a control as previously described. CAX1 and sCAX1 were expressed in KY160 (Hirschi, 1999). CAX1-T33A and CAX1-S25A were expressed in KY14 (this work).
Surface-sterilized tobacco seeds were germinated and grown as previously described (Hirschi, 1999). For ion sensitivity analysis, 14 d after plating, the seedlings were transferred to half-strength MS medium (Murashige and Skoog, 1962) containing 1% (w/v) agar supplemented with the appropriate metal salt. To make media deficient in Ca2+, standard half-strength MS medium including 1% (w/v) agar was prepared, except that CaCl2 was omitted. For the transport assays, tobacco seeds germinated on the standard medium were transferred to soil, and then the 4-week-old plants were transferred to a hydroponic system containing Hoagland's solution (Hoagland and Arnon, 1950).
DNA and RNA isolation: Southern and northern blot analysis
Tobacco genomic DNA was extracted from leaf tissue as previously described by Paterson et al. (1993). DNA (5–10 µg) was digested with EcoRI or BamHI and separated by electrophoresis and blotted onto Hybond N+ membrane (Amersham Biosciences, NJ, USA). Total RNA was extracted from tobacco leaves as previously described (Hirschi, 1999). RNA samples were treated with DNase to minimize any contamination of genomic DNA. The sequences of the CAX1 gene-specific primers used in reverse transcription PCR were 5'-AAAAAATCAGACCTCCGAGTGATTCAGAA-3' and 5'-CCTTTCTCCATTGTCTCTGCTTTGGAAA-3'. Total RNA (10 µg) was separated on 1% (v/v) agarose gel containing formaldehyde, blotted onto Hybond N+ membrane (Amersham), according to the manufacturer's instructions. PCR reactions were performed to make probes for both Southern and northern blot analysis, using a CAX1-specific primer set. The sequences of the CAX1 primers were 5'-ATGGCGGGAATCGTGACAGAGCC-3' and 5'-TTAACCCGTTTTAACTTTATTTG-3'. The membranes were prehybridized overnight at 60 °C in 7% (w/v) SDS and 0.25 M Na2HPO4, and then hybridized overnight at 60 °C in the same solution containing the probe labelled with 32P-dCTP using a random primed labelling kit (Invitrogen, CA, USA). Membranes were washed twice for 30 min each with 2x SSC and 1% SDS at 60 °C and then washed twice again for 30 min each with 0.1x SSC and 0.5% (w/v) SDS at 60 °C. Membranes were exposed to X-ray film at –80 °C.
Preparation of membrane vesicles and Ca2+ transport assay
Microsomal membrane vesicles were prepared from 8-week-old hydroponically grown plant root tissue and pretreated with 100 mM CaCl2 for 18 h before harvest. All membrane isolation steps were conducted at 4 °C as previously described (Hirschi, 1999). 45Ca2+ uptake was measured by a filtration assay as previously described (Pittman and Hirschi, 2001).
Calcium and mineral analysis
Four-week-old tobacco plants were transferred to hydroponic growth for 4 weeks in Hoagland's solution supplemented with 2 mM CaCl2. The roots were rinsed in water and harvested. The roots were dried at 70 °C for 3 d. At least 500 mg (dry weight) of root tissue was analysed individually; roots weighing less were pooled to yield the required weight as described previously (Hirschi, 1999). Total calcium and mineral contents per gram of dry mass were determined by using an inductively coupled plasma atomic emission spectrophotometer (Spectro, Kleve, Germany) as previously described (Lahner et al., 2003).
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CAX1 expression and the yeast ionome
The authors were interested in assessing the impact of expressing CAX variants on the yeast ionome. To summarize previous results, the CAX1-T33A variant made CAX1 constitutively active comparable with sCAX1 while a CAX1-S25A variant was no more active than CAX1 (Pittman et al., 2002a). In this study, these variants, the empty vector control, CAX1, and sCAX1 were expressed in the K667 (vcx1 pmc1 cnb1) mutant yeast strain, which is deficient in vacuolar Ca2+ transporters (Cunningham and Fink, 1996), and the cells were grown in liquid media supplemented with several elements to levels sufficient to facilitate detection in cell extracts by ICP-AES. As shown in Fig. 1A, sCAX1-expressing yeast cells accumulated Ca2+ approximately 4-fold compared with the vector control cells. The accumulation of calcium in the CAX1-T33A-expressing yeast cells was similar to that of sCAX1-expressing cells. Meanwhile CAX1- and CAX1-S25A-expressing yeast cells contained levels of calcium similar to the vector control cells (Fig. 1A). In addition to the increased calcium accumulation, nickel, zinc, iron, and magnesium levels were also increased in sCAX1- and CAX1-T33A-expressing yeast cells compared with the vector control cells (Fig. 1B). Again, these increases in other metals were not seen in the CAX1- and CAX1-S25A-expressing yeast cells. Other metal levels such as sodium, manganese, potassium, phosphorus, and copper were similar among all the yeast cells tested (Fig. 1C).
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CAX variants expressed in transgenic tobacco
To gain insights into the N-terminal autoinhibition of CAX1 in planta, empty vector controls, CAX1, sCAX1, CAX1-T33A, and CAX1-S25A were expressed in tobacco plants. It has previously been demonstrated that tobacco is an excellent plant model for assessing Ca2+/H+ antiport function because the morphological phenotypes which result from increased vacuolar Ca2+ loading, such as tip burning, are easily visualized (Hirschi, 1999; Shigaki et al., 2002). By contrast, the morphological differences when sCAX1 is expressed in Arabidopsis plants are quite subtle (Cheng et al., 2003). The tobacco cultivar KY160 line expressing 35S::sCAX1 was obtained from previous studies (Hirschi, 1999). In this study, 35S::CAX1 was expressed in tobacco cultivar KY160 and 35S::CAX1-T33A and 35S::CAX1-S25A were expressed in the tobacco cultivar KY14. Initially, it was determined that KY160 and KY14 were indistinguishable in the growth assays used here except for the difference in leaf colour and morphology (data not shown). Thus, the impact of the CAX1 variants could easily be compared without regard to the differences between KY160 and KY14. As mentioned previously, tobacco lines expressing sCAX1 were previously characterized (Hirschi, 1999). This sCAX1 expressing line is representative of the multiple independent transgenic lines previously characterized. Here, at least 10 different transgenic tobacco lines expressing CAX1, CAX1-T33A or CAX1-S25A were obtained. Southern analysis was performed and it was determined that all transgenic tobacco lines analysed contained multiple insertions (data not shown). Expression of CAX1, CAX1-T33A, and CAX1-S25A was initially measured by reverse transcription PCR (data not shown). Two independent transgenic lines expressing CAX1, CAX1-T33A, and CAX1-S25A with comparable expression levels to sCAX1 were confirmed by northern blot analysis (Fig. 2; lines 1 and 2). Together, these observations suggested that the expression levels were similar and there was no dramatic copy number difference among the transgenic lines. With these preliminary observations, the CAX1, CAX1-T33A, CAX1-S25A and sCAX1 expressing lines could confidently be compared and contrasted.
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Phenotypes of tobacco lines expressing CAX1 variants
Once the appropriate transgenic tobacco lines were identified, phenotypes were compared among CAX1, sCAX1, and the variants previously characterized exclusively in yeast (Hirschi, 1999; Pittman et al., 2002a). As reported earlier, transgenic tobacco seedlings that constitutively express the deregulated sCAX1 display hypersensitivity to ion imbalances, such as increased Mg2+ and Na+ and depleted Ca2+ (Hirschi, 1999). As shown in Fig. 3B, CAX1-T33A-expressing plants were extremely hypersensitive to Ca2+-depleted media in a manner similar to sCAX1-expressing lines. The growth of CAX1- and CAX1-S25A-expressing plants on the Ca2+-depleted media was comparable to vector control plants. In the Na+ tolerance growth assays, the same trend was observed. The sCAX1-expressing plants and CAX1-T33A-expressing plants were sensitive to the excess Na+ in the media while the CAX1-expressing plants and CAX1-S25A-expressing plants were not (Fig. 3C). In addition to the ion sensitivity, approximately 16% (6 out of 37) of the CAX1-T33A-expressing plants displayed the tip-burning Ca2+-deficient symptom associated with sCAX1 expression; however, this was not as dramatic as seen in sCAX1-expressing lines which displayed 100% tip-burning symptoms (data not shown). In soil, all CAX1- and CAX1-S25A-expressing plants grew in a manner indistinguishable from vector control lines.
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Tonoplast Ca2+/H+ antiport activity from tobacco lines expressing CAX1 variants
It has previously been shown that expression of sCAX1 increases tonoplast Ca2+/H+ activity at least 30% more in transgenic tobacco roots than in controls (Hirschi, 1999). In addition, the increased Ca2+ accumulation was observed primarily in tobacco roots rather than leaves (Hirschi, 1999). As with the ion sensitivity phenotypes, tonoplast Ca2+/H+ transport activity among vector control, sCAX1-, CAX1-, CAX1-T33A-, and CAX1-S25A-expressing lines were compared. Tonoplast-enriched vesicles were prepared from roots of hydroponically grown transgenic tobacco lines. A steady proton gradient (acid inside vesicles) was established by activation of the Mg2+-ATP-dependent H+-ATPase. sCAX1-expressing vesicles showed significant Ca2+/H+ antiport activity. Vector-expressing vesicles showed very low Ca2+/H+ antiport activity. The CAX1-T33A-expressing line had moderate Ca2+ uptake activity compared to that expressing sCAX1, and was approximately 64% of the sCAX1-expressing line (Fig. 4). However, in the tonoplast vesicles prepared from CAX1-expressing and CAX1-S25A-expressing lines, this uptake assay could not measure any higher Ca2+/H+ antiport activity than vector control (data not shown). The increase in Ca2+/H+ antiport activity was consistently measured only in the sCAX1- and CAX1-T33A-expressing lines.
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Metal accumulation in tobacco lines expressing-CAX1 variants
Calcium levels were measured in the transgenic tobacco roots to examine whether altered Ca2+/H+ antiport activity impacted the accumulation of Ca2+ in tobacco roots. As demonstrated previously (Hirschi, 1999), sCAX1-expressing plants accumulated approximately three times more calcium in root tissue than the vector control lines (Fig. 5A). A slightly lower amount of calcium was accumulated in the CAX1-T33A-expressing plants as they contained approximately twice as much total calcium in roots as did the vector controls (Fig. 5A). Meanwhile, the vector controls, CAX1- and CAX1-S25A-expressing plants contained similar calcium levels (Fig. 5A). These results are in general agreement with yeast ionome results presented earlier (Fig. 1A). Comparison of other mineral profiles revealed that potassium, magnesium, and manganese in sCAX1- and CAX1-T33A- expressing tobacco line were increased more than 2-fold, while zinc and/or phosphorus content also increased substantially in the sCAX1- and CAX1-T33A-expressing lines (Fig. 5B). Although concentrations of other minerals were slightly increased in the CAX1- and CAX1-S25A-expressing plants, they were more similar to control plants (Fig. 5B).
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Discussion |
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Previously, yeast assays have been used to suggest that CAX1 is regulated by an N-terminal autoinhibitory domain. Here, it has been established that these yeast assays provide a robust platform for predicting activity of CAX1 variants in transgenic plants. As far as is known, this type of cross-platform evaluation of N-terminal autoinhibition of plant transporters has not been previously reported. Furthermore, for the first time, it has been confirmed that N-terminal-mediated regulation of a Ca2+ transporter occurs in planta.
The yeast ionome: a new phenotype to characterize plant transporter function
The ionome provides a rigorous tool to examine the elemental profiles of various samples, be it a plant tissue or a yeast colony (Lahner et al., 2003; Hirschi, 2003; Eide et al., 2005). Given that the expression of many plant transporters in yeast does not generate a visual phenotype, the reproducibility and cost of these yeast ionome assays affords a new phenotype to infer transport function. It has been demonstrated here that measuring the yeast ionome from cells expressing variants of the Arabidopsis CAX1 transporter can be a rapid and reproducible inference of transporter function (Fig. 1). For example, sCAX1 and CAX1-T33A expressing yeast cells both demonstrated high tonoplast Ca2+/H+ activity and accumulated significant higher cellular calcium, whereas yeast cells expressing vector, CAX1 or the CAX1-S25A variant displayed negligible tonoplast Ca2+/H+ transport and lower total cellular calcium levels (Pittman et al., 2002a; Fig. 1A). As the mutant yeast strain used is lacking the endogenous tonoplast Ca2+/H+ antiporter VCX1, this lack of detectable activity in the vector control and CAX1-/CAX1-S25A-expressing strains was expected. Previously, ion competition and metal-dependent proton transport studies were used to suggest that sCAX1 can transport Ni2+, and possibly Zn2+ but not Mn2+ (Shigaki et al., 2003, 2005). In these ionome studies sCAX1- and CAX1-T33A- expressing yeast cells also contained significantly increased levels of nickel, and zinc (Fig. 1B). The increase of nickel and zinc in sCAX1- and CAX1-T33A-expressing lines is in an agreement with our published transport data (Shigaki et al., 2003, 2005; Fig. 1B). In addition to nickel and zinc, magnesium and iron accumulated to higher levels than control in sCAX1-expressing and T33A-expressing yeast cells (Fig. 1B). The increase of iron and magnesium could be an indirect result of increased Ca2+ transport. However, a previous yeast ionome study suggests that the homeostatic mechanisms that control the levels of different elements are interconnected. For example, changes in nickel levels correlate with changes of magnesium in the yeast ionome (Eide et al., 2005). Indeed, this could explain our observation that the increased magnesium in cells was correlated with increased nickel. While the increased levels of nickel and zinc in the sCAX1 and CAX1-T33A-expressing yeast cells were detectable, the much higher 4-fold increase in calcium in these cells confirms that sCAX1 is predominantly a Ca2+ transporter.
Phosphorus, potassium, and manganese levels did not significantly alter in the sCAX1- and CAX1-T33A-expressing yeast cells, yet following expression of these CAX1 variants in tobacco, these three metals were significantly increased. In addition, zinc and iron levels were slightly increased in these lines. Previous analysis has indicated that K+ is very poorly transported by sCAX1 (Park et al., 2005b) while Mn2+ and Fe2+ are not transported (Shigaki et al., 2003; J Pittman and K Hirschi, unpublished results), although sCAX1 does have some Zn2+ transport activity (Shigaki et al., 2005). Previously, increased levels of magnesium, zinc, iron, and manganese were observed in sCAX1-expressing tomato fruits, while phosphorus and potassium were not tested (Park et al., 2005b). This is generally consistent with the observations in sCAX1- and CAX1-T33A-expressing tobacco, although the iron and zinc increases are more subtle in tobacco. This may be due to tissue–type differences. The increased accumulation of these metals in the transgenic tobacco plants could be an indirect effect of increased vacuolar Ca2+/H+ transport activity that may lead to perturbed cytosolic Ca2+ levels which, in turn, may regulate other metal transporters (Cheng et al., 2005). This result also highlights the differences that may exist between unicellular yeast and plants with regard to homeostatic regulation for certain metals. However, the trend in calcium content following expression of the various CAX1 constructs was identical between yeast and plant. Thus our experimental approach and results suggest that in heterologous expression studies, the yeast ionome profiles can be used as an initial indicator of Arabidopsis transporter function.
CAX1 N-terminal autoinhibition in planta
It has been demonstrated here that transport activity of the full-length CAX1 protein is regulated in transgenic plants compared with significant deregulated transport activity of the N-terminal truncated sCAX1 or the N-terminal point mutant CAX1-T33A. It is suggested that, as in yeast, this down-regulation of Arabidopsis CAX1 is due to an autoinhibitory mechanism caused by protein conformation modification at the N-terminus (Pittman et al., 2002a). As observed previously, high level expression of sCAX1 in tobacco causes Ca2+ deficiency-like phenotypes such as apical burning and hypersensitivity to ion imbalances that are due to excessive and deregulated Ca2+ transport into the vacuole by sCAX1 (Hirschi, 1999). When the sCAX1 plants are grown on Ca2+-depleted conditions, the enhanced vacuolar accumulation of trace concentrations of Ca2+ acquired by the plant prevent any Ca2+ being acquired to other cellular locations where it is essential. The sensitivity to Na+ stress is also due to cytosolic Ca2+ deficiency caused by excessive vacuolar Ca2+ accumulation (Hirschi, 1999; Cheng et al., 2004b). By comparison, CAX1 was relatively inactive when expressed in transgenic tobacco plants. For example, none of the Ca2+-deficient symptoms associated with sCAX1 expression were seen in the CAX1-expressing plants (Fig. 3). Furthermore, increased Ca2+/H+ activity was not detected from the vesicles isolated from the CAX1 expressing transgenic roots (Fig. 4). Thus, the presence of the N-terminal domain of CAX1 appeared to autoinhibit the protein and prevent Ca2+ transport activity in tobacco. This also suggests that, unlike when expressed in Arabidopsis, mechanisms are not present in tobacco to switch on the autoinhibited Arabidopsis CAX1 (see below). Expression of the N-terminal mutated variants of CAX1 produced phenotypes in tobacco that correlated with their phenotypes in yeast cells. The CAX1-S25A variant, like CAX1, displayed no increased Ca2+/H+ activity when expressed in either yeast cells or tobacco plants (Figs 1, 2, 4
). Conversely, expressing the CAX1-T33A variant produced phenotypes in both yeast and tobacco (Fig. 3); however, direct measurement of Ca2+ transport activity of CAX1-T33A from the transgenic tobacco plants was lower than sCAX1 (64%) and the percentage of plants displaying apical burning was reduced (16%). Furthermore, the plants expressing-CAX1-T33A variants accumulated less total calcium than sCAX1 lines, but significantly higher levels than CAX1, CAX1-S25A, and vector lines (Fig. 5A). Previous experiments in yeast found that substitution of Thr-33 with another residue (either Ala, Asp, Ser or Glu) caused a gain of transport activity, leading to the suggestion that this residue plays a role in the proposed autoinhibitory mechanism (Pittman et al., 2002a). While substitution of Ser-25 with Ala did not lead to a gain of transport activity, it has been shown previously that a Ser-25 to Asp substitution caused a gain of activity; hence it is proposed that this residue could be a candidate for phosphorylation as a mechanism to activate full-length CAX1 Ca2+/H+ antiport activity (Pittman et al., 2002a). Further analysis will be required to ascertain the exact functions of these CAX1 N-terminal domain residues.
N-terminal autoinhibition is not unique to CAX transporters. For example, the plant autoinhibited Ca2+-ATPases (ACAs) also have an N-terminal autoinhibitory domain which is equivalent in structure and function to the C-terminal regulatory domain of animal calmodulin-stimulated Ca2+-ATPases (Hwang et al., 2000; Bonza et al., 2004). Studies using a Ca2+-ATPase deficient yeast mutant have demonstrated that plant ACA pumps can functionally complement the mutant only if the N-terminal calmodulin binding/autoinhibitory domain is deleted or if single residues required for autoinhibition have been mutated (Baekgaard et al., 2005). It is clear that calmodulin can stimulate Ca2+-ATPase activity in membrane fractions isolated from plants, and it has also been demonstrated that a peptide corresponding to the autoinhibitory domain of ACA8 can inhibit Ca2+-ATPase activity of a truncated form of the pump in membrane fractions from Arabidopsis suspension cells (Luoni et al., 2004). However, the ACA studies have not been extended by functionally expressing the deregulated ACA clones in a plant system to examine this autoinhibition in planta, while our work suggests that removal, or modification, of the CAX1 N-terminal domains will activate these transporters when expressed in the plant.
Several signalling molecules have been implicated in regulating plant transporters (Hwang et al., 2000; Cheng et al., 2004a). Calmodulin binding to the N-terminus of ACA2 causes activation, whereas the pump can be inhibited by a Ca2+-dependent protein kinase (Hwang et al., 2000). With the CAX transporters similar models have been posited where various regulators, such as the protein kinase SOS2, may either activate or repress transporter function (Cheng et al., 2004a, b). Our work here suggests the presence of functional N-terminal autoinhibitory domains in planta and by extension the cognate signalling molecules. These results may also suggest that the regulatory CAX1 activating proteins, which do not appear to be conserved in yeast, may likewise not be conserved amongst plant species, otherwise it might have been expected that expression of full-length CAX1 in tobacco would provide transport activity. It will be interesting to test in the future whether co-expression of CAX1 and an activator protein in tobacco causes an increase in Ca2+ transport activity. Our working hypothesis therefore is that high level expression of these regulatory modules is an additional means of altering transport function.
Applications for plant improvement
Previous studies have demonstrated that expression of the Arabidopsis Ca2+/H+ antiporter sCAX1 can be used to increase Ca2+ content in several important crops (Park et al., 2004, 2005a, b). In tomatoes, tempering sCAX1 expression levels by using a weaker promoter resulted in more fruit calcium and a prolonged shelf life, while only modestly compromising plant growth and development (Park et al., 2005b). These advantageous alterations in tomato plant growth suggest that further modulation of the expression and activity of a Ca2+/H+ transporter can be used to enhance crop growth. For example, expression in tomatoes of CAX4, which appears to have weaker Ca2+/H+ antiport activity than sCAX1, provided a modest increase in the calcium content without deleterious phenotypes (Park et al., 2005b). The data presented here suggest that, rather than altering expression levels, the N-terminal autoinhibitory domain can be modified to modulate activity. Modifications like the CAX1-T33A mutation in CAX1 could provide important tools for calcium fortification. These variants remain active and thus have higher levels of calcium accumulation (Fig. 5), but less deleterious effects such as the apical burning associated with sCAX1 expression. Our long-range goal is to engineer CAX variants which are active but produce no deleterious phenotypes.
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The N-terminal autoinhibition in the Ca2+/H+ transporter CAX1 has been intensively documented using yeast assays. In this study, these studies have been extended to show that N-terminal autoinhibition does indeed occur in planta. These findings provide a general platform to modify plant transporter activity.
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Acknowledgements |
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This work was supported by the US Department of Agriculture/Agricultural Research Service (under Cooperative Agreement 58-6250-6001) and by the National Science Foundation (grant NSF 0344350).
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Bækgaard L, Fuglsang AT, Palmgren MG. Regulation of plant plasma membrane H+- and Ca2+-ATPases by terminal domains. Journal of Bioenergetics and Biomembranes (2005) 37:369–374.[CrossRef][Web of Science][Medline]
Blumwald E, Poole RJ. Kinetics of Ca2+/H+ antiport in isolated tonoplast vesicles form storage tissue of Beta vulgaris L. Plant Physiology (1986) 80:727–731.
Bonza MC, Luoni L, De Michelis MI. Functional expression in yeast of N-deleted form of At-ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana, and characterization of a hyperactive mutant. Planta (2004) 218:814–823.[CrossRef][Web of Science][Medline]
Cheng NH, Liu J, Nelson S, Hirschi KD. Characterization of CXIP4, a novel Arabidopsis protein that activates the H+/Ca2+ antiporter, CAX1. FEBS Letters (2004a) 559:99–106.[CrossRef][Web of Science][Medline]
Cheng NH, Pittman JK, Barkla BJ, Shigaki T, Hirschi KD. The Arabidopsis cax1 mutant exhibits impaired ion homeostasis, development, and hormonal responses and reveals interplay among vacuolar transporters. The Plant Cell (2003) 15:347–364.
Cheng NH, Pittman JK, Shigaki T, Hirschi KD. Characterization of CAX4: an Arabidopsis H+/cation antiporter. Plant Physiology (2001) 128:1245–1254.[CrossRef][Web of Science]
Cheng NH, Pittman JK, Shigaki T, Lachmansingh J, LeClere S, Lahner B, Salt DE, Hirschi KD. Functional association of Arabidopsis CAX1 and CAX3 is required for normal growth and ion homeostasis. Plant Physiology (2005) 138:2048–2060.
Cheng NH, Pittman JK, Zhu K, Hirschi KD. The protein kinase SOS2 activates the Arabidopsis H+/Ca2+ antiporter CAX1 to integrate calcium transport and salt tolerance. Journal of Biological Chemistry (2004b) 279:2922–2926.
Cunningham KW, Fink GR. Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPase in Saccharomyces cerevisiae. Molecular and Cellular Biology (1996) 16:2226–2237.[Abstract]
Curran AC, Hwang I, Corbin J, Matinez S, Rayle D, Sze H, Harper JF. Autoinhibition of a calmodulin-dependent calcium pump involves a structure in stalk that connects the transmembrane domain to the ATPase catalytic domain. Journal of Biological Chemistry (2000) 275:30301–30308.
Eide DJ, Clark S, Nair TM, Gehl M, Gribskov M, Guerinot ML, Harper JF. Characterization of the yeast ionome: a genome-wide analysis of nutrient mineral and trace element homeostasis in Saccharomyces cerevisiae. Genome Biology (2005) 6:R77.[CrossRef][Medline]
Ettinger WF, Clear AM, Fanning KJ, Ml Peck. Identification of a Ca2+/H+ antiport in the plant chloroplast thylakoid membrane. Plant Physiology (1999) 119:1379–1386.
Hwang I, Sze H, Harper JF. A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis. Proceedings of the National Academy of Sciences, USA (2000) 97:6224–6229.
Hirschi KD. Expression of arabidopsis CAX1 in tobacco: altered calcium homeostasis and increased stress sensitivity. The Plant Cell (1999) 11:2113–2122.
Hirschi KD. Strike while the ionome is hot: making the most of plant genomic advances. Trends in Biotechnology (2003) 21:520–521.[CrossRef][Web of Science][Medline]
Hirschi KD, Korenkov V, Wilganowski N, Wagner G. Expression of Arabidopsis CAX2 in tobacco: altered metal accumulation and increased manganese tolerance. Plant Physiology (2000) 124:125–134.
Hirschi KD, Zhen R-G, Cunningham KW, Rea PA, Fink GR. CAX1, an H+/Ca2+ antiporter from Arabidopsis. Proceedings of the National Academy of Sciences, USA (1996) 93:8782–8786.
Hoagland DR, Arnon DI. The water-culture method for growing plants without soil. California Agricultural Experiment Station Circular (1950) 347:1–32.
Kamiya T, Akahori T, Ashikari M, Maesshima M. Expression of the vacuolar Ca2+/H+ exchanger, OsCAX1a, in rice: cell and age specificity of expression and enhancement by Ca2+. Plant and Cell Physiology (2005) 47:96–106.[CrossRef][Web of Science][Medline]
Kasai M, Muto S. Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves. Journal of Membrane Biology (1990) 114:133–142.[CrossRef][Web of Science][Medline]
Lahner B, Gong J, Mahmoudian M, et al. Genomic scale profiling of nutrient and trace elements in Arabidopsis thaliana. Nature Biotechnology (2003) 21:1215–1221.[CrossRef][Web of Science][Medline]
Luo GZ, Wang HW, Huang J, Tian AG, Wang YJ, Zhang JS, Chen SY. A putative plasma membrane cation/proton antiporter from soybean confers salt tolerance in Arabidopsis. Plant Molecular Biology (2005) 59:809–820.[CrossRef][Web of Science][Medline]
Luoni L, Meneghelli S, Bonza MC, De Michelis MI. Auto-inhibition of Arabidopsis thaliana plasma membrane Ca2+-ATPase involves an interaction of the N-terminus with the small cytoplasmic loop. FEBS Letters (2004) 574:20–24.[CrossRef][Web of Science][Medline]
Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiology (1962) 15:473–497.[CrossRef]
Nathan DF, Vos MH, Lindquist S. Identification of SSF1 and HCH1 as multicopy suppressors of a Saccharomyces cerevisiae Hsp90 loss-of-function mutation. Proceedings of the National Academy of Sciences, USA (1999) 96:1409–1414.
Paterson AH, Brubaker CL, Wendel JF. A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis. Plant Molecular Biology Reporter (1993) 11:122–127.
Park S, Cheng NH, Pittman JK, Yoo KS, Park J, Smith RH, Hirschi KD. Increased calcium levels and prolonged shelf life in tomatoes expressing Arabidopsis H+/Ca2+ transporters. Plant Physiology (2005b) 139:1194–1206.
Park S, Kang TS, Kim CK, Han JS, Kim S, Smith RH, Pike LM, Hirschi KD. Genetic manipulation for enhancing calcium content in potato tuber. Journal of Agricultural and Food Chemistry (2005a) 53:5598–5603.[CrossRef][Web of Science][Medline]
Park S, Kim C, Pike L, Smith R, Hirschi KD. Increased calcium in carrots by expression of an Arabidopsis H+/Ca2+ transporter. Molecular Breeding (2004) 14:275–282.[CrossRef][Web of Science]
Pittman JK, Hirschi KD. Regulation of CAX1, an Arabidopsis Ca2+/H+ antiporter: identification of an N-terminal autoinhibitory domain. Plant Physiology (2001) 127:1020–1029.
Pittman JK, Shigaki T, Cheng N, Hirschi KD. Mechanism of N-terminal autoinhibition in the Arabidopsis Ca2+/H+ antiporter CAX1. Journal of Biological Chemistry (2002a) 277:26452–26459.
Pittman JK, Shigaki T, Marshall JL, Morris JL, Cheng NH, Hirschi KD. Functional and regulatory analysis of the Arabidopsis thaliana CAX2 cation transporter. Plant Molecular Biology (2004) 56:959–971.[CrossRef][Web of Science][Medline]
Pittman JK, Sreevidya CS, Shigaki T, Ueoka-Nakanishi H, Hirschi KD. Distinct N-terminal regulatory domains of Ca2+/H+ antiporters. Plant Physiology (2002b) 130:1054–1062.
Qi BS, Li CG, Chen YM, Lu PL, Hao FS, Shen GM, Chen J, Wang XC. Functional analysis of rice Ca2+/H+ antiporter OsCAX3 in yeast and its subcellular localization in plant. Progress in Biochemistry and Biophysics (2005) 32:876–881. (in Chinese).[Web of Science]
Sanders D, Brownlee C, Harper JF. Communicating with calcium. The Plant Cell (1999) 11:691–706.
Shigaki T, Barkla BJ, Miranda-Vergara MC, Zhao J, Pantoja O, Hirschi KD. Identification of a crucial histidine involved in metal transport activity in the Arabidopsis cation/H+ exchanger CAX1. Journal of Biological Chemistry (2005) 280:30136–30142.
Shigaki T, Hirschi KD. Diverse functions and molecular properties emerging for CAX cation/H+ exchangers in plants. Plant Biology (2006) 8:419–429.[CrossRef][Medline]
Shigaki T, Pittman JK, Hirschi KD. Manganese specificity determinants in the Arabidopsis metal/H+ antiporter CAX2. Journal of Biological Chemistry (2003) 278:6610–6617.
Shigaki T, Sreevidya C, Hirschi KD. Analysis of the Ca2+ domain in the Arabidopsis H+/Ca2+ antiporters CAX1 and CAX3. Plant Molecular Biology (2002) 50:475–483.[CrossRef][Web of Science][Medline]
Sze H, Liang F, Hwang I, Curran AC, Harper JF. Diversity and regulation of plant Ca2+ pumps: insights from expression in yeast. Annual Review of Plant Physiology and Plant Molecular Biology (2000) 51:433–462.[CrossRef][Web of Science][Medline]
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