Increased calcium levels and prolonged shelf life in tomatoes expressing Arabidopsis H+/Ca2+ transporters

Here we demonstrate that fruit from tomato (Lycopersicon esculentum) plants expressing Arabidopsis (Arabidopsis thaliana) H(+)/cation exchangers (CAX) have more calcium (Ca2+) and prolonged shelf life when compared to controls. Previously, using the prototypical CAX1, it has been demonstrated that, in yeast (Saccharomyces cerevisiae) cells, CAX transporters are activated when the N-terminal autoinhibitory region is deleted, to give an N-terminally truncated CAX (sCAX), or altered through specific manipulations. To continue to understand the diversity of CAX function, we used yeast assays to characterize the putative transport properties of CAX4 and N-terminal variants of CAX4. CAX4 variants can suppress the Ca2+ hypersensitive yeast phenotypes and also appear to be more specific Ca2+ transporters than sCAX1. We then compared the phenotypes of sCAX1- and CAX4-expressing tomato lines. The sCAX1-expressing tomato lines demonstrate increased vacuolar H(+)/Ca2+ transport, when measured in root tissue, elevated fruit Ca2+ level, and prolonged shelf life but have severe alterations in plant development and morphology, including increased incidence of blossom-end rot. The CAX4-expressing plants demonstrate more modest increases in Ca2+ levels and shelf life but no deleterious effects on plant growth. These findings suggest that CAX expression may fortify plants with Ca2+ and may serve as an alternative to the application of CaCl2 used to extend the shelf life of numerous agriculturally important commodities. However, judicious regulation of CAX transport is required to assure optimal plant growth.     

In planta regulation of the Arabidopsis Ca(2+)/H(+) antiporter CAX1

Vacuolar localized Ca(2+)/H(+) exchangers such as Arabidopsis thaliana cation exchanger 1 (CAX1) play important roles in Ca(2+) 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 Ca(2+)/H(+) antiport activity. Furthermore, transgenic plants expressing sCAX1 display increased Ca(2+) accumulation and heightened activity of vacuolar Ca(2+)/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 Ca(2+)/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 Ca(2+) transporter in transgenic plants. Engineering the autoinhibitory domain thus provides a strategy to enhance transport function to affect agronomic traits.     

Functional association of Arabidopsis CAX1 and CAX3 is required for normal growth and ion homeostasis

Cation levels within the cytosol are coordinated by a network of transporters. Here, we examine the functional roles of calcium exchanger 1 (CAX1), a vacuolar H+/Ca2+ transporter, and the closely related transporter CAX3. We demonstrate that like CAX1, CAX3 is also localized to the tonoplast. We show that CAX1 is predominately expressed in leaves, while CAX3 is highly expressed in roots. Previously, using a yeast assay, we demonstrated that an N-terminal truncation of CAX1 functions as an H+/Ca2+ transporter. Here, we use the same yeast assay to show that full-length CAX1 and full-length CAX3 can partially, but not fully, suppress the Ca2+ hypersensitive yeast phenotype and coexpression of full-length CAX1 and CAX3 conferred phenotypes not produced when either transporter was expressed individually. In planta, CAX3 null alleles were modestly sensitive to exogenous Ca2+ and also displayed a 22% reduction in vacuolar H+-ATPase activity. cax1/cax3 double mutants displayed a severe reduction in growth, including leaf tip and flower necrosis and pronounced sensitivity to exogenous Ca2+ and other ions. These growth defects were partially suppressed by addition of exogenous Mg2+. The double mutant displayed a 42% decrease in vacuolar H+/Ca2+ transport, and a 47% decrease in H+-ATPase activity. While the ionome of cax1 and cax3 lines were modestly perturbed, the cax1/cax3 lines displayed increased PO4(3-), Mn2+, and Zn2+ and decreased Ca2+ and Mg2+ in shoot tissue. These findings suggest synergistic function of CAX1 and CAX3 in plant growth and nutrient acquisition.     

Characterization of CXIP4, a novel Arabidopsis protein that activates the H+/Ca2+ antiporter, CAX1

Precise regulation of calcium transporters is essential for modulating the Ca2+ signaling network that is involved in the growth and adaptation of all organisms. The Arabidopsis H+/Ca2+ antiporter, CAX1, is a high capacity and low affinity Ca2+ transporter and several CAX1-like transporters are found in Arabidopsis. When heterologously expressed in yeast, CAX1 is unable to suppress the Ca2+ hypersensitivity of yeast vacuolar Ca2+ transporter mutants due to an N-terminal autoinhibition mechanism that prevents Ca2+ transport. Using a yeast screen, we have identified CAX nteracting Protein 4 (CXIP4) that activated full-length CAX1, but not full-length CAX2, CAX3 or CAX4. CXIP4 encodes a novel plant protein with no bacterial, fungal, animal, or mammalian homologs. Expression of a GFP-CXIP4 fusion in yeast and plant cells suggests that CXIP4 is targeted predominantly to the nucleus. Using a yeast growth assay, CXIP4 activated a chimeric CAX construct that contained specific portions of the N-terminus of CAX1. Together with other recent studies, these results suggest that CAX1 is regulated by several signaling molecules that converge on the N-terminus of CAX1 to regulate H+/Ca2+ antiport.     

Characterization of Arabidopsis Ca2+/H+ exchanger CAX3

Plant calcium (Ca(2+)) gradients, millimolar levels in the vacuole and micromolar levels in the cytoplasm, are regulated in part by high-capacity vacuolar cation/H(+) exchangers (CAXs). Several CAX transporters, including CAX1, appear to contain an approximately 40-amino acid N-terminal regulatory region (NRR) that modulates transport through N-terminal autoinhibition. Deletion of the NRR from several CAXs (sCAX) enhances function in plant and yeast expression assays; however, to date, there are no functional assays for CAX3 (or sCAX3), which is 77% identical and 91% similar in sequence to CAX1. In this report, we create a series of truncations in the CAX3 NRR and demonstrate activation of CAX3 in both yeast and plants by truncating a large portion (up to 90 amino acids) of the NRR. Experiments with endomembrane-enriched vesicles isolated from yeast expressing activated CAX3 demonstrate that the gene encodes Ca(2+)/H(+) exchange with properties distinct from those of CAX1. The phenotypes produced by activated CAX3-expressing in transgenic tobacco lines are also distinct from those produced by sCAX1-expressing plants. These studies demonstrate shared and unique aspects of CAX1 and CAX3 transport and regulation.     

Regulation of CAX1, an Arabidopsis Ca2+/H+ Antiporter. Identification of an N-Terminal Autoinhibitory Domain

Regulation of Ca(2+) transport determines the duration of a Ca(2+) signal, and hence, the nature of the biological response. Ca(2+)/H+ antiporters such as CAX1 (cation exchanger 1), play a key role in determining cytosolic Ca(2+) levels. Analysis of a full-length CAX1 clone suggested that the CAX1 open reading frame contains an additional 36 amino acids at the N terminus that were not found in the original clone identified by suppression of yeast (Saccharomyces cerevisiae) vacuolar Ca(2+) transport mutants. The long CAX1 (lCAX1) could not suppress the yeast Ca(2+) transport defects despite localization to the yeast vacuole. Calmodulin could not stimulate lCAX1 Ca(2+)/H+ transport in yeast; however, minor alterations in the 36-amino acid region restored Ca(2+)/H+ transport. Sequence analysis suggests that a 36-amino acid N-terminal regulatory domain may be present in all Arabidopsis CAX-like genes. Together, these results suggest a structural feature involved in regulation of Ca(2+)/H+ antiport.     

Structural determinants of Ca2+ transport in the Arabidopsis H+/Ca2+ antiporter CAX1

Ca(2+) levels in plants, fungi, and bacteria are controlled in part by H(+)/Ca(2+) exchangers; however, the relationship between primary sequence and biological activity of these transporters has not been reported. The Arabidopsis H(+)/cation exchangers, CAX1 and CAX2, were identified by their ability to suppress yeast mutants defective in vacuolar Ca(2+) transport. CAX1 has a much higher capacity for Ca(2+) transport than CAX2. An Arabidopsis thaliana homolog of CAX1, CAX3, is 77% identical (93% similar) and, when expressed in yeast, localized to the vacuole but did not suppress yeast mutants defective in vacuolar Ca(2+) transport. Chimeric constructs and site-directed mutagenesis showed that CAX3 could suppress yeast vacuolar Ca(2+) transport mutants if a nine-amino acid region of CAX1 was inserted into CAX3 (CAX3-9). Biochemical analysis in yeast showed CAX3-9 had 36% of the H(+)/Ca(2+) exchange activity as compared with CAX1; however, CAX3-9 and CAX1 appear to differ in their transport of other ions. Exchanging the nine-amino acid region of CAX1 into CAX2 doubled yeast vacuolar Ca(2+) transport but did not appear to alter the transport of other ions. This nine-amino acid region is highly variable among the plant CAX-like transporters. These findings suggest that this region is involved in CAX-mediated Ca(2+) specificity.     

Analysis of the Ca2+ domain in the Arabidopsis H+/Ca2+ antiporters CAX1 and CAX3

Ca²⁺ levels in plants are controlled in part by H⁺/Ca²⁺ exchangers. Structure/function analysis of the Arabidopsis H⁺/cation exchanger, CAX1, revealed that a nine amino acid region (87–95) is involved in CAX1-mediated Ca²⁺ specificity. CAX3 is 77% identical (93% similar) to CAX1, and when expressed in yeast, localizes to the vacuole but does not suppress yeast mutants defective in vacuolar Ca²⁺ transport. Transgenic tobacco plants expressing CAX3 containing the 9 amino acid Ca²⁺ domain (Cad) from CAX1 (CAX3-9) displayed altered stress sensitivities similar to CAX1-expressing plants, whereas CAX3-9-expressing plants did not have any altered stress sensitivities. A single leucine-to-isoleucine change at position 87 (CAX3-I) within the Cad of CAX3 allows this protein to weakly transport Ca²⁺ in yeast (less than 10% of CAX1). Site-directed mutagenesis of the leucine in the CAX3 Cad demonstrated that no amino acid change tested could confer more activity than CAX3-I. Transport studies in yeast demonstrated that the first three amino acids of the CAX1 Cad could confer twice the Ca²⁺ transport capability compared to CAX3-I. The entire Cad of CAX3 (87–95) inserted into CAX1 abolishes CAX1-mediated Ca²⁺ transport. However, single, double, or triple amino acid replacements within the native CAX1 Cad did not block CAX1 mediated Ca²⁺ transport. Together these findings suggest that other domains within CAX1 and CAX3 influence Ca²⁺ transport. This study has implications for the ability to engineer CAX-mediated transport in plants by manipulating Cad residues.     

Identification of a crucial histidine involved in metal transport activity in the Arabidopsis cation/H+ exchanger CAX1

In plants, yeast, and bacteria, cation/H+ exchangers (CAXs) have been shown to translocate Ca2+ and other metal ions utilizing the H+ gradient. The best characterized of these related transporters is the plant vacuolar localized CAX1. We have used site-directed mutagenesis to assess the impact of altering the seven histidine residues to alanine within Arabidopsis CAX1. The mutants were expressed in a Saccharomyces cerevisiae strain that is sensitive to Ca2+ and other metals. By utilizing a yeast growth assay, the H338A mutant was the only mutation that appeared to alter Ca2+ transport activity. The CAX1 His338 residue is conserved among various CAX transporters and may be located within a filter for cation selection. We proceeded to mutate His338 to every other amino acid residue and utilized yeast growth assays to estimate the transport properties of the 19 CAX mutants. Expression of 16 of these His338 mutants could not rescue any of the metal sensitivities. However, expression of H338N, H338Q, and H338K allowed for some growth on media containing Ca2+. Most interestingly, H338N exhibited increased tolerance to Cd2+ and Zn2+. Endomembrane fractions from yeast cells were used to measure directly the transport of H338N. Although the H338N mutant demonstrated 25% of the wild type Ca2+/H+ transport, it showed an increase in transport for both Cd2+ and Zn2+ reflected in a decrease in the Km for these substrates. This study provides insights into the CAX cation filter and novel mechanisms by which metals may be partitioned across membranes.     

The protein kinase SOS2 activates the Arabidopsis H(+)/Ca(2+) antiporter CAX1 to integrate calcium transport and salt tolerance

The regulation of ions within cells is an indispensable component of growth and adaptation. The plant SOS2 protein kinase and its associated Ca(2+) sensor, SOS3, have been demonstrated to modulate the plasma membrane H(+)/Na(+) antiporter SOS1; however, how these regulators modulate Ca(2+) levels within cells is poorly understood. Here we demonstrate that SOS2 regulates the vacuolar H(+)/Ca(2+) antiporter CAX1. Using a yeast growth assay, co-expression of SOS2 specifically activated CAX1, whereas SOS3 did not. CAX1-like chimeric transporters were activated by SOS2 if the chimeric proteins contained the N terminus of CAX1. Vacuolar membranes from CAX1-expressing cells were made to be H(+)/Ca(2+)-competent by the addition of SOS2 protein in a dose-dependent manner. Using a yeast two-hybrid assay, SOS2 interacted with the N terminus of CAX1. In each of these yeast assays, the activation of CAX1 by SOS2 was SOS3-independent. In planta, the high level of expression of a deregulated version of CAX1 caused salt sensitivity. These findings suggest multiple functions for SOS2 and provide a mechanistic link between Ca(2+) and Na(+) homeostasis in plants.     

Evidence of differential pH regulation of the Arabidopsis vacuolar Ca2+/H+ antiporters CAX1 and CAX2.

The Arabidopsis Ca(2+)/H(+) antiporters cation exchanger (CAX) 1 and 2 utilise an electrochemical gradient to transport Ca(2+) into the vacuole to help mediate Ca(2+) homeostasis. Previous whole plant studies indicate that activity of Ca(2+)/H(+) antiporters is regulated by pH. However, the pH regulation of individual Ca(2+)/H(+) antiporters has not been examined. To determine whether CAX1 and CAX2 activity is affected by pH, Ca(2+)/H(+) antiport activity was measured in vacuolar membrane vesicles isolated from yeast heterologously expressing either transporter. Ca(2+) transport by CAX1 and CAX2 was regulated by cytosolic pH and each transporter had a distinct cytosolic pH profile. Screening of CAX1/CAX2 chimeras identified an amino acid domain within CAX2 that altered the pH-dependent Ca(2+) transport profile so that it was almost identical to the pH profile of CAX1. Results from mutagenesis of a specific His residue within this domain suggests a role for this residue in pH regulation.     

The Arabidopsis cax1 mutant exhibits impaired ion homeostasis,development, and hormonal responses and reveals interplay among vacuolar transporters

The Arabidopsis Ca(2+)/H(+) transporter CAX1 (Cation Exchanger1) may be an important regulator of intracellular Ca(2+) levels. Here, we describe the preliminary localization of CAX1 to the tonoplast and the molecular and biochemical characterization of cax1 mutants. We show that these mutants exhibit a 50% reduction in tonoplast Ca(2+)/H(+) antiport activity, a 40% reduction in tonoplast V-type H(+)-translocating ATPase activity, a 36% increase in tonoplast Ca(2+)-ATPase activity, and increased expression of the putative vacuolar Ca(2+)/H(+) antiporters CAX3 and CAX4. Enhanced growth was displayed by the cax1 lines under Mn(2+) and Mg(2+) stress conditions. The mutants exhibited altered plant development, perturbed hormone sensitivities, and altered expression of an auxin-regulated promoter-reporter gene fusion. We propose that CAX1 regulates myriad plant processes and discuss the observed phenotypes with regard to the compensatory alterations in other transporters.     

Functional Studies of Split Arabidopsis Ca2+/H+ Exchangers

In plants, high capacity tonoplast cation/H⁺ antiport is mediated in part by a family of cation exchanger (CAX) transporters. Functional association between CAX1 and CAX3 has previously been shown. In this study we further examine the interactions between CAX protein domains through the use of nonfunctional halves of CAX transporters. We demonstrate that a protein coding for an N-terminal half of an activated variant of CAX1 (sCAX1) can associate with the C-terminal half of either CAX1 or CAX3 to form a functional transporter that may exhibit unique transport properties. Using yeast split ubiquitin, in planta bimolecular fluorescence complementation, and gel shift experiments, we demonstrate a physical interaction among the half proteins. Moreover, the half-proteins both independently localized to the same yeast endomembrane. Co-expressing variants of N- and C-terminal halves of CAX1 and CAX3 in yeast suggested that the N-terminal region mediates Ca²⁺ transport, whereas the C-terminal half defines salt tolerance phenotypes. Furthermore, in yeast assays, auto-inhibited CAX1 could be differentially activated by CAX split proteins. The N-terminal half of CAX1 when co-expressed with CAX1 activated Ca²⁺ transport, whereas co-expressing C-terminal halves of CAX variants with CAX1 conferred salt tolerance but no apparent Ca²⁺ transport. These findings demonstrate plasticity through hetero-CAX complex formation as well as a novel means to engineer CAX transport.     

Mutations in the Ca2+/H+ transporter CAX1 increase CBF/DREB1 expression and the cold-acclimation response in Arabidopsis

Transient increases in cytosolic free calcium concentration ([Ca2+]cyt) are essential for plant responses to a variety of environmental stimuli, including low temperature. Subsequent reestablishment of [Ca2+]cyt to resting levels by Ca2+ pumps and antiporters is required for the correct transduction of the signal [corrected]. C-repeat binding factor/dehydration responsive element binding factor 1 (Ca2+/H+) antiporters is required for the correct transduction of the signal. We have isolated a cDNA from Arabidopsis that corresponds to a new cold-inducible gene, rare cold inducible4 (RCI4), which was identical to calcium exchanger 1 (CAX1), a gene that encodes a vacuolar Ca2+/H+ antiporter involved in the regulation of intracellular Ca2+ levels. The expression of CAX1 was induced in response to low temperature through an abscisic acid-independent pathway. To determine the function of CAX1 in Arabidopsis stress tolerance, we identified two T-DNA insertion mutants, cax1-3 and cax1-4, that display reduced tonoplast Ca2+/H+ antiport activity. The mutants showed no significant differences with respect to the wild type when analyzed for dehydration, high-salt, chilling, or constitutive freezing tolerance. However, they exhibited increased freezing tolerance after cold acclimation, demonstrating that CAX1 plays an important role in this adaptive response. This phenotype correlates with the enhanced expression of CBF/DREB1 genes and their corresponding targets in response to low temperature. Our results indicate that CAX1 ensures the accurate development of the cold-acclimation response in Arabidopsis by controlling the induction of CBF/DREB1 and downstream genes.     

Expression of Arabidopsis CAX1 in tobacco: altered calcium homeostasis and increased stress sensitivity

Calcium (Ca(2)+) efflux from the cytosol modulates Ca(2+) concentrations in the cytosol, loads Ca(2+) into intracellular compartments, and supplies Ca(2+) to organelles to support biochemical functions. The Ca(2+)/H(+) antiporter CAX1 (for CALCIUM EXCHANGER 1) of Arabidopsis is thought to be a key mediator of these processes. To clarify the regulation of CAX1, we examined CAX1 RNA expression in response to various stimuli. CAX1 was highly expressed in response to exogenous Ca(2+). Transgenic tobacco plants expressing CAX1 displayed symptoms of Ca(2+) deficiencies, including hypersensitivity to ion imbalances, such as increased magnesium and potassium concentrations, and to cold shock, but increasing the Ca(2+) in the media abrogated these sensitivities. Tobacco plants expressing CAX1 also demonstrated increased Ca(2+) accumulation and altered activity of the tonoplast-enriched Ca(2+)/H(+) antiporter. These results emphasize that regulated expression of Ca(2+)/H(+) antiport activity is critical for normal growth and adaptation to certain stresses.     

玉米亚硫酸氧化酶基因的克隆及其结构与表达分析

为明确亚硫酸氧化酶(sulfite oxidase,SO)基因的结构特征和进化关系及其在玉米不同组织器官发育过程中的表达和分布特性,采用RACE技术克隆了玉米SO基因(ZmSO)的全长cDNA。序列分析表明,获得的ZmSO全长1 492bp,其中5′-UTR 160bp,3′-UTR 138bp,开放阅读框为1 194bp,编码397个氨基酸组成的蛋白质。对该基因编码氨基酸保守结构域的分析发现,ZmSO包含1个钼辅因子结合域、1个自身二聚化域和1个过氧化物体靶信号序列。系统进化分析显示,SO在进化上较为保守,玉米与其它植物的SO相似性较高。荧光定量RT-PCR分析表明,在玉米成株期,根、茎、叶、雄花和幼穗中,ZmSO在根部表达丰度最低,在叶片和幼穗中表达量较高。酶活性测定结果显示,不同器官中SO活性与其mRNA转录水平上的表达趋势相似。     

Distinct N-terminal regulatory domains of Ca(2+)/H(+) antiporters

The regulation of intracellular Ca(2+) levels is achieved in part by high-capacity vacuolar Ca(2+)/H(+) antiporters. An N-terminal regulatory region (NRR) on the Arabidopsis Ca(2+)/H(+) antiporter CAX1 (cation exchanger 1) has been shown previously to regulate Ca(2+) transport by a mechanism of N-terminal auto-inhibition. Here, we examine the regulation of other CAX transporters, both within Arabidopsis and from another plant, mung bean (Vigna radiata), to ascertain if this mechanism is commonly used among Ca(2+)/H(+) antiporters. Biochemical analysis of mung bean VCAX1 expressed in yeast (Saccharomyces cerevisiae) showed that N-terminal truncated VCAX1 had approximately 70% greater antiport activity compared with full-length VCAX1. A synthetic peptide corresponding to the NRR of CAX1, which can strongly inhibit Ca(2+) transport by CAX1, could not dramatically inhibit Ca(2+) transport by truncated VCAX1. The N terminus of Arabidopsis CAX3 was also shown to contain an NRR. Additions of either the CAX3 or VCAX1 regulatory regions to the N terminus of an N-terminal truncated CAX1 failed to inhibit CAX1 activity. When fused to N-terminal truncated CAX1, both the CAX3 and VCAX1 regulatory regions could only auto-inhibit CAX1 after mutagenesis of specific amino acids within this NRR region. These findings demonstrate that N-terminal regulation is present in other plant CAX transporters, and suggest distinct regulatory features among these transporters.