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Bio-degradation is the most affordable method of azo dye decontamination, while its drawbacks such as aromatic amines accumulation and low degradation efficiency must be overcome. In this study, a novel mechanism of azo dye degradation by a fungus was discovered. At a concentration of 400 mg/L, the decolorization efficiency of Acid Red 73 (AR73) by Aspergillus tabacinus LZ-M was 90.28%. Metabolite analysis and transcriptome sequencing analysis revealed a self-redox process of AR73 degradation, where the electrons generated in carbon oxidation were transferred to the reduction of -C-N = and -NN. The metabolites, 2-hydroxynaphthalene and N-phenylnitrous amide were mineralized into CO 2 through catechol pathway and a glycolytic process. Furthermore, the mineralization ratio of dye was computed to be 31.8% by the carbon balance and electron balance. By using comparative transcriptome, a novel decoloring enzyme Ord95 was discovered in unknown genes through gene cloning. It hydrolyzed AR73 into 2-hydroxynaphthalene and N-phenylnitrous amide, containing a glutathione S-transferase domain with three arginines as key active sites. Here the new mechanism of azo dye degradation was discovered with identification of a novel enzyme in Aspergillus tabacinus LZ-M., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Some insects can degrade both natural and synthetic plastic polymers, their host and gut microbes play crucial roles in this process. However, there is still a scientific gap in understanding how the insect adapted to the polystyrene (PS) diet from natural feed. In this study, we analyzed diet consumption, gut microbiota responses, and metabolic pathways of Tenebrio molitor larvae exposed to PS and corn straw (CS)., Results: T. molitor larvae were incubated under controlled conditions (25 ± 1 °C, 75 ± 5% humidity) for 30 days by using PS foam with weight-, number-, and size-average molecular weight (Mw, Mn, and Mz) of 120.0, 73.2, and 150.7 kDa as a diet, respectively. The larvae exhibited lower PS consumption (32.5%) than CS (52.0%), and these diets had no adverse effects on their survival. The gut microbiota structures, metabolic pathways, and enzymatic profiles of PS- and CS-fed larvae showed similar responses. The gut microbiota of larvae analysis indicated Serratia sp., Staphylococcus sp., and Rhodococcus sp. were associated with both PS and CS diets. Metatranscriptomic analysis revealed that xenobiotics, aromatic compounds, and fatty acid degradation pathways were enriched in PS- and CS-fed groups; laccase-like multicopper oxidases, cytochrome P450, monooxygenase, superoxidase, and dehydrogenase were involved in lignin and PS degradation. Furthermore, the upregulated gene lac640 in both PS- and CS-fed groups was overexpressed in E. coli and exhibited PS and lignin degradation ability., Conclusions: The high similarity of gut microbiomes adapted to biodegradation of PS and CS indicated the plastics-degrading ability of the T. molitor larvae originated through an ancient mechanism that degrades the natural lignocellulose. Video Abstract., (© 2023. The Author(s).)

Bogia coconut syndrome (BCS) is one of the lethal yellowing (LY)-type diseases associated with phytoplasma presence that are seriously threatening coconut cultivation worldwide. It has recently emerged, and is rapidly spreading in northern parts of the island of New Guinea. BCS-associated phytoplasmas collected in different regions were compared in terms of 16S rRNA gene sequences, revealing high identity among them represented by strain BCS-Bo R . Comparative analysis of the 16S rRNA gene sequences revealed that BCS-Bo R shared less than a 97.5 % similarity with other species of 'Candidatus Phytoplasma', with a maximum value of 96.08 % (with strain LY; GenBank accession no. U18747). This result indicates the necessity and propriety of a novel taxon for BCS phytoplasmas according to the recommendations of the IRPCM. Phylogenetic analysis was also conducted on 16S rRNA gene sequences, resulting in a monophyletic cluster composed of BCS-Bo R and other LY-associated phytoplasmas. Other phytoplasmas on the island of New Guinea associated with banana wilt and arecanut yellow leaf diseases showed high similarities to BCS-Bo R and were closely related to BCS phytoplasmas. Based on the uniqueness of their 16S rRNA gene sequences, a novel taxon 'Ca.Phytoplasma noviguineense' is proposed for these phytoplasmas found on the island of New Guinea, with strain BCS-Bo R (GenBank accession no. LC228755) as the reference strain. The novel taxon is described in detail, including information on the symptoms of associated diseases and additional genetic features of the secY gene and rp operon.

The Pacific countries are small in land mass and therefore represent one of the most fragile ecosystems. Due to the isolation of these island counties, these are home to unique species of plants and animals as well as crop varieties and landraces. Biosafety issues in the Pacific countries, therefore, require special attention to take these factors into account. The issues are shared with other small island nations such as the Caribbean countries. Although most Pacific countries do not have scientific capacity to develop genetically modified organisms (GMOs), they are inadvertently introduced from the developed world. As the countries do not have appropriate capacity to monitor the introduction and commerce of GMO's, it is imperative to establish biosafety legislation and capacity by pooling the resources within the Pacific countries.

Deregulated expression of an Arabidopsis H⁺/Ca²⁺ antiporter (sCAX1) in agricultural crops increases total calcium (Ca²⁺) but may result in yield losses due to Ca²⁺ deficiency-like symptoms. Here we demonstrate that co-expression of a maize calreticulin (CRT, a Ca²⁺ binding protein located at endoplasmic reticulum) in sCAX1-expressing tobacco and tomato plants mitigated these adverse effects while maintaining enhanced Ca²⁺ content. Co-expression of CRT and sCAX1 could alleviate the hypersensitivity to ion imbalance in tobacco plants. Furthermore, blossom-end rot (BER) in tomato may be linked to changes in CAX activity and enhanced CRT expression mitigated BER in sCAX1 expressing lines. These findings suggest that co-expressing Ca²⁺ transporters and binding proteins at different intracellular compartments can alter the content and distribution of Ca²⁺ within the plant matrix.

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.

Phytoremediation is a cost-effective and minimally invasive technology to cleanse soils contaminated with heavy metals. However, few plant species are suitable for phytoremediation of metals such as cadmium (Cd). Genetic engineering offers a powerful tool to generate plants that can hyperaccumulate Cd. An Arabidopsis CAX1 mutant (CAXcd), which confers enhanced Cd transport in yeast, was ectopically expressed in petunia to evaluate whether the CAXcd expression would enhance Cd tolerance and accumulation in planta. The CAXcd-expressing petunia plants showed significantly greater Cd tolerance and accumulation than the controls. After being treated with either 50 or 100μM CdCl(2) for 6 weeks, the CAXcd-expressing plants showed more vigorous growth compared with controls, and the transgenic plants accumulated significantly more Cd (up to 2.5-fold) than controls. Moreover, the accumulation of Cd did not affect the development and morphology of the CAXcd-expressing petunia plants until the flowering and ultimately the maturing of seeds. Therefore, petunia has the potential to serve as a model species for developing herbaceous, ornamental plants for phytoremediation., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

CAtion/H(+) eXchangers (CAXs) are integral membrane proteins that transport Ca(2+) or other cations by exchange with protons. While several yeast and plant CAX proteins have been characterized, no functional analysis of a vertebrate CAX homologue has yet been reported. In this study, we further characterize a CAX from yeast, VNX1, and initiate characterization of a zebrafish CAX (Cax1). Localization studies indicated that both Vnx1 and Cax1 proteins are found in endomembrane compartments. Biochemical characterization of endomembrane fractions from vnx1 mutant cells and zebrafish Cax1-expressing yeast cells suggested that both yeast and fish CAXs have Ca(2+)/H(+) antiport activities. Additionally, the vnx1 mutation was associated with heightened pH-sensitivity. In zebrafish embryos, cax1 was specifically expressed in neural crest cells. Morpholino knockdown of cax1 caused defects in neural crest development, including alterations in pigmentation, defects in jaw development, and reduction in expression of the neural crest marker, Pax7. Collectively, our findings provide insights into Vnx1 function and support an unexpected role of CAX transporters in animal growth and development.

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 Ca(2+) 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 Ca(2+) transport, whereas co-expressing C-terminal halves of CAX variants with CAX1 conferred salt tolerance but no apparent Ca(2+) transport. These findings demonstrate plasticity through hetero-CAX complex formation as well as a novel means to engineer CAX transport.

Internal compartmentalization of metals is an important metal tolerance mechanism in many organisms. In plants and fungi, sequestration into the vacuole is a major detoxification mechanism for metals. Cation transport into the vacuole can be mediated by CAX (cation exchanger) transporters. The Arabidopsis thaliana AtCAX2 transporter was shown previously to transport Ca(2+), Cd(2+) and Mn(2+). To assess the conservation of the functional and regulatory characteristics of CAX2-like transporters in higher plants, we have characterized AtCAX2 orthologues from Arabidopsis (AtCAX5), tomato (LeCAX2) and barley (HvCAX2). Substrate specificity and regulatory activity were assessed using a yeast heterologous-expression assay. Each CAX could transport Ca(2+) and Mn(2+) into the yeast vacuole, but they each had different cation transport kinetics. Most notably, there was variation in the regulation of the transporters. As found with AtCAX2 previously, only expression of an N-terminally truncated form of AtCAX5 in yeast was able to mediate Ca(2+) and Mn(2+) transport, indicating that activity may be controlled by an autoregulatory region at the N-terminus. In contrast, either full-length or truncated LeCAX2 could efficiently transport Ca(2+), although Mn(2+) transport was controlled by the N-terminus. HvCAX2 did not appear to possess an N-terminal regulatory domain. Expression of AtCAX2 was not significantly modulated by metal stress; however, AtCAX5 and HvCAX2 were transcriptionally up-regulated by high Mn(2+) treatment, and by Ca(2+) and Na(+) stress respectively. It is therefore apparent that, despite the high sequence identity between plant CAX2 orthologues, there is significant diversity in their functional characteristics, particularly with regard to regulatory mechanisms.

In plants, high capacity tonoplast cation/H(+) antiport is mediated in part by a family of CAX (cation exchanger) transporters. Functional association between CAX1 and CAX3 has previously been inferred; however, the nature of this interaction has not been established. Here we analyze the formation of "hetero-CAX" complexes and their transport properties. Co-expressing both CAX1 and CAX3 mediated lithium and salt tolerance in yeast, and these phenotypes could not be recapitulated by expression of deregulated versions of either transporter. Coincident expression of Arabidopsis CAX1 and CAX3 occurs during particular stress responses, flowering, and seedling growth. Analysis of cax1, cax3, and cax1/3 seedlings demonstrated similar stress sensitivities. When plants expressed high levels of both CAXs, alterations in transport properties were evident that could not be recapitulated by high level expression of either transporter individually. In planta coimmunoprecipitation suggested that a protein-protein interaction occurred between CAX1 and CAX3. In vivo interaction between the CAX proteins was shown using a split ubiquitin yeast two-hybrid system and gel shift assays. These findings demonstrate cation exchange plasticity through hetero-CAX interactions.

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.

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.

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.

To facilitate the characterization of plant genes, the Cre-loxP site-specific recombination system was adapted to make reporter vectors for plant expression studies. This system allows promoter fragments to be cloned into a small vector (univector) and subsequently recombined in vitro with binary vectors containing different reporter genes precisely at near-perfect efficiency. We have constructed univector-adapted vectors with three reporters, beta-glucuronidase, luciferase, and green fluorescent protein, and a BASTA-resistance gene for selection of plant transformants. Expression in plants using the new system was validated by comparison to conventional reporter vectors. These new vectors are efficient and economical alternatives to the other plant reporter vectors currently available. The royalty-free Cre-loxP system serves as a platform for the future expansion of recombination-based cloning vectors for plant research.

The vacuolar sequestration of metals is an important metal tolerance mechanism in plants. The Arabidopsis thaliana vacuolar transporters CAX1 and CAX2 were originally identified in a Saccharomyces cerevisiae suppression screen as Ca2+/H+ antiporters. CAX2 has a low affinity for Ca2+ but can transport other metals including Mn2+ and Cd2+. Here we demonstrate that unlike cax1 mutants, CAX2 insertional mutants caused no discernable morphological phenotypes or alterations in Ca2+/H+ antiport activity. However, cax2 lines exhibited a reduction in vacuolar Mn2+/H+ antiport and, like cax1 mutants, reduced V-type H+ -ATPase (V-ATPase) activity. Analysis of a CAX2 promoter beta-glucoronidase (GUS) reporter gene fusion confirmed that CAX2 was expressed throughout the plant and strongly expressed in flower tissue, vascular tissue and in the apical meristem of young plants. Heterologous expression in yeast identified an N-terminal regulatory region in CAX2, suggesting that Arabidopsis contains multiple cation/H+ antiporters with shared regulatory features. Furthermore, despite significant variations in morphological and biochemical phenotypes, cax1 and cax2 lines both significantly alter V-ATPase activity, hinting at coordinate regulation among transporters driven by H+ gradients and the V-ATPase.

The Ca(2+)-dependent protein phosphatase calcineurin is an important regulator of ion transporters from many organisms, including the Saccharomyces cerevisiae vacuolar Ca(2+)/H(+) exchanger Vcx1p. In yeast and plants, cation/H(+) exchangers are important in shaping cytosolic Ca(2+) levels involved in signal transduction and providing tolerance to potentially toxic concentrations of cations such as Ca(2+), Mn(2+) and Cd(2+). Previous genetic evidence suggested Vcx1p is negatively regulated by calcineurin. By utilizing direct transport measurements into vacuolar membrane vesicles, we demonstrate that Vcx1p is a low-affinity Ca(2+) transporter and may also function in Cd(2+) transport, but cannot transport Mn(2+). Furthermore, direct Ca(2+) transport by Vcx1p is calcineurin sensitive. Using a yeast growth assay, a mutant allele of VCX1 (VCX1-S204A/L208P), termed VCX1-M1, was previously found to confer strong Mn(2+) tolerance. Here we demonstrate that this Mn(2+) tolerance is independent of the Ca(2+)/Mn(2+)-ATPase Pmr1p and results from Mn(2+)-specific vacuolar transport activity of Vcx1-M1p. This Mn(2+) transport by Vcx1-M1p is calcineurin dependent, although the localization of Vcx1-M1p to the vacuole appears to be calcineurin independent. Additionally, we demonstrate that mutation of L208P alone is enough to confer calcineurin-dependent Mn(2+) tolerance. This study demonstrates that calcineurin can positively regulate the transport of cations by VCX1-M1p.

In plants and fungi, vacuolar transporters help remove potentially toxic cations from the cytosol. Metal/H(+) antiporters are involved in metal sequestration into the vacuole. However, the specific transport properties and the ability to manipulate these transporters to alter substrate specificity are poorly understood. The Arabidopsis thaliana cation exchangers, CAX1 and CAX2, can both transport Ca(2+) into the vacuole. There are 11 CAX-like transporters in Arabidopsis; however, CAX2 was the only characterized CAX transporter capable of vacuolar Mn(2+) transport when expressed in yeast. To determine the domains within CAX2 that mediate Mn(2+) specificity, six CAX2 mutants were constructed that contained different regions of the CAX1 transporter. One class displayed no alterations in Mn(2+) or Ca(2+) transport, the second class showed a reduction in Ca(2+) transport and no measurable Mn(2+) transport, and the third mutant, which contained a 10-amino acid domain from CAX1 (CAX2-C), showed no reduction in Ca(2+) transport and a complete loss of Mn(2+) transport. The subdomain analysis of CAX2-C identified a 3-amino acid region that is responsible for Mn(2+) specificity of CAX2. This study provides evidence for the feasibility of altering substrate specificity in a metal/H(+) antiporter, an important family of transporters found in a variety of organisms.

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.

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.

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.

Regulation of Ca(2+)/H(+) antiporters may be an important function in determining the duration and amplitude of cytosolic Ca(2+) oscillations. Previously the Arabidopsis Ca(2+)/H(+) transporter, CAX1 (cation exchanger 1), was identified by its ability to suppress yeast mutants defective in vacuolar Ca(2+) transport. Recently, a 36-amino acid N-terminal regulatory region on CAX1 has been identified that inhibits CAX1-mediated Ca(2+)/H(+) antiport. Here we show that a synthetic peptide designed against the CAX1 36 amino acids inhibited Ca(2+)/H(+) transport mediated by an N-terminal-truncated CAX1 but did not inhibit Ca(2+) transport by other Ca(2+)/H(+) antiporters. Ca(2+)/H(+) antiport activity measured from vacuolar-enriched membranes of Arabidopsis root was also inhibited by the CAX1 peptide. Through analyzing CAX chimeric constructs the region of interaction of the N-terminal regulatory region was mapped to include 7 amino acids (residues 56-62) within CAX1. The CAX1 N-terminal regulatory region was shown to physically interact with this 7-amino acid region by yeast two-hybrid analysis. Mutagenesis of amino acids within the N-terminal regulatory region implicated several residues as being essential for regulation. These findings describe a unique mode of antiporter autoinhibition and demonstrate the first detailed mechanisms for the regulation of a Ca(2+)/H(+) antiporter from any organism.

Ion compartmentalization is essential for plant growth and development. The Arabidopsis open reading frames for CAX1, CAX2, and CAX3 (cation exchangers 1, 2, and 3) were previously identified as transporters that may modulate ion fluxes across the vacuolar membrane. To understand the diversity and role of H(+)/cation transporters in controlling plant ion levels, another homolog of the CAX genes, CAX4, was cloned from an Arabidopsis cDNA library. CAX4 is 53% identical to CAX1 at the amino acid level, 42% identical to CAX2, and 54% identical to CAX3. CAX4 transcripts appeared to be expressed at low levels in all tissues and levels of CAX4 RNA increased after Mn(2+), Na(+), and Ni(2+) treatment. An N-terminal CAX4-hemagglutinin fusion appeared to localize to both yeast and plant vacuolar membranes. When expressed in yeast, CAX4, like CAX3, failed to suppress the Ca(2+) sensitivity of yeast strains deficient in vacuolar Ca(2+) transport. Several modifications to CAX4 allowed the protein to transport Ca(2+). Addition of amino acids to the N terminus of CAX4 and CAX3 caused both transporters to suppress the sensitivity of yeast strains deficient in vacuolar Ca(2+) transport. These findings suggest that CAX transporters may modulate their ion transport properties through alterations at the N terminus.