Arabidopsis Ca2 Research Articles

Substrate profiling of the Arabidopsis Ca2+-dependent protein kinase AtCPK4 and its Ricinus communis ortholog RcCDPK1

AtCPK4 and AtCPK11 are Arabidopsis thaliana Ca2+-dependent protein kinase (CDPK) paralogs that have been reported to positively regulate abscisic acid (ABA) signal transduction by phosphorylating ABA-responsive transcription factor-4 (AtABF4). By contrast, RcCDPK1, their closest Ricinus communis ortholog, participates in the control of anaplerotic carbon flux in developing castor oil seeds by catalyzing inhibitory phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser451. LC-MS/MS revealed that AtCPK4 and RcCDPK1 transphosphorylated several common, conserved residues of AtABF4 and its castor ortholog, TRANSCRIPTION FACTOR RESPONSIBLE FOR ABA REGULATON. Arabidopsis atcpk4/atcpk11 mutants displayed an ABA-insensitive phenotype that corroborated the involvement of AtCPK4/11 in ABA signaling. A kinase-client assay was employed to identify additional AtCPK4/RcCDPK1 targets. Both CDPKs were separately incubated with a library of 2095 peptides representative of Arabidopsis protein phosphosites; five overlapping targets were identified including PLANT INTRACELLULAR RAS-GROUP-RELATED LEUCINE-RICH REPEAT PROTEIN-9 (AtPIRL9) and the E3-ubiquitin ligase ARABIDOPSIS TOXICOS EN LEVADURA 6 (AtATL6). AtPIRL9 and AtATL6 residues phosphorylated by AtCPK4/RcCDPK1 conformed to a CDPK recognition motif that was conserved amongst their respective orthologs. Collectively, this study provides evidence for novel AtCPK4/RcCDPK1 substrates, which may help to expand regulatory networks linked to Ca2+- and ABA-signaling, immune responses, and central carbon metabolism.

Substrate Profiling of the Arabidopsis Ca2+-Dependent Protein Kinase Atcpk4 and its Ricinus Communis Ortholog Rccdpk1

Substrate Profiling of the Arabidopsis Ca2+-Dependent Protein Kinase Atcpk4 and its Ricinus Communis Ortholog Rccdpk1

Arabidopsis Ca2+-ATPases 1, 2, and 7 in the endoplasmic reticulum contribute to growth and pollen fitness.

Generating cellular Ca2+ signals requires coordinated transport activities from both Ca2+ influx and efflux pathways. In Arabidopsis (Arabidopsis thaliana), multiple efflux pathways exist, some of which involve Ca2+-pumps belonging to the Autoinhibited Ca2+-ATPase (ACA) family. Here, we show that ACA1, 2, and 7 localize to the endoplasmic reticulum (ER) and are important for plant growth and pollen fertility. While phenotypes for plants harboring single-gene knockouts (KOs) were weak or undetected, a triple KO of aca1/2/7 displayed a 2.6-fold decrease in pollen transmission efficiency, whereas inheritance through female gametes was normal. The triple KO also resulted in smaller rosettes showing a high frequency of lesions. Both vegetative and reproductive phenotypes were rescued by transgenes encoding either ACA1, 2, or 7, suggesting that all three isoforms are biochemically redundant. Lesions were suppressed by expression of a transgene encoding NahG, an enzyme that degrades salicylic acid (SA). Triple KO mutants showed elevated mRNA expression for two SA-inducible marker genes, Pathogenesis-related1 (PR1) and PR2. The aca1/2/7 lesion phenotype was similar but less severe than SA-dependent lesions associated with a double KO of vacuolar pumps aca4 and 11. Imaging of Ca2+ dynamics triggered by blue light or the pathogen elicitor flg22 revealed that aca1/2/7 mutants display Ca2+ transients with increased magnitudes and durations. Together, these results indicate that ER-localized ACAs play important roles in regulating Ca2+ signals, and that the loss of these pumps results in male fertility and vegetative growth deficiencies.

The Arabidopsis Ca2+-Dependent Protein Kinase CPK12 Is Involved in Plant Response to Salt Stress.

CDPKs (Ca2+-Dependent Protein Kinases) are very important regulators in plant response to abiotic stress. The molecular regulatory mechanism of CDPKs involved in salt stress tolerance remains unclear, although some CDPKs have been identified in salt-stress signaling. Here, we investigated the function of an Arabidopsis CDPK, CPK12, in salt-stress signaling. The CPK12-RNA interference (RNAi) mutant was much more sensitive to salt stress than the wild-type plant GL1 in terms of seedling growth. Under NaCl treatment, Na+ levels in the roots of CPK12-RNAi plants increased and were higher than levels in GL1 plants. In addition, the level of salt-elicited H2O2 production was higher in CPK12-RNAi mutants than in wild-type GL1 plants after NaCl treatment. Collectively, our results suggest that CPK12 is required for plant adaptation to salt stress.

Arabidopsis Ca2+-dependent nuclease AtCaN2 plays a negative role in plant responses to salt stress

Eukaryotic nucleases are involved in processes such as DNA restriction digestion, repair, recombination, transposition, and programmed cell death (PCD). Studies on the role of nucleases have mostly focused on PCD during plant development, while the information on nucleases involved in responses to different abiotic stress conditions remains limited. Here, we identified a Ca2+-dependent nuclease, AtCaN2, in Arabidopsis thaliana and characterized its activity, expression patterns, and involvement in plant responses to salt stress. AtCaN2 showed a dual endonuclease and exonuclease activity, being able to degrade circular plasmids, RNA, single-stranded DNA, and double-stranded DNA. Expression analysis showed that AtCaN2 was strongly induced in senescent siliques and by salt stress. Overexpression of AtCaN2 decreased the plant tolerance to salt stress conditions, leading to an excessive H2O2 accumulation. However, an atcan2 mutant showed better tolerance to salt stress and a lower level of H2O2 accumulation. Moreover, the expression of several genes (AtAPX1, AtGPX8, and AtSOD1), encoding reactive oxygen species-scavenging enzymes (ascorbate peroxidase 1, glutathione peroxidase 8, and superoxide dismutase 1, respectively), was highly induced in the atcan2 mutant under salt stress conditions. In addition, salt-stress-induced cell death was increased in the AtCaN2-overexpressing transgenic plant but decreased in the atcan2 mutant. On the basis of these findings, we conclude that AtCaN2 plays a negative role in plant tolerance to salt stress. A AtCaN2 knock out could reduce ROS accumulation, decrease ROS-induced PCD, and improve overall plant tolerance.

Overexpression of Constitutively Active OsRab11 in Plants Enhances Tolerance to High Salinity Levels

Rab proteins are key regulators of intracellular trafficking between specific compartments in a cell. Among them, Rab11, a widely conserved sub-group, mainly regulates plasma membrane (PM) trafficking. Previously, we reported that Oryza sativa Rab11 (OsRab11) plays an important role in the intracellular trafficking from the trans-Golginetwork (TGN) to the plasma membrane (PM) and prevacuolar compartments (PVCs), and in the plant’s response to high salt stress. In this study, when the constitutively active mutant of OsRab11, (CA OsRab11(Q73L)) was co-transformed with Arabidopsis Ca2+-ATPase8-GFP (ACA8-GFP) or sporamin-GFP (Spo-GFP) into Arabidopsis protoplasts, the PM or vacuolar trafficking proportion of the reporter proteins was highly increased. Transgenic Arabidopsis plants overexpressing (OE) CA OsRab11(Q73L) exhibited enhanced tolerance to high salt stress and exogenous abscisic acid (ABA) compared to Col plants. Moreover, certain stress-responsive genes were expressed under high salt stress and ABA treatment in OEOsRab11(Q73L) plants. Thus, these results suggest that the active conformation of OsRab11 may be required to modulate plant responses to salt and ABA via the regulation of the expression of stress-responsive genes.

Ca(2+) -regulated and diurnal rhythm-regulated Na(+) /Ca(2+) exchanger AtNCL affects flowering time and auxin signalling in Arabidopsis.

Calcium (Ca(2+) ) is vital for plant growth, development, hormone response and adaptation to environmental stresses, yet the mechanisms regulating plant cytosolic Ca(2+) homeostasis are not fully understood. Here, we characterize an Arabidopsis Ca(2+) -regulated Na(+) /Ca(2+) exchanger AtNCL that regulates Ca(2+) and multiple physiological processes. AtNCL was localized to the tonoplast in yeast and plant cells. AtNCL appeared to mediate sodium (Na(+) ) vacuolar sequestration and meanwhile Ca(2+) release. The EF-hand domains within AtNCL regulated Ca(2+) binding and transport of Ca(2+) and Na(+) . Plants with diminished AtNCL expression were more tolerant to high CaCl2 but more sensitive to both NaCl and auxin; heightened expression of AtNCL rendered plants more sensitive to CaCl2 but tolerant to NaCl. AtNCL expression appeared to be regulated by the diurnal rhythm and suppressed by auxin. DR5::GUS expression and root responses to auxin were altered in AtNCL mutants. The auxin-induced suppression of AtNCL was attenuated in SLR/IAA14 and ARF6/8 mutants. The mutants with altered AtNCL expression also altered flowering time and FT and CO expression; FT may mediate AtNCL-regulated flowering time change. Therefore, AtNCL is a vacuolar Ca(2+) -regulated Na(+) /Ca(2+) exchanger that regulates auxin responses and flowering time.

The Arabidopsis Ca2 +-dependent protein kinase CPK27 is required for plant response to salt-stress

Ca2+-dependent protein kinases (CDPKs) play vital roles in plant adaptations to environmental challenges. The precise regulatory mechanism of CDPKs in mediating salt stress still remains unclear, although several CDPK members have been identified to be involved in salt stress accumulation in various plants, such as Arabidopsis thaliana and Oryza sativa. Here, we investigated the function of an Arabidopsis CDPK, CPK27, in salt stress-signaling. CPK27 is a membrane-localized protein kinase; its expression was induced by NaCl. cpk27-1, a T-DNA insertion mutant of CPK27, was much more sensitive to salt stress than wild-type plants in terms of seed germination and post-germination seedling growth. In ion-flux assay, cpk27-1 mutants exhibited a lower capacity than wild-type plants to extrude Na+ and import H+ after a long-term salt treatment (110mM NaCl for 10days). Moreover, the content of Na+ was higher and K+ was lower in cpk27-1 mutants than in wild-type plants under salt stress. In addition, the level of salt-elicited H2O2 production was higher in cpk27-1 mutants than in wild-type plants Col after a short-term NaCl shock and long-term salt treatment. Collectively, our results suggest that CPK27 is required for plant adaptation to salt stress.

The plasma membrane-localised Ca2+-ATPase ACA8 plays a role in sucrose signalling involved in early seedling development in Arabidopsis

Arabidopsis Ca (2+) -ATPase ACA8 plays a role in sucrose signalling during early seedling development by integrating developmental signals with carbon source availability. Calcium (Ca(2+)) is an essential signal transduction element in eukaryotic organisms. Changes in the levels of intracellular Ca(2+) affect multiple developmental processes in plants, including cell division, polar growth, and organogenesis. Here, we report that the plasma-membrane-localised Arabidopsis Ca(2+)-ATPase ACA8 plays a role in sucrose signalling during early seedling development. Disruption of the ACA8 gene elevated the expression of genes that encode transporters for Ca(2+) efflux. The seedlings that carried a T-DNA insertion mutation in ACA8 experienced water stress during early development. This response was unrelated to inadequate osmoregulatory responses and was most likely caused by disruption of cell membrane integrity and severe ion leakage. In addition, aca8-1 seedlings displayed a significant decline in photosynthetic performance and arrested root growth after removal of sucrose from the growth medium. The two phenomena resulted from impaired photosynthesis, reduced cell proliferation in the root meristem and the sucrose control of cell-cycle events. All of the stress-response phenotypes were rescued when expression of ACA8 was restored in aca8-1 mutant. Taken together, our results indicate that ACA8-mediated Ca(2+) signalling contributes to modulate early seedling development and coordinates root development with nutrient availability.

ATP protects against FITC labeling of Solanum lycopersicon and Arabidopsis thaliana Ca2+-ATPase ATP binding domains

Ca(2+)-ATPases are integral membrane proteins that actively transport Ca(2+) against substantial concentration gradients in eukaryotic cells. This active transport is energized by coupling ion translocation with ATP hydrolysis. In order to better understand this coupling mechanism, we studied the nucleotide specificities of isolated ATP binding domains (ABDs) of Solanum lycopersicon Ca(2+)-ATPase (LCA), a type IIA non-calmodulin regulated P-type pump found in tomato plants that is very similar to mammalian sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), and Arabidopsis Ca(2+)-ATPase, isoform 2 (ACA2), a type IIB calmodulin regulated P-type ATPase found in the endoplasmic reticulum of Arabidopsis cells. We used nucleotide protection against FITC labeling as a measure of binding since both LCA and ACA contained the KGAP(S,V,F)E motif, which has been shown to be modified by fluorescein isothiocyanate (FITC) in P-type pumps from animal cells. We demonstrated that the heterologously expressed GST-tagged ABDs from both LCA and ACA2 were modified by FITC and that ATP protects against this modification. Moreover, GTP was able to reduce, but not eliminate, the level of FITC labeling in both ABD constructs, suggesting that these plant pumps may also bind GTP with low affinity, which is in contrast to mammalian SERCA and PMCA type pumps which do not bind GTP.

Bifurcation of Arabidopsis NLR Immune Signaling via Ca2+-Dependent Protein Kinases

Nucleotide-binding domain leucine-rich repeat (NLR) protein complexes sense infections and trigger robust immune responses in plants and humans. Activation of plant NLR resistance (R) proteins by pathogen effectors launches convergent immune responses, including programmed cell death (PCD), reactive oxygen species (ROS) production and transcriptional reprogramming with elusive mechanisms. Functional genomic and biochemical genetic screens identified six closely related Arabidopsis Ca2+-dependent protein kinases (CPKs) in mediating bifurcate immune responses activated by NLR proteins, RPS2 and RPM1. The dynamics of differential CPK1/2 activation by pathogen effectors controls the onset of cell death. Sustained CPK4/5/6/11 activation directly phosphorylates a specific subgroup of WRKY transcription factors, WRKY8/28/48, to synergistically regulate transcriptional reprogramming crucial for NLR-dependent restriction of pathogen growth, whereas CPK1/2/4/11 phosphorylate plasma membrane-resident NADPH oxidases for ROS production. Our studies delineate bifurcation of complex signaling mechanisms downstream of NLR immune sensors mediated by the myriad action of CPKs with distinct substrate specificity and subcellular dynamics.

The Calmodulin-Binding Transcription Factor SIGNAL RESPONSIVE1 is a Novel Regulator of Glucosinolate Metabolism and Herbivory Tolerance in Arabidopsis

The Arabidopsis Ca(2+)/calmodulin (CaM)-binding transcription factor SIGNAL RESPONSIVE1 (AtSR1/CAMTA3) was previously identified as a key negative regulator of plant immune responses. Here, we report a new role for AtSR1 as a critical component of plant defense against insect herbivory. Loss of AtSR1 function impairs tolerance to feeding by the generalist herbivore Trichoplusia ni as well as wound-induced jasmonate accumulation. The susceptibility of the atsr1 mutant is associated with decreased total glucosinolate (GS) levels. The two key herbivory deterrents, indol-3-ylmethyl (I3M) and 4-methylsulfinylbutyl (4MSOB), showed the most significant reductions in atsr1 plants. Further, changes in AtSR1 transcript levels led to altered expression of several genes involved in GS metabolism including IQD1, MYB51 and AtST5a. Overall, our results establish AtSR1 as an important component of plant resistance to insect herbivory as well as one of only three described proteins involved in Ca(2+)/CaM-dependent signaling to function in the regulation of GS metabolism, providing a novel avenue for future investigations of plant-insect interactions.

Arabidopsis Ca<sup>2+</sup>-dependent protein kinase CPK3 mediates relationship of putative inositol triphosphate receptor with slow-type anion channel

It has been suggested in Arabidopsis thaliana (L.) Heynh. cv. Columbia that, contrary to 30 μM abscisic acid (ABA), 20 μM ABA induces guard cell Ca2+ mobilization through activating phosphoinositide-specific phospholipase C (PI-PLC)-dependent inositol 1,4,5-triphosphate (IP3) production. Here, it was investigated whether Ca2+-dependent protein kinase, CPK3 or CPK6 would mediate ABA-induced stomatal closure downstream of IP3 production. In the knockout cpk3-1 mutant, the PLC inhibitor (U73122) adjusted 20 μM ABA-induced stomatal closure to the extent observed in the knockout cpk6-1 and cpk3-1cpk6-1 mutants and the wild type, whereas, in the wild type, the inhibitor of IP3-induced Ca2+ mobilization, xestospongin C (XeC), adjusted this closure to the extent observed in the cpk3-1 mutant. The Ca2+ buffer, EGTA and XeC positively interacted with the slow anion channel blocker, anthracene-9-carboxylic acid (9-AC) to inhibit 20 μM ABA-induced stomatal closure, which was suppressed in the dexamethasone-inducible AtPLC1 antisense transgene or the knockout cpk3-1, cpk6-1, cpk3-1cpk6-1 and NADPH oxidase atrbohD/F mutants. Discrete concentrations of 9-AC or another slow anion channel blocker (probenecid) negatively interacted with the Ca2+ buffer, BAPTA or the inhibitor of cyclic ADP-ribose-induced Ca2+ mobilization, ruthenium red, to inhibit 30 μM ABAinduced stomatal closure in the wild type but not in the cpk6-1, cpk3-1cpk6-1 and atrbohD/F mutants. Based on so far revealed features of the tested compounds and plant materials, interpretation of the results confirmed that guard cell ABA concentration discriminates between two Ca2+ mediations and outlined that one of them sequentially implicates CPK6, PLC1, a putative IP3 receptor homologue, CPK3, and the slow anion channel, whereas the other one excludes AtPLC1-dependent IP3 production and CPK3.

Arabidopsis Ca2+-dependent protein kinase CPK3 mediates relationship of putative inositol triphosphate receptor with slow-type anion channel

It has been suggested in Arabidopsis thaliana (L.) Heynh. cv. Columbia that, contrary to 30 μM abscisic acid (ABA), 20 μM ABA induces guard cell Ca2+ mobilization through activating phosphoinositide-specific phospholipase C (PI-PLC)-dependent inositol 1,4,5-triphosphate (IP3) production. Here, it was investigated whether Ca2+-dependent protein kinase, CPK3 or CPK6 would mediate ABA-induced stomatal closure downstream of IP3 production. In the knockout cpk3-1 mutant, the PLC inhibitor (U73122) adjusted 20 μM ABA-induced stomatal closure to the extent observed in the knockout cpk6-1 and cpk3-1cpk6-1 mutants and the wild type, whereas, in the wild type, the inhibitor of IP3-induced Ca2+ mobilization, xestospongin C (XeC), adjusted this closure to the extent observed in the cpk3-1 mutant. The Ca2+ buffer, EGTA and XeC positively interacted with the slow anion channel blocker, anthracene-9-carboxylic acid (9-AC) to inhibit 20 μM ABA-induced stomatal closure, which was suppressed in the dexamethasone-inducible AtPLC1 antisense transgene or the knockout cpk3-1, cpk6-1, cpk3-1cpk6-1 and NADPH oxidase atrbohD/F mutants. Discrete concentrations of 9-AC or another slow anion channel blocker (probenecid) negatively interacted with the Ca2+ buffer, BAPTA or the inhibitor of cyclic ADP-ribose-induced Ca2+ mobilization, ruthenium red, to inhibit 30 μM ABAinduced stomatal closure in the wild type but not in the cpk6-1, cpk3-1cpk6-1 and atrbohD/F mutants. Based on so far revealed features of the tested compounds and plant materials, interpretation of the results confirmed that guard cell ABA concentration discriminates between two Ca2+ mediations and outlined that one of them sequentially implicates CPK6, PLC1, a putative IP3 receptor homologue, CPK3, and the slow anion channel, whereas the other one excludes AtPLC1-dependent IP3 production and CPK3.

Improved watermelon quality using bottle gourd rootstock expressing a Ca2+/H+ antiporter

Bottle gourd (Lagenaria siceraria Standl.) has been commonly used as a source of rootstock for watermelon. To improve its performance as a rootstock without adverse effects on the scion, the bottle gourd was genetically engineered using a modified Arabidopsis Ca2+/H+ exchanger sCAX2B. This transporter provides enhanced Ca2+ substrate specificity and decreased Mn2+ transport capability. Our previous work demonstrated that sCAX2B bottle gourds were more robust and nutrient dense than controls. Here, several cucurbit crops were test-grafted onto the transgenic bottle gourd to determine its effect on the scions. The grafted watermelons and melons onto the transgenic rootstocks appeared to show more robust growth than the controls 35 days after greenhouse transplanting. Watermelon fruits with the watermelon/transgenic bottle gourd (scion/rootstock) combination demonstrated higher osmotic pressure and more soluble solids than controls. These results suggest that sCAX2B expression in the bottle gourd rootstock facilitates improved watermelon quality through the translocation of nutrients and/or water toward enhancing the biomass of scion.

Interaction between Arabidopsis Ca2+/H+ Exchangers CAX1 and CAX3

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.

Evidence of differential pH regulation of the Arabidopsis vacuolar Ca 2+/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.

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.

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

Abstract Regulation of Ca2+ transport determines the duration of a Ca2+ signal, and hence, the nature of the biological response. Ca2+/H+ antiporters such as CAX1 (cation exchanger 1), play a key role in determining cytosolic Ca2+ 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 Ca2+ transport mutants. The long CAX1 (lCAX1) could not suppress the yeast Ca2+ transport defects despite localization to the yeast vacuole. Calmodulin could not stimulate lCAX1 Ca2+/H+ transport in yeast; however, minor alterations in the 36-amino acid region restored Ca2+/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 Ca2+/H+ antiport.