Knockout of Multiple Arabidopsis Cation/H+ Exchangers Suggests Isoform-Specific Roles in Metal Stress Response,Germination and Seed Mineral Nutrition

Cation/H⁺ exchangers encoded by CAX genes play an important role in the vacuolar accumulation of metals including Ca²⁺ and Mn²⁺. Arabidopsis thaliana CAX1 and CAX3 have been previously shown to differ phylogenetically from CAX2 but the physiological roles of these different transporters are still unclear. To examine the functions and the potential of redundancy between these three cation transporters, cax1/cax2 and cax2/cax3 double knockout mutants were generated and compared with wild type and cax single knockouts. These double mutants had equivalent metal stress responses to single cax mutants. Both cax1 and cax1/cax2 had increased tolerance to Mg stress, while cax2 and cax2/cax3 both had increased sensitivity to Mn stress. The cax1/cax2 and cax2/cax3 mutants did not exhibit the deleterious developmental phenotypes previously seen with the cax1/cax3 mutant. However, these new double mutants did show alterations in seed germination, specifically a delay in germination time. These alterations correlated with changes in nutrient content within the seeds of the mutants, particularly the cax1/cax2 mutant which had significantly higher seed content of Ca and Mn. This study indicates that the presence of these Arabidopsis CAX transporters is important for normal germination and infers a role for CAX proteins in metal homeostasis within the seed.     

Arabidopsis sos1 mutant in a salt-tolerant accession revealed an importance of salt acclimation ability in plant salt tolerance

An analysis of the salinity tolerance of 354 Arabidopsis thaliana accessions showed that some accessions were more tolerant to salt shock than the reference accession, Col-0, when transferred from 0 to 225 mM NaCl. In addition, several accessions, including Zu-0, showed marked acquired salt tolerance after exposure to moderate salt stress. It is likely therefore that Arabidopsis plants have at least two types of tolerance, salt shock tolerance and acquired salt tolerance. To evaluate a role of well-known salt shock tolerant gene SOS1 in acquired salt tolerance, we isolated a sos1 mutant from ion-beam-mutagenized Zu-0 seedlings. The mutant showed severe growth inhibition under salt shock stress owing to a single base deletion in the SOS1 gene and was even more salt sensitive than Col-0. Nevertheless, it was able to survive after acclimation on 100 mM NaCl for 7 d followed by 750 mM sorbitol for 20 d, whereas Col-0 became chlorotic under the same conditions. We propose that genes for salt acclimation ability are different from genes for salt shock tolerance and play an important role in the acquisition of salt or osmotic tolerance.     

Characterization and primary functional analysis of a bamboo NAC gene targeted by miR164b

Key message

PeSNAC1 , a stress-related NAC1 from Phyllostachys edulis , was characterized. Ectopic expression in Arabidopsis indicated that PeSNAC1 together with ped -miR164b participated in the regulation of organ boundaries and stress tolerance.

Abstract

NAC (NAM, ATAF1/2 and CUC2) participates in many different processes regulating plant growth, development, and stress response. A total of 125 NAC genes have been predicted in moso bamboo (Phyllostachys edulis), but their roles are poorly understood. PeSNAC1 targeted by ped-miR164b was focused for further study. The cleavage of PeSNAC1 mRNA guided by ped-miR164b was validated using RLM-5′ RACE. Tissue-specific expression analysis demonstrated that ped-miR164b had a declining trend from root, sheath, leaf, to that of stem, which was opposite to that of PeSNAC1. Transgenic Arabidopsis plants overexpressing either PeSNAC1 (OX-PeSNAC1) or, ped-miR164b (OX-ped-miR164b) driven by the CaMV35S promoter were generated. OX-ped-miR164b plants showed similar phenotype of cuc2 mutants whose growth was seriously suppressed. Compared with Col-0, sense OX-PeSNAC1 plants grew rapidly and flowered earlier, whereas antisense plants grew slowly and exhibited delayed flowering. Sense OX-PeSNAC1 plants had the greatest number of lateral roots, while antisense OX-PeSNAC1 and OX-ped-miR164b plants had fewer lateral roots than Col-0. Under NaCl and PEG6000 stresses, survival rates were higher and F _v/F _m values declined more slowly in sense OX-PeSNAC1 plants than in Col-0, with lower survival rates and a more rapid decrease in F _v/F _m values conversely observed in antisense OX-PeSNAC1 and OX-ped-miR164b plants. These findings indicated that ped-miR164b-targeted PeSNAC1 may play key roles in plant development and tolerance to salinity and drought stresses.

     

Vacuolar Ca2+/H+ transport activity is required for systemic phosphate homeostasis involving shoot-to-root signaling in Arabidopsis

Calcium ions (Ca(2+)) and Ca(2+)-related proteins mediate a wide array of downstream processes involved in plant responses to abiotic stresses. In Arabidopsis (Arabidopsis thaliana), disruption of the vacuolar Ca(2+)/H(+) transporters CAX1 and CAX3 causes notable alterations in the shoot ionome, including phosphate (P(i)) content. In this study, we showed that the cax1/cax3 double mutant displays an elevated P(i) level in shoots as a result of increased P(i) uptake in a miR399/PHO2-independent signaling pathway. Microarray analysis of the cax1/cax3 mutant suggests the regulatory function of CAX1 and CAX3 in suppressing the expression of a subset of shoot P(i) starvation-responsive genes, including genes encoding the PHT1;4 P(i) transporter and two SPX domain-containing proteins, SPX1 and SPX3. Moreover, although the expression of several PHT1 genes and PHT1;1/2/3 proteins is not up-regulated in the root of cax1/cax3, results from reciprocal grafting experiments indicate that the cax1/cax3 scion is responsible for high P(i) accumulation in grafted plants and that the pht1;1 rootstock is sufficient to moderately repress such P(i) accumulation. Based on these findings, we propose that CAX1 and CAX3 mediate a shoot-derived signal that modulates the activity of the root P(i) transporter system, likely in part via posttranslational regulation of PHT1;1 P(i) transporters.     

Metallothioneins BcMT1 and BcMT2 from Brassica campestris enhance tolerance to cadmium and copper and decrease production of reactive oxygen species in Arabidopsis thaliana

Aims

Metallothioneins are cysteine-rich, metal-binding proteins, but their exact functions are not fully understood. In this study, we isolated two metallothionein genes, BcMT1 and BcMT2 from Brassica campestris to increase our understanding of metal tolerance mechanisms in Brassica plants.

Methods

Semi-quantitative RT-PCR was used to analyze expression of the two BcMTs genes. BcMT1 and BcMT2 were ectopically expressed in Arabidopsis thaliana. Quantitative real-time RT-PCR and GUS-staining method were used to select transgenic Arabidopsis plants. Cd and Cu concentrations were analyzed by flame atomic absorption spectrometry. Histochemical detection of H₂O₂ and O₂ ^?? were conducted by 3,3-diaminobenzidine and nitroblue tetrazoliu-staining methods.

Results

BcMT1 is expressed predominantly in roots, whereas BcMT2 is expressed mainly in leaves of B. campestris. Expression of BcMT1 was induced by both Cd and Cu, but expression of BcMT2 was enhanced only by Cd. Ectopic expression of BcMT1 and BcMT2 in Arabidopsis thaliana enhanced the tolerance to Cd and Cu and increased the Cu concentration in the shoots of the transgenic plants. Transgenic Arabidopsis accumulated less reactive oxygen species (ROS) than wild-type plants.

Conclusions

BcMT1 and BcMT2 increased Cd and Cu tolerance in transgenic Arabidopsis, and decreased production of Cd- and Cu-induced ROS, thereby protecting plants from oxidative damage.     

Diverse phosphate and auxin transport loci distinguish phosphate tolerant from sensitive Arabidopsis accessions

Phosphorus (P) is an essential element for plant growth often limiting agroecosystems. To identify genetic determinants of performance under variable phosphate (Pi) supply, we conducted genome-wide association studies on five highly predictive Pi starvation response traits in 200 Arabidopsis (Arabidopsis thaliana) accessions. Pi concentration in Pi-limited organs had the strongest, and primary root length had the weakest genetic component. Of 70 trait-associated candidate genes, 17 responded to Pi withdrawal. The PHOSPHATE TRANSPORTER1 gene cluster on chromosome 5 comprises PHT1;1, PHT1;2, and PHT1;3 with known impact on P status. A second locus featured uncharacterized endomembrane-associated auxin efflux carrier encoding PIN-LIKES7 (PILS7) which was more strongly suppressed in Pi-limited roots of Pi-starvation sensitive accessions. In the Col-0 background, Pi uptake and organ growth were impaired in both Pi-limited pht1;1 and two pils7 T-DNA insertion mutants, while Pi -limited pht1;2 had higher biomass and pht1;3 was indistinguishable from wild-type. Copy number variation at the PHT1 locus with loss of the PHT1;3 gene and smaller scale deletions in PHT1;1 and PHT1;2 predicted to alter both protein structure and function suggest diversification of PHT1 is a key driver for adaptation to P limitation. Haplogroup analysis revealed a phosphorylation site in the protein encoded by the PILS7 allele from stress-sensitive accessions as well as additional auxin-responsive elements in the promoter of the “stress tolerant” allele. The former allele’s inability to complement the pils7-1 mutant in the Col-0 background implies the presence of a kinase signaling loop controlling PILS7 activity in accessions from P-rich environments, while survival in P-poor environments requires fine-tuning of stress-responsive root auxin signaling.

A series of insertion/deletion nucleotide polymorphisms at PHOSPHATE TRANSPORTER1 and PIN-LIKES7 loci confer natural variation in low phosphate tolerance in 200 Arabidopsis accessions.     

Functional and regulatory analysis of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> CAX2 cation transporter

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 -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.     

Hydrogen peroxide-induced salt tolerance in the <Emphasis Type="Italic">Arabidopsis</Emphasis> salicylate-deficient transformants <Emphasis Type="Italic">NahG</Emphasis>

The effect of hydrogen peroxide treatment on the salt tolerance of wild-type Arabidopsis thaliana L. plants (Col-0) and plants transformed with the bacterial salicylate hydroxylase gene (NahG) was studied. The base tolerance to salt stress caused by 200 mM of NaCl in solution culture was higher in plants with the NahG genotype in comparison with the wild-type plants. Growth inhibition was observed for wild-type plants under the action of exogenous hydrogen peroxide, which was not observed for the NahG transformants; salt tolerance increased in the both types of plants after treatment, which was assessed based on the growth indicators and the ability to preserve the chlorophyll pool following NaCl treatment. The content of endogenous Н2О2 in the leaves of wild-type plants increased significantly following exogenous hydrogen peroxide treatment and salt stress, while it practically did not change in the leaves of the NahG genotype. The SOD activity increased in both genotypes after treatment with exogenous hydrogen peroxide, and remained at an elevated level after salt stress in comparison with the nontreated plants. Furthermore, the catalase activity increased in leaves of the salicylate-deficient genotype but not in the Col-0 genotype. The guaiacol peroxidase activity increased in plants of both genotypes under the action of hydrogen peroxide and salt stress, with the NahG plants demonstrating a higher degree of increase. The Н₂О₂ treatment facilitated the increase of the proline content in leaves of the plants of both genotypes under conditions of salt stress. It was concluded that there were hydrogen peroxide signal transduction pathways in Arabidopsis plants that were salicylic acid independent and that the antioxidant system functioned more effectively in salicylate-deficient Arabidopsis plants.     

Isolation and characterization of two sorbitol transporter gene promoters in micropropagated apple plants (Malus?×?domestica) regulated by drought stress

The role of polyol transporters in stress tolerance in plants have been elucidated by many studies. Sorbitol transporter genes MdSOT3, MdSOT4 and MdSOT5 in apple plants, which are important for sorbitol loading and unloading, are regulated by drought stress. To further confirm the role of sorbitol transporters in stress tolerance, the constructs harboring MdSOT3 and MdSOT5 genes were introduced into wild type Arabidopsis plants (Col-0) and the Arabidopsis transformed with MdSOT3 or MdSOT5 performed higher drought stress tolerance compared to WT. In order to further understand how sorbitol transporters are involved in drought tolerance in apple plants, upstream regions of sorbitol transporter genes were isolated from apple plant source leaves by Anchored PCR from genomic DNA obtained, and then were used to drive expression of the GUS reporter in tobacco plants. The results showed that the longest fragments of MdSOT3 and MdSOT5 promoters induced the highest GUS activity under drought stress conditions. Additionally, fragments of these promoters that contain cis-acting elements known to be involved in stress response also induced GUS activity under drought stress. Taken together, our data suggest that increased MdSOT3 and MdSOT5 activity, through cis-acting elements in the promoters of these genes, play important roles in imparting tolerance to drought in micropropagated apple plants.     

A Medicago truncatula EF-Hand Family Gene,MtCaMP1, Is Involved in Drought and Salt Stress Tolerance

Background

Calcium-binding proteins that contain EF-hand motifs have been reported to play important roles in transduction of signals associated with biotic and abiotic stresses. To functionally characterize gens of EF-hand family in response to abiotic stress, an MtCaMP1 gene belonging to EF-hand family from legume model plant Medicago truncatula was isolated and its function in response to drought and salt stress was investigated by expressing MtCaMP1 in Arabidopsis.

Methodology/Principal Findings

Transgenic Arabidopsis seedlings expressing MtCaMP1exhibited higher survival rate than wild-type seedlings under drought and salt stress, suggesting that expression of MtCaMP1 confers tolerance of Arabidopsis to drought and salt stress. The transgenic plants accumulated greater amounts of Pro due to up-regulation of P5CS1 and down-regulation of ProDH than wild-type plants under drought stress. There was a less accumulation of Na⁺ in the transgenic plants than in WT plants due to reduced up-regulation of AtHKT1 and enhanced regulation of AtNHX1 in the transgenic plants compared to WT plants under salt stress. There was a reduced accumulation of H₂O₂ and malondialdehyde in the transgenic plants than in WT plants under both drought and salt stress.

Conclusions/Significance

The expression of MtCaMP1 in Arabidopsis enhanced tolerance of the transgenic plants to drought and salt stress by effective osmo-regulation due to greater accumulation of Pro and by minimizing toxic Na⁺ accumulation, respectively. The enhanced accumulation of Pro and reduced accumulation of Na⁺ under drought and salt stress would protect plants from water default and Na⁺ toxicity, and alleviate the associated oxidative stress. These findings demonstrate that MtCaMP1 encodes a stress-responsive EF-hand protein that plays a regulatory role in response of plants to drought and salt stress.     

Vacuolar CAX1 and CAX3 Influence Auxin Transport in Guard Cells via Regulation of Apoplastic pH

CATION EXCHANGERs CAX1 and CAX3 are vacuolar ion transporters involved in ion homeostasis in plants. Widely expressed in the plant, they mediate calcium transport from the cytosol to the vacuole lumen using the proton gradient across the tonoplast. Here, we report an unexpected role of CAX1 and CAX3 in regulating apoplastic pH and describe how they contribute to auxin transport using the guard cell’s response as readout of hormone signaling and cross talk. We show that indole-3-acetic acid (IAA) inhibition of abscisic acid (ABA)-induced stomatal closure is impaired in cax1, cax3, and cax1/cax3. These mutants exhibited constitutive hypopolarization of the plasma membrane, and time-course analyses of membrane potential revealed that IAA-induced hyperpolarization of the plasma membrane is also altered in these mutants. Both ethylene and 1-naphthalene acetic acid inhibited ABA-triggered stomatal closure in cax1, cax3, and cax1/cax3, suggesting that auxin signaling cascades were functional and that a defect in IAA transport caused the phenotype of the cax mutants. Consistent with this finding, chemical inhibition of AUX1 in wild-type plants phenocopied the cax mutants. We also found that cax1/cax3 mutants have a higher apoplastic pH than the wild type, further supporting the hypothesis that there is a defect in IAA import in the cax mutants. Accordingly, we were able to fully restore IAA inhibition of ABA-induced stomatal closure in cax1, cax3, and cax1/cax3 when stomatal movement assays were carried out at a lower extracellular pH. Our results suggest a network linking the vacuolar cation exchangers to apoplastic pH maintenance that plays a crucial role in cellular processes.Stomata are pores at the surface of the leaves, gating water loss and gas exchange between plants and the atmosphere. One stoma is formed by two specialized guard cells that are able to modulate their size and shape to control stomatal aperture in response to various signals, including water status, hormonal stimuli, CO₂ levels, light, or temperature (Kwak et al., 2008). These stomatal movements are regulated by ion fluxes in guard cells, the changes in the osmoticum status being compensated by water movement, which modifies the cell’s volume. Ion transport between the cell and ion stores (vacuole, apoplastic space) must be therefore tightly controlled, and any change in the guard cell’s ability to regulate this can compromise its faculty to trigger stomatal movement.Calcium ion (Ca²⁺) is one ion that regulates stomatal movements, and its cytosolic concentration is controlled by both influx, via plasma membrane channels, and release from internal stores such as vacuoles and the endoplasmic reticulum. Calcium transport from the vacuole is ensured, at least in part, by members of the Cation Exchanger (CAX) family (Punshon et al., 2012). Six members of this family are found in Arabidopsis (Arabidopsis thaliana); all use a proton gradient generated by the vacuolar H⁺-ATPase (VHA) or the vacuolar pyrophosphatase (AVP1) to energize their activity. CAX1 and CAX3 are the closest homologs within the family and have been proposed to play similar roles in Ca²⁺ homeostasis (Zhao et al., 2008). However, biochemical characterization highlighted differences in their respective rates of Ca²⁺ transport, and they have been proposed to function as heterodimers, with unique properties associated with this structure (Cheng et al., 2005).Among common phenotypes of cax1 and cax3, an increased sensitivity to abscisic acid (ABA; Zhao et al., 2008) suggests a function for these transporters in modulating hormone signaling. ABA is well known for its role in triggering stomatal closure, whereas auxin, ethylene, or cytokinins can counteract its effect. Auxin in particular is also essential in governing plant development, including root architecture, tropisms and polarity, apical dominance, tissue differentiation, and plant development. Tight control of its distribution throughout the plant is achieved via ubiquitous and specific expression of members of three transporter families, acting together in mediating indole-3-acetic acid (IAA) fluxes (Krecek et al., 2009).The unique pattern of auxin distribution is predominately due to the asymmetrical localization of members of the PIN-FORMED (PIN) family of auxin exporters (Zazímalová et al., 2010). In Arabidopsis, this family comprises eight members, whose spatiotemporal expression is responsible for the auxin gradient observed in many plant tissues (Paponov et al., 2005). In addition, most members of the ATP-binding cassette (ABC)-type family of exporter ABCB (ABCB/multidrug resistance/phosphoglycoprotein) have been shown to mediate auxin export from the cell (Geisler and Murphy, 2006). Auxin import is mainly ensured by (1) active transport of IAA by members of the AUX1/LAX family proteins (Geisler and Murphy, 2006), and (2) passive diffusion across the plasma membrane. AUX1 activity was demonstrated to be pH-dependent (Yang et al., 2006), IAA transport being optimal at acidic pH (5.5–6), and dramatically reduced at higher values. It is interesting that passive, pH-dependent IAA diffusion across the plasma membrane also accounts for an important part of IAA transport and signaling. At apoplastic pH (5.5), between 10% and 25% of IAA is protonated (Yang et al., 2006), which allows for free diffusion of IAA through the membrane. In contrast, the ratio between protonated and deprotonated IAA (IAAH/IAA⁻) falls to 1% to 5% when pH exceeds 6.5, preventing it from being passively transported into the cytoplasm (Yang et al., 2006). These two aspects make control of the apoplastic pH crucial in the regulation of auxin signaling, as it modulates all the known routes of IAA import. Such a tight pH constraint is ensured by plasma membrane-localized Arabidopsis H+-ATPases (AHA; Haruta et al., 2010) that transport protons from the cytosol to the extracellular space.Our work presents the characterization of two vacuolar transporters’ abilities to modulate the apoplastic pH, and therefore contribute to proper auxin transport and signaling. Our results highlight the effects of mutations in CAX1 and CAX3 in plant development and in stomatal functioning, providing new insights for understanding hormone signaling in plants as well as plant adaptation to stress conditions via hormone cross talk.     

A review on transporters in salt tolerant mangroves

Key message

The role of transporters in imparting salt tolerance to mangroves is not yet understood. Identification of the role of transporters in halophytes is promising, as far as the development of genetically engineered salt tolerant crops is concerned.

Abstract

Mangroves are models for stress tolerance and they provide a reservoir for some of the novel genes and proteins, involved in salt tolerance. Biochemical or physiological mechanisms contribute to salt tolerance depending on variations in the environment. A great deal of research on salinity tolerance of plants, probes into water relations, photosynthesis, and accumulation of various in-organic ions and organic metabolites. The ability of the plant to react to high salinity depends on the genes that are expressed during stress. The mechanism of salinity tolerance becomes complicated when the responses of plants varies with salinity and environmental conditions. During the onset and development of salt stress within a plant, major processes such as photosynthesis, protein synthesis and lipid metabolisms are affected. The present review attempts to dissect out the role of transporters in salt tolerance of mangroves.     

Early Zn2+-induced effects on membrane potential account for primary heavy metal susceptibility in tolerant and sensitive Arabidopsis species

Background and Aims

Uptake of heavy metals by plant root cells depends on electro-physiological parameters of the plasma membrane. In this study, responses of the plasma membrane in root cells were analysed where early reactions to the metal ion-induced stress are localized. Three different Arabidopsis species with diverse strategies of their adaptation to heavy metals were compared: sensitive Arabidopsis thaliana and tolerant A. halleri and A. arenosa.

Methods

Plants of A. thaliana Col-0 ecotype and plants of A. arenosa and A. halleri originating from natural metallicolous populations were exposed to high concentrations of Zn²⁺. Plants were tested for root growth rate, cellular tolerance, plant morphology and cell death in the root apex. In addition, the membrane potential (E_M) of mature cortical root cells and changes in the pH of the liquid culture media were measured.

Key Results

Primary roots of A. halleri and A. arenosa plants grew significantly better at increased Zn²⁺ concentrations than A. thaliana plants. Elevated Zn²⁺ concentrations in the culture medium induced rapid changes in E_M. The reaction was species-specific and concentration-dependent. Arabidopsis halleri revealed the highest insensitivity of the plasma membrane and the highest survival rate under prolonged treatment with extra-high concentrations. Plants were able to effectively adjust the pH in the control, but much less at Zn²⁺-induced lower pH.

Conclusions

The results indicate a similar mode of early reaction to Zn²⁺, but with different extent in tolerant and sensitive species of Arabidopsis. The sensitivity of A. thaliana and a high tolerance of A. halleri and A. arenosa were demonstrated. Plasma membrane depolarization was lowest in the hyperaccumulator A. halleri and highest in A. thaliana. This indicates that rapid membrane voltage changes are an excellent tool to monitor the effects of heavy metals.