A plasma membrane-localized transporter remobilizes aleurone layer magnesium for seed germination in rice.

The aleurone layer in cereal grains acts as a major reservoir of essential mineral nutrients, significantly influencing seed germination. However, the molecular mechanism underlying the redistribution of nutrients from the aleurone layer in the germinating seed is still not well understood. Here, in rice, we identified a plasma membrane (PM) localized magnesium transporter, MAGNESIUM RELEASE TRANSPORTER 3 (MGR3), is critical for seed germination. OsMGR3 is predominantly expressed in the aleurone layer cells of endosperm, facilitating magnesium remobilization during germination. Non-invasive Micro-test Technology assay data demonstrated that the loss-of-function of OsMGR3 restrained magnesium efflux from the aleurone layer. In the embryo/endosperm grafting experiment, we observed that the mutation of OsMGR3 in the aleurone layer suppressed the growth and differentiation of the embryo during germination. Furthermore, magnesium fluorescence imaging revealed the osmgr3 mutant seeds showed impaired exportation of aleurone layer-stored magnesium to the embryo, consequently delaying germination. Importantly, we discovered that disrupting OsMGR3 could inhibit pre-harvest sprouting without affecting rice yield and quality. Therefore, the magnesium efflux transporter OsMGR3 in the aleurone layer represents a promising genetic target for future agronomic trait improvement.

Two magnesium transporters in the chloroplast inner envelope essential for thylakoid biogenesis in Arabidopsis.

Magnesium (Mg2+ ) serves as a cofactor for a number of photosynthetic enzymes in the chloroplast, and is the central atom of the Chl molecule. However, little is known about the molecular mechanism of Mg2+ transport across the chloroplast envelope. Here, we report the functional characterization of two transport proteins in Arabidopsis: Magnesium Release 8 (MGR8) and MGR9, of the ACDP/CNNM family, which is evolutionarily conserved across all lineages of living organisms. Both MGR8 and MGR9 genes were expressed ubiquitously, and their encoded proteins were localized in the inner envelope of chloroplasts. Mutations of MGR8 and MGR9 together, but neither of them alone, resulted in albino ovules and chlorotic seedlings. Further analysis revealed severe defects in thylakoid biogenesis and assembly of photosynthetic complexes in the double mutant. Both MGR8 and MGR9 functionally complemented the growth of the Salmonella typhimurium mutant strain MM281, which lacks Mg2+ uptake capacity. The embryonic and early seedling defects of the mgr8/mgr9 double mutant were rescued by the expression of MGR9 under the embryo-specific ABI3 promoter. The partially rescued mutant plants were hypersensitive to Mg2+ deficient conditions and contained less Mg2+ in their chloroplasts than wild-type plants. Taken together, we conclude that MGR8 and MGR9 serve as Mg2+ transporters and are responsible for chloroplast Mg2+ uptake.

Danger-Associated Peptides Interact with PIN-Dependent Local Auxin Distribution to Inhibit Root Growth in Arabidopsis.

Plant elicitor peptides (Peps) are damage/danger-associated molecular patterns that are perceived by the receptor-like kinases, PEPR1 and PEPR2, to enhance innate immunity and to inhibit root growth in Arabidopsis (Arabidopsis thaliana). Here, we show that Arabidopsis Pep1 inhibits root growth in a PEPR2-dependent manner, which is accompanied by swelling epidermal and cortex cells and root hair formation in the transition zone (TZ). These Pep1-induced changes were mimicked by exogenous auxin application and were suppressed in the auxin perception mutants transport inhibitor response1 (tir1) and tir1 afb1 afb2 Pep1-induced auxin accumulation in the TZ region preceded cell expansion in roots. Because local auxin distribution depends on PIN-type auxin transporters, we examined Pep1-PEPR-induced root growth inhibition in several pin mutants and found that pin2 was highly sensitive but pin3 was less sensitive to Pep1. The pin2 pin3 double mutant was as sensitive to Pep1 treatment as wild-type plants. Pep1 reduced the abundance of PIN2 in the plasma membrane through activating endocytosis while increasing PIN3 expression in the TZ, leading to changes in local auxin distribution and inhibiting root growth. These results suggest that Pep-PEPR signaling undergoes crosstalk with auxin accumulation to control cell expansion and differentiation in roots during immune responses.

An Arabidopsis vasculature distributed metal tolerance protein facilitates xylem magnesium diffusion to shoots under high-magnesium environments.

Magnesium (Mg2+ ) is an essential metal for plant growth; however, its over-accumulation in cells can be cytotoxic. The metal tolerance protein family (MTP) belongs to an ubiquitous family of cation diffusion facilitator (CDF) proteins that export divalent metal cations for metal homeostasis and tolerance in all organisms. We describe here the identification of MTP10 to be critical for xylem Mg homeostasis in Arabidopsis under high Mg2+ conditions. The Arabidopsis plant contains 12 MTP genes, and only knockout of MTP10 decreased the tolerance of high-Mg stress. The functional complementation assays in a Mg2+ -uptake-deficient bacterial strain MM281 confirmed that MTP10 conducted Mg2+ transport. MTP10 is localized to the plasma membrane of parenchyma cells around the xylem. Reciprocal grafting analysis further demonstrated that MTP10 functions in the shoot to determine the shoot growth phenotypes under high Mg2+ conditions. Moreover, compared to the wild type, the mtp10 mutant accumulated more Mg2+ in xylem sap under high-Mg stress. This study reveals that MTP10 facilitates Mg2+ diffusion from the xylem to shoots and thus determines Mg homeostasis in shoot vascular tissues during high-Mg stress.

An ICln homolog contributes to osmotic and low-nitrate tolerance by enhancing nitrate accumulation in Arabidopsis.

Nitrate (NO3- ) is a source of plant nutrients and osmolytes, but its delivery machineries under osmotic and low-nutrient stress remain largely unknown. Here, we report that AtICln, an Arabidopsis homolog of the nucleotide-sensitive chloride-conductance regulatory protein family (ICln), is involved in response to osmotic and low-NO3- stress. The gene AtICln, encoding plasma membrane-anchored proteins, was upregulated by various osmotic stresses, and its disruption impaired plant tolerance to osmotic stress. Compared with the wild type, the aticln mutant retained lower anions, particularly NO3- , and its growth retardation was not rescued by NO3- supply under osmotic stress. Interestingly, this mutant also displayed growth defects under low-NO3 stress, which were accompanied by decreases in NO3- accumulation, suggesting that AtICln may facilitate the NO3- accumulation under NO3- deficiency. Moreover, the low-NO3- hypersensitive phenotype of aticln mutant was overridden by the overexpression of NRT1.1, an important NO3- transporter in Arabidopsis low-NO3- responses. Further genetic analysis in the plants with altered activity of AtICln and NRT1.1 indicated that AtICln and NRT1.1 play a compensatory role in maintaining NO3- homeostasis under low-NO3- environments. These results suggest that AtICln is involved in cellular NO3- accumulation and thus determines osmotic adjustment and low-NO3- tolerance in plants.

Danger-Associated Peptides Close Stomata by OST1-Independent Activation of Anion Channels in Guard Cells.

The plant elicitor peptides (Peps), a family of damage/danger-associated molecular patterns (DAMPs), are perceived by two receptors, PEPR1 and PEPR2, and contribute to plant defense against pathogen attack and abiotic stress. Here, we show that the Peps-PEPR signaling pathway functions in stomatal immunity by activating guard cell anion channels in Arabidopsis thaliana The mutant plants lacking both PEPR1 and PEPR2 (pepr1 pepr2) displayed enhanced bacterial growth after being sprayed with Pseudomonas syringae pv tomato (Pst) DC3000, but not after pathogen infiltration into leaves, implicating PEPR function in stomatal immunity. Indeed, synthetic Arabidopsis Peps (AtPeps) effectively induced stomatal closure in wild-type but not pepr1 pepr2 mutant leaves, suggesting that the AtPeps-PEPR signaling pathway triggers stomatal closure. Consistent with this finding, patch-clamp recording revealed AtPep1-induced activation of anion channels in the guard cells of wild-type but not pepr1 pepr2 mutant plants. We further identified two guard cell-expressed anion channels, SLOW ANION CHANNEL1 (SLAC1) and its homolog SLAH3, as functionally overlapping components responsible for AtPep1-induced stomatal closure. The slac1 slah3 double mutant, but not slac1 or slah3 single mutants, failed to respond to AtPep1 in stomatal closure assays. Interestingly, disruption of OPEN STOMATA1 (OST1), an essential gene for abscisic acid-triggered stomatal closure, did not affect the AtPep1-induced anion channel activity and stomatal response. Together, these results illustrate a DAMP-triggered signaling pathway that, unlike the flagellin22-FLAGELLIN-SENSITIVE2 pathway, triggers stomata immunity through an OST1-independent mechanism.

Transcription Factor WRKY33 Mediates the Phosphate Deficiency-Induced Remodeling of Root Architecture by Modulating Iron Homeostasis in Arabidopsis Roots.

The remodeling of root architecture is regarded as a major development to improve the plant's adaptivity to phosphate (Pi)-deficient conditions. The WRKY transcription factors family has been reported to regulate the Pi-deficiency-induced systemic responses by affecting Pi absorption or transportation. Whether these transcription factors act as a regulator to mediate the Pi-deficiency-induced remodeling of root architecture, a typical local response, is still unclear. Here, we identified an Arabidopsis transcription factor, WRKY33, that acted as a negative regulator to mediate the Pi-deficiency-induced remodeling of root architecture. The disruption of WRKY33 in wrky33-2 mutant increased the plant's low Pi sensitivity by further inhibiting the primary root growth and promoting the formation of root hair. Furthermore, we revealed that WRKY33 negatively regulated the remodeling of root architecture by controlling the transcriptional expression of ALMT1 under Pi-deficient conditions, which further mediated the Fe3+ accumulation in root tips to inhibit the root growth. In conclusion, this study demonstrates a previously unrecognized signaling crosstalk between WRKY33 and the ALMT1-mediated malate transport system to regulate the Pi deficiency responses.

The Arabidopsis phosphatase PP2C49 negatively regulates salt tolerance through inhibition of AtHKT1;1.

Type 2C protein phosphatases (PP2Cs) are the largest protein phosphatase family. PP2Cs dephosphorylate substrates for signaling in Arabidopsis, but the functions of most PP2Cs remain unknown. Here, we characterized PP2C49 (AT3G62260, a Group G PP2C), which regulates Na+ distribution under salt stress and is localized to the cytoplasm and nucleus. PP2C49 was highly expressed in root vascular tissues and its disruption enhanced plant tolerance to salt stress. Compared with wild type, the pp2c49 mutant contained more Na+ in roots but less Na+ in shoots and xylem sap, suggesting that PP2C49 regulates shoot Na+ extrusion. Reciprocal grafting revealed a root-based mechanism underlying the salt tolerance of pp2c49. Systemic Na+ distribution largely depends on AtHKT1;1 and loss of function of AtHKT1;1 in the pp2c49 background overrode the salt tolerance of pp2c49, resulting in salt sensitivity. Furthermore, compared with plants overexpressing PP2C49 in the wild-type background, plants overexpressing PP2C49 in the athtk1;1 mutant background were sensitive to salt, like the athtk1;1 mutants. Moreover, protein-protein interaction and two-voltage clamping assays demonstrated that PP2C49 physically interacts with AtHKT1;1 and inhibits the Na+ permeability of AtHKT1;1. This study reveals that PP2C49 negatively regulates AtHKT1;1 activity and thus determines systemic Na+ allocation during salt stress.

Choline transporter-like 1 (CTL1) positively regulates apical hook development in etiolated Arabidopsis seedlings.

Ethylene is a gaseous phytohormone that is perceived by two-component histidine kinase-type receptors. Recent studies identified choline transporter-like 1 (CTL1) essential for Arabidopsis growth and development, including apical hook development in the etiolated seedlings. Here, we report that CTL1 contributes to apical hook development by enhancing ethylene response. The expression of CTL1 was highly correlated with the intensity of ethylene response and was enriched in the apical hook, cotyledon tip and hypocotyl. Genetic analysis showed that the dark-grown ctl1 mutant displayed a defect in ethylene-induced apical hook development as compared with the wild type. Accordingly, the expression of ethylene signaling reporter EBS::GUS in ctl1 mutant was greatly reduced in leaves, apical hook, hypocotyl and root, suggesting that the disruption of CTL1 impairs the ethylene signaling. Furthermore, protein-protein interaction assays demonstrated that CTL1 may interact with ethylene receptors, including ETR1, ETR2, ERS1, ERS2. Importantly, the abundance of CTL1 was diminished when ETR1 was disrupted upon ethylene response. Taken together, our results suggest that CTL1 functions as a positive regulator in ethylene signaling which in turn contributes to apical hook development of etiolated plant seedlings.

Vacuolar Phosphate Transporters Contribute to Systemic Phosphate Homeostasis Vital for Reproductive Development in Arabidopsis.

Vacuolar storage of phosphate (Pi) is essential for Pi homeostasis in plants. Recent studies have identified a family of vacuolar Pi transporters, VPTs (PHT5s), responsible for vacuolar sequestration of Pi. We report here that both VPT1 and VPT3 contribute to cytosol-to-vacuole Pi partitioning. Although VPT1 plays a predominant role, VPT3 is particularly important when VPT1 is absent. Our data suggested that the vpt1 vpt3 double mutant was more defective in Pi homeostasis than the vpt1 single mutant, as indicated by Pi accumulation capacity, vacuolar Pi influx, subcellular Pi allocation, and plant adaptability to changing Pi status. The remaining member of the VPT family, VPT2 (PHT5;2), did not appear to contribute to Pi homeostasis in such assays. Particularly interesting is the finding that the vpt1 vpt3 double mutant was impaired in reproductive development with shortened siliques and impaired seed set under sufficient Pi, and this phenotype was not found in the vpt1 vpt2 and vpt2 vpt3 double mutants. Measurements of Pi contents revealed Pi over-accumulation in the floral organs of vpt1 vpt3 as compared with the wild type. Further analysis identified excess Pi in the pistil as inhibitory to pollen tube growth, and thus seed yield, in the mutant plants. Reducing the Pi levels in culture medium or mutation of PHO1, a Pi transport protein responsible for root-shoot transport, restored the seed set of vpt1 vpt3 Thus, VPTs, through their function in vacuolar Pi sequestration, control the fine-tuning of systemic Pi allocation, which is particularly important for reproductive development.

A Defective Vacuolar Proton Pump Enhances Aluminum Tolerance by Reducing Vacuole Sequestration of Organic Acids.

Plants cope with aluminum (Al) toxicity by secreting organic acids (OAs) into the apoplastic space, which is driven by proton (H+) pumps. Here, we show that mutation of vacuolar H+-translocating adenosine triphosphatase (H+-ATPase) subunit a2 (VHA-a2) and VHA-a3 of the vacuolar H+-ATPase enhances Al resistance in Arabidopsis (Arabidopsis thaliana). vha-a2 vha-a3 mutant plants displayed less Al sensitivity with less Al accumulation in roots compared to wild-type plants when grown under excessive Al3+ Interestingly, in response to Al3+ exposure, plants showed decreased vacuolar H+ pump activity and reduced expression of VHA-a2 and VHA-a3, which were accompanied by increased plasma membrane H+ pump (PM H+-ATPase) activity. Genetic analysis of plants with altered PM H+-ATPase activity established a correlation between Al-induced increase in PM H+-ATPase activity and enhanced Al resistance in vha-a2 vha-a3 plants. We determined that external OAs, such as malate and citrate whose secretion is driven by PM H+-ATPase, increased with PM H+-ATPase activity upon Al stress. On the other hand, elevated secretion of malate and citrate in vha-a2 vha-a3 root exudates appeared to be independent of OAs metabolism and tolerance of phosphate starvation but was likely related to impaired vacuolar sequestration. These results suggest that coordination of vacuolar H+-ATPase and PM H+-ATPase dictates the distribution of OAs into either the vacuolar lumen or the apoplastic space that, in turn, determines Al tolerance capacity in plants.

Arabidopsis choline transporter-like 1 (CTL1) regulates secretory trafficking of auxin transporters to control seedling growth.

Auxin controls a myriad of plant developmental processes and plant response to environmental conditions. Precise trafficking of auxin transporters is essential for auxin homeostasis in plants. Here, we report characterization of Arabidopsis CTL1, which controls seedling growth and apical hook development by regulating intracellular trafficking of PIN-type auxin transporters. The CTL1 gene encodes a choline transporter-like protein with an expression pattern highly correlated with auxin distribution and is enriched in shoot and root apical meristems, lateral root primordia, the vascular system, and the concave side of the apical hook. The choline transporter-like 1 (CTL1) protein is localized to the trans-Golgi network (TGN), prevacuolar compartment (PVC), and plasma membrane (PM). Disruption of CTL1 gene expression alters the trafficking of 2 auxin efflux transporters-Arabidopsis PM-located auxin efflux transporter PIN-formed 1 (PIN1) and Arabidopsis PM-located auxin efflux transporter PIN-formed 3 (PIN3)-to the PM, thereby affecting auxin distribution and plant growth and development. We further found that phospholipids, sphingolipids, and other membrane lipids were significantly altered in the ctl1 mutant, linking CTL1 function to lipid homeostasis. We propose that CTL1 regulates protein sorting from the TGN to the PM through its function in lipid homeostasis.

Danger-Associated Peptide Regulates Root Growth by Promoting Protons Extrusion in an AHA2-Dependent Manner in Arabidopsis.

Plant elicitor peptides (Peps) are damage/danger-associated molecular patterns (DAMPs) that are derived from precursor proteins PROPEPs and perceived by a pair of leucine-rich repeat receptor-like kinases (LRR-RLKs), PEPR1 and PEPR2, to enhance innate immunity and to inhibit root growth in Arabidopsis thaliana. In this study, we show that Arabidopsis Pep1 inhibits the root growth by interfering with pH signaling, as acidic condition increased, but neutral and alkaline conditions decreased the Pep1 effect on inhibiting the root growth. The perception of Pep1 to PEPRs activated the plasma membrane-localized H+-ATPases (PM H+-ATPases) -the pump proton in plant cell-to extrude the protons into apoplast, and induced an overly acidic environment in apoplastic space, which further promoted the cell swelling in root apex and inhibited root growth. Furthermore, we revealed that pump proton AUTOINHIBITED H+-ATPase 2 (AHA2) physically interacted with PEPR2 and served downstream of the Pep1-PEPRs signaling pathway to regulate Pep1-induced protons extrusion and root growth inhibition. In conclusion, this study demonstrates a previously unrecognized signaling crosstalk between Pep1 and pH signaling to regulate root growth.

Type A2 BTB Members Decrease the ABA Response during Seed Germination by Affecting the Stability of SnRK2.3 in Arabidopsis.

The Arabidopsis genome comprises eighty genes encoding BTB (broad-complex, tramtrack, and bric-a-brac) family proteins that are characterized with the BTB domain and that potentially serve as substrate adaptors for cullin-based E3-ligases. In addition to the BTB domain, most BTB proteins also contain various other interaction motifs that probably act as target recognition elements. Here, we report three members of the BTB-A2 subfamily that distinctly only contain the BTB domain, BTB-A2.1, BTB-A2.2, and BTB-A2.3, that negatively regulates abscisic acid (ABA) signaling in Arabidopsis. BTB-A2.1, BTB-A2.2, and BTB-A2.3 encoded cytoplasm- and nucleus-localized proteins and displayed highly overlapping expression patterns in Arabidopsis tissues. Disruption of these three genes, but not single or double mutants, resulted in a decrease in ABA-induced inhibition of seed germination. Further analyses demonstrated the expression levels of these three genes were up-regulated by ABA, and their mutation increased ABA signalling. Importantly, protein-protein interaction assays showed that these three BTB-A2 proteins physically interacted with SnRK2.3. Moreover, biochemical and genetic assays indicated that BTB-A2.1, BTB-A2.2, and BTB-A2.3 decreased the stability of SnRK2.3 and attenuated the SnRK2.3 responsible for the ABA hypersensitive phenotype of seed germination. This report thus reveals that BTB-A2s serve as negative regulators for balancing the intensity of ABA signaling during seed germination.

Danger-Associated Peptide Regulates Root Immune Responses and Root Growth by Affecting ROS Formation in Arabidopsis.

Plant elicitor peptides (Peps) are damage/danger-associated molecular patterns (DAMPs) that are perceived by a pair of receptor-like kinases, PEPR1 and PEPR2, to enhance innate immunity and induce the growth inhibition of root in Arabidopsis thaliana. In this study, we show that PEPR1 and PEPR2 function vitally in roots to regulate the root immune responses when treating the roots with bacterial pathogen Pst DC3000. PEPR2, rather than PEPR1, played a predominant role in the perception of Pep1 in the roots and further triggered a strong ROS accumulation-the substance acts as an antimicrobial agent or as a secondary messenger in plant cells. Consistently, seedlings mutating two major ROS-generating enzyme genes, respiratory burst oxidase homologs D and F (RBOHD and RBOHF), abolished the root ROS accumulation and impaired the growth inhibition of the roots induced by Pep1. Furthermore, we revealed that botrytis-induced kinase 1 (BIK1) physically interacted with PEPRs and RBOHD/F, respectively, and served downstream of the Pep1-PEPRs signaling pathway to regulate Pep1-induced ROS production and root growth inhibition. In conclusion, this study demonstrates a previously unrecognized signaling crosstalk between Pep1 and ROS signaling to regulate root immune response and root growth.

FERONIA interacts with ABI2-type phosphatases to facilitate signaling cross-talk between abscisic acid and RALF peptide in Arabidopsis.

Receptor-like kinase FERONIA (FER) plays a crucial role in plant response to small molecule hormones [e.g., auxin and abscisic acid (ABA)] and peptide signals [e.g., rapid alkalinization factor (RALF)]. It remains unknown how FER integrates these different signaling events in the control of cell growth and stress responses. Under stress conditions, increased levels of ABA will inhibit cell elongation in the roots. In our previous work, we have shown that FER, through activation of the guanine nucleotide exchange factor 1 (GEF1)/4/10-Rho of Plant 11 (ROP11) pathway, enhances the activity of the phosphatase ABA Insensitive 2 (ABI2), a negative regulator of ABA signaling, thereby inhibiting ABA response. In this study, we found that both RALF and ABA activated FER by increasing the phosphorylation level of FER. The FER loss-of-function mutant displayed strong hypersensitivity to both ABA and abiotic stresses such as salt and cold conditions, indicating that FER plays a key role in ABA and stress responses. We further showed that ABI2 directly interacted with and dephosphorylated FER, leading to inhibition of FER activity. Several other ABI2-like phosphatases also function in this pathway, and ABA-dependent FER activation required PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)-A-type protein phosphatase type 2C (PP2CA) modules. Furthermore, suppression of RALF1 gene expression, similar to disruption of the FER gene, rendered plants hypersensitive to ABA. These results formulated a mechanism for ABA activation of FER and for cross-talk between ABA and peptide hormone RALF in the control of plant growth and responses to stress signals.

Calcineurin B-Like Proteins CBL4 and CBL10 Mediate Two Independent Salt Tolerance Pathways in Arabidopsis.

In Arabidopsis, the salt overly sensitive (SOS) pathway, consisting of calcineurin B-like protein 4 (CBL4/SOS3), CBL-interacting protein kinase 24 (CIPK24/SOS2) and SOS1, has been well defined as a crucial mechanism to control cellular ion homoeostasis by extruding Na+ to the extracellular space, thus conferring salt tolerance in plants. CBL10 also plays a critical role in salt tolerance possibly by the activation of Na+ compartmentation into the vacuole. However, the functional relationship of the SOS and CBL10-regulated processes remains unclear. Here, we analyzed the genetic interaction between CBL4 and CBL10 and found that the cbl4 cbl10 double mutant was dramatically more sensitive to salt as compared to the cbl4 and cbl10 single mutants, suggesting that CBL4 and CBL10 each directs a different salt-tolerance pathway. Furthermore, the cbl4 cbl10 and cipk24 cbl10 double mutants were more sensitive than the cipk24 single mutant, suggesting that CBL10 directs a process involving CIPK24 and other partners different from the SOS pathway. Although the cbl4 cbl10, cipk24 cbl10, and sos1 cbl10 double mutants showed comparable salt-sensitive phenotype to sos1 at the whole plant level, they all accumulated much lower Na+ as compared to sos1 under high salt conditions, suggesting that CBL10 regulates additional unknown transport processes that play distinct roles from the SOS1 in Na+ homeostasis.

Vacuolar Phosphate Transporter 1 (VPT1) Affects Arsenate Tolerance by Regulating Phosphate Homeostasis in Arabidopsis.

Arsenate [As(V)] is toxic to nearly all organisms. Soil-borne As(V) enters plant cells mainly through the plasma membrane-localized phosphate (Pi) transporter PHT1 family proteins due to its chemical similarity to Pi. We report here that VPT1, a major vacuolar phosphate transporter which contributes to vacuolar Pi sequestration, is associated with As(V) tolerance in Arabidopsis. vpt1 mutants displayed enhanced tolerance to As(V) toxicity, whereas plants overexpressing VPT1 were more sensitive to As(V) as compared with the wild-type plants. Measurements of arsenic content indicated that vpt1 mutants accumulated less arsenic and, in contrast, up-regulating VPT1 expression contributed to higher levels of arsenic accumulation in plants. To examine further how VPT1 may modulate arsenic contents in plants, we surveyed the expression patterns of all the PHT1 family members that play roles in As(V) uptake, and found that many of the PHT1 genes were down-regulated in the vpt1 mutant as compared with the wild type under Pi-sufficient conditions, but not when Pi levels were low in the medium. Interestingly, As(V) sensitivity assays indicated that As(V) resistance in vpt1 mutants was prominent only under Pi-sufficient but not under Pi-deficient conditions. These results suggest that under Pi-sufficient conditions, loss of VPT1 leads to elevated levels of Pi in the cytosol, which in turn suppressed the expression of PHT1-type transporters and reduced accumulation of arsenic.

A vacuolar phosphate transporter essential for phosphate homeostasis in Arabidopsis.

Inorganic phosphate (Pi) is stored in the vacuole, allowing plants to adapt to variable Pi availability in the soil. The transporters that mediate Pi sequestration into vacuole remain unknown, however. Here we report the functional characterization of Vacuolar Phosphate Transporter 1 (VPT1), an SPX domain protein that transports Pi into the vacuole in Arabidopsis. The vpt1 mutant plants were stunted and consistently retained less Pi than wild type plants, especially when grown in medium containing high levels of Pi. In seedlings, VPT1 was expressed primarily in younger tissues under normal conditions, but was strongly induced by high-Pi conditions in older tissues, suggesting that VPT1 functions in Pi storage in young tissues and in detoxification of high Pi in older tissues. As a result, disruption of VPT1 rendered plants hypersensitive to both low-Pi and high-Pi conditions, reducing the adaptability of plants to changing Pi availability. Patch-clamp analysis of isolated vacuoles showed that the Pi influx current was severely reduced in vpt1 compared with wild type plants. When ectopically expressed in Nicotiana benthamiana mesophyll cells, VPT1 mediates vacuolar influx of anions, including Pi, SO4(2-), NO3(-), Cl(-), and malate with Pi as that preferred anion. The VPT1-mediated Pi current amplitude was dependent on cytosolic phosphate concentration. Single-channel analysis showed that the open probability of VPT1 was increased with the increase in transtonoplast potential. We conclude that VPT1 is a transporter responsible for vacuolar Pi storage and is essential for Pi adaptation in Arabidopsis.

Vacuolar Proton Pyrophosphatase Is Required for High Magnesium Tolerance in Arabidopsis.

Magnesium (Mg2+) is an essential nutrient in all organisms. However, high levels of Mg2+ in the environment are toxic to plants. In this study, we identified the vacuolar-type H⁺-pyrophosphatase, AVP1, as a critical enzyme for optimal plant growth under high-Mg conditions. The Arabidopsis avp1 mutants displayed severe growth retardation, as compared to the wild-type plants upon excessive Mg2+. Unexpectedly, the avp1 mutant plants retained similar Mg content to wild-type plants under either normal or high Mg conditions, suggesting that AVP1 may not directly contribute to Mg2+ homeostasis in plant cells. Further analyses confirmed that the avp1 mutant plants contained a higher pyrophosphate (PPi) content than wild type, coupled with impaired vacuolar H⁺-pyrophosphatase activity. Interestingly, expression of the Saccharomyces cerevisiae cytosolic inorganic pyrophosphatase1 gene IPP1, which facilitates PPi hydrolysis but not proton translocation into vacuole, rescued the growth defects of avp1 mutants under high-Mg conditions. These results provide evidence that high-Mg sensitivity in avp1 mutants possibly resulted from elevated level of cytosolic PPi. Moreover, genetic analysis indicated that mutation of AVP1 was additive to the defects in mgt6 and cbl2 cbl3 mutants that are previously known to be impaired in Mg2+ homeostasis. Taken together, our results suggest AVP1 is required for cellular PPi homeostasis that in turn contributes to high-Mg tolerance in plant cells.