Conserved mechanism for vacuolar magnesium sequestration in yeast and plant cells.

Magnesium (Mg2+) is an essential nutrient for all life forms. In fungal and plant cells, the majority of Mg2+ is stored in the vacuole but mechanisms for Mg2+ transport into the vacuolar store are not fully understood. Here we demonstrate that members of ancient conserved domain proteins (ACDPs) from Saccharomyces cerevisiae and Arabidopsis thaliana function in vacuolar Mg2+ sequestration that enables plant and yeast cells to cope with high levels of external Mg2+. We show that the yeast genome (as well as other fungal genomes) harbour a single ACDP homologue, referred to as MAM3, that functions specifically in vacuolar Mg2+ accumulation and is essential for tolerance to high Mg. In parallel, vacuolar ACDP homologues were identified from Arabidopsis and shown to complement the yeast mutant mam3Δ. An Arabidopsis mutant lacking one of the vacuolar ACDP homologues displayed hypersensitivity to high-Mg conditions and accumulated less Mg in the vacuole compared with the wild type. Taken together, our results suggest that conserved transporters mediate vacuolar Mg2+ sequestration in fungal and plant cells to maintain cellular Mg2+ homeostasis in response to fluctuating Mg2+ levels in the environment.

Four plasma membrane-localized MGR transporters mediate xylem Mg2+ loading for root-to-shoot Mg2+ translocation in Arabidopsis.

Magnesium (Mg2+), an essential structural component of chlorophyll, is absorbed from the soil by roots and transported to shoots to support photosynthesis in plants. However, the molecular mechanisms underlying root-to-shoot Mg2+ translocation remain largely unknown. We describe here the identification of four plasma membrane (PM)-localized transporters, named Mg2+ release transporters (MGRs), that are critical for root-to-shoot Mg transport in Arabidopsis. Functional complementation assays in a Mg2+-uptake-deficient bacterial strain confirmed that these MGRs conduct Mg2+ transport. PM-localized MGRs (MGR4, MGR5, MGR6, and MGR7) were expressed primarily in root stellar cells and participated in the xylem loading step of the long-distance Mg2+ transport process. In particular, MGR4 and MGR6 played a major role in shoot Mg homeostasis, as their loss-of-function mutants were hypersensitive to low Mg2+ but tolerant to high Mg2+ conditions. Reciprocal grafting analysis further demonstrated that MGR4 functions in the root to determine shoot Mg2+ accumulation and physiological phenotypes caused by both low- and high-Mg2+ stress. Taken together, our study has identified the long-sought transporters responsible for root-to-shoot Mg2+ translocation in plants.

Plant Membrane Transport Research in the Post-genomic Era.

Membrane transport processes are indispensable for many aspects of plant physiology including mineral nutrition, solute storage, cell metabolism, cell signaling, osmoregulation, cell growth, and stress responses. Completion of genome sequencing in diverse plant species and the development of multiple genomic tools have marked a new era in understanding plant membrane transport at the mechanistic level. Genes coding for a galaxy of pumps, channels, and carriers that facilitate various membrane transport processes have been identified while multiple approaches are developed to dissect the physiological roles as well as to define the transport capacities of these transport systems. Furthermore, signaling networks dictating the membrane transport processes are established to fully understand the regulatory mechanisms. Here, we review recent research progress in the discovery and characterization of the components in plant membrane transport that take advantage of plant genomic resources and other experimental tools. We also provide our perspectives for future studies in the field.

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 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.

Mechanistic analysis of AKT1 regulation by the CBL-CIPK-PP2CA interactions.

Arabidopsis K+ transporter 1 (AKT1) participates in K+ uptake in roots, especially under low-K conditions. We recently identified a Ca²âº signaling pathway consisting of multiple calcineurin B-like calcium sensors (CBLs) and multiple target kinases (CBL-interacting protein kinases or CIPKs) that phosphorylate and activate AKT1, whereas a specific PP2C-type phosphatase inactivates CIPK-dependent AKT1 activity. In this study, we analyzed the interactions between PP2Cs and the CBL-CIPK pathway and found previously unsuspected mechanisms underlying the CBL-CIPK-PP2C signaling processes. The interaction between the CIPKs and PP2Cs involves the kinase domain of the CIPK component, in addition to the protein phosphatase interacting motif (PPI) in the regulatory domain. Furthermore, specific CBLs physically interact with and inactivate PP2C phosphatases to recover the CIPK-dependent AKT1 channel activity. These findings provide further insights into the signaling network consisting of CBL-CIPK-PP2C interactions in the activation of the AKT1 channel.

A rice high-affinity potassium transporter (HKT) conceals a calcium-permeable cation channel.

Plant high-affinity K(+) transport (HKT) proteins are so named because of their relation to bacterial and fungal transporters that mediate high-affinity K(+) uptake. The view that HKT family members are sodium-selective uniporters or sodium-potassium symporters is widely held. We have found that one of the rice HKT proteins also functions as a Ca(2+)-permeable cation channel that conducts current carried by a wide range of monovalent and divalent cations. The HKT rice gene, named OsHKT2;4, is expressed in several cell types, including root hairs and vascular parenchyma cells. The protein is localized to the plasma membrane, thereby providing a mechanism for cation uptake and extrusion. This finding goes against firmly entrenched dogma in showing that HKT proteins can function as both ion carriers and channels. The study further extends the function of HKT proteins to Ca(2+)-linked processes and, in so doing, defines a previously undescribed type of Ca(2+)-permeable cation channels in plants. The work also raises questions about the evolutionary changes in this protein family following the divergence of monocots and dicots.

A protein phosphorylation/dephosphorylation network regulates a plant potassium channel.

Potassium (K(+)) is an essential nutrient for plant growth and development. Plants often adapt to low K(+) conditions by increasing their K(+) uptake capability. Recent studies have led to the identification of a calcium signaling pathway that enables plants to act in this capacity. Calcium is linked to two calcineurin B-like calcium sensors (CBLs) and a target kinase (CBL-interacting protein kinase 23 or CIPK23) that, in turn, appears to phosphorylate and activate the potassium channel, Arabidopsis K(+) transporter 1 (AKT1), responsible for K(+) uptake in roots. Here, we report evidence that this regulatory mechanism is more elaborate than earlier envisaged. The recently described pathway is part of an extensive network whereby several CBLs interact with multiple CIPKs in the activation of the potassium channel, AKT1. The physical interactions among the CBL, CIPK, and AKT1 components provide a mechanism for specifying the members of the CBL and CIPK families functional in AKT1 regulation. The interaction between the CIPKs and AKT1 was found to involve the kinase domain of the CIPK component and the ankyrin repeat domain of the channel. Furthermore, we identified a 2C-type protein phosphatase that physically interacts and inactivates the AKT1 channel. These findings provide evidence that the calcium-sensitive CBL and CIPK families together with 2C-type protein phosphatases form a protein phoshporylation/dephosphorylation network that regulates the AKT1 channel for K(+) transport in plants.

Modulation of hepatocellular swelling-activated K+ currents by phosphoinositide pathway-dependent protein kinase C.

K(+) channels participate in the regulatory volume decrease (RVD) accompanying hepatocellular nutrient uptake and bile formation. We recently identified KCNQ1 as a molecular candidate for a significant fraction of the hepatocellular swelling-activated K(+) current (I(KVol)). We have shown that the KCNQ1 inhibitor chromanol 293B significantly inhibited RVD-associated K(+) flux in isolated perfused rat liver and used patch-clamp techniques to define the signaling pathway linking swelling to I(KVol) activation. Patch-electrode dialysis of hepatocytes with solutions that maintain or increase phosphatidylinositol 4,5-bisphosphate (PIP(2)) increased I(KVol), whereas conditions that decrease cellular PIP(2) decreased I(KVol). GTP and AlF(4)(-) stimulated I(KVol) development, suggesting a role for G proteins and phospholipase C (PLC). Supporting this, the PLC blocker U-73122 decreased I(KVol) and inhibited the stimulatory response to PIP(2) or GTP. Protein kinase C (PKC) is involved, because K(+) current was enhanced by 1-oleoyl-2-acetyl-sn-glycerol and inhibited after chronic PKC stimulation with phorbol 12-myristate 13-acetate (PMA) or the PKC inhibitor GF 109203X. Both I(KVol) and the accompanying membrane capacitance increase were blocked by cytochalasin D or GF 109203X. Acute PMA did not eliminate the cytochalasin D inhibition, suggesting that PKC-mediated I(KVol) activation involves the cytoskeleton. Under isotonic conditions, a slowly developing K(+) current similar to I(KVol) was activated by PIP(2), lipid phosphatase inhibitors to counter PIP(2) depletion, a PLC-coupled alpha(1)-adrenoceptor agonist, or PKC activators and was depressed by PKC inhibition, suggesting that hypotonicity is one of a set of stimuli that can activate I(KVol) through a PIP(2)/PKC-dependent pathway. The results indicate that PIP(2) indirectly activates hepatocellular KCNQ1-like channels via cytoskeletal rearrangement involving PKC activation.

Contribution of a time-dependent and hyperpolarization-activated chloride conductance to currents of resting and hypotonically shocked rat hepatocytes.

Hepatocellular Cl- flux is integral to maintaining cell volume and electroneutrality in the face of the many transport and metabolic activities that describe the multifaceted functions of these cells. Although a significant volume-regulated Cl- current (VRAC) has been well described in hepatocytes, the Cl- channels underlying the large resting anion conductance have not been identified. We used a combination of electrophysiological and molecular approaches to describe potential candidates for this conductance. Anion currents in rat hepatocytes and WIF-B and HEK293T cells were measured under patch electrode-voltage clamp. With K+-free salts of Cl- comprising the major ions externally and internally, hyperpolarizing steps between -40 and -140 mV activated a time-dependent inward current in hepatocytes. Steady-state activation was half-maximal at -63 mV and 28-38% of maximum at -30 to -45 mV, previously reported hepatocellular resting potentials. Gating was dependent on cytosolic Cl-, shifting close to 58 mV/10-fold change in Cl- concentration. Time-dependent inward Cl- currents and a ClC-2-specific RT-PCR product were also observed in WIF-B cells but not HEK293T cells. All cell types exhibited typical VRAC in response to dialysis with hypertonic solutions. DIDS (0.1 mM) inhibited the hepatocellular VRAC but not the inward time-dependent current. Antibodies against the COOH terminus of ClC-2 reacted with a protein between 90 and 100 kDa in liver plasma membranes. The results demonstrate that rat hepatocytes express a time-dependent inward Cl- channel that could provide a significant depolarizing influence in the hepatocyte.

Involvement of NADPH oxidase in hydrogen peroxide accumulation by Aspergillus niger elicitor-induced Taxus chinensis cell cultures.

After determining that hydrogen peroxide (H2O2) accumulation induced by a fungal elicitor from Aspergillus niger was from the superoxide dismutase-catalyzed dismutation of superoxide radical, the site of H2O2 generation in cell suspension cultures of Taxus chinensis was studied. The results showed that 90% and 10% of the elicitor-induced H2O2 accumulation respectively appeared in intracellular and extracellular fractions of cells, and that the elicitor-induced H2O2 accumulation in protoplasts and plasma membranes was similar to that in intact cells, indicating that the site of H2O2 accumulation was plasma membranes but not in extracellular fraction of Taxus cells. The H2O2 forming enzyme was also investigated. The elicitor-induced H2O2 accumulation in intact cells was not changed by loss of apoplastic peroxidase (POD) by the washing, and the H2O2 accumulation in plasma membranes was inhibited by the mammalian neutrophil NAD(P)H oxidase inhibitor diphenylene iodonium (DPI), but was slightly affected by exogenous POD and its inhibitor. Furthermore, in plasma membranes, the H2O2 accumulation was more significantly enhanced by NADPH than by NADH, and the former was more obviously decreased by DPI than the latter. The present results show that NADPH oxidase in plasma membranes is involved in H2O2 accumulation in fungal elicitor-induced Taxus chinensis cell cultures.

Hydrogen peroxide from the oxidative burst is not involved in the induction of taxol biosynthesis in Taxus chinensis cells.

In cell suspension cultures of Taxus chinensis, 40 mg/l fungal elicitor from Aspergillus niger and 20 microM HgCl2 elicited 5.7 and 3.6 mg/l taxol, which was a 9-fold and 5-fold increase vs. compared with the control, respectively. The fungal elicitor induced hydrogen peroxide (H2O2) accumulation but HgCl2 did not, indicating that H2O2 was not necessary for enhancement of taxol induced by elicitor. Compared with the treatment with fungal elicitor alone, exogenous catalase, ascorbic acid, diphenylene iodonium and superoxide dismutase induced a 0.45, 0.4, 0.7 and 1.4-fold H2O2, but elicited taxol production, which was 0.98, 1.2, 1.1 and 0.9-fold, respectively, vs. non-treated cells Elicitor-induced taxol production was not accorded with the amount of H2O2 production.