Genes encoding the vacuolar Na+/H+ exchanger and flower coloration

Vacuolar pH plays an important role in flower coloration: an increase in the vacuolar pH causes blueing of flower color. In the Japanese morning glory (Ipomoea nil or Pharbitis nil), a shift from reddish-purple buds to blue open flowers correlates with an increase in the vacuolar pH. We describe details of the characterization of a mutant that carries a recessive mutation in the Purple (Pr) gene encoding a vacuolar Na+/H+ exchanger termed InNHX1. The genome of I. nil carries one copy of the Pr (or InNHX1) gene and its pseudogene, and it showed functional complementation to the yeast nhx1 mutation. The mutant of I. nil, called purple (pr), showed a partial increase in the vacuolar pH during flower-opening and its reddish-purple buds change into purple open flowers. The vacuolar pH in the purple open flowers of the mutant was significantly lower than that in the blue open flowers. The InNHX1 gene is most abundantly expressed in the petals at around 12 h before flower-opening, accompanying the increase in the vacuolar pH for the blue flower coloration. No such massive expression was observed in the petunia flowers. Since the NHX1 genes that promote the transport of Na+ into the vacuoles have been regarded to be involved in salt tolerance by accumulating Na+ in the vacuoles, we can add a new biological role for blue flower coloration in the Japanese morning glory by the vacuolar alkalization.     

Characterization of a novel Na+/H+ antiporter gene InNHX2 and comparison of InNHX2 with InNHX1, which is responsible for blue flower coloration by increasing the vacuolar pH in the Japanese morning glory

The reddish-purple buds of the wild-type Japanese morning glory (Ipomoea nil) change into blue open flowers, and the shift in the flower coloration correlates with an increase in the vacuolar pH of the flower epidermal cell. In the mutant deficient in the InNHX1 gene for the vacuolar Na(+)/H(+) antiporter, the vacuolar alkalization occurs only partially, and reddish-purple buds become purple open flowers. While most of the plant NHX genes characterized are generally expressed in leaves, stems and roots and induced by NaCl treatment, the InNHX1 gene is expressed predominantly in the flower limbs at around 12 h before flower opening. It is expressed very sparsly in leaves, stems and roots, and no induction occurs in response to NaCl treatment. Here, we identified a novel vacuolar Na(+)/H(+) antiporter gene InNHX2, which is expressed in leaves, stems and roots and is induced in response to NaCl treatment. In addition, relatively higher expression of InNHX2 was observed in the flower limbs shortly before flower opening. We also discovered that both the InNHX1 and InNHX2 proteins can catalyze both Na(+) and K(+) transport into vacuoles. These results suggest that InNHX2 performs dual functions: to confer salt tolerance on the plant and to promote partial vacuolar alkalization in the petals. The implication is that the InNHX2 protein is probably one of the components responsible for converting reddish-purple buds into purple open flowers by partially increasing the vacuolar pH in the absence of major InNHX1 activity.     

Regulation and reversibility of vacuolar H(+)-ATPase

Arabidopsis thaliana vacuolar H(+)-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H(+)-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (DeltamuH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (DeltapH). On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (DeltaPsi). Chimeric vacuolar membranes showed ATP synthesis coupled with DeltamuH established by V-PPase. The ATP synthesis was sensitive to bafilomycin A(1) and exhibited two apparent K(m) values for ADP. These results indicate that V-ATPase is a reversible enzyme. The ATP synthesis was not observed in the presence of nigericin, which dissipates DeltapH but not DeltaPsi, suggesting that DeltapH is essential for ATP synthesis.     

The Arabidopsis Na+/H+ antiporters NHX1 and NHX2 control vacuolar pH and K+ homeostasis to regulate growth, flower development, and reproduction

Intracellular Na(+)/H(+) (NHX) antiporters have important roles in cellular pH and Na(+), K(+) homeostasis. The six Arabidopsis thaliana intracellular NHX members are divided into two groups, endosomal (NHX5 and NHX6) and vacuolar (NHX1 to NHX4). Of the vacuolar members, NHX1 has been characterized functionally, but the remaining members have largely unknown roles. Using reverse genetics, we show that, unlike the single knockouts nhx1 or nhx2, the double knockout nhx1 nhx2 had significantly reduced growth, smaller cells, shorter hypocotyls in etiolated seedlings and abnormal stamens in mature flowers. Filaments of nhx1 nhx2 did not elongate and lacked the ability to dehisce and release pollen, resulting in a near lack of silique formation. Pollen viability and germination was not affected. Quantification of vacuolar pH and intravacuolar K(+) concentrations indicated that nhx1 nhx2 vacuoles were more acidic and accumulated only 30% of the wild-type K(+) concentration, highlighting the roles of NHX1 and NHX2 in mediating vacuolar K(+)/H(+) exchange. Growth under added Na(+), but not K(+), partly rescued the flower and growth phenotypes. Our results demonstrate the roles of NHX1 and NHX2 in regulating intravacuolar K(+) and pH, which are essential to cell expansion and flower development.     

欧洲山杨液泡膜Na+/H+ 反向运输体的性质鉴定

植物液泡膜Na /H 反向运输体可将细胞质中的Na 转运到液泡内储存,以减少胞内Na 的毒性.但木本植物如杨树是否有同样的机制目前还不清楚.以欧洲山杨的愈伤组织为材料,捣碎破碎愈伤组织细胞,经过差速离心和不连续蔗糖梯度离心得到纯化的欧洲山杨液泡微囊.通过液泡V-ATPase建立质子梯度,该液泡能够利用此梯度调控Na 的转运,表明液泡膜上存在Na /H 反向运输体活性(表观米氏常数Km是11.4mmol/L).Na /H 反向运输体的抑制剂——氨氯吡嗪咪能明显抑制转运体的活性.该Na /H 反向运输体也可以转运K ,但亲和能力比Na 低30%.该结果首次证明木本植物的液泡膜上存在Na /H 反向运输体.初步功能研究表明,愈伤组织在盐胁迫条件下,Na /H 反向运输体活性明显下降,提示该机制可能与山杨不耐盐有关.     

Sodium and sulfate ion transport in yeast vacuoles

The intra-luminal acidic pH of endomembrane organelles is established by a proton pump, vacuolar H(+)-ATPase (V-ATPase), in combination with other ion transporter(s). The proton gradient (DeltapH) established in yeast vacuolar vesicles decreased and reached the lower value after the addition of alkaline cations including Na(+). As expected, the uptake of (22)Na(+) was coupled with DeltapH generated by V-ATPase. Disruption of NHX1 or NHA1, encoding known Na(+)/H(+) antiporters, did not result in the loss of (22)Na(+) uptake or the alkaline cation-dependent DeltapH decrease. Upon the addition of sulfate ions, the V-ATPase-dependent DeltapH in the vacuolar vesicles increased, but the membrane potential (DeltaPsi) decreased. Consistent with this observation, radioactive sulfate was transported into the vesicles with a K(m) value of 0.07 mM. The transport activity was unaffected upon disruption of the putative genes coding for homologues of plasma membrane sulfate transporters. These results indicate that the vacuoles exhibit unique Na(+)/H(+) antiport and sulfate transport, which regulate the luminal pH and ion homeostasis in yeast.     

Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa.

Na+/H+ exchanger catalyzes the countertransport of Na+ and H+ across membranes. We isolated a rice cDNA clone the deduced amino acid sequence of which had homology with a putative Na+/H+ exchanger in Saccharomyces cerevisiae, NHX1. The sequence contains 2330 bp with an open reading frame of 1608 bp. The deduced amino acid sequence is similar to that of NHX1 and NHE isoforms in mammals, and shares high similarity with the sequences within predicted transmembrane segments and an amiloride-binding domain. The expression of the gene was increased by salt stress. These results suggest that the product of the novel gene, OsNHX1, functions as a Na+/H+ exchanger, and plays important roles in salt tolerance of rice.     

Vacuolar proton pumps and aquaporins involved in rapid internode elongation of deepwater rice

Rapid growth of the submerged shoots of deepwater rice is essential for survival during the rainy season. We investigated changes in the expression of vacuolar H(+)-ATPase (V-ATPase), H(+)-pyrophosphatase (V-PPase), and aquaporins under submerged conditions. The amounts of vacuolar proton pumps, which support the active transport of ions into the vacuoles, were maintained on a membrane protein basis in the developing vacuoles. Among the six isogenes of V-PPase, OsVHP1;3 was markedly enhanced by submersion. The gene expression of efficient water channels, OsTIP1;1, OsTIP2;2, OsPIP1;1, OsPIP2;1, and OsPIP2;2, was markedly enhanced by submersion. The increase in aquaporin expression might support quick elongation of internodes. The mRNA levels of OsNIP2;2 and OsNIP3;1, which transport silicic and boric acids respectively, clearly decreased. The present study indicates that internodes of deepwater rice upregulate vacuolar proton pumps and water channel aquaporins and downregulate aquaporins that allow permeation of the substrates that suppress internode growth.     

Ferric ions involved in the flower color development of the Himalayan blue poppy, Meconopsis grandis

The Himalayan blue poppy, Meconopsis grandis, has sky blue-colored petals, although the anthocyanidin nucleus of the petal pigment is cyanidin. The blue color development in this blue poppy involving ferric ions was therefore studied. We analyzed the vacuolar pH, and the organic and inorganic components of the colored cells. A direct measurement by a proton-selective microelectrode revealed that the vacuolar pH value was 4.8. The concentrations of the total anthocyanins in the colored cells were around 5mM, and ca. three times more concentrated flavonols were detected. Fe was detected by atomic analysis of the colored cells, and the ratio of Fe to anthocyanins was ca. 0.8 eq. By mixing the anthocyanin, flavonol and metal ion components in a buffered aq. solution at pH 5.0, we were able to reproduce the same blue color; the visible absorption spectrum and CD were identical to those in the petals, with Fe(3+), Mg(2+) and flavonol being essential for the blue color. The blue pigment in Meconopsis should be a new type of metal complex pigment that is different from a stoichiometric supramolecular pigment such as commelinin or protocyanin.     

Identification and characterization of Vnx1p, a novel type of vacuolar monovalent cation/H+ antiporter of Saccharomyces cerevisiae

We identified and characterized Vnx1p, a novel vacuolar monovalent cation/H+ antiporter encoded by the open reading frame YNL321w from Saccharomyces cerevisiae. Despite the homology of Vnx1p with other members of the CAX (calcium exchanger) family of transporters, Vnx1p is unable to mediate Ca2+ transport but is a low affinity Na+/H+ and K+/H+ anti-porter with a Km of 22.4 and 82.2 mm for Na+ and K+, respectively. Sequence analyses of Vnx1p revealed the absence of key amino acids shown to be essential for Ca2+/H+ exchange. vnx1Delta cells displayed growth inhibition when grown in the presence of hygromycin B or NaCl. Vnx1p activity was found in the vacuoles and shown to be dependent on the electrochemical potential gradient of H+ generated by the action of the V-type H+-ATPase. The presence of Vnx1p at the vacuolar membrane was further confirmed with cells expressing a VNX1::GFP chimeric gene. Similar to Nhx1p, the prevacuolar compartment-bound Na+/H+ antiporter, the vacuole-bound Vnx1p appears to play roles in the regulation of ion homeostasis and cellular pH.     

Rice Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter Nhx1 partially complements the alkali-metal-cation sensitivity of yeast strains lacking three sodium transporters

A triple mutant strain of Saccharomyces cerevisiae lacking its own Na+-ATPases and Na+/H+ antiporters (enal-4delta nha1delta nhx1delta) was used for the expression of the Oryza sativa NHX1 gene encoding a putative vacuolar Na+/H+ exchanger. Upon expression in yeast cells, the OsNhx 1p is not a transport system specific only for sodium cations but it has a broad substrate specificity for at least four alkali metal cations (Na+, Li+, K+ and Rb+) and is able to substitute for the endogenous yeast ScNhx1 antiporter. Its activity contributes to sequestration of alkali metal cations in intracellular vesicles.     

A vacuolar iron transporter in tulip, TgVit1, is responsible for blue coloration in petal cells through iron accumulation

Blue color in flowers is due mainly to anthocyanins, and a considerable part of blue coloration can be attributed to metal-complexed anthocyanins. However, the mechanism of metal ion transport into vacuoles and subsequent flower color development has yet to be fully explored. Previously, we studied the mechanism of blue color development specifically at the bottom of the inner perianth in purple tulip petals of Tulipa gesneriana cv. Murasakizuisho. We found that differences in iron content were associated with the development of blue- and purple-colored cells. Here, we identify a vacuolar iron transporter in T. gesneriana ( TgVit1 ), and characterize the localization and function of this transporter protein in tulip petals. The amino acid sequence of TgVit1 is 85% similar that of the Arabidopsis thaliana vacuolar iron transporter AtVIT1, and also showed similarity to the AtVIT1 homolog in yeast, Ca²⁺-sensitive cross-complementer 1 (CCC1). The gene TgVit1 was expressed exclusively in blue-colored epidermal cells, and protein levels increased with increasing mRNA expression and blue coloration. Transient expression experiments revealed that TgVit1 localizes to the vacuolar membrane, and is responsible for the development of the blue color in purple cells. Expression of TgVit1 in yeast rescued the growth defect of ccc1 mutant cells in the presence of high concentrations of FeSO₄. Our results indicate that TgVit1 plays an essential role in blue coloration as a vacuolar iron transporter in tulip petals. These results suggest a new role for involvement of a vacuolar iron transporter in blue flower color development.     

Differential Induction of Two Glucoamylases in Candida pelliculosa

Rapid growth of the submerged shoots of deepwater rice is essential for survival during the rainy season. We investigated changes in the expression of vacuolar H⁺-ATPase (V-ATPase), H⁺-pyrophosphatase (V-PPase), and aquaporins under submerged conditions. The amounts of vacuolar proton pumps, which support the active transport of ions into the vacuoles, were maintained on a membrane protein basis in the developing vacuoles. Among the six isogenes of V-PPase, OsVHP1;3 was markedly enhanced by submersion. The gene expression of efficient water channels, OsTIP1;1, OsTIP2;2, OsPIP1;1, OsPIP2;1, and OsPIP2;2, was markedly enhanced by submersion. The increase in aquaporin expression might support quick elongation of internodes. The mRNA levels of OsNIP2;2 and OsNIP3;1, which transport silicic and boric acids respectively, clearly decreased. The present study indicates that internodes of deepwater rice upregulate vacuolar proton pumps and water channel aquaporins and downregulate aquaporins that allow permeation of the substrates that suppress internode growth.     

ATP-dependent uptake of 5-hydroxytryptamine by secretory granules isolated from thyroid parafollicular cells.

The current study was done to test the hypotheses that parafollicular granules contain a vacuolar ATPase (V-ATPase) similar to that found in chromaffin granules, that the transport of H+ into granules mediated by this enzyme drives the granular uptake of 5-hydroxytryptamine (5-HT, serotonin), and that secretagogues stimulate both the acidification of parafollicular granules and their ability to take up 5-HT by opening an anion channel in the granular membrane. Our studies indicate that parafollicular granules contain a V-ATPase that is antigenically similar to that of the V-ATPase of adrenal chromaffin granules; however, the parafollicular granular membrane differs from that of chromaffin granules in permeability to Cl- and K+. The membranes of granules derived from resting parafollicular cells appear to be relatively impermeable to Cl- but permeable to K+. Parafollicular granules (and ghosts derived from them) manifest ATP-dependent transmembrane transport of 5-HT. This transport is more dependent on the pH difference (delta pH) than on the membrane potential component of the proton electrochemical gradient across the granular membrane. Transport of 5-HT is thus inhibited more by exposure of parafollicular granules to agents, such as nigericin, that collapse delta pH than by those, such as valinomycin, that decrease transmembrane difference in potential. ATP-dependent uptake of 5-HT by granules isolated from secretagogue-stimulated parafollicular cells is greater than that into granules isolated from unstimulated cells. Since secretagogues open a Cl- channel in parafollicular granule membranes, which enhances acidification of the granules, the facilitation of 5-HT uptake by secretagogues is probably due to an increase in delta pH.     

V-ATPase, ScNhx1p and yeast vacuole fusion

Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos. It is a central cellular reaction that plays important roles in signal transduction, protein sorting and subcellular compartmentation. Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summarized in this article. It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhx1p are key components of the vacuole fusion machinery in yeast. Yeast ScNhx1p regulates vacuole fusion by controlling the luminal pH. V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast. Fission defects are epistatic to fusion defects. Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast, the fusion reaction does not need the transport activity but requires the physical presence of the proton pump. V0, the membrane-integral sector of the V-ATPase, forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the V0trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.     

Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue.

Intact vacuoles were isolated from petals of Hippeastrum and Tulipa (Wagner G.J. and Siegelman, H.W. (1975) Science 190, 1298--1299). The ATPase activity of fresh vacuole suspensions was found to be 2--3 times that of protoplasts from the same tissue. 70--80% of the ATPase activity of intact vacuoles was recovered in tonoplast preparations. The antibiotic Dio-9 at 6mug/10(6) vacuoles or protoplasts causes 40% inhibition. However, only the protoplast ATPase is sensitive to oligomycin. N,N'-dicyclohexylcarbodiimide (DCCD) slightly stimulates ATPase activity in both vacuole and protoplast suspensions, whereas ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDAC) strongly inhibits. Spectrophotometric studies show that in the petal the vacuolar contents have a pH of 4.0 for Tuplipa and 4.3 for Hippeastrum, whereas the intact isolated vacuole has an internal pH of 7.0 (in pH 8.0 buffer) for (Tulipa and about 7.3 for Hippeastrum. Internal ion concentrations of 150, 46, 30, 30 and 6 mM were found for K+, Na+, Mg2+, Cl-, and Ca2+ respectively, which are about the same as those in protoplasts.     

Precursors and Enzymatic Development of Caucas Flavor Components

Although coloration in plants is ascribable to both the accumulation of anthocyanin pigments in vacuoles and to the acidification of vacuolar pH, the environmental factors causing the decrease in vacuolar pH are unknown. We found that blue-light irradiation of buckwheat seedlings using light-emitting diodes caused reddening on the surface of the hypocotyls. It has also been reported that light stimulation induces an accumulation of anthocyanin pigments. However, here we confirmed for the first time on the basis of real-time PCR analysis that light stimulation simultaneously triggers expression of the genes coding for subunit A of vacuolar H⁺-ATPase (V-ATPase) and vacuolar H⁺-pyrophosphatase (V-PPase).