Sepal color variation of Hydrangea macrophylla and vacuolar pH measured with a proton-selective microelectrode

Sepal color of hydrangea varies with the environmental conditions. Although chemical and biological studies on this color variation have a long history, little correct knowledge has been generated about color development. All colored sepals contain the same anthocyanin, delphinidin 3-glucoside. Thus, there must be some other system for developing the wide variety of colors. In hydrangea sepals the cells of the epidermis are colorless and only the second layer of cells contain pigment. We prepared protoplasts without any color change during enzyme treatment of sepals and measured the vacuolar pH of each of the colored cells. We could correlate the color of a single hydrangea cell with its vacuolar pH using a combination of micro-spectrophotometry and a proton-selective microelectrode. Values for the vacuolar pH of blue (lambda vismax: 589 nm) and red cells (lambda vismax: 537 nm) were 4.1 and 3.3, respectively, the vacuolar pH of blue cells being significantly higher.     

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.     

Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin

Flower color is mainly determined by anthocyanins. Rosa hybrida lacks violet to blue flower varieties due to the absence of delphinidin-based anthocyanins, usually the major constituents of violet and blue flowers, because roses do not possess flavonoid 3',5'-hydoxylase (F3'5'H), a key enzyme for delphinidin biosynthesis. Other factors such as the presence of co-pigments and the vacuolar pH also affect flower color. We analyzed the flavonoid composition of hundreds of rose cultivars and measured the pH of their petal juice in order to select hosts of genetic transformation that would be suitable for the exclusive accumulation of delphinidin and the resulting color change toward blue. Expression of the viola F3'5'H gene in some of the selected cultivars resulted in the accumulation of a high percentage of delphinidin (up to 95%) and a novel bluish flower color. For more exclusive and dominant accumulation of delphinidin irrespective of the hosts, we down-regulated the endogenous dihydroflavonol 4-reductase (DFR) gene and overexpressed the Irisxhollandica DFR gene in addition to the viola F3'5'H gene in a rose cultivar. The resultant roses exclusively accumulated delphinidin in the petals, and the flowers had blue hues not achieved by hybridization breeding. Moreover, the ability for exclusive accumulation of delphinidin was inherited by the next generations.     

Structure of anthocyanin from the blue petals of Phacelia campanularia and its blue flower color development

The dicaffeoyl anthocyanin, phacelianin, was isolated from blue petals of Phacelia campanularia. Its structure was determined to be 3-O-(6-O-(4'-O-(6-O-(4'-O-beta-d-glucopyranosyl-(E)-caffeoyl)-beta-d-glucopyranosyl)-(E)-caffeoyl)-beta-d-glucopyranosyl)-5-O-(6-O-malonyl-beta-d-glucopyranosyl)delphinidin. The CD of the blue petals of the phacelia showed a strong negative Cotton effect and that of the suspension of the colored protoplasts was the same, indicating that the chromophores of phacelianin may stack intermolecularly in an anti-clockwise stacking manner in the blue-colored vacuoles. In a weakly acidic aqueous solution, phacelianin displayed the same blue color and negative Cotton effect in CD as those of the petals. However, blue-black colored precipitates gradually formed without metal ions. A very small amount of Al(3+) or Fe(3+) may be required to stabilize the blue solution. Phacelianin may take both an inter- and intramolecular stacking form and shows the blue petal color by molecular association and the co-existence of a small amount of metal ions. We also isolated a major anthocyanin from the blue petals of Evolvulus pilosus and revised the structure identical to phacelianin.     

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.     

Solid-state 13C and 15N NMR study of the low pH forms of bacteriorhodopsin

The visible absorption of bacteriorhodopsin (bR) is highly sensitive to pH, the maximum shifting from 568 nm (pH 7) to approximately 600 nm (pH 2) and back to 565 nm (pH 0) as the pH is decreased further with HCl. Blue membrane (lambda max greater than 600 nm) is also formed by deionization of neutral purple membrane suspensions. Low-temperature, magic angle spinning 13C and 15N NMR was used to investigate the transitions to the blue and acid purple states. The 15N NMR studies involved [epsilon-15N]lysine bR, allowing a detailed investigation of effects at the Schiff base nitrogen. The 15N resonance shifts approximately 16 ppm upfield in the neutral purple to blue transition and returns to its original value in the blue to acid purple transition. Thus, the 15N shift correlates directly with the color changes, suggesting an important contribution of the Schiff base counterion to the "opsin shift". The results indicate weaker hydrogen bonding in the blue form than in the two purple forms and permit a determination of the contribution of the weak hydrogen bonding to the opsin shift at a neutral pH of approximately 2000 cm-1. An explanation of the mechanism of the purple to blue to purple transition is given in terms of the complex counterion model. The 13C NMR experiments were performed on samples specifically 13C labeled at the C-5, C-12, C-13, C-14, or C-15 positions in the retinylidene chromophore. The effects of the purple to blue to purple transitions on the isotropic chemical shifts for the various 13C resonances are relatively small. It appears that bR600 consists of at least four different species. The data confirm the presence of 13-cis- and all-trans-retinal in the blue form, as in neutral purple dark-adapted bR. All spectra of the blue and acid purple bR show substantial inhomogeneous broadening which indicates additional irregular distortions of the protein lattice. The amount of distortion correlates with the variation of the pH, and not with the color change.     

Purification and characterization of a purple acid phosphatase from rat spleen

An acid phosphatase species which is activated by Fe2+ was purified 3,700-fold from rat spleen by chromatography on columns containing Blue-Sepharose, concanavalin A-Sepharose, Sephadex G-100, and CM-Sephadex. The enzyme hydrolyzed aryl phosphates, nucleoside di- and triphosphates, phosphoproteins, and thiamine pyrophosphate with Km values of 10(-4) to 10(-3) M at an optimal pH of 5.0-5.8. Co-purification of the acid phosphatase and acid phosphoprotein phosphatase indicated that they were identical. The purified enzyme was glycoprotein in nature, showing four heterogeneous forms on acid polyacrylamide gel electrophoresis (pI values, 7.8, 8.0, 8.3, and 8.5), but it gave a molecular weight of 33,000 on sodium dodecyl sulfate-gel electrophoresis and gel permeation chromatography. The enzyme had a purple color (lambda max 545 nm) and contained 2 iron atoms per enzyme molecule. Among reductants, ascorbic acid and Fe2+ were the best activators, although their combined effect was not additive. Fe2+ and ascorbic acid both changed the purple enzyme into the same active form (lambda max 515 nm), giving almost the same kinetic constants for substrates and for inhibitors such as molybdate, phosphate and fluoride. However, low concentrations of Fe2+, from 0.01 mM to 1.0 mM, immediately and reversibly activated the enzyme, whereas high concentrations of ascorbic acid over 1 mM were required for maximal activation, which was slow and irreversible.     

Anatomical and biochemical studies of bicolored flower development in Muscari latifolium

The inflorescence of the broad-leafed grape hyacinth, Muscari latifolium, shows an interesting, two-tone appearance with the upper flowers being pale blue and the lower ones purple. To elucidate the mechanism of the differential color development, anatomical research was carried out and a cytological study of the colored protoplasts in which the shapes of the cells accumulating anthocyanin were observed by scanning electron microscopy. Next, vacuolar pH was recorded using a pH meter with a micro combination pH electrode, and the sap’s metal-ion content was measured by inductively coupled plasma mass spectrometry. The anthocyanin and co-pigment composition was determined by high-performance liquid chromatography (HPLC). Chemical analyses reveal that the difference in metal-ion content of the two parts was not great. The vacuolar pHs of the upper and lower flowers were 5.91 and 5.84, respectively, with the difference being nonsignificant. HPLC results indicate that the dihydroflavonol and flavonol contents are also very similar in the two sorts of flower. However, the upper flowers contained only delphinidin, whereas the lower flowers also contained cyanidin. The total anthocyanin content in the lower flowers was 4.36 mg g^?1, which is approximately seven times higher than in the upper flowers, while the delphinidin content is four times higher. Quantitative real-time PCR analysis established that the two-tone flower was a result of different expressions of the F3′5′H, F3′H and DFR genes, and these lead to different amounts of anthocyanin.     

The involvement of tonoplast proton pumps and Na+(K+)/H+ exchangers in the change of petal color during flower opening of Morning Glory, Ipomoea tricolor cv. Heavenly Blue

The petal color of morning glory, Ipomoea tricolor cv. Heavenly Blue, changes from purplish red to blue during flower opening. This color change is caused by an unusual increase in vacuolar pH from 6.6 to 7.7 in the colored adaxial and abaxial cells. To clarify the mechanism underlying the alkalization of epidermal vacuoles in the open petals, we focused on vacuolar H+-ATPase (V-ATPase), H+-pyrophosphatase (V-PPase) and an isoform of Na+/H+ exchanger (NHX1). We isolated red and blue protoplasts from the petals in bud and fully open flower, respectively, and purified vacuolar membranes. The membranes contained V-ATPase, V-PPase and NHX1, which were immunochemically detected, with relatively high transport activity. NHX1 could be detected only in the vacuolar membranes prepared from flower petals and its protein level was the highest in the colored petal epidermis of the open flower. These results suggest that the increase of vacuolar pH in the petals during flower opening is due to active transport of Na+ and/or K+ from the cytosol into vacuoles through a sodium- or potassium-driven Na+(K+)/H+ exchanger NXH1 and that V-PPase and V-ATPase may prevent the over-alkalization. This systematic ion transport maintains the weakly alkaline vacuolar pH, producing the sky-blue petals.     

Intra-vacuolar reserves of membranes during stomatal closure: the possible role of guard cell vacuoles estimated by 3-D reconstruction

Stomatal apertures are regulated by morphological changes in guard cells which have been associated with guard cell vacuolar structures. To investigate the contribution of guard cell vacuoles to stomatal movement, we examined the dynamics of vacuolar membrane structures in guard cells and evaluated the changes in vacuolar volumes and surface areas during stomatal movement. Using a transgenic Arabidopsis line expressing green fluorescent protein (GFP)-AtVAM3, we have found that the guard cell vacuolar structures became complicated during stomatal closure with the appearance of numerous intra-vacuolar membrane structures. A three-dimensional (3-D) reconstruction using our originally developed software, REANT (reconstructor and analyzer of 3-D structure), and photobleaching analysis revealed the continuity of the vacuolar structures, even when they appeared to be compartmented in confocal images of closed stomata. Furthermore, calculations of the surface area by REANT revealed an increase in vacuolar surface area during stomatal closure but a decrease in the surface area of the guard cells. Movement of a vital staining dye, FM4-64, to the vacuolar membrane was accelerated during ABA-induced stomatal closure in Vicia faba. These results suggest that the guard cell vacuoles store some portion of the excess membrane materials produced during stomatal closure as intra-vacuolar structures.     

The purple to blue transition of bacteriorhodopsin is accompanied by a loss of the hexagonal lattice and a conformational change

X-ray diffraction measurements show that in contrast to the purple membrane, the bacteriorhodopsin molecules are not organized in a hexagonal lattice in the deionized blue membrane. Addition of Ca2+ restores both the purple color and the normal (63 A) hexagonal protein lattice. In the blue state, the circular dichroism spectrum in the visible has the typical exciton features indicating that a trimeric structure is retained. Time-resolved linear dichroism measurements show that the blue patch rotates in aqueous suspension with a mean correlation time of 11 ms and provide no evidence for rotational mobility of bacteriorhodopsin within the membrane. The circular dichroism spectra of the blue and the Ca2+-regenerated purple state in the far-UV are different, indicating a small change in secondary structure. The thermal stability of the blue membrane is much smaller than that of the purple membrane. At pH 5.0, the irreversible denaturation transition of the blue form has a midpoint at 61 degrees C. The photocycle of the blue membrane (lambda ex 590 nm) has an L intermediate around 540 nm whose decay is slowed down into the millisecond time range (5 ms). Light-dark adaptation in the blue membrane is rapid with an exponential decay time of 38 s at 25 degrees C. The purple to blue transition apparently involves a conformational change in the protein leading to a change in the aggregation state from a highly ordered and stable hexagonal lattice to a disordered array of thermally more labile trimers.(ABSTRACT TRUNCATED AT 250 WORDS)     

A bacteriorhodopsin analog reconstituted with a nonisomerizable 13-trans retinal derivative displays light insensitivity.

With the aim of preparing a light-insensitive bacteriorhodopsin-like pigment, bacterio-opsin expressed in Escherichia coli was treated in phospholipid-detergent micelles with the retinal analog II, in which the C13-C14 trans-double bond cannot isomerize due to inclusion in a cyclopentene ring. The formation of a complex with a fine structure (lambda max, 439 nm) was first observed. This partially converted over a period of 12 days to a bacteriorhodopsin-like chromophore (ebR-II) with lambda max, 555 nm. An identical behavior has been observed previously upon reconstitution of bleached purple membrane with the analog II. Purification by gel filtration gave pure ebR-II with lambda max, 558 nm, similar to that of light-adapted bacterio-opsin reconstituted with all-trans retinal (ebR-I). Spectrophotometric titration of ebR-II as a function of pH showed that the purple to blue transition of bacteriorhodopsin at acidic pH was altered, and the apparent pKa of Schiff base deprotonation at alkaline pH was lowered by 2.4 units, relative to that of ebR-I. ebR-II showed no light-dark adaptation, no proton pumping, and no intermediates characteristic of the bacteriorhodopsin photocycle. In addition, the rates of reaction with hydroxylamine in the dark and in the light were similar. These results show, as expected, that isomerization of the C13-C14 double bond is required for bacteriorhodopsin function and that prevention of this isomerization confers light insensitivity.     

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.     

Control of bacteriorhodopsin color by chloride at low pH. Significance for the proton pump mechanism

The chromophore of bacteriorhodopsin undergoes a transition from purple (570 nm absorbance maximum) to blue (605 nm absorbance maximum) at low pH or when the membrane is deionized. The blue form was stable down to pH 0 in sulfuric acid, while 1 M NaCl at pH 0 completely converted the pigment to a purple form absorbing maximally at 565 Other acids were not as effective as sulfuric in maintaining the blue form, and chloride was the best anion for converting blue membrane to purple membrane at low pH. The apparent dissociation constant for Cl- was 35 mM at pH 0, 0.7 M at pH 1 and 1.5 M at pH 2. The pH dependence of apparent Cl- binding could be modeled by assuming two different types of chromophore-linked Cl- binding site, one pH-dependent. Chemical modification of bacteriorhodopsin carboxyl groups (probably Asp-96, -102 and/or -104) by 1-ethyl-3-dimethlyaminopropyl carbodiimide, Lys-41 by dansyl chloride, or surface arginines by cyclohexanedione had no effect on the conversion of blue to purple membrane at pH 1. Fourier transform infrared difference spectroscopy of chloride purple membrane minus acid blue membrane showed the protonation of a carboxyl group (trough at 1392 cm -1 and peak at 1731 cm -1). The latter peak shifted to 1723 cm -1 in D2O. Ultraviolet difference spectroscopy of chloride purple membrane minus acid blue membrane showed ionization of a phenolic group (peak at 243 nm and evidence for a 295 nm peak superimposed on a tryptophan perturbation trough). This suggests the possibility of chloride-induced proton transfer from a tyrosine phenolic group to a carboxylate side-chain. We propose a mechanism for the purple to acid blue to chloride purple transition based on these results and the proton pump model of Braiman et al. (Biochemistry 27 (1988) 8516-8520).     

Aluminum-induced ethylene production is associated with inhibition of root elongation in Lotus japonicus L

Inhibition of root elongation by toxic aluminum (Al(3+)) occurs rapidly and is one of the most distinct and earliest symptoms of Al toxicity. To elucidate mechanism underlying Al(3+)-induced inhibition of root elongation, we investigated the involvement of ethylene in Al(3+)-induced inhibition of root elongation using the legume model plants Lotus japonicus and Medicago truncatula. Root elongation of L. japonicus and M. truncatula was rapidly inhibited by exposure to AlCl(3). A similar rapid inhibition of root elongation by the ethylene-releasing substance, ethephon, and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), was also observed. The Al(3+)-induced inhibition of root elongation was substantially ameliorated in the presence of antagonists of ethylene biosynthesis [Co(2+) and aminoethoxyvinylglycine (AVG)]. Al(3+) increased the activity of ACC oxidase (ACO), and induced a rapid evolution of ethylene from root apices and expression of genes of ACC synthase (ACS) and ACO. These findings suggest that induction of ethylene evolution resulting from up-regulation of ACS and ACO plays a critical role in Al(3+)-induced inhibition of root elongation.     

Ferriprotoporphyrin IX mediated oxygen activation/insertion reactions: the anerobic reduction of the aniline-Fe3+-microperoxidase-8 complex by NADH

A spectrophotometric study of the reduction of the Fe3+ microperoxidase-8-aniline (Fe3+-MP-8-An) complex has been carried out. Addition of NADH to a solution of Fe3+-MP-8-An under strictly anerobic conditions results in the formation of a species with lambda max = 414 nm (Fe3+-MP-8-An lambda max 407 nm). The kinetics of formation of this species show an induction period (tau) which follows saturation kinetics with respect to [aniline] with Km(app) = 2.2 x 10(-3) mol dm-3, i.e., close to that obtained in the preceding paper from O2 consumption kinetics mediated by MP-8. Addition of an anerobic solution of the NADH reduced MP-8-An complex, to a saturated O2 solution at pH 12 in the presence of 0.5 mM NADH and aniline 10 mM results in the virtual elimination of the induction phase, which has previously characterized O2 consumption kinetics in ferriprotoporphyrin IX oxygen activation systems. The Arrhenius activation energy for the reduction of the Fe3+-MP-8-An complex is close to that observed for the first reductive step in the cyt P-450 O2 activation cycle. Anerobic reduction of Fe3+-MP-8 by sodium dithionite in 20% MeOH/Aq at pH 8 followed by anerobic titration of the Fe2+-MP-8 (lambda max 420.5 nm) with aniline at pH 12 gives rise to a species lambda max 415 with KD for the process = 4.4 x 10(-3) mol dm-3 (+/- 1.2 x 10(-3) mol dm-3).     

Potential role of annexin AnnAt1 from Arabidopsis thaliana in pH-mediated cellular response to environmental stimuli

Plant annexins, Ca(2+)- and membrane-binding proteins, are probably implicated in the cellular response to stress resulting from acidification of cytosol. To understand how annexins can contribute to cellular ion homeostasis, we investigated the pH-induced changes in the structure and function of recombinant annexin AnnAt1 from Arabidopsis thaliana. The decrease of pH from 7.0 to 5.8 reduced the time of the formation of ion channels by AnnAt1 in artificial lipid membranes from 3.5 h to 15-20 min and increased their unitary conductance from 32 to 63 pS. These changes were accompanied by an increase in AnnAt1 hydrophobicity as revealed by hydrophobicity predictions, by an increase in fluorescence of 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS) bound to AnnAt1 and fluorescence resonance energy transfer from AnnAt1 tryptophan residues to TNS. Concomitant lipid partition of AnnAt1 at acidic pH resulted in its partial protection from proteolytic digestion. Secondary structures of AnnAt1 determined by circular dichroism and infrared spectroscopy were also affected by lowering the pH from 7.2 to 5.2. These changes were characterized by an increase in beta-sheet content at the expense of alpha-helical structures, and were accompanied by reversible formation of AnnAt1 oligomers as probed by ultracentrifugation in a sucrose gradient. A further decrease of pH from 5.2 to 4.5 or lower led to the formation of irreversible aggregates and loss of AnnAt1 ionic conductance. Our findings suggest that AnnAt1 can sense changes of the pH milieu over the pH range from 7 to 5 and respond by changes in ion channel conductance, hydrophobicity, secondary structure of the protein and formation of oligomers. Further acidification irreversibly inactivated AnnAt1. We suggest that the pH-sensitive ion channel activity of AnnAt1 may play a role in intracellular ion homeostasis.     

Elicitor-induced cytoskeletal rearrangement relates to vacuolar dynamics and execution of cell death: in vivo imaging of hypersensitive cell death in tobacco BY-2 cells

Disintegration of the vacuolar membrane (VM) has been proposed to be a crucial event in various types of programmed cell death (PCD) in plants. However, its regulatory mechanisms are mostly unknown. To obtain new insights on the regulation of VM disintegration during hypersensitive cell death, we investigated the structural dynamics and permeability of the VM, as well as cytoskeletal reorganization during PCD in tobacco BY-2 cells induced by a proteinaceous elicitor, cryptogein. From sequential observations, we have identified the following remarkable events during PCD. Stage 1: bulb-like VM structures appear within the vacuolar lumen and the cortical microtubules are disrupted, while the cortical actin microfilaments are bundled. Simultaneously, transvacuolar strands including endoplasmic microtubules and actin microfilaments are gradually disrupted and the nucleus moves from the center to the periphery of the cell. Stage 2: cortical actin microfilament bundles and complex bulb-like VM structures disappear. The structure of the large central vacuole becomes simpler, and small spherical vacuoles appear. Stage 3: the VM is disintegrated and a fluorescent dye, BCECF, leaks out of the vacuoles just prior to PCD. Application of an actin polymerization inhibitor facilitates both the disappearance of bulb-like vacuolar membrane structures and induction of cell death. These results suggest that the elicitor-induced reorganization of actin microfilaments is involved in the regulation of hypersensitive cell death via modification of the vacuolar structure to induce VM disintegration.     

Protein phosphorylation and binding of a 14-3-3 protein in Vicia guard cells in response to ABA

Under drought stress, ABA promotes stomatal closure to prevent water loss. Although protein phosphorylation plays an important role in ABA signaling, little is known about these processes at the biochemical level. In this study, we searched for substrates of protein kinases in ABA signaling through the binding of a 14-3-3 protein to phosphorylated proteins using Vicia guard cell protoplasts. ABA induced binding of a 14-3-3 protein to proteins with molecular masses of 61, 43 and 39 kDa, with the most remarkable signal for the 61 kDa protein. The ABA-induced binding to the 61 kDa protein occurred only in guard cells, and reached a maximum within 3 min at 1 microM ABA. The 61 kDa protein localized in the cytosol. ABA induced the binding of endogenous vf14-3-3a to the 61 kDa protein in guard cells. Autophosphorylation of ABA-activated protein kinase (AAPK), which mediates anion channel activation, and ABA-induced phosphorylation of the 61 kDa protein showed similar time courses and similar sensitivities to the protein kinase inhibitor K-252a. AAPK elicits the binding of the 14-3-3 protein to the 61 kDa protein in vitro when AAPK in guard cells was activated by ABA. The phosphorylation of the 61 kDa protein by ABA was not affected by the NADPH oxidase inhibitor, H(2)O(2), W-7 or EGTA. From these results, we conclude that the 61 kDa protein may be a substrate for AAPK and that the 61 kDa protein is located upstream of H(2)O(2) and Ca(2+), or on Ca(2+)-independent signaling pathways in guard cells.     

A grape berry (Vitis vinifera L.) cation/proton antiporter is associated with berry ripening

We have cloned and characterized VvNHX1, a gene encoding a vacuolar cation/H(+) antiporter from Vitis vinifera cv. Cabernet Sauvignon. VvNHX1 belongs to the vacuolar NHX protein family and showed high similarity to other known vacuolar antiporters. The expression of VvNHX1 partially complements the salt- and hygromycin-sensitive phenotypes of an ena1-4 nhx1 yeast strain. Immunoblots of vacuoles of yeast expressing a VvNHX1, together with the expression of a VvNHX1-GFP (green fluorescent protein) chimera demonstrated that VvNHX1 localized to the vacuoles. VvNHX1 displayed low affinity K(+)/H(+) and Na(+)/H(+) exchange activities (12.8 and 40.2 mM, respectively). The high levels of expression of VvNHX1 during the véraison and post-véraison stages would indicate that the increase in vacuolar K(+) accumulation, mediated by VvNHX1, is needed for vacuolar expansion. This process, together with the rapid accumulation of reducing sugars, would drive water uptake to the berry and the concomitant berry size increase, typical of the post-véraison stage of growth.