Biomedical subjects

K S Schumaker

Publications and source records attributed to K S Schumaker.

9 recordsLinked to original sources

PubMed1998

Viviparous-5 encodes phytoene desaturase, an enzyme essential for abscisic acid (ABA) accumulation and seed development in maize.

The role of synthesis in the regulation of abscisic acid accumulation was investigated in the developing maize seed. To do this, expression and regulation of the abscisic acid biosynthetic enzyme phytoene desaturase were examined. Comparison of the gene sequence encoding phytoene desaturase and its transcript in the wild-type and viviparous-5 mutant showed that the mutant gene contains multiple insertions and deletions, resulting in the synthesis of a larger transcript. In addition, the 55-kDa phytoene desaturase protein was not detectable in the viviparous-5 mutant, indicating that this phenotype results from a mutation at the phytoene desaturase locus. Levels of phytoene desaturase transcript and protein were compared to abscisic acid levels during development to determine whether phytoene desaturase might regulate abscisic acid accumulation. In the endosperm, transcript levels were initially high and declined during late maturation and dormancy, while protein levels remained high throughout development. In the embryo, transcript levels were low and constant, while protein levels declined. Both temporal and tissue-specific expression of phytoene desaturase were unrelated to abscisic acid levels. An abscisic acid mutant (viviparous-2) deficient in phytoene desaturation was used to determine whether the wild-type protein encoded by Viviparous-2 regulates phytoene desaturase. Phytoene desaturase transcript and protein levels were compared in wild-type and viviparous-2 mutant embryos and endosperm. Normalized levels of phytoene desaturase were similar in wild-type and mutant tissues, suggesting that the wild-type Viviparous-2 protein does not regulate phytoene desaturase transcript or protein levels.

Abscisic Acid↗

PubMed1996

G proteins regulate dihydropyridine binding to moss plasma membranes.

The role of calcium as an activator and regulator of many biological processes is linked to the ability of the cell to rapidly change its cytoplasmic calcium levels. Calcium acts as an intracellular messenger in hormone-induced bud formation during the development of the moss Physcomitrella patens. Calcium transport and ligand binding studies have implicated plasma membrane-localized 1, 4-dihydropyridine (DHP)-sensitive calcium channels in this increase in cellular calcium. To understand the regulation of the moss calcium channel, we investigated the involvement of GTP binding regulatory proteins (G proteins). Guanosine 5'-(gamma-thio)triphosphate (GTPgammaS), a nonhydrolyzable GTP analog that locks G proteins into their active state, stimulated DHP binding to high affinity receptors in the moss plasma membrane. DHP binding was measured as the ability of the DHP agonist Bay K8644 or the DHP antagonist nifedipine to compete with the DHP arylazide [3H]azidopine for binding to moss plasma membranes. G protein stimulation of binding was seen when competition was carried out with either nifedipine or Bay K8644. G proteins regulated the rates of association and dissociation of bound [3H]azidopine, and stimulation was dependent on GTPgammaS concentration. Guanosine 5'-(beta-thio)diphosphate, a GDP analog that locks G proteins into their inactivated state, did not affect the dose dependence of either the agonist or the antagonist. These results suggest that G proteins may act via a membrane-delimited pathway to regulate calcium channels in the moss plasma membrane.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1995

1,4-Dihydropyridine binding sites in moss plasma membranes. Properties of receptors for a calcium channel antagonist.

An increase in cytoplasmic calcium is an early event in hormone (cytokinin)-induced vegetative bud formation in the moss Physcomitrella patens. Whole cell and calcium transport studies have implicated 1,4-dihydropyridine-sensitive calcium channels in this increase in cellular calcium. To understand the molecular nature of the dihydropyridine-sensitive calcium channel, we have established conditions for the binding of the arylazide 1,4-dihydropyridine, [3H]azidopine, to its receptor in moss plasma membranes. [3H]Azidopine bound specifically in a saturable and reversible manner. The KD for [3H]azidopine binding was 5.2 nM and the Bmax was 35.6 pmol/mg of protein. Association and dissociation of the receptor and [3H]azidopine were temperature-dependent, and association varied as a function of pH. Binding was inhibited by dihydropyridine, phenylalkylamine, and benzothiazepine calcium channel blockers, bepridil, lanthanum, and N-ethylmaleimide. [3H]Azidopine binding was stimulated by cations including calcium, strontium, manganese, and barium. [3H]Azidopine binding was also stimulated by cytokinin with a Km value for kinetin of 0.13 nM. These studies utilize a simple plant system to provide a biochemical framework for understanding calcium regulation during development and have implications for understanding mechanisms of signal transduction in plants.

Azides↗

PubMed1993

Cytokinin stimulates dihydropyridine-sensitive calcium uptake in moss protoplasts.

Ca2+ influx through dihydropyridine (DHP)-sensitive Ca2+ channels is thought to be an early event in cytokinin-induced bud formation in moss protonema because DHP antagonists inhibit bud formation in the presence of cytokinin and DHP agonists stimulate bud formation in the absence of cytokinin [Conrad, P. A. & Helper, P. K. (1988) Plant Physiol. 86, 684-687]. In the present study, we established the presence of a DHP-sensitive Ca2+ transport system by measuring 45Ca2+ influx into moss protoplasts. Ca2+ influx was stimulated by external KCl (up to 5 mM), indicating that transport is voltage-dependent. K(+)-induced Ca2+ influx was DHP-sensitive with > 50% inhibition at 500 nM nifedipine. Ca2+ influx was stimulated by increasing concentrations of the DHP Ca2+ channel agonist Bay K8644 with half-maximal effects at 25 nM; this stimulation was seen only in the absence of K+, suggesting that the agonist works preferentially on polarized membranes. Ca2+ influx was also inhibited by phenylalkylamines (verapamil) and benzothiazepines (diltiazem). The phytohormone 6-benzylaminopurine consistently stimulated Ca2+ influx with a Km value of 1 nM, whereas adenine, indoleacetic acid, and gibberellic acid had no effect on Ca2+ transport. The cytokinins kinetin and trans-zeatin caused a greater stimulation of Ca2+ influx and induced more bud formation than did 6-benzylaminopurine. These results indicate that Ca2+ is taken up into moss protoplasts through voltage-dependent DHP-sensitive Ca2+ channels on the plasma membrane and that one of the cytokinin effects in the induction of bud formation is regulation of this plasma membrane Ca2+ channel.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1990

Solubilization and reconstitution of the oat root vacuolar h/ca exchanger.

Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H(+)/Ca(2+) antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [DeltapH] dependent). The first step toward purification and identification of the H(+)/Ca(2+) antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H(+)/Ca(2+) antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A DeltapH (acid inside) was generated in the proteoliposomes with an NH(4)Cl gradient (NH(4) (+) (in) >> NH(4) (+) (out)) as determined by methylamine uptake. Fundamental properties of DeltapH dependent calcium uptake such as the K(m) for calcium ( approximately 15 micromolar) and the sensitivity to inhibitors such as N,N'-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H(+)/Ca(2+) antiporter has been reconstituted in active form.

Journal Article↗

PubMed1987

Inositol 1,4,5-trisphosphate releases Ca2+ from vacuolar membrane vesicles of oat roots.

In plant cells, transient changes in cytoplasmic Ca2+ levels can modulate numerous developmental processes. Ca2+ is accumulated in the vacuole via a H+/Ca2+ antiport system that is energized by the tonoplast H+-pumping ATPase. Inositol 1,4,5-triphosphate (InsP3), but not inositol 1,4-bisphosphate, myo-inositol 1-phosphate, or fructose 2,6-bisphosphate, caused a transient reduction of Ca2+ levels in tonoplast vesicles. The decrease was dependent on InsP3 concentration (Km apparent = 0.6 microM). The InsP3-induced Ca2+ release was blocked by the Ca2+ antagonist, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate-HCl. These results suggest that the vacuolar membrane is one target site for InsP3 action and that InsP3 may operate as a second messenger in the mobilization of intracellular Ca2+ in plant cells.

Calcium↗

PubMed1987

Decrease of pH Gradients in Tonoplast Vesicles by NO(3) and Cl: Evidence for H-Coupled Anion Transport.

Chloride or nitrate decreased a pH gradient (measured as [(14)C]methylamine accumulation) in tonoplast-enriched vesicles. The DeltapH decrease was dependent on the anion concentration. These effects are independent of the anion-sensitive H(+)-ATPase of the tonoplast, since the pH gradient (acid inside) was imposed artificially using a pH jump or a K(+) gradient and nigericin. 4,4'-Diisothiocyano-2,2'-stilbene disulfonic acid partially prevented the decrease in pH gradient induced by Cl(-). Two possible models to account for this anion-dependent decrease of DeltapH are: (a) H(+) loss is accompanied by Cl(-) or NO(3) (-) efflux from the vesicles via H(+)/anion symport systems on the tonoplast and (b) H(+) loss is accompanied by Cl(-) or NO(3) (-) uptake into the vesicles via H(+)/anion antiport systems. Depending on the requirements and conditions of the cell, these two systems would serve to either mobilize Cl(-) and NO(3) (-) stored in the vacuole for use in the cytoplasm or to drive anions into the vacuole. Chloride or nitrate also decreased a pH gradient in fractions containing plasma membrane and Golgi, implying that these membranes may have similar H(+)-coupled anion transport systems.

Journal Article↗

PubMed1986

Calcium transport into the vacuole of oat roots. Characterization of H+/Ca2+ exchange activity.

Calcium (Ca2+) is sequestered into vacuoles of oat root cells through a H+/Ca2+ antiport system that is driven by the proton-motive force of the tonoplast H+-translocating ATPase. The antiport has been characterized directly by imposing a pH gradient in tonoplast-enriched vesicles. The pH gradient was imposed by diluting K+-loaded vesicles into a K+-free medium. Nigericin induced a K+/H+ exchange resulting in a pH gradient of 2 (acid inside). The pH gradient was capable of driving 45Ca2+ accumulation. Ca2+ uptake was tightly coupled to H+ loss as increasing Ca2+ levels progressively dissipated the steady state pH gradient. Ca2+ uptake displayed saturation kinetics with a Km(app) for Ca2+ of 10 microM. The relative affinity of the antiporter for transport of divalent cations was Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. La3+ or Mn2+ blocked Ca2+ uptake possibly by occupying the Ca2+-binding site. Ruthenium red (I50 = 40 microM) and N,N'-dicyclohexylcarbodiimide (I50 = 3 microM) specifically inhibited the H+/Ca2+ antiporter. When driven by pH jumps, the H+/Ca2+ exchange generated a membrane potential, interior positive, as shown by [14C]SCN accumulation. Furthermore, Ca2+ uptake was stimulated by an imposed negative membrane potential. The results support a simple model of one Ca2+ taken up per H+ lost. The exchange transport can be reversed, as a Ca2+ gradient (Ca2+in greater than Ca2+out) was effective in forming a pH gradient (acid inside). We suggest that the H+/Ca2+ exchange normally transports Ca2+ into the vacuole; however, under certain conditions, Ca2+ may be released into the cytoplasm via this antiporter.

Acridine Orange↗

PubMed1985

A Ca/H Antiport System Driven by the Proton Electrochemical Gradient of a Tonoplast H-ATPase from Oat Roots.

Two types of ATP-dependent calcium (Ca(2+)) transport systems were detected in sealed microsomal vesicles from oat roots. Approximately 80% of the total Ca(2+) uptake was associated with vesicles of 1.11 grams per cubic centimeter and was insensitive to vanadate or azide, but inhibited by NO(3) (-). The remaining 20% was vanadate-sensitive and mostly associated with the endoplasmic reticulum, as the transport activity comigrated with an endoplasmic reticulum marker (antimycin A-insensitive NADH cytochrome c reductase), which was shifted from 1.11 to 1.20 grams per cubic centimeter by Mg(2+).Like the tonoplast H(+)-ATPase activity, vanadate-insensitive Ca(2+) accumulation was stimulated by 20 millimolar Cl(-) and inhibited by 10 micromolar 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid or 50 micromolar N,N'-dicyclohexylcarbodiimide. This Ca(2+) transport system had an apparent K(m) for Mg-ATP of 0.24 millimolar similar to the tonoplast ATPase. The vanadate-insensitive Ca(2+) transport was abolished by compounds that eliminated a pH gradient and Ca(2+) dissipated a pH gradient (acid inside) generated by the tonoplast-type H(+)-ATPase. These results provide compelling evidence that a pH gradient generated by the H(+)-ATPase drives Ca(2+) accumulation into right-side-out tonoplast vesicles via a Ca(2+)/H(+) antiport. This transport system was saturable with respect to Ca(2+) (K(m) apparent = 14 micromolar). The Ca(2+)/H(+) antiport operated independently of the H(+)-ATPase since an artifically imposed pH gradient (acid inside) could also drive Ca(2+) accumulation. Ca(2+) transport by this system may be one major way in which vacuoles function in Ca(2+) homeostasis in the cytoplasm of plant cells.

Journal Article↗