Characterization of two maize HSP90 heat shock protein genes: expression during heat shock, embryogenesis, and pollen development.

We have isolated two genes from Zea mays encoding proteins of 82 and 81 kD that are highly homologous to the Drosophila 83-kD heat shock protein gene and have analyzed the structure and pattern of expression of these two genes during heat shock and development. Southern blot analysis and hybrid select translations indicate that the highly homologous hsp82 and hsp81 genes are members of a small multigene family composed of at least two and perhaps three or more gene family members. The deduced amino acid sequence of these proteins based on the nucleotide sequence of the coding regions shows 64-88% amino acid homology to other hsp90 family genes from human, yeast, Drosophila, and Arabidopsis. The promoter regions of both the hsp82 and hsp81 genes contain several heat shock elements (HSEs), which are putative binding sites for heat shock transcription factor (HSF) commonly found in the promoters of other heat shock genes. Gene-specific oligonucleotide probes were synthesized and used to examine the mRNA expression patterns of the hsp81 and hsp82 genes during heat shock, embryogenesis, and pollen development. The hsp81 gene is only mildly heat inducible in leaf tissue, but is strongly expressed in the absence of heat shock during the pre-meiotic and meiotic prophase stages of pollen development and in embryos, as well as in heat-shocked embryos and tassels. The hsp82 gene shows strong heat inducibility at heat-shock temperatures (37-42 degrees C) and in heat shocked embryos and tassels but is only weakly expressed in the absence of heat shock. Promoter-GUS reporter gene fusions made and analyzed by transient expression assays in Black Mexican Sweet (BMS) Maize protoplasts also indicate that the hsp82 and hsp81 are regulated differentially. The hsp82 promoter confers strong heat-inducible expression of the GUS reporter gene in heat-treated cells (60- to 80-fold over control levels), whereas the hsp81 promoter is only weakly heat inducible (5- to 10-fold over control levels).

Intron-mediated enhancement of heterologous gene expression in maize.

Chimeric genes containing the coding sequence for bacterial chloramphenicol acetyl transferase (CAT) have been introduced by electroporation into maize protoplasts (Black Mexican Sweet) and transient expression monitored by enzyme assays. Levels of CAT expression were enhanced 12-fold and 20-fold respectively by the inclusion of maize alcohol dehydrogenase-1 introns 2 and 6 in the chimeric construct. This enhancement was seen when the intron was placed within the 5' translated region but not when it was located upstream of the promoter or within the 3' untranslated region. Deletion of exon sequences adjacent to intron 2 abolished its ability to mediate enhancement of CAT gene expression. Northern analysis of protoplasts electroporated with intron constructs revealed elevated levels of CAT mRNA. However, this elevation was insufficient to account for the increased enzyme activity. One explanation of these results is that splicing affects both the quantity and quality of mRNA.

Genetically transformed maize plants from protoplasts.

Genetically transformed maize plants were obtained from protoplasts treated with recombinant DNA. Protoplasts that were digested from embryogenic cell suspension cultures of maize inbred A188 were combined with plasmid DNA containing a gene coding for neomycin phosphotransferase (NPT II) next to the 35S promoter region of cauliflower mosaic virus. A high voltage electrical pulse was applied to the protoplasts, which were then grown on filters placed over feeder layers of maize suspension cells (Black Mexican Sweet) and selected for growth in the presence of kanamycin. Selected cell lines showed NPT II activity. Plants were regenerated from transformed cell lines and grown to maturity. Southern analysis of DNA extracted from callus and plants indicated the presence of the NPT II gene.

Characterization of in vitro proton pumping by microsomal vesicles isolated from corn coleoptiles.

Corn (Zea mays L. cv Golden Cross Bantam) coleoptile microsomal vesicles have been isolated which are capable of ATP-driven H(+)-transport as measured by [(14)C]methylamine accumulation and quinacrine fluorescence quenching. Formation of the pH gradient in vitro shows a high specificity for ATP.Mg, is temperature-sensitive, exhibits a pH optimum at 7.5, and is inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Of the divalent cations tested, Mn(2+) is almost as effective as Mg(2+), while Ca(2+) is ineffective. Excess divalent cations, particularly Ca(2+), reduces the pH gradient. H(+) transport is strongly promoted by anions, especially chloride, while potassium does not affect pump activity. Studies with (36)Cl(-) indicate that ATP-driven H(+) transport into the vesicles is associated with chloride uptake. Both carbonyl cyanide-m-chlorophenylhydrazone and the anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, inhibit methylamine accumulation and (36)Cl(-) uptake. Proton pumping is also blocked by diethyl stilbestrol and N,N'-dicyclohexylcarbodiimide, but is insensitive to oligomycin and vanadate. These properties of the pump are inconsistent with either a mitochondrial or plasma membrane origin.

Localization of the proton pump of corn coleoptile microsomal membranes by density gradient centrifugation.

Previous studies characterizing an ATP-dependent proton pump in microsomal membrane vesicles of corn coleoptiles led to the conclusion that the proton pump was neither mitochondrial nor plasma membrane in origin (Mettler, Mandala, Taiz 1982 Plant Physiol 70: 1738-1742). To facilitate positive identification of the vesicles, corn coleoptile microsomal membranes were fractionated on linear sucrose and dextran gradients, with ATP-dependent [(14)C]methylamine uptake as a probe for proton pumping. On sucrose gradients, proton pumping activity exhibited a density of 1.11 grams/cubic centimeter and was coincident with the endoplasmic reticulum (ER). In the presence of high magnesium, the ER shifted to a heavier density, while proton pumping activity showed no density shift. On linear dextran gradients, proton pumping activity peaked at a lighter density than the ER. The proton pump appears to be electrogenic since both [(14)C]SCN(-) uptake and (36)Cl(-) uptake activities coincided with [(14)C] methylamine uptake on dextran gradients. On the basis of density and transport properties, we conclude that the proton pumping vesicles are probably derived from the tonoplast. Nigericin-stimulated ATPase activity showed a broad distribution which did not coincide with any one membrane marker.

Oxidation of NADH in Glyoxysomes by a Malate-Aspartate Shuttle.

Glyoxysomes isolated from germinating castor bean endosperm accumulate NADH by beta-oxidation of fatty acids. By utilizing the glutamate: oxaloacetate aminotransferase and malate dehydrogenase present in glyoxysomes and mitochondria, reducing equivalents could be transferred between the organelles by a malate-aspartate shuttle. The addition of aspartate plus alpha-ketoglutarate to purified glyoxysomes brought about a rapid oxidation of accumulated NADH, and the oxidation was prevented by aminooxyacetate, an inhibitor of aminotransferase activity. Citrate synthetase activity in purified glyoxysomes could be coupled readily to glutamate: oxaloacetate aminotransferase activity as a source of oxaloacetate, but coupling to malate dehydrogenase and malate resulted in low rates of citrate formation. Glyoxysomes purified in sucrose or Percoll gradients were permeable to low molecular weight compounds. No evidence was obtained for specific transport mechanisms for the proposed shuttle intermediates. The results support a revised model of gluconeogenic metabolism incorporating a malate-aspartate shuttle in the glyoxysomal pathway.

Isolation and partial characterization of vacuoles from tobacco protoplasts.

Protoplasts from suspension-cultured cells of Nicotiana glutinosa L. were lysed in 0.3 molar sorbitol in 2 millimolar ethylenediaminetetraacetate-tris(hydroxymethyl) aminomethane (pH 7.5) to release intact vacuoles. The vacuoles were purified by centrifugation in a Ficoll step gradient. About 11% of the vacuoles and 13% of the acid phosphatase activity was recovered in the purified vacuole fraction, suggesting that the vacuole is the major site for acid phosphatase in these cells. NADH-cytochrome c reductase, malate dehydrogenase, and cytochrome c oxidase activities were reduced during vacuole purification. The majority of the adenosine 5'-triphosphate (ATP) hydrolytic activity of purified vacuoles was associated with nonspecific acid phosphatase and not with a transport ATPase. As judged by acid phosphatase distribution and electron microscopy, the effective density of vacuoles in a sucrose gradient was low (less than 1.1 grams per cubic centimeter), although an unequivocal estimate of the vacuole or tonoplast density was not possible from the experiments conducted.

Isolation and characterization of the protein body membrane of castor beans.

Intact protein bodies were isolated from dry castor bean seeds (Ricinus communis L.) after homogenization in nonaqueous medium. After repeated washing with glycerol to remove trapped lipid globules, the soluble matrix proteins were removed by the addition of aqueous buffer. The membrane remained attached to the insoluble protein crystalloids and was subsequently released by sonication. Purification of the membrane vesicles in a sucrose gradient produced a single band at a density of 1.21 grams per cubic centimeter. Treatment with 6 molar urea, 1 molar KCl, or 0.25 molar galactose had no effect on the equilibrium density of the membrane. Electron microscopy revealed a highly pure and uniform collection of membrane vesicles. No enzyme activity was specifically associated with the membrane. Sodium dodecyl sulfate gel electrophoresis of the protein body fractions showed that the membrane contained unique proteins, two of which were glycosylated. The membrane contained 153 nanomoles of phospholipid per milligram of protein. The composition of the phosphoglycerides was 51% ethanolamine, 41% choline, 8% inositol, and a trace of serine.

Ion transport in isolated protoplasts from tobacco suspension cells: I. General characteristics.

An investigation was conducted into the feasibility of using enzymically isolated protoplasts from suspension-cultured cells of Nicotiana glutinosa L. to study ion transport. Transport of K(+) ((86)Rb), (36)Cl(-), H(2) (32)PO(4) (-) and (45)Ca(2+) from 1 millimolar salt solutions was determined after separation of intact protoplasts from nonabsorbed tracers by centrifugation through a Ficoll step gradient. Influx of K(+), Cl(-), and H(2)PO(4) (-) measured over a 30-minute period was reduced (up to 99%) by respiratory inhibitors such as 5 micrograms per milliliter oligomycin, 0.1 millimolar dinitrophenol, 0.1 millimolar cyanide, or N(2) gas. In contrast, Ca(2+) influx was not tightly coupled to respiratory energy production. The influx of K(+) was highest between pH 6.5 and 7.5 whereas the influx of H(2)PO(4) (-) and Cl(-) was greatest between pH 4.5 and 5.5. Influx of K(+) and Cl(-) was maximal at 35 and 45 C, respectively, and was almost completely inhibited below 10 C. Fusicoccin (0.01 millimolar) stimulated K(+) influx by more than 200% but had no effect on the influx of either Cl(-) or H(2)PO(4) (-). Apparent H(+) efflux, as measured by decrease in solution pH, was enhanced by K(+), stimulated further by 0.01 millimolar fusicoccin, and inhibited by 0.1 millimolar dinitrophenol or 5 micrograms per milliliter oligomycin. The measured ionic fluxes into protoplasts were similar to those obtained with intact cultured cells. The results indicate that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast, and that it is feasible to use isolated protoplasts for studies on ion transport.

Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells: II. Selectivity and Kinetics.

Protoplasts were enzymically isolated from suspension cultured cells of Nicotiana glutinosa L. and aspects of transport selectivity and kinetics were studied. In the presence of Ca(2+), transport was selective for K(+) ((86)Rb) over Na(+). (36)Cl(-) transport was inhibited by Br(-) or I(-) but not by H(2)PO(4) (-). The kinetic data for short term (30 minutes) K(+) influx over the range of 0.05 to 100 millimolar KCl were complex but similar to those observed in other plant tissues. In contrast, the kinetic data for Cl(-) and H(2) (32)PO(4) (-) over the same concentration range were different from those observed for K(+), and could be accounted for by a single isotherm in the range of 0.05 to 4 millimolar and by an almost linear increase in influx rate above 4 millimolar. The kinetic data for Cl(-) transport into intact cultured cells were identical in character to those observed for isolated protoplasts. The results support the view that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast.