A role for the AKT1 potassium channel in plant nutrition.

In plants, potassium serves an essential role as an osmoticum and charge carrier. Its uptake by roots occurs by poorly defined mechanisms. To determine the role of potassium channels in planta, we performed a reverse genetic screen and identified an Arabidopsis thaliana mutant in which the AKT1 channel gene was disrupted. Roots of this mutant lacked inward-rectifying potassium channels and displayed reduced potassium (rubidium-86) uptake. Compared with wild type, mutant plants grew poorly on media with a potassium concentration of 100 micromolar or less. These results and membrane potential measurements suggest that the AKT1 channel mediates potassium uptake from solutions that contain as little as 10 micromolar potassium.

Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis.

The alpha subunit of a prototypical heterotrimeric GTP-binding protein (G protein), which is encoded by a single gene (GPA1) in Arabidopsis, is a modulator of plant cell proliferation. gpa1 null mutants have reduced cell division in aerial tissues throughout development. Inducible overexpression of GPA1 in Arabidopsis confers inducible ectopic cell division. GPA1 overexpression in synchronized BY-2 cells causes premature advance of the nuclear cycle and the premature appearance of a division wall. Results from loss of function and ectopic expression and activation of GPA1 indicate that this subunit is a positive modulator of cell division in plants.

A calcium-dependent protein kinase with a regulatory domain similar to calmodulin.

Calcium can function as a second messenger through stimulation of calcium-dependent protein kinases. A protein kinase that requires calcium but not calmodulin or phospholipids for activity has been purified from soybean. The kinase itself binds calcium with high affinity. A complementary DNA clone for this kinase has been identified; it encodes a protein with a predicted molecular mass of 57,175 daltons. This protein contains a catalytic domain similar to that of calmodulin-dependent kinases and a calmodulin-like region with four calcium binding domains (EF hands). The predicted structure of this kinase explains its direct regulation via calcium binding and establishes it as a prototype for a new family of calcium-regulated protein kinases.

Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array.

Oligonucleotide microarrays, also called "DNA chips," are currently made by a light-directed chemistry that requires a large number of photolithographic masks for each chip. Here we describe a maskless array synthesizer (MAS) that replaces the chrome masks with virtual masks generated on a computer, which are relayed to a digital micromirror array. A 1:1 reflective imaging system forms an ultraviolet image of the virtual mask on the active surface of the glass substrate, which is mounted in a flow cell reaction chamber connected to a DNA synthesizer. Programmed chemical coupling cycles follow light exposure, and these steps are repeated with different virtual masks to grow desired oligonucleotides in a selected pattern. This instrument has been used to synthesize oligonucleotide microarrays containing more than 76,000 features measuring 16 microm 2. The oligonucleotides were synthesized at high repetitive yield and, after hybridization, could readily discriminate single-base pair mismatches. The MAS is adaptable to the fabrication of DNA chips containing probes for thousands of genes, as well as any other solid-phase combinatorial chemistry to be performed in high-density microarrays.

Potassium uptake supporting plant growth in the absence of AKT1 channel activity: Inhibition by ammonium and stimulation by sodium.

A transferred-DNA insertion mutant of Arabidopsis that lacks AKT1 inward-rectifying K+ channel activity in root cells was obtained previously by a reverse-genetic strategy, enabling a dissection of the K+-uptake apparatus of the root into AKT1 and non-AKT1 components. Membrane potential measurements in root cells demonstrated that the AKT1 component of the wild-type K+ permeability was between 55 and 63% when external [K+] was between 10 and 1,000 microM, and NH4+ was absent. NH4+ specifically inhibited the non-AKT1 component, apparently by competing for K+ binding sites on the transporter(s). This inhibition by NH4+ had significant consequences for akt1 plants: K+ permeability, 86Rb+ fluxes into roots, seed germination, and seedling growth rate of the mutant were each similarly inhibited by NH4+. Wild-type plants were much more resistant to NH4+. Thus, AKT1 channels conduct the K+ influx necessary for the growth of Arabidopsis embryos and seedlings in conditions that block the non-AKT1 mechanism. In contrast to the effects of NH4+, Na+ and H+ significantly stimulated the non-AKT1 portion of the K+ permeability. Stimulation of akt1 growth rate by Na+, a predicted consequence of the previous result, was observed when external [K+] was 10 microM. Collectively, these results indicate that the AKT1 channel is an important component of the K+ uptake apparatus supporting growth, even in the "high-affinity" range of K+ concentrations. In the absence of AKT1 channel activity, an NH4+-sensitive, Na+/H+-stimulated mechanism can suffice.

A transgene encoding a plasma membrane H+-ATPase that confers acid resistance in Arabidopsis thaliana seedlings.

Proton pumps (H+-ATPases) are the primary active transport systems in the plasma membrane of higher plant cells. These enzymes are encoded by a large gene family expressed throughout the plant, with specific isoforms directed to various specialized cells. While their involvement in membrane energetics has been suggested by a large body of biochemical and physiological studies, a genetic analysis of their role in plants has not yet been performed. We report here that mutant Arabidopsis thaliana plants containing a phloem-specific transgene encoding a plasma membrane H+-ATPase with an altered carboxy terminus show improved growth at low pH during seedling development. These observations provide the first genetic evidence for a role of the plasma membrane H+-ATPase in cytoplasmic pH homeostasis in plants.

Purification of integral plasma membrane proteins by reverse-phase high performance liquid chromatography.

Following dissolution in anhydrous trifluoroacetic acid, plasma membrane isolated from two eukaryotic species was directly injected onto a reverse-phase high performance liquid chromatograph column. Upon development with a 60 to 100% (v/v) linear gradient of ethanol containing 0.1% trifluoroacetic acid, most of the polypeptides eluted without retention. Only the lipids and very hydrophobic proteins were retained and resolved. Most noticeable among retained proteins was the Mr 100,000 catalytic polypeptide of each species' primary plasma membrane cation pump, the Na+,K+-ATPase of pig kidney and the H+-ATPase of Neurospora crassa hyphae. This simple 60-min procedure yielded nearly pure ATPase starting from crude membranes and in a completely volatile solvent, without detergent. When fungal plasma membranes were phosphorylated in vitro with [gamma-32P]ATP prior to injection, protein kinase activity was observed and this resulted in the phosphorylation of the H+-ATPase as well as of several other less-abundant hydrophobic membrane proteins. This procedure is useful as an alternative method for the rapid characterization of those membrane-associated polypeptides that contain several hydrophobic, transmembrane sequences.

Purification of the Neurospora crassa plasma membrane H+-ATPase by high-pressure liquid chromatography in the presence of sodium dodecyl sulfate.

Current methods for purifying the Mr 100,000 H+-ATPase from the plasma membrane of fungi and higher plants rely on detergent solubilization followed by density gradient centrifugation. The procedure yields catalytically active enzyme of high purity but takes several days, and the yields are low. For chemical studies on the primary structure of this enzyme, an alternative more rapid procedure was sought. In this paper a method which uses a high-performance gel filtration column in the presence of sodium dodecyl sulfate to purify nanomole quantities of the enzyme in only 30 min is described. With this procedure the enzyme was isolated free of other protein contaminants, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration was performed on a high-pressure liquid chromatograph equipped with a diode array spectrophotometric detector, allowing spectral analysis of the membrane proteins. An ultraviolet absorption spectrum of the plasma membrane Mr 104,000 H+-ATPase revealed an absorption peak at ca. 275 nm that is consistent with its content of aromatic amino acids.

In vitro cytokinin binding to a particulate fraction of tobacco cells.

At least two types of cytokinin-binding sites are present in a particulate fraction of tobacco (Nicotiana tabacum L.) cells that sediments at 80,000 x g. The major binding component has a low affinity towards cytokinins, is resistant to heating at 100°C, and is not specific for biologically active cytokinin analogues. The second site occurs in much lower frequency, is heat labile, shows high affinity towards cytokinins, and is specific for biologically active analogs of the hormone. The testing for binding specificity was mainly performed with a series of halogenated benzyladenine derivatives having a wide range of biological activities. The low-affinity binding site shows some of the same features as talcum powder, a non-biological material which binds cytokinins in a non-specific fashion. The properties of the high-affinity binding site are consistent with the expected characteristics of a cytokinin receptor. However, the role of the observed high-affinity binding site with regard to the biological action of cytokinins is not yet known.

The synthesis and biological properties of 8-azido-N (6)-benzyladenine, a potential photoaffinity reagent for cytokinin.

A cytokinin photoaffinity reagent, 8-azido-N (6)-benzyladenine (8N3BA), was synthesized from 8-bromoadenosine via azide replacement, benzylation at N-1, rearrangement to the N-6-benzyl derivative and acid hydrolysis. The compound thus obtained was found to have full cytokinin activity in the moss and tobacco cell-suspension bioassays. Photolysis of 8N3BA was accomplished with long and short-wavelength ultraviolet light and produced compounds which had very little or no biological activity in the two bioassays. In-vivo photolysis of 8N3BA caused loss of the cytokinin activity of this compound in moss protonemata. This result was similar to earlier ones where the biological response of moss protonemata to benzyladenine was reversed following removal of the hormone by a short rinse with water.

Isolation and sequence of tryptic peptides from the proton-pumping ATPase of the oat plasma membrane.

In crude extracts of plant tissue, the M(r) = 100,000 proton-pumping ATPase constitutes less than 0.01% of the total cell protein. A large-scale purification procedure is described that has been used to obtain extensive protein sequence information from this enzyme. Plasma membrane vesicles enriched in ATPase activity were obtained from extracts of oat roots by routine differential and density gradient centrifugation. Following a detergent wash, the ATPase was resolved from other integral membrane proteins by size fractionation at 4 degrees C in the presence of lithium dodecyl sulfate. After carboxymethylation of cysteine residues and removal of detergent, the ATPase was digested with trypsin and resultant peptide fragments separated by reverse phase high performance liquid chromatography. Peptides were recovered with high yield and were readily sequenced by automated Edman degradation on a gas-phase sequencer. Of the eight peptides sequenced, six showed strong homology with known amino acid sequences of the fungal proton-pumping and other cation-transporting ATPases.

Phosphorylation of the plasma-membrane H(+)-ATPase of oat roots by a calcium-stimulated protein kinase.

When plasma-membrane vesicles isolated from oat (Avena sativa L.) root cells were incubated with [γ-(32)P]ATP, the H(+)-ATPase was found to be phosphorylated at serine and threonine residues. Phosphotyrosine was not detected. Endogenous ATPase kinase activity was also observed in plasma-membrane vesicles isolated from potato (Solanum tuberosum L.) root cells as well as from yeast (Saccharomyces cerevisiae). Identity of the phosphorylated oat root Mr=100 000 polypeptide as the ATPase was confirmed using conventional glycerol density-gradient centrifugation to purify the native enzyme and by a new procedure for purifying the denatured polypeptide using reversephase high-performance liquid chromatography. Kinase-mediated phosphorylation of the oat root plasma-membrane H(+)-ATPase was stimulated by the addition of low concentrations of Ca(2+) and by a decrease in pH, from 7.2 to 6.2. These results demonstrate that kinase-mediated phosphorylation of the H(+)-ATPase is a plausible mechanism for regulating activity. They further indicate that changes in the cytoplasmic [Ca(2+)] and pH are potentially important elements in modulating the kinase-mediated phosphorylation.

Inhibition and Labeling of the Plant Plasma Membrane H-ATPase with N-Ethylmaleimide.

H(+)-ATPase activity in plasma membranes isolated from Avena sativa root cells is inhibited by N-ethylmaleimide, a covalent modifier of protein sulfhydryl groups. The rate of inhibition is reduced by ADP, MgADP, and MgATP, but even at 40 millimolar ADP the enzyme is only partially protected against inactivation. When plasma membranes are treated wth N-[2-(3)H]ethylmaleimide and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, prominent radioactive bands appear at M(r)=100,000 and several other positions. However, only radioactivity in the M(r)=100,000 protein is reduced by the presence of MgADP. These results provide independent evidence that the M(r)=100,000 polypeptide which is observed in purified preparations of the enzyme is the catalytic subunit of the H(+)-ATPase. When tryptic peptides are produced from N-[2-(3)H]ethylmaleimide labeled M(r)=100,000 protein and separated by reverse phase high performance liquid chromatography, two radioactive peaks are observed for which N-[2-(3)H]ethylmaleimide incorporation is reduced in the presence of MgADP.

Immunocytological localization of an epitope-tagged plasma membrane proton pump (H(+)-ATPase) in phloem companion cells.

In higher plants, the plasma membrane proton pump (H(+)-ATPase) is encoded by a surprisingly large multigene family whose members are expressed in different tissues. Using an 18-amino acid epitope tag derived from the animal oncogene c-Myc, we have performed immunocytolocalization measurements of the protein expressed by one member of this family, AHA3 (Arabidopsis H(+)-ATPase isoform 3). Immunofluorescence studies with tissue sections of transgenic plants have revealed that c-Myc-tagged AHA3 is restricted to the plasma membrane of phloem companion cells, whereas other AHA isoproteins are more widely distributed in the plasma membrane of other cell types. Electron microscopy with immunogold-labeled tissue sections suggests that there is a high concentration of proton pumps in the plasma membrane of companion cells but a much lower concentration in the plasma membrane of sieve elements. Due to plasmodesmata connecting the plasma membrane of these two adjacent cell types, it is likely that the proton motive force generated by the proton pump in companion cells can serve to power the uptake of sugar by proton-coupled symporters in either the companion cell or sieve element cell. The abundance of the proton pump in the plasma membrane of companion cells supports an apoplastic model for phloem loading in which the metabolic energy that drives sugar uptake is consumed by AHA3 at the companion cell plasma membrane. These experiments with a genetically altered integral plasma membrane protein demonstrate the utility of using a short c-Myc sequence as an epitope tag in Arabidopsis. Furthermore, our results demonstrate that, using genes encoding individual members of a gene family, it is possible to label plasma membrane proteins immunologically in specific, differentiated cell types of higher plants.

Unusual membrane-associated protein kinases in higher plants.

Plant genomes encode a variety of protein kinases, and while some are functional homologues of animal and fungal kinases, others have a novel structure. This review focuses on three groups of unusual membrane-associated plant protein kinases: receptor-like protein kinases (RLKs), calcium-dependent protein kinases (CDPKs), and histidine protein kinases. Animal RLKs have a putative extracellular domain, a single transmembrane domain, and a protein kinase domain. In plants, all of the RLKs identified thus far have serine/threonine signature sequences, rather than the tyrosine-specific signature sequences common to animals. Recent genetic experiments reveal that some of these plant kinases function in development and pathogen resistance. The CDPKs of plants and protozoans are composed of a single polypeptide with a protein kinase domain fused to a C-terminal calmodulin-like domain containing four calcium-binding EF hands. No functional plant homologues of protein kinase C or Ca2+/calmodulin-dependent protein kinase have been identified, and no animal or fungal CDPK homologues have been identified. Recently, histidine kinases have been shown to participate in signaling pathways in plants and fungi. ETR1, an Arabidopsis histidine kinase homologue with three transmembrane domains, functions as a receptor for the plant hormone ethylene. G-protein-coupled receptors, which often serve as hormone receptors in animal systems, have not yet been identified in plants.

Modification of the Neurospora crassa plasma membrane [H+]-ATPase with N,N'-dicyclohexylcarbodiimide.

The [H+]-ATPase of the Neurospora plasma membrane is composed of a single Mr = 104,000 polypeptide (B. J. Bowman, F. Blasco, and C. W. Slayman, J. Biol. Chem. (1981) 256, 12343-12349). The carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) inactivates the ATPase with pseudo-first order kinetics, suggesting that one site on the enzyme is involved. The rate constant for inactivation at pH 7.5 and 30 degrees C is approximately 1000 M-1 min-1, similar to values reported for the DCCD-binding proteolipid of F0-F1-type [H+]-ATPases and for the sarcoplasmic reticulum [Ca+2]-ATPase. Although hydrophobic carbodiimides are inhibitory at micromolar concentrations, a hydrophilic analogue, 1-ethyl-3-(dimethylaminopropyl)-carbodiimide, is completely inactive even at millimolar concentrations. This result implies that the DCCD-reactive site is located in a lipophilic environment. [14C]DCCD is incorporated into the Mr = 104,000 polypeptide at a rate similar to the rate of inactivation. There is no evidence for a separate low molecular weight DCCD-binding proteolipid. Using quantitative amino acid analysis, we established that complete inhibition occurs at a stoichiometry of 0.4 mol of DCCD/mol of polypeptide. Overall, the results are consistent with the idea that DCCD reacts with a single amino acid residue of the Neurospora [H+]-ATPase, thereby blocking ATP hydrolysis and proton translocation.

Solubilization of the receptor for N-1-naphthylphthalamic Acid.

A receptor protein for the auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA), has been solubilized from corn coleoptile membranes using Triton X-100. [(3)H]NPA binding activity of the receptor was compared in soluble and membrane-bound states. Both activities are abolished by treatment with trypsin. Differences between the two are observed in pH optima and rates of heat inactivation.At pH 5, the membrane-bound and solubilized receptors have similar affinity for NPA (K(d) about 10(-7) molar). Solubilization results in a 10-fold increase in affinity for the low-affinity auxin analogs, alpha- and beta-naphthaleneacetic acid (K(d) about 10(-5) molar). There is no measurable competition by indoleacetic acid (K(d) > 10(-3) molar) for [(3)H]NPA binding to membranes, but there is competition with the solubilized receptor (K(d) for IAA about 10(-5) molar). There is no measurable competition with benzoic acid in either preparation. These observations of an affinity of auxin analogs for the solubilized NPA receptor raise the interesting possibility that the NPA binding site is one conformation of an auxin binding site involved in polar auxin transport.

Auxin uptake and action of N-1-naphthylphthalamic acid in corn coleoptiles.

The validity of a chemiosmotic hypothesis for uptake of weak acids as an explanation for the accumulation of auxin by cells has been explored further by comparing the uptake of indole-3-acetic acid (IAA) by 1-mm segments of corn (Zea mays L.) coleoptiles with that of benzoic acid and two neutral indoles, indoleethanol and indoleacetonitrile, which do not ionize. These substances, while structurally related to IAA lack both auxin activity and polar transport. Uptake of IAA and benzoic acid increase with decreasing external pH, whereas the uptake of the two neutral indoles is independent of external pH.Although metabolism of IAA, during 90 min or less, is minimal and without significant effect on its uptake, metabolism of benzoic acid appears responsible for the apparent saturation of benzoic acid uptake at high concentrations. An inhibitor of auxin transport, N-1-naphthylphathalamic acid (NPA), stimulates uptake of IAA but has no effect on uptake of either benzoic acid or the two neutral indoles. Thus, NPA does not affect the driving forces for accumulation of weak acids but probably specifically decreases the flux of the auxin anions relative to undissociated auxin. Since the electrochemical potential of auxin anions is usually higher in than outside cells, blocking the anion flux with NPA would enhance auxin uptake. Azide, which abolishes accumulation of both IAA and benzoic acid, may simply collapse the pH gradient across the plasma membrane.In the absence of NPA, increasing concentrations of auxins or the analogoue ß-naphthaleneacetic acid (ß-NAA) exert two opposing effects on the uptake of IAA-depression and stimulation. Stimulation results from saturating the anion flux. With uptake fully stimulated by NPA, however, increasing concentrations of auxins or analogues only depress uptake of [(3)H]IAA. These results are consistent with more than one path for auxin transport each with a different dependence on concentration. In depressing NPA-stimulated IAA uptake, the effectiveness of ß-NAA≧IAA≫α-NAA≫ benzoic acid, a specificity similar to that of an auxin binding site in vitro that has been implicated by others in auxin transport. The results support the general hypothesis that cellular auxin uptake and polar transport through tissues are chemiosmotically coupled to the electrochemical potential of auxin and protons.