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R E Sharp

Publications and source records attributed to R E Sharp.

At least 19 recordsLinked to original sources
PubMed2006

Comparing regional transcript profiles from maize primary roots under well-watered and low water potential conditions.

Regionally distinct elongation responses to water stress in the maize primary root tip have been observed in the past. A genetic basis for such differential responses has been demonstrated. Normalized bar-coded cDNA libraries were generated for four regions of the root tip, 0-3 mm (R1), 3-7 mm (R2), 7-12 mm (R3), and 12-20 mm (R4) from the root apex, and transcript profiles for these regions were sampled. This permitted a correlation between transcript nature and regional location for 15 726 expressed sequence tags (ESTs) that, in approximately equal numbers, derived from three conditions of the root: water stress (water potential: -1.6 MPa) for 5 h and for 48 h, respectively, and well watered (5 h and 48 h combined). These normalized cDNA libraries provided 6553 unigenes. An analysis of the regional representation of transcripts showed that populations were largely unaffected by water stress in R1, correlating with the maintenance of elongation rates under water stress known for R1. In contrast, transcript profiles in regions 2 and 3 diverged in well-watered and water-stressed roots. In R1, transcripts for translation and cell cycle control were prevalent. R2 was characterized by transcripts for cell wall biogenesis and cytoskeleton formation. R3 and R4 shared prevalent groups of transcripts responsible for defence mechanisms, ion transport, and biogenesis of secondary metabolites. Transcripts which were followed for 1, 6, and 48 h of water stress showed distinct region-specific changes in absolute expression and changes in regulated functions.

Expressed Sequence Tags↗
PubMed2005

The maize root transcriptome by serial analysis of gene expression.

Serial Analysis of Gene Expression was used to define number and relative abundance of transcripts in the root tip of well-watered maize seedlings (Zea mays cv FR697). In total, 161,320 tags represented a minimum of 14,850 genes, based on at least two tags detected per transcript. The root transcriptome has been sampled to an estimated copy number of approximately five transcripts per cell. An extrapolation from the data and testing of single-tag identifiers by reverse transcription-PCR indicated that the maize root transcriptome should amount to at least 22,000 expressed genes. Frequency ranged from low copy number (2-5, 68.8%) to highly abundant transcripts (100-->1,200; 1%). Quantitative reverse transcription-PCR for selected transcripts indicated high correlation with tag frequency. Computational analysis compared this set with known maize transcripts and other root transcriptome models. Among the 14,850 tags, 7,010 (47%) were found for which no maize cDNA or gene model existed. Comparing the maize root transcriptome with that in other plants indicated that highly expressed transcripts differed substantially; less than 5% of the most abundant transcripts were shared between maize and Arabidopsis (Arabidopsis thaliana). Transcript categories highlight functions of the maize root tip. Significant variation in abundance characterizes transcripts derived from isoforms of individual enzymes in biochemical pathways.

Base Sequence↗
PubMed2001

Modification of expansin transcript levels in the maize primary root at low water potentials.

We previously demonstrated that maintenance of cell elongation in the apical region of maize primary roots at low water potentials (psi(w)) was associated with an increase in expansin activity and extractable expansin protein. Here, we characterized the spatial pattern of expansin gene expression along the growing maize root and studied the effect of low psi(w) on expansin gene expression. Roots were divided into three segments: apical 0 to 5 mm, subapical 5 to 10 mm, and non-growing 10 to 20 mm. Of the five expansin genes expressed in control roots, two alpha-expansins (Exp1 and Exp5) and two beta-expansins (ExpB2 and ExpB8) are expressed specifically in the growing region, whereas expression of beta-expansin ExpB6 is shifted basipetally. After seedlings were transplanted to vermiculite with a psi(w) of -1.6 MPa, transcripts for Exp1, Exp5, and ExpB8 rapidly accumulated in the apical region of the root. These mRNA changes correlated with the maintenance of root elongation and increases in wall extensibility found previously. The beta-expansins ExpB2 and ExpB6 showed distinctive patterns of expression and responses to low psi(w,) indicative of distinctive functions. Inhibition of abscisic acid (ABA) accumulation at low psi(w) (by fluridone treatment) had no effect on expansin expression, except that ExpB2 transcript level showed a minor dependence on ABA. Gene-specific regulation of alpha- and beta-expansin mRNA pools likely contributes to growth alterations of the maize (Zea mays) root as it adapts to a low psi(w), but these changes do not appear to be mediated by changes in ABA content.

Abscisic Acid↗
PubMed2000

Heme redox potential control in de novo designed four-alpha-helix bundle proteins.

The effects of various mechanisms of metalloporphyrin reduction potential modulation were investigated experimentally using a robust, well-characterized heme protein maquette, synthetic protein scaffold H10A24 [¿CH(3)()CONH-CGGGELWKL.HEELLKK.FEELLKL.AEERLKK. L-CONH(2)()¿(2)](2). Removal of the iron porphyrin macrocycle from the high dielectric aqueous environment and sequestration within the hydrophobic core of the H10A24 maquette raises the equilibrium reduction midpoint potential by 36-138 mV depending on the hydrophobicity of the metalloporphyrin structure. By incorporating various natural and synthetic metalloporphyrins into a single protein scaffold, we demonstrate a 300-mV range in reduction potential modulation due to the electron-donating/withdrawing character of the peripheral macrocycle substituents. Solution pH is used to modulate the metalloporphyrin reduction potential by 160 mV, regardless of the macrocycle architecture, by controlling the protonation state of the glutamate involved in partial charge compensation of the ferric heme. Attempts to control the reduction potential by inserting charged amino acids into the hydrophobic core at close proximity to the metalloporphyrin lead to varied success, with H10A24-L13E lowering the E(m8.5) by 40 mV, H10A24-E11Q raising it by 50 mV, and H10A24-L13R remaining surprisingly unaltered. Modifying the charge of the adjacent metalloporphyrin, +1 for iron(III) protoporphyrin IX or neutral for zinc(II) protoporphyrin IX resulted in a loss of 70 mV [Fe(III)PPIX](+) - [Fe(III)PPIX](+) interaction observed in maquettes. Using these factors in combination, we illustrate a 435-mV variation of the metalloporphyrin reduction midpoint potential in a simple heme maquette relative to the about 800-mV range observed for natural cytochromes. Comparison between the reduction potentials of the heme maquettes and other de novo designed heme proteins reveals global trends in the E(m) values of synthetic cytochromes.

Amino Acid Sequence↗
PubMed2000

Growth of Arabidopsis thaliana seedlings under water deficit studied by control of water potential in nutrient-agar media.

We have characterized the growth responses of Arabidopsis thaliana seedlings to water deficit. To manipulate the water potential, we developed a method whereby the nutrient-agar medium could be supplemented with polyethylene glycol (PEG 8000); PEG was introduced into gelled media by diffusion, which produced media with water potential as low as -1.6 MPa. For dark-grown plants, hypocotyl growth had a hyperbolic dependence on water potential, and was virtually stopped by -1 MPa. In contrast, primary root elongation was stimulated by moderate deficit and even at -1.6 MPa was not significantly less than the control. That these results did not depend on a direct effect of PEG was attested by obtaining indistinguishable results when a dialysis membrane impermeable to PEG was placed between the medium and the seedlings. For light-grown seedlings, moderate deficit also stimulated primary root elongation and severe deficit reduced elongation only partially. These changes in elongation were paralleled by changes in root system dry weight. At moderate deficit, lateral root elongation and initiation were unaffected and at higher stress levels both were inhibited. Primary root diameter increased steadily with time in well-watered controls and under water deficit increased transiently before stabilizing at a diameter that was inversely proportional to the deficit. Along with stimulated primary root elongation, moderate water deficit also stimulated the rate of cell production. Thus, A. thaliana responds to water deficit vigorously, which enhances its use as a model to uncover mechanisms underlying plant responses to water deficit.

Agar↗
PubMed2000

Endogenous ABA maintains shoot growth in tomato independently of effects on plant water balance: evidence for an interaction with ethylene.

To examine whether the reduced shoot growth of abscisic acid (ABA)-deficient mutants of tomato is independent of effects on plant water balance, flacca and notabilis were grown under controlled-humidity conditions so that their leaf water potentials were equal to or higher than those of well-watered wild-type plants throughout development. Most parameters of shoot growth remained markedly impaired and root growth was also greatly reduced. Additional experiments with flacca showed that shoot growth substantially recovered when wild-type levels of ABA were restored by treatment with exogenous ABA, even though improvement in leaf water potential was prevented. The ability of applied ABA to increase growth was greatest for leaf expansion, which was restored by 75%. The ethylene evolution rate of growing leaves was doubled in flacca compared to the wild type and treatment with silver thiosulphate to inhibit ethylene action partially restored shoot growth. The results demonstrate that normal levels of endogenous ABA are required to maintain shoot development, particularly leaf expansion, in well-watered tomato plants, independently of effects on plant water balance. The impairment of shoot growth caused by ABA deficiency is at least partly attributable to ethylene.

Abscisic Acid↗
PubMed2000

Abscisic acid accumulation maintains maize primary root elongation at low water potentials by restricting ethylene production.

Previous work showed that primary root elongation in maize (Zea mays L.) seedlings at low water potentials (psi(w)) requires the accumulation of abscisic acid (ABA) (R.E. Sharp, Y. Wu, G.S. Voetberg, I.N. Saab, M.E. LeNoble [1994] J Exp Bot 45: 1743-1751). The objective of the present study was to determine whether the inhibition of elongation in ABA-deficient roots is attributable to ethylene. At a psi(w) of -1.6 MPa, inhibition of root elongation in dark-grown seedlings treated with fluridone to impose ABA deficiency was largely prevented with two inhibitors of ethylene synthesis (aminooxyacetic acid and aminoethoxyvinylglycine) and one inhibitor of ethylene action (silver thiosulfate). The fluridone treatment caused an increase in the rate of ethylene evolution from intact seedlings. This effect was completely prevented with aminooxyacetic acid and also when ABA was supplied at a concentration that restored the ABA content of the root elongation zone and the root elongation rate. Consistent results were obtained when ABA deficiency was imposed using the vp5 mutant. Both fluridone-treated and vp5 roots exhibited additional morphological symptoms of excess ethylene. The results demonstrate that an important role of ABA accumulation in the maintenance of root elongation at low psi(w) is to restrict ethylene production.

Abscisic Acid↗
PubMed1999

Effect of inhibitors on the ubiquinone binding capacity of the primary energy conversion site in the Rhodobacter capsulatus cytochrome bc(1) complex.

A key issue concerning the primary conversion (Q(O)) site function in the cytochrome bc(1) complex is the stoichiometry of ubiquinone/ubihydroquinone occupancy. Previous evidence suggests that the Q(O) site is able to accommodate two ubiquinone molecules, the double occupancy model [Ding, H., Robertson, D. E., Daldal, F., and Dutton, P. L. (1992) Biochemistry 31, 3144-3158]. In the recently reported crystal structures of the cytochrome bc(1) complex, no electron density was identified in the Q(O) site that could be ascribed to ubiquinone. To provide further insight into this issue, we have manipulated the cytochrome bc(1) complex Q(O) site occupancy in photosynthetic membranes from Rhodobacter capsulatus by using inhibitor titrations and ubiquinone extraction to modulate the amount of ubiquinone bound in the site. The nature of the Q(O) site occupants was probed via the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectra to modulation of Q(O) site occupancy. Diphenylamine (DPA) and methoxyacrylate (MOA)-stilbene are known Q(O) site inhibitors of the cytochrome bc(1) complex. Addition of stoichiometric concentrations of MOA-stilbene or excess DPA to cytochrome bc(1) complexes with natural levels of ubiquinone elicits the same change in the [2Fe-2S] cluster EPR spectra; the g(x)() resonance broadens and shifts from 1. 800 to 1.783. This is exactly the same signal as that obtained when there is only one ubiquinone present in the Q(O) site. Furthermore, addition of MOA-stilbene or DPA to the cytochrome bc(1) complex depleted of ubiquinone does not alter the [2Fe-2S] cluster EPR spectral line shapes, which remain indicative of one ubiquinone or zero ubiquinones in the Q(O) site, with broad g(x)() resonances at 1. 783 or 1.765, respectively. The results are quite consistent with the Q(O) site double occupancy model, in which MOA-stilbene and DPA inhibit by displacing one, but not both, of the Q(O) site ubiquinones.

Diphenylamine↗
PubMed1999

Mechanisms for regulating electron transfer in multi-centre redox proteins.

Protein-mediated electron transfer is a key process in nature. Many of the proteins involved in such electron transfers are complex and contain a number of redox-active cofactors. The very complexity of these multi-centre redox proteins has made it difficult to fully understand the various electron transfer events they catalyse. This is sometimes because the electron transfer steps themselves are gated or coupled to other processes such as proton transfer. However, with the molecular structures of many of these proteins now available it is possible to probe these electron transfer reactions at the molecular level. It is becoming apparent that many of these multi-centre redox proteins have rather subtle and elegant ways for regulating electron transfer. The purpose of this article is to illustrate how nature has used different approaches to control electron transfer in a number of different systems. Illustrative examples include: thermodynamic control of electron transfer in flavocytochromes b(2) and P450 BM3; a novel control mechanism involving calmodulin-binding-dependent electron transfer in neuronal nitric oxide synthase; the probable gating of electron transfer by ATP hydrolysis in nitrogenase; conformational gating of electron transfer in cytochrome cd(1); the regulation of electron transfer by protein dynamics in the cytochrome bc(1) complex; and finally the coupling of electron transfer to proton transfer in cytochrome c oxidase.

Animals↗
PubMed1999

Ubiquinone binding capacity of the Rhodobacter capsulatus cytochrome bc1 complex: effect of diphenylamine, a weak binding QO site inhibitor.

Diphenylamine (DPA), a known inhibitor of polyene and isoprene biosynthesis, is shown to inhibit flash-activatable electron transfer in photosynthetic membranes of Rhodobacter capsulatus. DPA is specific to the QO site of ubihydroquinone:cytochrome c oxidoreductase, where it inhibits not only reduction of the [2Fe-2S]2+ cluster in the FeS subunit and subsequent cytochrome c reduction but also heme bL reduction in the cytochrome b subunit. In both cases, the kinetic inhibition constant (Ki) is 25 +/- 10 microM. A novel aspect of the mode of action of DPA is that complete inhibition is established without disturbing the interaction between the reduced [2Fe-2S]+ cluster and the QO site ubiquinone complement, as observed from the electron paramagnetic resonance (EPR) spectral line shape of the reduced [2Fe-2S] cluster, which remained characteristic of two ubiquinones being present. These observations imply that DPA is behaving as a noncompetitive inhibitor of the QO site. Nevertheless, at higher concentrations (>10 mM), DPA can interfere with the QO site ubiquinone occupancy, leading to a [2Fe-2S] cluster EPR spectrum characteristic of the presence of only one ubiquinone in the QO site. Evidently, DPA can displace the more weakly bound of the two ubiquinones in the site, but this is not requisite for its inhibiting action.

Binding Sites↗
PubMed1999

Primary steps in the energy conversion reaction of the cytochrome bc1 complex Qo site.

The primary energy conversion (Qo) site of the cytochrome bc1 complex is flanked by both high- and low-potential redox cofactors, the [2Fe-2S] cluster and cytochrome bL, respectively. From the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectral g(x)-band and line shape to the degree and type of Qo site occupants, we have proposed a double-occupancy model for the Qo site by ubiquinone in Rhodobacter capsulatus membrane vesicles containing the cytochrome bc1 complex. Biophysical and biochemical experiments have confirmed the double occupancy model and from a combination of these results and the available cytochrome bc1 crystal structures we suggest that the two ubiquinone molecules in the Qo site serve distinct catalytic roles. We propose that the strongly bound ubiquinone, termed Qos, is close to the [2Fe-2S] cluster, where it remains tightly associated with the Qo site during turnover, serving as a catalytic cofactor; and the weaker bound ubiquinone, Qow, is distal to the [2Fe-2S] cluster and can exchange with the membrane Qpool on a time scale much faster than the turnover, acting as the substrate. The crystallographic data demonstrates that the FeS subunit can adopt different positions. Our own observations show that the equilibrium position of the reduced FeS subunit is proximal to the Qo site. On the basis of this, we also report preliminary results modeling the electron transfer reactions that can occur in the cytochrome bc1 complex and show that because of the strong distance dependence of electron transfer, significant movement of the FeS subunit must occur in order for the complex to be able to turn over at the experimental observed rates.

Animals↗
PubMed1998

Design, synthesis, and characterization of a photoactivatable flavocytochrome molecular maquette.

We report the construction of a synthetic flavo-heme protein that incorporates two major physiological activities of flavoproteins: light activation of flavin analogous to DNA photolyase and rapid intramolecular electron transfer between the flavin and heme cofactors as in several oxidoreductases. The functional tetra-alpha-helix protein comprises two 62-aa helix-loop-helix subunits. Each subunit contains a single cysteine to which flavin (7-acetyl-10-methylisoalloxazine) is covalently attached and two histidines appropriately positioned for bis-his coordination of heme cofactors. Both flavins and hemes are situated within the hydrophobic core of the protein. Intramolecular electron transfer from flavosemiquinone generated by photoreduction from a sacrificial electron donor in solution was examined between protoporphyrin IX and 1-methyl-2-oxomesoheme XIII. Laser pulse-activated electron transfer from flavin to meso heme occurs on a 100-ns time scale, with a favorable free energy of approximately -100 meV. Electron transfer from flavin to the lower potential protoporphyrin IX, with an unfavorable free energy, can be induced after a lag phase under continuous light illumination. Thus, the supporting peptide matrix provides an excellent framework for the positioning of closely juxtaposed redox groups capable of facilitating intramolecular electron transfer and begins to clarify in a simplified and malleable system the natural engineering of flavoproteins.

Amino Acid Sequence↗
PubMed1998

Non-inhibiting perturbation of the primary energy conversion site (Qo site) in Rhodobacter capsulatus ubihydroquinone: cytochrome c oxidoreductase (cytochrome bc1 complex).

Ethanol added to Rhodobacter capsulatus chromatophore membranes containing the cytochrome bc1 complex effectively uncouples the sensitivity of the [2Fe-2S] cluster EPR spectrum to the number and redox state of ubiquinone/ubihydroquinone within the Qo site. Ethanol has no effect upon the rate of catalysis, leading to a non-inhibiting perturbation of cytochrome bc1 function. We suggest that displacement occurs by ethanol acting from the aqueous phase to successfully compete with the Qo site ubiquinones and water to hydrogen bond the N(epsilon)H atom(s) of the coordinating [2Fe-2S] cluster histidines.

Binding Sites↗
PubMed1996

Modulation of flavocytochrome b2 intraprotein electron transfer via an interdomain hinge region.

The two domains of flavocytochrome b2 are connected by a typical hinge peptide. To probe the role of the hinge in modulating the efficiency of intraprotein electron transfer between these two domains, a number of mutant enzymes with truncated hinge regions were previously constructed and characterized [Sharp, Chapman and Reid (1996) Biochemistry 35, 891-899]. In the present study two mutant enzymes with elongated hinge regions have been constructed (HI3 and HI6) to further our understanding of the controlling influence of hinge length and primary structure on intraprotein electron transfer. Modification of the hinge had little effect on the lactate dehydrogenase activity of the enzyme, as was evident from steady-state experiments using ferricyanide as electron acceptor and from pre-steady-state experiments monitoring flavin reduction. However, the hinge insertions lowered the enzyme's effectiveness as a cytochrome c reductase. This effect results from a defect at the first interdomain electron-transfer step (FMNH2 --> haem electron transfer), where the rate constants for haem reduction in HI3 and HI6 were 50- and 100-fold lower than the corresponding value for the wild-type enzyme. Preservation of structural integrity within the hinge region is apparently essential for efficient intraprotein electron transfer.

Amino Acid Sequence↗
PubMed1996

Interaction of cytochrome c with flavocytochrome b2.

Flavocytochrome b2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction. At 25 degrees C, 0.10 M ionic strength, and saturating L-lactate concentration, the turnover rate is 207 s-1 [per cytochrome c reduced; Miles, C. S., Rouviere, N., Lederer, F., Mathews, F. S., Reid, G. A., Black, M. T., & Chapman, S. K. (1992) Biochem. J. 285, 187-192]. The second-order rate constant for cytochrome c reduction in the pre-steady-state has been determined by stopped-flow spectrophotometry to be 34.8 (+/- 0.9) muM-1 s-1 in the presence of 10 mM L-lactate. This rate constant has been found to be dependent entirely on the rate of complex formation, the electron-transfer rate in the pre-formed complex being in excess of 1000 s-1. Inhibition of the pre-steady-state reduction of cytochrome c by either zinc-substituted cytochrome c or ferrocytochrome c has led to the estimation of a Kd for the catalytically competent complex of 8 microM, and from this the dissociation rate constant of 280 s-1, a value much less than the actual electron-transfer rate. The inhibition observed is only partial which indicates that electron transfer from the 1:1 complex to another cytochrome c can occur and that alternative electron transfer sites exist. The cytochrome c binding site proposed by Tegoni et al. [Tegoni, M., White, S. A., Roussel, A., Mathews, F. S. & Cambillau, C. (1993) Proteins 16, 408-422] has been tested using site-directed mutagenesis. Mutations designed to affect the complex stability and putative electron-transfer pathway had little effect, suggesting that the primary cytochrome c binding site on flavocytochrome b2 lies elsewhere. The combination of tight binding and multiple electron-transfer sites gives flavocytochrome b2 a low K(m) and a high kcat, maximizing its catalytic efficiency. In the steady-state, the turnover rate is therefore largely limited by other steps in the catalytic cycle, a conclusion which is discussed in the preceding paper in this issue [Daff, S., Ingledew, W. J., Reid, G. A., & Chapman, S. K. (1996) Biochemistry 35, 6345-6350].

Base Sequence↗
PubMed1996

Deletions in the interdomain hinge region of flavocytochrome b2: effects on intraprotein electron transfer.

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase, EC 1.1.2.3) are connected by a typical hinge peptide. To probe the importance of the structural integrity of the hinge region for efficient intraprotein electron transfer, three mutant enzymes have been constructed: H delta 3 [Sharp, R. E., White, P., Chapman, S. K., & Reid, G. A. (1994) Biochemistry 33, 5115-5120], H delta 6, and H delta 9 in which three, six, and nine amino acids, respectively, were deleted from the hinge region. Intraprotein electron transfer was investigated by steady-state and stopped-flow kinetic analyses. All three hinge-deletion enzymes remained good L-lactate dehydrogenases, as was evident from steady-state experiments with ferricyanide as the electron acceptor and from stopped-flow experiments monitoring flavin reduction. The global effect of these deletions is to lower the enzyme's effectiveness as a cytochrome c reductase. This property of H delta 6 and H delta 9 flavocytochromes b2 is manifested at the first interdomain electron-transfer step (fully reduced FMN-->heme electron transfer), where the rate of heme reduction is the same within experimental error as the steady-state rate of cytochrome c reduction. Thus, interdomain electron transfer is rate limiting in the case of these two hinge-deletion enzymes compared to the wild-type enzyme, where alpha H abstraction from C-2 of L-lactate still contributes substantially to rate limitation. The situation for H delta 3 is more complicated, with more than one interdomain electron-transfer step being affected. Kinetic data, along with the measured deuterium kinetic isotope effects, are discussed in the context of the flavocytochrome b2 catalytic cycle and show that complete structural integrity within the hinge region is essential for efficient interdomain communication.

Amino Acid Sequence↗