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Biomedical subjects

R E Sharp

Publications and source records attributed to R E Sharp.

At least 37 records · Page 2Linked to original sources
PubMed1996

Growth maintenance of the maize primary root at low water potentials involves increases in cell-wall extension properties, expansin activity, and wall susceptibility to expansins.

Previous work on the growth biophysics of maize (Zea mays L.) primary roots suggested that cell walls in the apical 5 mm of the elongation zone increased their yielding ability as an adaptive response to low turgor and water potential (psi w). To test this hypothesis more directly, we measured the acid-induced extension of isolated walls from roots grown at high (-0.03 MPa) or low (-1.6 MPa) psi w using an extensometer. Acid-induced extension was greatly increased in the apical 5 mm and was largely eliminated in the 5- to 10-mm region of roots grown at low psi w. This pattern is consistent with the maintenance of elongation toward the apex and the shortening of the elongation zone in these roots. Wall proteins extracted from the elongation zone possessed expansin activity, which increased substantially in roots grown at low psi w. Western blots likewise indicated higher expansin abundance in the roots at low psi w. Additionally, the susceptibility of walls to expansin action was higher in the apical 5 mm of roots at low psi w than in roots at high psi w. The basal region of the elongation zone (5-10 mm) did not extend in response to expansins, indicating that loss of susceptibility to expansins was associated with growth cessation in this region. Our results indicate that both the increase in expansin activity and the increase in cell-wall susceptibility to expansins play a role in enhancing cell-wall yielding and, therefore, in maintaining elongation in the apical region of maize primary roots at low psi w.

Cell Wall↗
PubMed1995

Mutation to glutamine of histidine 373, the catalytic base of flavocytochrome b2 (L-lactate dehydrogenase).

Flavocytochrome b2 catalyzes the two-electron oxidation of L-lactate. Reducing equivalents are transferred first to FMN then to heme b2 in the same subunit, finally to cytochrome c or a non-physiological acceptor. The enzyme's three-dimensional structure, when analyzed in the light of existing mechanistic knowledge, suggested that His 373 is the active site base which initiates the substrate chemical transformation by abstracting the lactate alpha-proton. We report here the properties of a mutant enzyme with glutamine substituted histidine at position 373. The mutated enzyme preparations show a 10(4)-fold decrease in catalytic activity. We find that most of this residual activity can be eliminated by treatments with: 1) fluoropyruvate, an affinity label for His 373; and 2) 2- hydroxy-3-butynoate, a suicide reagent which normally forms an adduct with FMN but in this case leaves the bulk of the prosthetic group intact. Furthermore, although spectral titrations do not detect any binding of oxalate, this reagent inhibits the mutant enzyme with the same kinetic behaviour as for the wild-type enzyme. We conclude that the enzyme preparations contain about 1 in 10(4) molecules of wild-type flavocytochrome b2; this is probably due to codon misreading during biosynthesis. Thus the H373Q enzyme displays at most 10(5)-fold less activity than the wild-type enzyme. We report values for the spectrally determined binding constants of sulfite, pyruvate and D-lactate for the mutant enzyme. Finally, we show that 2,6-dichlorophenol indophenol, which is a 10-fold more sensitive routine electron acceptor than ferricyanide, accepts electrons only from heme b2 and not from the flavin.

2,6-Dichloroindophenol↗
PubMed1994

Role of the interdomain hinge of flavocytochrome b2 in intra- and inter-protein electron transfer.

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase, EC 1.1.2.3) are connected by a hinge peptide. Kinetics experiments [White, P., Manson, F. D. C., Brunt, C. E., Chapman, S. K., & Reid, GA. (1993) Biochem. J. 291, 89-94] have illustrated the importance for efficient interdomain electron transfer of maintaining the structural integrity of the hinge. To probe the role of the hinge in a more subtle manner, we have constructed a mutant enzyme, H delta 3, which has a three amino acid deletion in the hinge region. Intra- and inter-protein electron transfer within H delta 3 flavocytochrome b2 and the H delta 3:cytochrome c redox complex was investigated by steady-state and stopped-flow kinetics analysis. The H delta 3 mutant enzyme remains a good L-lactate dehydrogenase, as is evident from steady-state experiments with ferricyanide as electron acceptor (40% less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (15% less active than wild-type enzyme). The global effect of the deletion is to lower the enzyme's effectiveness as a cytochrome c reductase. This property of the H delta 3 enzyme is manifested at two electron-transfer steps on the catalytic cycle of flavocytochrome b2. First, the rate of heme reduction has fallen 5-fold in H delta 3 compared with the wild-type enzyme (from 445 to 91 s-1), due to poor interdomain electron transfer from flavin to heme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Effect of inhibition of abscisic Acid accumulation on the spatial distribution of elongation in the primary root and mesocotyl of maize at low water potentials.

Previous work showed that accumulation of endogenous abscisic acid (ABA) acts both to maintain primary root growth and inhibit shoot growth in maize seedlings at low water potentials (psi(w)) (IN Saab, RE Sharp, J Pritchard, GS Voetberg [1990] Plant Physiol 93: 1329-1336). In this study, we have characterized the growth responses of the primary root and mesocotyl of maize (Zea mays L. cv FR27 x FRMo 17) to manipulation of ABA levels at low psi(w) with a high degree of spatial resolution to provide the basis for studies of the mechanism(s) of ABA action. In seedlings growing at low psi(w) and treated with fluridone to inhibit carotenoid (and ABA) biosynthesis, ABA levels were decreased in all locations of the root and mesocotyl growing zones compared with untreated seedlings growing at the same psi(w). In the root, low psi(w) (-1.6 megapascals) caused a shortening of the growing zone, as reported previously. The fluridone treatment was associated with severe inhibition of root elongation rate, which resulted from further shortening of the growing zone. In the mesocotyl, low psi(w) (-0.3 megapascal) also resulted in a shortened growing zone. In contrast with the primary root, however, fluridone treatment prevented most of the inhibition of elongation and the shortening of the growing zone. Final cell length measurements indicated that the responses of both root and mesocotyl elongation to ABA manipulation at low psi(w) involve large effects on cell expansion. Measurements of the relative changes in root and shoot water contents and dry weights after transplanting to a psi(w) of -0.3 megapascal showed that the maintenance of shoot elongation in fluridone-treated seedlings was not attributable to increased water or seed-reserve availability resulting from inhibition of root growth. The results suggest a developmental gradient in tissue responsiveness to endogenous ABA in both the root and mesocotyl growing zones. In the root, the capacity for ABA to protect cell expansion at low psi(w) appears to decrease with increasing distance from the apex. In the mesocotyl, in contrast, the accumulation of ABA at low psi(w) appears to become increasingly inhibitory to expansion as cells are displaced away from the meristematic region.

Journal Article↗
PubMed1992

Peroxidase activity in the leaf elongation zone of tall fescue : I. Spatial distribution of ionically bound peroxidase activity in genotypes differing in length of the elongation zone.

Cessation of cell expansion has been associated with cell wall cross-linking reactions catalyzed by peroxidase. This study utilized two genotypes of tall fescue (Festuca arundinacea Schreb.) that differ in length of the leaf elongation zone to investigate the relationship between ionically bound peroxidase activity and the spatial distribution of leaf elongation. Peroxidase activity was also localized histochemically in transverse sections of the leaf blade using 3,3' -diaminobenzidine. Soluble or soluble plus ionically bound peroxidase activities were extracted from homogenized segments of the elongating leaf blade and assayed spectrophotometrically. Activity of the ionically bound fraction, expressed per milligram fresh weight or per microgram protein, increased as cells were displaced through the distal half of the elongation zone, corresponding to the region in which the elongation rate declined. In both genotypes, the initial increase in activity preceded the onset of growth deceleration by about 10 hours. In the basal region where elongation began, histochemical localization showed that peroxidase activity was found only in vascular tissues. As cells were displaced farther through the elongation zone, peroxidase activity appeared in walls of other longitudinally continuous tissues such as the epidermis and bundle sheaths. Increase in ionically bound peroxidase activity and changes in localization of peroxidase activity occurred at comparable developmental stages in the two genotypes. The results indicate that cessation of elongation followed an increase in cell wall peroxidase activity.

Journal Article↗
PubMed1992

Peroxidase Activity in the Leaf Elongation Zone of Tall Fescue : II. Spatial Distribution of Apoplastic Peroxidase Activity in Genotypes Differing in Length of the Elongation Zone.

Previous work suggested that cell wall peroxidase activity increased as cells were displaced through the elongation zone in leaf blades of tall fescue (Festuca arundinacea Schreb.). In this study, two genotypes that differ in length of the elongation zone were used to examine the relationship between peroxidase activity in apoplastic fluid of intact leaf blade segments and the spatial distribution of leaf growth. Apoplastic fluid was extracted by vacuum infiltration and centrifugation, and peroxidase activity was assayed spectrophotometrically. Isoelectric focusing was used to characterize the isoforms of apoplastic peroxidase within the region of elongation and in the region of secondary cell wall deposition, which is distal to the elongation zone. A striking correlation was found in each genotype between both the location and timing of increase in apoplastic peroxidase activity and the onset of growth deceleration. Only cationic isoforms of apoplastic peroxidase could be identified in the elongation zone, whereas additional anionic isoforms appeared in the region of secondary cell wall deposition. We conclude that cessation of elongation growth in tall fescue leaf blades is likely to be related to the secretion of cationic isoforms of peroxidase into the cell wall.

Journal Article↗
PubMed1991

Spatial distribution of turgor and root growth at low water potentials.

Spatial distributions of turgor and longitudinal growth were compared in primary roots of maize (Zea mays L. cv FR27 x FRMo 17) growing in vermiculite at high (-0.02 megapascals) or low (-1.6 megapascals) water potential. Turgor was measured directly using a pressure probe in cells of the cortex and stele. At low water potential, turgor was greatly decreased in both tissues throughout the elongation zone. Despite this, longitudinal growth in the apical 2 millimeters was the same in the two treatments, as reported previously. These results indicate that the low water potential treatment caused large changes in cell wall yielding properties that contributed to the maintenance of root elongation. Further from the apex, longitudinal growth was inhibited at low water potential despite only slightly lower turgor than in the apical region. Therefore, the ability to adjust cell wall properties in response to low water potential may decrease with cell development.

Journal Article↗
PubMed1991

Growth of the Maize Primary Root at Low Water Potentials : III. Role of Increased Proline Deposition in Osmotic Adjustment.

Seedlings of maize (Zea mays L. cv WF9 x Mo 17) growing at low water potentials in vermiculite contained greatly increased proline concentrations in the primary root growth zone. Proline levels were particularly high toward the apex, where elongation rates have been shown to be completely maintained over a wide range of water potentials. Proline concentration increased even in quite mild treatments and reached 120 millimolal in the apical millimeter of roots growing at a water potential of -1.6 megapascal. This accounted for almost half of the osmotic adjustment in this region. Increases in concentration of other amino acids and glycinebetaine were comparatively small. We have assessed the relative contributions of increased rates of proline deposition and decreased tissue volume expansion to the increases in proline concentration. Proline content profiles were combined with published growth velocity distributions to calculate net proline deposition rate profiles using the continuity equation. At low water potential, proline deposition per unit length increased by up to 10-fold in the apical region of the growth zone compared to roots at high water potential. This response accounted for most of the increase in proline concentration in this region. The results suggest that osmotic adjustment due to increased proline deposition plays an important role in the maintenance of root elongation at low water potentials.

Journal Article↗
PubMed1990

Increased endogenous abscisic Acid maintains primary root growth and inhibits shoot growth of maize seedlings at low water potentials.

Roots of maize (Zea mays L.) seedlings continue to grow at low water potentials that cause complete inhibition of shoot growth. In this study, we have investigated the role of abscisic acid (ABA) in this differential growth sensitivity by manipulating endogenous ABA levels as an alternative to external applications of the hormone. An inhibitor of carotenoid biosynthesis (fluridone) and a mutant deficient in carotenoid biosynthesis (vp 5) were used to reduce the endogenous ABA content in the growing zones of the primary root and shoot at low water potentials. Experiments were performed on 30 to 60 hour old seedlings that were transplanted into vermiculite which had been preadjusted to water potentials of approximately -1.6 megapascals (roots) or -0.3 megapascals (shoots). Growth occurred in the dark at near-saturation humidity. Results of experiments using the inhibitor and mutant approaches were very similar. Reduced ABA content by either method was associated with inhibition of root elongation and promotion of shoot elongation at low water potentials, compared to untreated and wild-type seedlings at the same water potential. Elongation rates and ABA contents at high water potential were little affected. The inhibition of shoot elongation at low water potential was completely prevented in fluridone-treated seedlings during the first five hours after transplanting. The results indicate that ABA accumulation plays direct roles in both the maintenance of primary root elongation and the inhibition of shoot elongation at low water potentials.

Journal Article↗
PubMed1990

Growth of the Maize Primary Root at Low Water Potentials : II. Role of Growth and Deposition of Hexose and Potassium in Osmotic Adjustment.

Primary roots of maize (Zea mays L. cv WF9 x Mo17) seedlings growing in vermiculite at various water potentials exhibited substantial osmotic adjustment in the growing region. We have assessed quantitatively whether the osmotic adjustment was attributable to increased net solute deposition rates or to slower rates of water deposition associated with reduced volume expansion. Spatial distributions of total osmotica, soluble carbohydrates, potassium, and water were combined with published growth velocity distributions to calculate deposition rate profiles using the continuity equation. Low water potentials had no effect on the rate of total osmoticum deposition per unit length close to the apex, and caused decreased deposition rates in basal regions. However, rates of water deposition decreased more than osmoticum deposition. Consequently, osmoticum deposition rates per unit water volume were increased near the apex and osmotic potentials were lower throughout the growing region. Because the stressed roots were thinner, osmotic adjustment occurred without osmoticum accumulation per unit length. The effects of low water potential on hexose deposition were similar to those for total osmotica, and hexose made a major contribution to the osmotic adjustment in middle and basal regions. In contrast, potassium deposition decreased at low water potentials in close parallel with water deposition, and increases in potassium concentration were small. The results show that growth of the maize primary root at low water potentials involves a complex pattern of morphogenic and metabolic events. Although osmotic adjustment is largely the result of a greater inhibition of volume expansion and water deposition than solute deposition, the contrasting behavior of hexose and potassium deposition indicates that the adjustment is a highly regulated process.

Journal Article↗
PubMed1988

Growth of the maize primary root at low water potentials : I. Spatial distribution of expansive growth.

Seedlings of maize (Zea mays L. cv WF9 x Mo 17) were grown in vermiculite at various water potentials. The primary root continued slow rates of elongation at water potentials which completely inhibited shoot growth. To gain an increased understanding of the root growth response, we examined the spatial distribution of growth at various water potentials. Time lapse photography of the growth of marked roots revealed that inhibition of root elongation at low water potentials was not explained by a proportional decrease in growth along the length of the growing zone. Instead, longitudinal growth was insensitive to water potentials as low as - 1.6 megapascal close to the root apex, but was inhibited increasingly in more basal locations such that the length of the growing zone decreased progressively as the water potential decreased. Cessation of longitudinal growth occurred in tissue of approximately the same age regardless of spatial location or water status, however. Roots growing at low water potentials were also thinner, and analysis revealed that radial growth rates were decreased throughout the elongation zone, resulting in greatly decreased rates of volume expansion.

Journal Article↗
PubMed1987

Leaf magnesium alters photosynthetic response to low water potentials in sunflower.

We grew sunflower (Helianthus annuus L.) plants in nutrient solutions having nutritionally adequate but low or high Mg(2+) concentrations and determined whether photosynthesis was effected as leaf water potentials (psi(w)) decreased. Leaf Mg contents were 3- to 4-fold higher in the plants grown in high Mg(2+) concentrations (10 millimolar) than in those grown in low concentrations (0.25 millimolar). These contents were sufficient to support maximum growth, plant dry weight, and photosynthesis, and the plants appeared normal. As low psi(w) developed, photosynthesis was inhibited but moreso in high Mg leaves than in low Mg leaves. The effect was particularly apparent under conditions of light- and CO(2)-saturation, indicating that the chloroplast capacity to fix CO(2) was altered. The differential inhibition observed in leaves of differing Mg contents was not observed in leaves having differing K contents, suggesting that the effect may have been specific for Mg. Because Mg(2+) inhibits photophosphorylation and coupling factor activities at concentrations likely to occur as leaves dehydrate, Mg may play a role in the inhibition of chloroplast reactions at low psi(w), especially in leaves such as sunflower that markedly decrease in water content as psi(w) decreases.

Journal Article↗
PubMed1986

Photosynthesis at low water potentials in sunflower: lack of photoinhibitory effects.

The losses in chloroplast capacity to fix CO(2) when photosynthesis is reduced at low leaf water potential (psi(1)) have been proposed to result from photoinhibition. We investigated this possibility in soil-grown sunflower (Helianthus annuus L. cv IS894) using gas exchange techniques to measure directly the influence of light during dehydration on the in situ chloroplast capacity to fix CO(2). The quantum yield for CO(2) fixation as well as the rate of light- and CO(2)-saturated photosynthesis were strongly inhibited at low psi(1). The extent of inhibition was the same whether the leaves were exposed to high or to low light during dehydration. When intercellular partial pressures of CO(2) were decreased to the compensation point, which was lower than the partial pressures resulting from stomatal closure, the inhibition of the quantum yield was also unaffected. Photoinhibition could be observed only after high light exposures were imposed under nonphysiological low CO(2) and O(2) where both photosynthesis and photorespiration were suppressed. The experiments are the first to test whether gas exchange at low psi(1) is affected by potentially photoinhibitory conditions and show that the loss in chloroplast capacity to fix CO(2) was entirely the result of a direct effect of water availability on chloroplast function and not photoinhibition.

Journal Article↗
PubMed1984

Kok effect and the quantum yield of photosynthesis : light partially inhibits dark respiration.

The linear response of photosynthesis to light at low photon flux densities is known to change abruptly in the vicinity of the light compensation point so that the quantum yield seems to decrease as radiation increases. We studied this ;Kok effect' in attached sunflower (Helianthus annuus L. cv IS894) leaves using gas exchange techniques. The effect was present even though respiration was constant in the dark. It was observed at a similar photon flux density (7 to 11 micromole photons per square meter per second absorbed photosynthetically active radiation) despite a wide range of light compensation points as well as rates of photosynthesis. The effect was not apparent when photorespiration was inhibited at low pO(2) (1 kilopascal), but this result was complicated because dark respiration was quite O(2)-sensitive and was partially suppressed under these conditions. The Kok effect was observed at saturating pCO(2) and, therefore, could not be explained by a change in photorespiration. Instead, the magnitude of the effect varied as dark respiration varied in a single leaf, and was minimized when dark respiration was minimized, indicating that a partial suppression of dark respiration by light is responsible. Quantum yields measured at photon flux densities between 0 and 7 to 11 micromole photons per square meter per second, therefore, represent the combined yields of photosynthesis and of the suppression of a component of dark respiration by light. This leads to an overestimate of the quantum yield of photosynthesis. In view of these results, quantum yields of photosynthesis must be measured (a) when respiration is constant in the dark, and (b) when dark respiration has been inhibited either at low pO(2) to eliminate most of the light-induced suppression of dark respiration or at photon flux densities above that required to saturate the light-induced suppression of dark respiration. Significant errors in quantum yields of photosynthesis can result in leaves exhibiting this respiratory behavior if these principles are not followed.

Journal Article↗