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Biomedical subjects

I Zelitch

Publications and source records attributed to I Zelitch.

At least 19 recordsLinked to original sources
PubMed2001

Travels in a world of small science.

As a boy, I read Sinclair Lewis's Arrowsmithand dreamed of doing research of potential benefit to society. I describe the paths of my scientific career that followed. Several distinguished scientists served as my mentors and I present their profiles. Much of my career was in a small department at a small institution where independent researchers collaborated informally. I describe the unique method of carrying on research there. My curiosity about glycolate metabolism led to unraveling the enzymatic mechanism of the glycolate oxidase reaction and showing the importance of H(2)O(2) as a byproduct. I discovered enzymes catalyzing the reduction of glyoxylate and hydroxypyruvate. I found alpha-hydroxysulfonates were useful competitive inhibitors of glycolate oxidase. In a moment of revelation, I realized that glycolate metabolism was an essential part of photorespiration, a process that lowers net photosynthesis in C(3) plants. I added inhibitors of glycolate oxidase to leaves and showed: (1) glycolate was synthesized only in light as an early product of photosynthetic CO(2) assimilation, (2) the rate of glycolate oxidation consumed a sizable fraction of net photosynthesis in C(3) but not in C(4) plants, and (3) that glycolate metabolism increased greatly at higher temperatures. For a while I studied the control of stomatal opening in leaves, and this led to the finding that potassium ions are a key solute in guard cells. I describe experiments that show that when photorespiration rates are high, as occurs at higher temperatures, genetically increasing leaf catalase activity reduces photorespiration and increases net photosythetic CO(2) assimilation.

Journal Article↗
PubMed1998

Manipulation of catalase levels produces altered photosynthesis in transgenic tobacco plants.

Constructs containing the cDNAs encoding the primary leaf catalase in Nicotiana or subunit 1 of cottonseed (Gossypium hirsutum) catalase were introduced in the sense and antisense orientation into the Nicotiana tabacum genome. The N. tabacum leaf cDNA specifically overexpressed CAT-1, the high catalatic [corrected] form, activity. Antisense constructs reduced leaf catalase specific activities from 0.20 to 0.75 times those of wild type (WT), and overexpression constructs increased catalase specific activities from 1.25 to more than 2.0 times those of WT. The NADH-hydroxypyruvate reductase specific activity in transgenic plants was similar to that in WT. The effect of antisense constructs on photorespiration was studied in transgenic plants by measuring the CO2 compensation point (gamma) at a leaf temperature of 38 degrees C. A significant linear increase was observed in gamma with decreasing catalase (at 50% lower catalase activity gamma increased 39%). There was a significant temperature-dependent linear decrease in gamma in transgenic leaves with elevated catalase compared with WT leaves (at 50% higher catalase gamma decreased 17%). At 29 degrees C, gamma also decreased with increasing catalase in transgenic leaves compared with WT leaves, but the trend was not statistically significant. Rates of dark respiration were the same in WT and transgenic leaves. Thus, photorespiratory losses of CO2 were significantly reduced with increasing catalase activities at 38 degrees C, indicating that the stoichiometry of photorespiratory CO2 formation per glycolate oxidized normally increases at higher temperatures because of enhanced peroxidation.

Alcohol Oxidoreductases↗
PubMed1992

Factors affecting expression of enhanced catalase activity in a tobacco mutant with o(2)-resistant photosynthesis.

Tobacco (Nicotiana tabacum) mutants with 40 to 50% more catalase activity than wild type show O(2)-resistant photosynthesis under conditions of high photorespiration. More than 90% of the population of mutant plants of an M(7) and M(8) generation had enhanced catalase activity, and nearly 40% had activities >3 standard deviations above the mean of wild type. Superoxide dismutase activity was the same in mutant and wild-type leaves. The greater photosynthetic rate of mutant leaves previously observed in the laboratory was confirmed with field-grown plants that showed significantly higher rates (8%) than wild type during 8 days of measurements during a 19-day period of active growth. The tip region of expanding mutant leaves had higher catalase activity than the base of the lamina, and photosynthesis was O(2) resistant in 42% O(2) in the tip compared with the base, thus further supporting the hypothesis that there is a biochemical linkage between these traits. Plants grown in high light (270 micromole photons per square meter per second) had greater catalase activity and an activity ratio of mutant to wild type of 1.45 compared with 1.22 for those grown in low light (130 micromole photons per square meter per second). After acclimation for 3 weeks, plants transferred from low to high light showed increasing activities, and after 5 days the activity ratio of mutant to wild type was the same as in plants acclimated in higher light. The role of enhanced catalase activity in reducing photorespiratory CO(2) is discussed.

Journal Article↗
PubMed1991

Leaf catalase mRNA and catalase-protein levels in a high-catalase tobacco mutant with o(2)-resistant photosynthesis.

Experiments were conducted with a tobacco (Nicotiana tabacum) mutant with 40 to 50% greater catalase activity than wild type that is associated with a novel form of O(2)-resistant photosynthesis. The apparent K(m) for H(2)O(2) was the same in mutant and wild-type leaf extracts. Tobacco RNAs were hybridized with Nicotiana sylvestris catalase cDNA, and a threefold greater steady-state level of catalase mRNA was found in mutant leaves. Steady-state levels of ribulose-1,5-bisphosphate carboxylase small subunit mRNA were similar in mutant and wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified catalase showed that the protein concentration in the band corresponding to catalase was higher in the mutant than in the wild type. Separation of leaf catalase proteins by isoelectric focusing revealed the presence of five major bands and one minor band of activity. The distribution of the catalase activity among these forms was similar in mutant and wild type, although the total activity was higher in the mutant in all five major bands. The results indicate that the enhanced catalase activity in mutant leaves is caused by an increase in synthesis of catalase protein and that this trait is mediated at the nucleic acid level.

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PubMed1990

Further studies on o(2)-resistant photosynthesis and photorespiration in a tobacco mutant with enhanced catalase activity.

The increase in net photosynthesis in M(4) progeny of an O(2)-resistant tobacco (Nicotiana tabacum) mutant relative to wild-type plants at 21 and 42% O(2) has been confirmed and further investigated. Self-pollination of an M(3) mutant produced M(4) progeny segregating high catalase phenotypes (average 40% greater than wild type) at a frequency of about 60%. The high catalase phenotype cosegregated precisely with O(2)-resistant photosynthesis. About 25% of the F(1) progeny of reciprocal crosses between the same M(3) mutant and wild type had high catalase activity, whether the mutant was used as the maternal or paternal parent, indicating nuclear inheritance. In high-catalase mutants the activity of NADH-hydroxypyruvate reductase, another peroxisomal enzyme, was the same as wild type. The mutants released 15% less photorespiratory CO(2) as a percent of net photosynthesis in CO(2)-free 21% O(2) and 36% less in CO(2)-free 42% O(2) compared with wild type. The mutant leaf tissue also released less (14)CO(2) per [1-(14)C]glycolate metabolized than wild type in normal air, consistent with less photorespiration in the mutant. The O(2)-resistant photosynthesis appears to be caused by a decrease in photorespiration especially under conditions of high O(2) where the stoichiometry of CO(2) release per glycolate metabolized is expected to be enhanced. The higher catalase activity in the mutant may decrease the nonenzymatic peroxidation of keto-acids such as hydroxypyruvate and glyoxylate by photorespiratory H(2)O(2).

Journal Article↗
PubMed1990

Physiological investigations of a tobacco mutant with o(2)-resistant photosynthesis and enhanced catalase activity.

Experiments are described further indicating that O(2)-resistant photosynthesis observed in a tobacco (Nicotiana tabacum) mutant with enhanced catalase activity is associated with decreased photorespiration under conditions of high photorespiration relative to net photosynthesis. The effects on net photosynthesis of (a) increasing O(2) concentrations from 1% to 42% at low CO(2) (250 microliters CO(2) per liter), and (b) of increasing O(2) concentrations from 21% to 42% at high CO(2) (500 microliters CO(2) per liter) were investigated in M(6) progeny of mutant and wild-type leaf discs. The mutant displayed a progressive increase in net photosynthesis relative to wild type with increasing O(2) and the faster rate at 42% O(2) was completely reversed on returning to 21% O(2). The photosynthetic rate by the mutant was similar to wild type in 21% and 42% O(2) at 500 microliters CO(2) per liter, and a faster rate by the mutant was restored on returning to 250 microliters CO(2) per liter. The results are consistent with a lowered release of photorespiratory CO(2) by the mutant because greater catalase activity inhibits the chemical decarboxylation of alpha-keto acids by peroxisomal H(2)O(2). Higher catalase activity was observed in the tip and middle regions of expanding leaves than in the basal area. On successive selfing of mutant plants with enhanced catalase activity, the percent of plants with this phenotype increased from 60% in M(4) progeny to 85% in M(6) progeny. An increase was also observed in the percent of plants with especially high catalase activity (averaging 1.54 times wild type) on successive selfings suggesting that homozygosity for enhanced catalase activity was being approached.

Journal Article↗
PubMed1989

Selection and characterization of tobacco plants with novel o(2)-resistant photosynthesis.

Plants were obtained with novel O(2)-resistant photosynthetic characteristics. At low CO(2) (250-350 muL CO(2) L(-1)) and 30 degrees C when O(2) was increased from 1% to 21% to 42%, the ratio of net CO(2) uptake in O(2)-resistant whole plants or leaf discs compared to wild type increased progressively, and this was not related to stomatal opening. Dihaploid plantlets regenerated from anther culture were initially screened and selected for O(2)-resistant growth in 42% O(2)/160 muL CO(2) L(-1) and 0.18% of the plantlets showed O(2)-resistant photosynthesis. About 30% of the progeny (6 of 19 plants) of the first selfing of a fertile plant derived from a resistant dihaploid plant had O(2)-resistant photosynthesis, and after a second selfing this increased to 50% (6 of 12 plants). In 21% O(2) and low CO(2), net photosynthesis of the resistant plants was about 15% greater on a leaf area basis than wild type. Net photosynthesis was compared in leaf discs at 30 and 38 degrees C in 21% O(2), and at the higher temperature O(2)-resistant plants showed still greater photosynthesis than wild type. The results suggest that the O(2)-resistant photosynthesis described here is associated with a decreased stoichiometry of CO(2) release under conditions of rapid photorespiration. This view was supported by the finding that leaves of O(2)-resistant plants averaged 40% greater catalase activity than wild type.

Journal Article↗
PubMed1988

Cell position and light influence C4 versus C3 patterns of photosynthetic gene expression in maize.

C4 plants such as maize partition photosynthetic activities in two morphologically distinct cell types, bundle sheath (BS) and mesophyll (M), which lie as concentric layers around veins. We show that both light and cell position relative to veins influence C4 photosynthetic gene expression. A pattern of gene expression characteristic of C3 plants [ribulose bisphosphate carboxylase (RuBPCase) and light-harvesting chlorophyll a/b binding protein in all photosynthetic cells] is observed in leaf-like organs such as husk leaves, which are sparsely vascularized. This pattern of gene expression reflects direct fixation of CO2 in the C3 photosynthetic pathway, as determined by O2 inhibition assays. Light induces a switch from C3-type to C4-type gene expression patterns in all leaves, primarily in cells that are close to a vein. We propose that light causes repression of RuBPCase expression in M cells, by a mechanism associated with the vascular system, and that this is an essential step in the induction of C4 photosynthesis.

Cell Compartmentation↗
PubMed1988

Synthesis of Glycolate from Pyruvate via Isocitrate Lyase by Tobacco Leaves in Light.

Tobacco (Nicotiana tabacum var Havana Seed) leaf discs were supplied tracer quantities of [2-(14)C]- and [3-(14)C]pyruvate for 60 minutes in steady state photosynthesis with 21% or 1% O(2), and the glycolate oxidase inhibitor alpha-hydroxy-2-pyridinemethanesulfonic acid was then added for 5 or 10 minutes to cause glycolate to accumulate. The [3-(14)C]pyruvate was converted directly to glycolate as shown by a 50% greater than equallabeled (14)C in C-2 of glycolate, and the fraction of (14)C in C-2 increased in 1% O(2) to 80% greater than equal-labeled. This suggests the pathway using pyruvate is less O(2)-dependent than the oxygenase reaction producing glycolate from the Calvin cycle. The formation of glycolate from pyruvate in the leaf discs was time-dependent and with [2-(14)C]- and [3-(14)C]pyruvate supplied leaf discs the C-2 of glyoxylate derived from C-2 of isocitrate was labeled asymmetrically in a manner similar to the asymmetrical labeling of C-2 of glycolate under a number of conditions. Thus glycolate was probably formed by the reduction of glyoxylate. Isocitric lyase activity of tobacco leaves was associated with leaf mitochondria, though most of the activity was in the supernatant fraction after differential centrifugation of leaf homogenates. The total enzyme activity was at least 35 micromoles per gram fresh weight per hour. The relative contribution of the pathway to the glycolate pool is unknown, but the results support the existence of a sequence of reactions leading to glycolate synthesis during photosynthesis with pyruvate, isocitrate, and glyoxylate as intermediates.

Journal Article↗
PubMed1988

A Nuclear Mutation in Nicotiana sylvestris Causing a Thiamine-Reversible Defect in Synthesis of Chloroplast Pigments.

We report the recovery of a nuclear recessive mutation in Nicotiana sylvestris (Spegazzini and Comes) producing a conditional disruption in the pathway for synthesis of chlorophyll a and b and carotenoids which is fully reversible by exogenous thiamine (0.3 micromolar). In the absence of supplemental thiamine, chlorophyll levels declined by 50% after 5 days, and fell to undetectable levels by 11 days. Mitochondrial (KCN sensitive) respiration rates remained normal in albino leaves (80% loss of chlorophyll), suggesting that chlorosis results primarily from a deficiency of thiamine in the chloroplasts. After thiamine removal, mutant plants produced at least 10 albino leaves with a substantial capacity for growth (0-15 centimeters; 70-fold increase in area), demonstrating sustained operation of many cellular functions in spite of chloroplast disruption. Activities of the plastid isozymes of phosphoglucomutase and phosphoglucoisomerase in albino leaves indicated that the decline in pigment synthesis does not result from a general loss of metabolic activity in chloroplast. Plastid pyruvate dehydrogenase from mutant and wild-type plants displayed a similar affinity for thiamine pyrophosphate, showing that chlorosis does not result from an alteration in this enzyme. Growth of albino leaves and ultrastructural evidence for thylakoid membranes in the chloroplasts suggest that a certain level of fatty acid synthesis is maintained after the interruption of pigment synthesis. Since thiamine deprivation is expected to block production of acetyl-coenzyme A from pyruvate by pyruvate dehydrogenase, acetyl-coenzyme A supporting fatty acid synthesis in albino leaves may be derived solely from mitochondrial acetate.

Journal Article↗
PubMed1987

Effects of CO(2) and O(2) on Photosynthesis and Growth of Autotrophic Tobacco Callus.

Mean inhibition of net photosynthesis in autotrophic tobacco callus by 21 and 40% O(2) was 30 and 47%, respectively, similar to intact leaves. Increasing CO(2) concentrations (500-2000 microliters per liter) produced a steady decline in percent inhibition at both O(2) concentrations, indicating that O(2) inhibition resulted primarily from photorespiration. Net photosynthetic rate was plotted as a function of CO(2) concentration at 1, 21, and 40% O(2) for calculation of kinetic constants. Values for V(max) were similar at all O(2) concentrations (x = 5.27 mumol CO(2) per gram fresh weight per hour), indicating that O(2) inhibition of net photosynthesis was fully reversible by CO(2). To determine whether CO(2) and O(2) produced similar effects on growth, autotrophic callus was incubated for three weeks in atmospheres of normal air, high CO(2), high O(2) and high CO(2)/high O(2). Growth in high CO(2) was nearly double that in normal air. High O(2) decreased growth significantly relative to air, but growth in high CO(2)/high O(2) was similar to that in air. Lack of CO(2) reversal of growth inhibition by O(2) indicates that prolonged exposure to high O(2) results in toxicity arising from a nonphotorespiratory source. Growth of heterotrophic callus (2% sucrose), however, was not inhibited by 40 or 60% O(2), suggesting that O(2) toxicity in autotrophic callus results primarily from disruption of photosynthetic functions.

Journal Article↗
PubMed1985

Recognition of Superior Photosynthetic Efficiency in the Field Using the CO(2)-Depletion Technique.

The relationship between CO(2) exchange rate (CER) and growth of crops in the field was investigated in Connecticut Broadleaf tobacco (Nicotiana tabacum) using the CO(2)-depletion technique. A particular objective was to determine if modest (i.e. <10%) varietal differences could be distinguished in mean CER. Statistical analysis of numerous CER values obtained over a wide range of irradiances during the course of the season indicated that differences of as little as 7% in the mean CER between varieties would be significant (n approximately 400). The usefulness of the CO(2)-depletion technique in detecting modest differences in photosynthetic efficiency has thus been demonstrated. These results are discussed in relation to the prospects for introducing and detecting genetic traits which would diminish photorespiration and increase CER and growth.

Journal Article↗
PubMed1982

Altered glycine decarboxylation inhibition in isonicotinic Acid hydrazide-resistant mutant callus lines and in regenerated plants and seed progeny.

Isonicotinic acid hydrazide (INH), an inhibitor of the photorespiratory pathway blocking the conversion of glycine to serine and CO(2), has been used as a selective agent to obtain INH-resistant tobacco (Nicotiana tabacum) callus cells. Of 22 cell lines that were INH-resistant, none were different from wild-type cells in their ability to take up [(3)H]INH or to oxidize INH to isonicotinic acid. In 7 of the 22 cell lines, INH resistance was associated with decreased inhibition of NAD-dependent glycine decarboxylation activity in isolated mitochondrial preparations. In the cell line that was most extensively investigated (I 24), this biochemical phenotype (exhibiting a 3-fold higher K(i) with INH) was observed in leaf mitochondria of regenerated plants and of plants produced from them by self-fertilization. After crosses between resistant and sensitive plants, the decreased inhibition of glycine decarboxylation was observed among F(2) and backcross progeny only in those plants previously identified as INH-resistant by callus growth tests. In contrast, in siblings identified as INH-sensitive, glycine decarboxylation was inhibited by INH at the wild-type level. This demonstration of the transfer of an altered enzyme property from callus to regenerated plants and through seed progeny fulfills an important requirement for the use of somatic cell genetics to produce biochemical mutants of higher plants.

Journal Article↗
PubMed1982

Relationship between Net CO(2) Assimilation and Dry Weight Accumulation in Field-Grown Tobacco.

To assess the variability of net photosynthetic CO(2) exchange per unit leaf area and to construct budgets for stands of field-grown tobacco (Nicotiana tabacum, Connecticut Broadleaf), a number of short-time measurements were made on all available leaf positions on two varieties using a hand-held transparent chamber for conducting gas exchange measurements on leaves. Measurements of net CO(2) exchange were carried out on 18 separate days during a 35-day period, beginning 22 days after the seedlings were transplanted to the field. Gas exchange assays on leaves were conducted under ambient conditions of temperature and light intensity at all times of day. Solar radiation was monitored throughout the period, and losses of respiratory CO(2) from stems, roots, and leaves (in the dark) were estimated. A simple model was proposed to relate daily total CO(2) input to irradiance and total leaf area. The total leaf area was assumed to be a function of day number. Dark respiratory losses accounted for 41% to 47% of total CO(2) assimilation. Analysis of variance indicated that the two varieties were not significantly different in whole plant rate of CO(2) fixation per unit of leaf area. CO(2) input was closely associated with leaf area within each variety. Throughout the experiment, the difference between the two varieties in total leaf area per plant was the largest single factor in determining net CO(2) inputs. The cumulative dry weight increase for each variety was similar to the prediction of net dry matter input obtained by gas exchange measurements, thus confirming the close relationship between total plant net CO(2) assimilation and dry weight yield.

Journal Article↗
PubMed1980

Isolation and Characterization of Glycine Hydroxamate-resistant Cell Lines of Nicotiana tabacum.

Seven lines of haploid Nicotiana tabacum tissue culture selected for resistance to normally toxic levels of the glycine analog glycine hydroxamate, a competitive inhibitor of the glycine decarboxylase reaction, were investigated. The presence of glycine hydroxamate greatly increased the intracellular concentration of both glycine and alanine in wild type and resistant cell lines, suggesting that the inhibitor blocks both glycine- and alanine-utilizing reactions. All the resistant cell lines, whether grown in the presence or absence of glycine hydroxamate, had high intracellular concentrations of the 12 free amino acids which were analyzed, including glycine and serine. (These lines averaged 3.6 times the total amino acid content of wild-type cells in the absence of the inhibitor). The resistant cell lines were indistinguishable from wild-type cell lines in their metabolism of radioactively labeled glycine hydroxamate and glycine. Comparison of the metabolism of radioactively labeled alanine, glycolate, and glyoxylate in wild-type and alpha resistant line also revealed no distinctive differences. Glycine decarboxylase activities were unaltered in the resistant cell lines. The cellular toxicity of glycine hydroxamate is considered in relation to (1) the competitive inhibition by glycine hydroxamate of the glycine- and alanine-utilizing enzymes and (2) the resultant imbalances caused by high intracellular concentrations of these amino acids. The significance of elevation of total free amino acid concentration in effecting resistance to the inhibitor is discussed.Plants were regenerated from 5 of these lines and callus cultures of explants were tested for glycine hydroxamate resistance. Plants from seedlings of two lines which retained the resistant characteristic in explanted callus did not have high amino acid levels in leaves.

Journal Article↗
PubMed1979

Inhibition of glycine decarboxylation and serine formation in tobacco by glycine hydroxamate and its effect on photorespiratory carbon flow.

Glycine hydroxamate is a competitive inhibitor of glycine decarboxylation and serine formation (referred to as glycine decarboxylase activity) in particulate preparations obtained from both callus and leaf tissue of tobacco. In preparations from tobacco callus tissues, the K(i) for glycine hydroxamate was 0.24 +/- 0.03 millimolar and the K(m) for glycine was 5.0 +/- 0.5 millimolar. The inhibitor was chemically stable during assays of glycine decarboxylase activity, but reacted strongly when incubated with glyoxylate. Glycine hydroxamate blocked the conversion of glycine to serine and CO(2)in vivo when callus tissue incorporated and metabolized [1-(14)C]glycine, [1-(14)C]glycolate, or [1-(14)C]glyoxylate. The hydroxamate had no effect on glyoxylate aminotransferase activities in vivo, and the nonenzymic reaction between glycine hydroxamate and glyoxylate did not affect the flow of carbon in the glycolate pathway in vivo. Glycine hydroxamate is the first known reversible inhibitor of the photorespiratory conversion of glycine to serine and CO(2).

Journal Article↗