Israel Zelitch

The Close Relationship Between Net Photosynthesis and Crop Yield

Crop yield is closely related to the net photosynthetic assimilation of CO2 throughout an entire season, but instantaneous measurements of photosynthesis may be misleading. Increasing the rates of net photosynthesis and translocation and enlarging the storage capacity by selection and breeding may bring about large increases in yield, especially in C3 species. (Accepted for publication 23 April 1982)

High Glycolate Oxidase Activity Is Required for Survival of Maize in Normal Air

A mutant in the maize (Zea mays) Glycolate Oxidase1 (GO1) gene was characterized to investigate the role of photorespiration in C4 photosynthesis. An Activator-induced allele of GO1 conditioned a seedling lethal phenotype when homozygous and had 5% to 10% of wild-type GO activity. Growth of seedlings in high CO2 (1%–5%) was sufficient to rescue the mutant phenotype. Upon transfer to normal air, the go1 mutant became necrotic within 7 d and plants died within 15 d. Providing [1-14C]glycolate to leaf tissue of go1 mutants in darkness confirmed that the substrate is inefficiently converted to 14CO2, but both wild-type and GO-deficient mutant seedlings metabolized [1-14C]glycine similarly to produce [14C]serine and 14CO2 in a 1:1 ratio, suggesting that the photorespiratory pathway is otherwise normal in the mutant. The net CO2 assimilation rate in wild-type leaves was only slightly inhibited in 50% O2 in high light but decreased rapidly and linearly with time in leaves with low GO. When go1 mutants were shifted from high CO2 to air in light, they accumulated glycolate linearly for 6 h to levels 7-fold higher than wild type and 11-fold higher after 25 h. These studies show that C4 photosynthesis in maize is dependent on photorespiration throughout seedling development and support the view that the carbon oxidation pathway evolved to prevent accumulation of toxic glycolate.

The photooxidation of glyoxylate by envelope-free spinach chloroplasts and its relation to photorespiration

Abstract Large quantities of CO2 are released within many photosynthesizing tissues in the light by the process of photorespiration. This CO2 arises largely from the carboxylcarbon atom of glycolate, which is synthesized in chloroplasts during photosynthesis. Glyoxylate is then produced by the glycolate oxidase reaction. The glyoxylate may be directly decarboxylated to CO2, but some investigators believe the glyoxylate must first be converted to glycine before CO2 is released during photorespiration. Spinach chloroplasts with their envelope membranes removed in dilute buffer solution have now been shown to carry out the oxidative decarboxylation of [1-14C]glyoxylate, in the presence of light and manganous ions in an atmosphere containing oxygen, to yield 1 mole each of 14CO2 and formate. Rates of enzymatic decarboxylation exceeding 50 μmoles of 14CO2 mg chlorophyll−1 hr−1 were obtained at pH 7.6; hydrogen peroxide is probably the oxidant in the reaction. Heated chloroplasts are inactive under the standard conditions and there is an almost absolute requirement for each of the components listed above. Conditions for some other nonenzymatic decarboxylations of glyoxylate have also been described. [1-14C]Glycine is decarboxylated by the enzymatic system at only 1% of the rate of [1-14C]glyoxylate. Maize chloroplast preparations are much less active than spinach chloroplasts. The high rates of CO2 produced by the spinach system directly from glyoxylate, as well as the need for light and oxygen, suggest that this reaction functions in photorespiration, and that CO2 arises during photorespiration without glycine as a mandatory intermediate.

The effect of glycidate, an inhibitor of glycolate synthesis, on photorespiration and net photosynthesis

Abstract Glycolate synthesis was inhibited 40–50% in illuminated tobacco leaf disks, which have rapid rates of photorespiration, when floated on 20 m m potassium glycidate (2,3-epoxypropionate), an epoxide similar in structure to glycolate. The inhibitor also decreased the release of photorespiratory CO 2 about 40%, and the specificity of glycidate was demonstrated by the 40–50% increase in rate of photosynthetic CO 2 uptake observed in its presence. The importance of glycolate synthesis and metabolism in the production of photorespiratory CO 2 and the role of glycolate in diminishing net photosynthesis in species with rapid rates of photorespiration was thus further confirmed. L -(or 2 S )-Glycidate was slightly more active than DL -glycidate, but glycidate was more effective as a specific inhibitor in leaf tissue than several other epoxide analogs of glycolate examined. The products of photosynthetic 14 O 2 fixation after 3 or 4 min of uptake were proportionately altered in the presence of glycidate, and the specific radioactivity of the [ 14 C]glycolate produced was closer to that of the 14 CO 2 supplied. Glycidate inhibited glycolate synthesis in tobacco leaf disks irreversibly, since the degree of inhibition was the same for at least 2 hr after the inhibitor solution was removed. Glycidate also blocked glycolate synthesis in maize leaf disks, tissue with low rates of photorespiration, but large increases in net photosynthesis were not observed in maize with glycidate, because glycolate synthesis is normally only about 10% as rapid in maize as in tobacco. The demonstration of increases in net photosynthesis of 40–50% when glycolate synthesis (and photorespiration) is blocked with glycidate indicates in an independent manner that the biochemical or genetic control of photorespiration should permit large increases in plant productivity in plant species possessing rapid rates of photorespiration.

Reduction of Transpiration of Leaves through Stomatal Closure Induced by Alkenylsuccinic Acids.

Low concentrations of long-chain alkenylsuccinic acids and certain of their derivatives inhibit stomatal opening in a tobacco-leaf disk assay, and when used as a spray. The monomethyl ester of decenylsuccinic acid is presently the most effective of this class of compounds. Stomatal closure induced with this substance is accompanied by a significant reduction in the rate of transpiration. These inhibitors probably close stomata by altering the permeability of the membranes of the guard cells.

A mutant of Nicotiana sylvestris deficient in serine glyoxylate aminotransferase activity : Callus induction and photorespiratory toxicity in regenerated plants.

SummaryA photorespiration mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity was isolated in the M2 generation following EMS mutagenesis. Mutants showing chlorosis in air and normal growth in 1% CO2 were fed [14C]-2-glycolate to examine the distribution of 14C among photorespiratory intermediates. Mutant strain NS 349 displayed a 9-fold increase in serine accumulation relative to wild-type controls. Enzyme assays revealed an absence of serine: glyoxylate aminotransferase (SGAT) activity in NS 349, whereas other peroxisomal enzymes were recovered at normal levels. Heterozygous siblings of NS 349 segregating air-sensitive M3 progeny in a 3∶1 ratio were shown to contain one half the normal level of SGAT activity, indicating that air sensitivity in NS 349 results from a single nuclear recessive mutation eliminating SGAT activity. Since toxicity of the mutation depends on photorespiratory activity, callus cultures of the mutant were initiated and maintained under conditions suppressing the formation of functional plastids. Plantlets regenerated from mutant callus were shown to retain the SGAT deficiency and conditional lethality in air. The utility of photorespiration mutants of tobacco as vehicles for genetic manipulation of ribulose bisphosphate carboxylase/oxygenase at the somatic cell level is discussed.

Distribution of catalase isoforms in Nicotiana tabacum

Abstract Total catalase activity per milligram protein averaged three-fold higher in leaves than in green stem or leaf midvein tissue of Nocotiana tabacum . The distribution of total activity between low-peroxidatic (LP-CAT) and enhanced-peroxidatic (EP-CAT) isoforms differed in the three tissues. In the leaf 85–90% of the total activity consisted of LP-CAT and EP-CAT was a minor form, whereas in the stem and midvein LP-CAT isoforms were less than 15% of the total and EP-CAT was the predominant isoform. The catalase mRNA levels were higher in the stem than in the leaves, which may result from the inability of the LP-CAT probe to distinguish between the two forms. The EP-CAT of stem was similar to EP-CAT of leaves in subunit size, specific activity, and in its ability to react with antibodies raised to leaf EP-CAT.

Photorespiratory toxicity in autotrophic cell cultures of a mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity

Procedures were devised for heterotrophic culture and autotrophic establishment of protoplast-derived cell cultures from the sat mutant of Nicotiana sylvestris Speg. et Comes lacking serine: glyoxylate aminotransferase (SGAT; EC 2.6.1.45) activity. Increasing photon flux rates (dark, 40, 80 μmol quanta·m-2·s-1) enhanced the growth rate of autotrophic (no sucrose) wild-type (WT) cultures in air and 1% CO2. Mutant cultures showed a similar response to light under conditions suppressing photorespiration (1% CO2), and maintained 65% of WT chlorophyll levels. In normal air, however, sat cultures developed severe photorespiratory toxicity, displaying a negligible rate of growth and rapid loss of chlorophyll to levels below 1% of WT. Low levels of sucrose (0.3%) completely reversed photorespiratory toxicity of the mutant cells in air. Mutant cultures maintained 75% of WT chlorophyll levels in air, displayed light stimulation of growth, and fixed 14CO2 at rates identical to WT. Autotrophic sat cultures accumulated serine to levels nearly nine-fold above that of WT cultures in air. Serine accumulated to similar levels in mixotrophic (0.3% sucrose) sat cultures in air, but had no deleterious effect on fixation of 14CO2 or growth, indicating that high levels of serine are not toxic, and that toxicity of the sat mutation probably stems from depletion of intermediates of the Calvin cycle. Autotrophic sat cultures were employed in selection experiments designed to identify spontaneous reversions restoring the capacity for growth in air. From a population of 678 000 sat colonies, 23 plantlets were recovered in which sustained growth in air resulted from reacquisition of SGAT activity. Twenty-two had SGAT levels between 25 and 50% of WT, but one had less than 10% of WT SGAT activity, and eventually developed symptoms typical of the sat mutant. The utility of autotrophic sat cultures for selection of chloroplast mutations diminishing the oxygenase activity of ribulose-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is discussed.

Travels in a world of small science

As a boy, I read Sinclair Lewiss 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 H2O2 as a byproduct. I discovered enzymes catalyzing the reduction of glyoxylate and hydroxypyruvate. I found α-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 C3 plants. I added inhibitors of glycolate oxidase to leaves and showed: (1) glycolate was synthesized only in light as an early product of photosynthetic CO2 assimilation, (2) the rate of glycolate oxidation consumed a sizable fraction of net photosynthesis in C3 but not in C4 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 CO2 assimilation.

Evidence for a Role for NAD(P)H Dehydrogenase in Concentration of CO2 in the Bundle Sheath Cell of Zea mays

Loss of a functional NAD(P)H dehydrogenase supercomplex impairs CO2 concentration in bundle sheath cells. Prior studies with Nicotiana and Arabidopsis described failed assembly of the chloroplastic NDH [NAD(P)H dehydrogenase] supercomplex by serial mutation of several subunit genes. We examined the properties of Zea mays leaves containing Mu and Ds insertions into nuclear gene exons encoding the critical o- and n-subunits of NDH, respectively. In vivo reduction of plastoquinone in the dark was sharply diminished in maize homozygous mutant compared to normal leaves but not to the extreme degree observed for the corresponding lesions in Arabidopsis. The net carbon assimilation rate (A) at high irradiance and saturating CO2 levels was reduced by one-half due to NDH mutation in maize although no genotypic effect was evident at very low CO2 levels. Simultaneous assessment of chlorophyll fluorescence and A in maize at low (2% by volume) and high (21%) O2 levels indicated the presence of a small, yet detectable, O2-dependent component of total linear photosynthetic electron transport in 21% O2. This O2-dependent component decreased with increasing CO2 level indicative of photorespiration. Photorespiration was generally elevated in maize mutant compared to normal leaves. Quantification of the proportion of total electron transport supporting photorespiration enabled estimation of the bundle sheath cell CO2 concentration (Cb) using a simple kinetic model of ribulose bisphosphate carboxylase/oxygenase function. The A versus Cb relationships overlapped for normal and mutant lines consistent with occurrence of strictly CO2-limited photosynthesis in the mutant bundle sheath cell. The results are discussed in terms of a previously reported CO2 concentration model [Laisk A, Edwards GE (2000) Photosynth Res 66: 199–224].