Chemical inhibition of the glycolate pathway in soybean leaf cells

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in α-hydroxypyridinemethanesulfonic acid increased ¹⁴CO₂ incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of ¹⁴CO₂ into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of ¹⁴C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of ¹⁴CO₂ into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O₂/CO₂ ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O₂, but these compounds increased the O₂ inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.     

Photosynthetic carbon metabolism in wild, primitive and cultivated forms of wheat at three levels of ploidy: Role of the glycolate pathway

¹⁴CO₂ assimilation was studied with diploid, tetraploid, hexaploidspecies of the genera Triticum and their wild relatives Aegilops.Attached mature leaves of 3–4 weekold plants were allowedto undergo photosynthesis under air at ambient temperature.The pattern of distribution of ¹⁴C was notably similar in Triticumand Aegilops species whatever the level of ploidy. Sucrose wasthe sink for photosynthetic carbon. ¹⁴C for sucrose synthesis was supplied either through the glycolatepathway by glycolate, the product of the photorespiration orby the Calvin cycle intermediates exported into the cytoplasm.Depending on the species, the glycolate pathway provided 40to 75%of the sucrose ¹⁴C. The higher labeling of sucrose was associated with the greaterparticipation of the glycolate pathway in the wild diploid (DD)A. squarrosa and in the cultivated hexaploid (AABBDD) T. aestivum.The results suggest that the expression of the male D genomeis dominant over the female AB genome in T. aestivum. In T. aestivum under ambient conditions lowering (low temperature)or hindering (1% O₂ ) photorespiration, sucrose labeling decreased,but serine and glycine labeling was favoured. We propose thatin wheat leaves, the role of photorespiration is to drain artof the carbon exported from the chloroplast as glycolate, towardssucrose synthesis. (Received March 16, 1979; )     

A Mathematical Model of Leaf Carbon Metabolism

A dynamic mechanistic mathematical model of C₃ leaf carbon metabolism,incorporating the Calvin cycle with starch and sucrose synthesisand degradation, is proposed. The model consists of a systemof non-linear differential equations based on biochemical andphysiological assumptions. An analytical steady-state solution,with interesting mathematical properties that are interpretedbiologically, is presented and shown to be stable. The systemequations are integrated numerically, and an approximate analyticalsolution to the sucrose sub-system is derived. Some resultsof the model are compared with experimental data. Mathematical model, Calvin cycle, leaf carbon metabolism, photosynthesis, differential equations     

Effect of Photorespiratory C2 Acids on CO2 Assimilation,PS II Photochemistry and the Xanthophyll Cycle in Maize

The photorespiration cycle plays an important role in avoiding carbon drainage from the Calvin cycle and in protecting plants from photoinhibition. The role of photorespiration is frequently underestimated in C(4) plants, since these are characterized by low photorespiration rates. The aim of this work was to study the relationship between CO(2) assimilation, PS II photochemistry and the xanthophyll cycle when the photorespiratory cycle is disrupted in Zea mays L. To this end, the photorespiration inhibitor phosphinothricin (PPT) was applied individually or together with the photorespiratory C(2) acids, glycolate and glyoxylate to maize leaves. Application of PPT alone led to the inhibition of CO(2) assimilation. Moreover, feeding with glycolate or glyoxylate enhanced the effect of PPT on CO(2) assimilation. Our results confirm that the avoidance of the accumulation of the photorespiratory metabolites glycolate, glyoxylate or phosphoglycolate, is of vital importance for coordinated functioning between the glycolate pathway and CO(2) assimilation. Relatively early changes in PS II photochemistry also took place when the photorespiratory cycle was interrupted. Thus, fluorescence photochemical quenching (qP) was slightly reduced (10%) due to the application of PPT together with glycolate or glyoxylate. A decrease in the efficiency of excitation-energy capture by open PS II reaction centres (F'v/F'm) and an increase in thermal energy dissipation (non-photochemical quenching, NPQ) were also measured. These observations are consistent with a limitation of activity of the Calvin cycle and a subsequent lower demand for reduction equivalents. The increase in NPQ is discussed on the basis of changes in the xanthophyll cycle in maize, which seem to provide a limited protective role to avoid photoinhibition when the glycolate pathway is blocked. We conclude that C(2) photorespiratory acids can act as physiological regulators between the photorespiratory pathway and the Calvin cycle in maize.     

Regulation of photorespiration in leaves: evidence that the fraction of ribulose bisphosphate oxygenated is conserved and stoichiometry fluctuates

Under steady-state conditions the combined system of the reductive photosynthetic cycle and the oxidative photorespiratory loop may be defined by two partitioning terms: the fraction of ribulose bisphosphate oxygenated and the fraction of glycolate carbon photorespired (the stoichiometry of photorespiration). A combination of physical and stereochemical methods [K.R. Hanson, and R. B. Peterson, (1985) Arch. Biochem. Biophys. 237,300-310] has been used to estimate these partitionings for tobacco leaf discs. Inverted discs, as compared to normally oriented discs, were found to have greater net photosynthesis; their ratio of photorespiration to net photosynthesis was less, and less of their glycolate carbon was photorespired. An eightfold reduction of irradiance below that of full sunlight for inverted discs in normal air at 32 degrees C reduced both photosynthesis and photorespiration about threefold but had little effect on the partitioning of ribulose bisphosphate and glycolate. Increasing the temperature from 22 to 40 degrees C for inverted discs in normal air and 1000 microE m-2 s-1 irradiance had little effect on net photosynthesis but increased the ratio of photorespiration to net photosynthesis almost threefold; ribulose bisphosphate partitioning was little changed but the fraction of glycolate carbon photorespired more than doubled. If field-grown plants respond to temperature in a similar fashion, genetic intervention to reduce the increase in photorespiration stoichiometry with temperature could increase total daily carbon assimilation and hence improve crop yields.     

The isotope effect in the glycine dehydrogenase reaction is the cause of the intramolecular isotope inhomogeneity of glucose carbon of starch synthesized during photorespiration

The isotope distribution of glucose-6-phosphate in the main pathways of its biosynthesis (in the processes of CO2 assimilation and photorespiration in the Calvin cycle and during resynthesis from the degradation products of lipids and proteins) was analyzed. For reconstructing the isotope distribution of glucoso-6-phosphate synthesized in the Calvin cycle during photorespiration, the functioning of the cycle with regard to its coupling with the glycolate chain, which together constitute the photorespiration chain, was considered. In the glycine dehydrogenase reaction of the glycolate cycle, there arises an isotope effect, which determines the distribution of isotopes in the glucose-6-phosphate and other photorespiration products. The isotope effect of the glycine dehydrogenase reaction increases at the expense of the exhaustion of glucose resources feeding the photorespiration chain. As a result, atoms C-3 and C-4 of glucose become enriched with the heavy isotope, and subsequent mixing of atoms and the specificity of interactions in the photorespiration chain lead to an isotope weighting of the other atoms and an uneven distribution of carbon isotopes in glucose-6-phosphate and other photorespiration products. A comparison of the glucose-6-phosphate isotope patterns in different pathways of the synthesis with the experimental data on the distribution of carbon isotopes in starch glucose of storing plant organs led to the conclusion that the starch resources are predominantly formed at the expense of glucose-6-phosphate of photorespiration. This is consistent with the earlier observed enhancement of photorespiration at the stage of plant maturation.     

CO2-Fixation, Glycolate Formation, and Enzyme Activities of Photorespiration and Photosynthesis during Greening and Senescence of Sunflower Cotyledons

Time-courses of ¹⁴CO₂-fixation and of enzyme activities involvedin photorespiration and photosynthesis were determined duringthe life span of cotyledons from sunflower seedlings (Helianthusannuus L.). Glycolate formation in vivo was estimated from theresults of combined labelling and inhibitor experiments. NADPH-glyceraldehyde-3-phosphatedehydrogenase, NADPH-glyoxylate reductase and chlorophyll werewell correlated with the time-course of ¹⁴CO₂-fixation (photosynthesis).There was, however, a considerable discrepancy between the developmentalsequence of photosynthesis and that of both ribulose-l,5-bisphosphatecarboxylase and glycolate oxidase. Furthermore, time-coursesof glycolate oxidase activity in vitro and of glycolate formationin vivo differed significantly. Therefore, the use of glycolateoxidase as a marker for the activity of photorespiration ingreening sunflower cotyledons may be questionable. Results from¹⁴CO₂-labelling experiments with cotyledons treated with theglycolate oxidase inhibitor 2-hydroxy butynoic acid suggestthat glycolate formation relative to CO₂-fixation is reducedin senescent cotyledons. Key words: Development, glycolate oxidase, photorespiration, ribulose-l,5-bisphosphate carboxylase, oxygenase     

Responses of Photosynthesis and Dark Respiration to Temperature

This analysis suggests that a model of the temperature dependenceof carbon exchange by a plant can be developed based upon absolutereaction rate theory. Component temperature-dependent physiologicalprocesses necessary to describe net photosynthesis over thebiological temperature range include the light reaction, darkreaction (carboxylase CO₂ uptake, oxygenase photorespiration)and mitochondrial dark respiration. An essential assumptionof the model is the reversibility of thermal inhibition. Supportingevidence for this assumption is provided within the biologicalrange. Thermodynamic constants were found to be strongly correlatedwith the thermal environment to which they were adapted. Therewas little difference in non-photorespiration thermodynamicconstants between C₂ andC₄species within thermal habitat types.The model shows the observed shift in temperature optima withlight intensity as a natural consequence of enzyme kineticsand absolute reaction rate theory. photosynthesis, photorespiration, dark respiration, temperature response, carbon exchange, mathematical model     

Photorespiration and Glycolate Metabolism: A Re-examination and Correlation of Some Previous Studies

Some previous studies of photorespiration and glycolate oxidation were re-examined and correlated by infra-red CO₂ analysis. Data about rate of photosynthesis and oxygen sensitivity indicated that complete inhibition of photosynthesis with 3-(3,4-dichlorophenyl)-1,1 dimethyl urea (DCMU) allowed dark respiration to continue in the light. Photorespiration was also inhibited. The oxygen sensitivity of glycolate-stimulated CO₂ production was found to be compatible with the proposal that glycolate is a substrate of photorespiration. Both `in vivo' and `in vitro' studies of the alga Nitella flexilis have revealed a pathway of glycolate oxidation similar to that of higher plants. DCMU inhibition of photosynthesis by Nitella gave results similar to those for the monocotyledons tested. Under very low light intensity, carbon dioxide compensation in corn was measurable but was not sensitive to high oxygen concentration. It appears that the lack of photorespiration in this plant is not the end result of efficient internal recycling of CO₂ to photosynthesis.     

光呼吸突变体研究进展

光呼吸(photorespiration)是绿色植物在光下吸收氧气并释放CO₂的过程。C₃植物光呼吸可消耗25%光合产物, 故合理改良光呼吸可望提高植物的光合效率。筛选与利用光呼吸突变体是研究光呼吸代谢及其功能的最为有效的途径。该文对光呼吸代谢途径、光呼吸突变体的筛选以及研究进展进行综述, 以期为深入探讨植物光呼吸的生物学功能及进行植物分子改良提供帮助。     

Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana

We introduced the Escherichia coli glycolate catabolic pathway into Arabidopsis thaliana chloroplasts to reduce the loss of fixed carbon and nitrogen that occurs in C(3) plants when phosphoglycolate, an inevitable by-product of photosynthesis, is recycled by photorespiration. Using step-wise nuclear transformation with five chloroplast-targeted bacterial genes encoding glycolate dehydrogenase, glyoxylate carboligase and tartronic semialdehyde reductase, we generated plants in which chloroplastic glycolate is converted directly to glycerate. This reduces, but does not eliminate, flux of photorespiratory metabolites through peroxisomes and mitochondria. Transgenic plants grew faster, produced more shoot and root biomass, and contained more soluble sugars, reflecting reduced photorespiration and enhanced photosynthesis that correlated with an increased chloroplastic CO(2) concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These effects are evident after overexpression of the three subunits of glycolate dehydrogenase, but enhanced by introducing the complete bacterial glycolate catabolic pathway. Diverting chloroplastic glycolate from photorespiration may improve the productivity of crops with C(3) photosynthesis.     

The Mechanism of Photosynthesis in the Tea Plant (Camellia sinensis L.)

Leaves of the tea plant photosynthesizing in ¹⁴CO₂ incorporatedmuch radioactivity into intermediates of the glycolate pathwayand little into C₄ acids. Increased O₂ in the atmosphere decreasedphotosynthesis, stimulated photorespiration, and increased theCO₂ compensation point. In air the rate of photorespirationwas 19% of net photosynthesis. These observations indicate aC₃ rather than a C₄ mechanism of photosynthesis.     

Ribulose bisphosphate oxygenase activity from freshly ruptured spinach chloroplasts

Suspensions of freshly lysed spinach chloroplasts, in which ribulose bisphosphate carboxylase displays an in vivo K_m [CO], exhibited a ribulose bisphosphate-dependent uptake of oxygen. The kinetic properties of this oxygenase activity were examined at air levels of CO₂ (10 μm) and O₂ (240 μm). The pH optimum was 8.6–8.8 and the K_M [ribulose bisphosphate] was 45 μm. At 240 μm O₂, the oxygenase activity is inhibited one-half by 25 μm CO₂. The apparent K_m(O₂) is large, somewhere between 1 and 2 atm. The phosphoglycolate phosphatase activity of the chloroplasts was in great excess, suggesting that phosphoglycolate formed by the oxygenase would be quickly hydrolyzed to glycolate for possible metabolism by photorespiration.A comparison of the pH dependence of both the carboxylase and oxygenase activities at air levels of CO₂ and O₂ suggests that the pH of the chloroplast stroma could regulate their relative activities and that the oxygenase activity is sufficient to account for glycolate production during photosynthesis. It is predicted that at pH 7.8, about 40% of the carbon assimilated by the Calvin cycle would go through glycolate.     

Excretion of glycolate during synchronous culture of Ankistrodesmus braunii in the presence of disalicylidenepropanediamine or hydroxypyridinemethanesulfonate

The rate of excretion of glycolate by the unicellular greenalga Ankistrodesmus braunii changes during its life cycle. Itis high in the main growth phase during the light period witha maximum 6 hr after the start of illumination, and low duringthe period of cell division in the dark. The glycolate excretion is stimulated by DSPD and HPMS, whilethe total ¹⁴CO₂-fixation is inhibited by DSPD and enhanced byHPMS. Changes in the effects of DSPD and HPMS on glycolate excretionas well as on photosynthetic ¹⁴CO₂-fixation during the courseof the algal life cycle were followed using the technique ofsynchronous culture. How far the change of glycolate excretion is due to a changeof glycolate oxidase activity during the life cycle and to achange of C₂-supply from the carbon reduction cycle is discussed.The effect of DSPD on glycolate excretion suggests a participationof ferredoxin in the glycolate pathway. (Received August 10, 1968; )     

Effects of Certain Inhibitors on Photorespiration by Wheat Leaf Segments

The effect on the carbon metabolism of wheat leaf segments ofcertain inhibitors of photorespiration was studied. Sodium 2-hydroxy-3-butynoatesupplied for 40 min resulted in accumulation of ¹⁴C in glycolicacid with only a 7% inhibition of photosynthesis; when suppliedfor 90 min, photosynthesis was inhibited by 47%. When ¹⁴CO₂was replaced by 1000 vpm ¹²CO₂, radioactivity in glycine decreasedbut increased more rapidly in sucrose with less release of ¹⁴CO₂.Isonicotinyl hydrazide (INH) inhibited photosynthesis from ¹⁴CO₂by 50% and glycine replaced sucrose as the main product. When,after 15 min, ¹⁴CO₂ was replaced by 150 vpm ¹²CO₂, in the presenceof INH less ¹⁴CO₂ was released, ¹⁴CO in glycine decreased moreslowly, and less [¹⁴CO]sucrose accumulated. Glycidate (potassium2,3-epoxypropionate) at 2 mM had no effect on photosyntheticrate and little effect on carbon metabolism; 20 mM glycidateinhibited photosynthesis by 64% and resulted in less radioactivityin glycine, more in phosphate esters, and less ¹⁴CO₂ released.When photosynthesis was measured in 1000 vpm CO₂ the inhibitorsgave smaller effects on metabolism than during photosynthesisfrom 150 vpm ¹⁴CO₂ but 20 mM glycidate still resulted in a 42%inhibition of photosynthesis. When U- [¹⁴CO]glycerate was appliedto leaf segments in air with 320 vpm ¹⁴CO₂ the total uptakeof glycerate was not changed by the inhibitors. INH and glycidateboth decreased the amount of glycerate metabolised. More ¹⁴COaccumulated in glycine in the presence of INH and in phosphateesters and serine in the presence of glycidate. Hydroxybutynoateincreased the production of glycolate from glycerate but didnot affect the total amount of glycerate metabolised. Although all three inhibitors affected photorespiratory metabolismnone stimulated photosynthesis. The results are consistent withthe main release of CO₂ in photorespiration arising from theconversion of glycine to serine.     

Glycine as a substrate for photorespiration

Substrates for photorespiration were examined by feeding ¹⁴Clabeled compounds to tobacco and corn leaf segments and by measuring¹⁴CO₂ evolution in light and darkness. CO₂ release in the darkwas rapid, but in light CO₂ release was slow due to refixationby photosynthesis. Carboxyl labeled glycine was more rapidlydecarboxylated than were glyoxylate, glycolate or serine. Hydroxypyridinemethanesulfonate, an inhibitor of glycolate oxidase, blocked CO₂ releasefrom glycolate but not from glycine. Isonicotynyl hydrazideblocked CO₂ release from both glycine and glycolate. DCMU blockedphotosynthetic refixation of the released CO₂, consequentlythe rates of CO₂ release in light and dark were about equal.It was concluded that CO₂ release during photo-respiration camefrom the conversion of 2 molecules of glycine to one serineand one CO₂. ¹⁴CO₂ release from glycine-l-¹⁴C in the dark or with DCMU inlight can be used as an assay for photorespiration ability. CO₂ release from glycine and glycolate by corn leaf segmentsin the dark proceeded at the rate of that in normal tobaccoleaf. This result, together with other work on O₂ exchange andenzymatic analysis, indicates that corn and other plants docarry on photorespiration, but it is not manifested by CO₂ releasein light. A yellow tobacco mutant, Consolation 402, had high rates ofphotorespiration by the ¹⁴CO₂ assay, nearly half (or more) asmany peroxisomes as chloroplasts, and high rates of CO₂ releasefrom glycine-l-¹⁴C or glycolate-l-¹⁴C. A common tobacco, BrightYellow, had lower rates of photorespiration, fewer visible peroxisomes,and slower decarboxylation of glycine and glycolate. The amount of ¹⁴CO₂ release from glycine-l-¹⁴C or glycolate-l-¹⁴Cincreased only slightly when the temperature was raised from25 to 35°C. ¹Parts of this work were abstracted at the Annual Meeting (April,1969) of Japanese Society of Plant Physiologists, Kanazawa ²Department of Biochemistry, Michigan State University, EastLansing, Michigan, U.S.A. (Received September 3, 1969; )     

根据光合作用能量代谢测定光呼吸的方法研究

本文根据光合作用和光呼吸途径能量代谢,通过改变外界CO2和O2浓度,计算卡尔文循环固定的CO2和光呼吸消耗的O2。结果表明,可以通过3种方法计算。方法1,测定在CO2饱和点（A）和正常CO2（A＇）浓度下吸收的CO2,得出光呼吸消耗的O2为：18／19（A-A＇）,卡尔文循环固定的CO2为：1／19（6A＋13A＇＋19Rd）。方法2,测定在不含O2的空气中（O）和正常O2（O’）浓度下释放的O2,得出光呼吸消耗的O2为：-13／5O-O＇-18／5Rd,卡尔文循环固定的CO2为：13／18（O＇—O）。方法3,测定在正常情况下吸收的CO2（A）和释放的O2（O＇）,得出光呼吸消耗的O2为：18（O＇—A＇）,卡尔文循环固定的CO2为：6O＇-5A＇＋Rd。测定在CO2饱和点和正常CO2浓度下吸收的CO2计算出水稻光呼吸释放的CO2占光合作用固定的24％-40％。     

Partial Reduction in Activities of Photorespiratory Enzymes in C3-C4 Intermediates of Alternanthera and Parthenium

The maximum catalytic activities of several photorespiratoryand photosynthetic enzymes were determined in leaf extractsof three C₃–C₄ intermediates (Alternanthera ficoides,A. tenella and Parthenium hysterophorus) and were compared tothose of C₃ (A. sessiles, Pisum sativum) and C₄ (A. pungens,Zea mays and Amaranthus hypochondriacus) species. The activitylevels of key photorespiratory enzymes, glycolate oxidase, catalase,NADH-hydroxypyruvate reductase and glycerate kinase were less(28 to 35% reduced) in intermediates than those of typical C₃species. Similarly, the activities of photorespiratory aminotransferasesin the C₃–C₄ intermediates were also partially reduced(23 to 37% reduction). The activities of phosphoenolpyruvatecarboxylase (PEPC), pyruvate, orthophosphate dikinase and NAD-malicenzyme were higher (2 to 7 times) in leaf extracts of the intermediatesthan those of C₃ species. But the ratios of PEPC/rubisco inthe C₃–C₄ intermediates were more like C₃ than C₄ species.We draw attention to the partial reduction in enzyme activityof photorespiratory metabolism, which could be an importantfactor for restriction of photorespiration in the C₃–C₄intermediate species, in addition to enzyme compartmentationand/or operation of a ‘C₄-like’ cycle Key words: C₃–C₄ intermediates, C₄ pathway, enzyme profile, glycolate metabolism, photorespiration, photosynthesis     

Leaf Carbon Dioxide Compensation Points at High Light Flux Densities

A model for leaf photosynthesis by C₃ plant species is usedto examine the inter-relations between carbon dioxide and oxygenconcentrations, and dark respiration rates at the leaf compensationpoint. The model is applied to data published on a range ofC₃ plant types. These data indicate a remarkable constancy inthe slope of linear relationship between the CO₂ and O₂ concentrationsat the leaf compensation point, indicating a constancy in theratio of photorespiration to photosynthesis across C₃ planttypes. The model is extended to deal with C₄ plants and used to interpretthe differences in the compensation point relationships of C₃and C₄ plants.