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     

Stimulation of Malate Synthesis by Glycolate in Tomato Leaves in Relation to CO2 Concentration

The mechanism by which malate synthesis from CO2 is increasedunder low concentrations of CO₂ was investigated in C₃ plants.A number of metabolites were administered to illuminated tomatoleaves, and their effects on the incorporation of ¹⁴CO₂ intomalate were determined. Compared with water as a control, glycolate,glyoxylate, D,L-glycerate, glycine, phosphoglycolate and L-serineincreased malate synthesis by factors of 6.8, 3.8, 3.3, 2.5,2.3 and 2.2, respectively. The effect of exogenous glycolateon malate synthesis from CO₂ was dependent on its concentrationup to 100 mu, but was independent of ambient CO₂ concentration.The feeding of l-¹⁴C-glycolate in the light indicated that glycolatestimulated the carbon flow from CO₂ to malate. The analysis of the products of ¹⁴CO₂ fixation in illuminatedleaves supplied with glycolate showed increases in malate andsugar and decreases in serine and phosphate esters. However,this stimulated malate synthesis ceased when malonate was suppliedsimultaneously with glycolate. Treatment with glycolate didnot affect the dark ¹⁴CO₂-fixation, but increased the ¹⁴C-malatesynthesis, with a corresponding decrease in ¹⁴C-aspartate and14^C-glutamate. These results suggest that exogenous glycolateactivates malate dehydrogenase in leaves, and that the increasedglycolate formation at low CO₂ concentrations is associatedwith the increased malate synthesis from CO₂. (Received January 12, 1981; Accepted May 20, 1981)     

Role of Carbonic Anhydrase and Identification of the Active Species of Inorganic Carbon Utilized for Photosynthesis in Chora corallina

Carbonic anhydrase (CA, EC. 4.2.1.1 [EC] ) activity in air-grown Characorallina was detected mainly in the intracellular fraction,most of which composed of chloroplasts and cytoplasmic gel,and not on the cell surface. Only minor levels of CA activity,on the basis of equivalent volumes, were detected in the cellsap and the cytoplasmic sol. The maximum rate of photosynthetic O₂ evolution by air-grownChara corallina at pH 6.0 was twice that at pH 7.6, while theapparent K_m for external inorganic carbon (C_i) at pH 7.6 wasabout three times that at pH 6.0. However, the apparent K_m(CO₂)was about three times larger at pH 6.0 than at pH 7.6. The K_m(C_i)-valueat pH 7.6 increased severalfold in the presence of acetazolamide(AZA), an inhibitor of CA, but no inhibition was observed atpH 6.0. The pH-dependence may be due to differences in the permeabilityof AZA at the given pH values. Fixation of ¹⁴CO₂ at 20 µMand of H¹⁴CO₃^– at 200 µM over the course of 5 swas very similar at pH 7.4. Addition of CA significantly suppressedthe photosynthetic ¹⁴CO₂-fixation but it stimulated the H¹⁴CO₃^–-fixation.This result indicates that free CO₂ is an active species ofC_i that is incorporated into the cell during photosynthesis. These results together suggest the following: (1) Free CO₂ isutilized for photosynthesis, (2) CA is mainly located insidethe cell and functions to increase the affinity for CO₂ in photosynthesisby facilitating the supply of CO₂ from the plasmalemma to thesite of CO₂-fixation. ³Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Chiba, 260 Japan. (Received December 9, 1988; Accepted March 22, 1989)     

Green Light Drives CO2 Fixation Deep within Leaves

Maximal ^l4CO₂-fixation in spinach occurs in the middle of thepalisade mesophyll [Nishio et al. (1993) Plant Cell 5: 953],however, ninety percent of the blue and red light is attenuatedin the upper twenty percent of a spinach leaf [Cui et al. (1991)Plant Cell Environ. 14: 493]. In this report, we showed thatgreen light drives ¹⁴CO₂-fixation deep within spinach leavescompared to red and blue light. Blue light caused fixation mainlyin the palisade mesophyll of the leaf, whereas red light drovefixation slightly deeper into the leaf than did blue light.¹⁴CO₂-fixation measured under green light resulted in less fixationin the upper epidermal layer (guard cells) and upper most palisademesophyll compared to red and blue light, but led to more fixationdeeper in the leaf than that caused by either red or blue light.Saturating white, red, or green light resulted in similar maximal¹⁴CO₂-fixation rates, whereas under the highest irradiance ofblue light given, carbon fixation was not saturated, but itasymptotically approached the maximal ¹⁴CO₂-fixation rates attainedunder the other types of light. The importance of green lightin photosynthesis is discussed. ¹Supported in part by grants from Competitive Research GrantsOffice, U.S. Department of Agriculture (Nos. 91-37100-6672 and93-37100-8855).     

Effects of disalicylidenepropandiamine and near far-red light on 14CO2-fixation in Chlorella cells

1) With Chlorella ellipsoidea cells, in the presence of 5x10^–6M DSPD, or in its absence, the amounts of ¹⁴CO₂ incorporatedin P-esters, serine-plus-glycine and alanine were larger underred light than under blue light, whereas blue light specificallyincreased ¹⁴CO₂-incorporation in aspartate, glutamate, malateand fumarate (blue light effect). The amount of total ¹⁴C fixedunder blue or red light was greatly decreased by the additionof DSPD. When the concentration of DSPD was raised to 5x10^–4M, practically no radioactivity was found, under blue or redlight, in aspartate, glutamate and fumarate. Radioactivity inalanine was greatly increased. Effects of higher concentrationof DSPD are explained as due to the inhibition of PEP carboxylaseactivity in Chlorella cells. 2) The percentage incorporation of ¹⁴C into aspartate and theother compounds mentioned above, under near infra-red illuminationwas significantly smaller than that under blue light and wasalmost equal to that under red light. These results along withthe effect of 5x10^–6 M DSPD, exclude the possibility thatcyclic photophosphorylation is involved in the "blue light effect"mechanism. (Received December 12, 1969; )     

Glycolate synthesis in a C3 C4 and intermediate photosynthetic plant type

Excised leaves of a C₃-photosynthetic type, Hordeum vulgare,a C₄-type, Panicum miliaceum, and an intermediate-type, Panicummilioides, were allowed to take up through their cut ends a1 mM solution of butyl hydroxybutynoate (BHB), an irreversibleinactivator of glycolate oxidase. After 30 to 60 min in BHB,extractable glycolate oxidase activity could not be detectedin the distal quarter of the leaf blades. Following this pretreatment,recovery of ¹⁴C-glycolate from ¹⁴CO₂ incorporated in a 10 minperiod was nearly maximal for each of the three plant types.Labeled glycolate was 51% of the total ¹⁴CO₂ incorporated forthe C₃-species, 36% for the intermediate-species, and 27% forthe C₄-species Increased labeling of glycolate was compensatedfor primarily by decreased labeling of the neutral and basicfractions for the C₃ and intermediate-type species. In the C₄-type,label decreased primarily in the neutral and insoluble fractions,but increased in the basic fraction. A lower rate of glycolatesynthesis is indicative of a lower rate of photorespirationand consistent with a lower O₂/CO₂ ratio present in the bundle-sheathcells of C₄-plants. We conclude that both decreased glycolatesynthesis and the refixation of photorespiratory-released CO₂are important in maintaining a lower rate of photorespirationin C₄-plants compared to C₃ plants. Intermediate glycolate synthesisin Panicum milioldes is consistent with its intermediate levelof O₂ inhibition of photosynthesis and intermediate rate ofphotorespiration. (Received May 6, 1978; )     

Biosynthetic mechanism of glycolate in Chromatium 6. Glycolate formation and metabolism under low O21

Under low O₂ (0.05 mM O₂), there was no measurable excretionof glycolate or glycine by Chromatium cells, unlike the caseof their incubation under high (0.7 mM) O₂ However, upon additionof non-radioactive glycolate and glycine to the suspension medium,there occurred a measurable incorporation of ¹⁴CO₂ into thesecompounds, which were then excreted extracellularly; the totalradioactivities measured were approximately 15% of the totalCO₂ fixed photosynthetically. This phenomenon could be as cribedto the dilution of the intracellular pools by the compoundsadded. The results indicate that under low O₂ the glycolatemolecules produced are metabolically further transformed inthe bacterial cells. The incorporation of ¹⁴CO₂ into the extracellularglycolate fraction was maximal at 0.3 mM glycolate in both highand low O₂. Presumably, glycolate formed in the bacterial cellsunder both the high and low O₂ is metabolized in a similar manner,although the excess glycolate and glycine molecules are rapidlyexcreted. During glycolate metabolism CO₂ was evolved from anisonicotinylhydrazide-sensitive reaction, suggesting that thepathway <glycolate glycine . CO₂ was similar in green plants.The results thus indicate that studies on glycolate and glycinemetabolism in the anaerobic bacterium, Chromatium, provide auseful model system for elucidating the mechanism of photorespirationin green plants. (Received May 19, 1978; )     

Biosynthetic mechanism of glycolate in Chromatium IV. Glycolate-glycine transformation

The metabolic transformation of glycolate to glycine occurringin photosynthesizing cells of Chromatium was investigated bythe radioisotopic technique and by amino acid analysis. By analyzingthe distribution of radiocarbon upon feeding [1-¹⁴C] glycolate,[2-¹⁴C] glyoxylate and [1-¹⁴C] glycine to bacterial cells, itwas demonstrated that glycolate is converted to glycinc viaglyoxylate, and both glycolate and glycine are excreted extracellularly.Although the formation of serine was barely detected by theabove two techniques in both N₂ and O₂ atmospheres, it was foundthat ¹⁴CO₂ is evolved quite markedly from both [1-¹⁴C] glycolateand [1-¹⁴C] glycine fed to the Chromatium cells. Analyticalresults of transient changes in amino acid compositions underatmospheric changes of N₂O₂ and by the addition of exogenousglycolate in N₂ confirm the notion that glycolate is convertedto glycine. Acidic amino acids (glutamic acid and aspartic acid)appear to take part in glycine formation as amino donors. Theformation of glycine from glycolate in a N₂ atmosphere suggeststhat an unknown glycolate dehydrogenation reaction may operatein the overall process. ¹ This is paper XXXVII in the series ‘Structure and Functionof Chloroplast Proteins’. Paper XXXVI is ref. (5). Theresearch was supported in part by grants from the Ministry ofEducation of Japan (No. 111912), the Toray Science Foundation(Tokyo) and the Naito Science Foundation (Tokyo). (Received July 14, 1976; )     

Regulation of CO2-fixation in Chlorella by light of varied wavelengths and intensities

1) The wavelength effects on ¹⁴CO₂-fixation by Chlorella cellswere studied, using monochromatic light of different light intensities. 2) Blue light (453 mµ) stimulated the incorporation of¹⁴C into aspartate, glutamate and malate. Red light (679 mµ),on the other hand, stimulated its incorporation into P-esters,free sugars and insoluble material. 3) The blue light effect was observed in the presence of CMUat concentrations completely suppressing ordinary photosyntheticCO₂-fixation. 4) The blue light effect in the presence of CMU was inducedat very low intensities. At 453 mµ, 300 erg cm^–2sec^–1 was sufficient for complete saturation. 5) Time courses of ¹⁴C-incorporation into individual compoundswere investigated. Irrespective of the wavelength of the illuminatinglight, the first stable CO₂-fixation product formed under weaklight (400–500 erg cm^–2 sec^–1) was citrulline.At higher light intensities (4,000–7,000 erg cm^–2sec^–1), PGA was the first stable CO₂-fixation product.The incorporation of ¹⁴C into citrulline was not inhibited byCMU. 6) Experimental results indicate that both blue light-inducedincorporation of ¹⁴C into amino and organic acids and the incorporationof ¹⁴C into citrulline induced by low intensity light are operatedby a mechanism(s) independent of ordinary photosynthetic CO₂-fixation.Possible effects of light regulating the carbon metabolism inalgal cells are discussed. (Received July 24, 1969; )     

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; )     

Biosynthetic mechanism of glycolate in Chromatium III. Effects of {alpha}-hydroxy-2-pyridinemethanesulfonate, glycidate and cyanide on glycolate excretion

Effects of -hydroxy-2-pyridinemethanesulfonate (-HPMS), 2,3-epoxypropionate(glycidate), and cyanide on the photosynthetic activities ofChromatiumwere studied. -HPMS stimulated photosynthetic CO₂fixation in the bacterial cells in both N₂ and O₂ environments.The formation and subsequent excretion of both glycolate andglycine in the O₂ atmosphere were markedly enhanced by -HPMS.In contrast to a recent report by Zelitch [Arch. Biochem. Biophys.163: 367–377 (1974) ] that glycidate specifically inhibitsglycolate formation in tobacco leaf disks, we found that ithad no influence on CO₂ fixation by Chromatium in either N₂or O₂ atmosphere, and that the synthesis and extracellular excretionof glycolate were markedly stimulated by glycidate treatment.Cyanide (0.01–1 mM) exerted a marked inhibitory effecton photosynthetic CO₂ fixation in N². In O₂ atmosphere, photosynthesiswas stimulated by 0.01 mM cyanide, and inhibited by it abovethis level. Both the incorporation of ¹⁴CO₂ into glycolate andthe total synthesis of glycolate in the light were also enhancedby 0.01 mM cyanide, and strongly inhibited above that concentration. ¹This is paper XXXVI in the series "Structure and Function ofChloroplast Proteins," and the research supported in part bygrants from the Ministry of Education of Japan (No. 111912),the Toray Science Foundation (Tokyo) and the Naito Science Foundation(Tokyo). (Received May 31, 1976; )     

Effect of oxygen on photosynthesis and biosynthesis of glycolate in photoheterotrophically grown cells of Rhodospirillum rubrum

Photosynthetic CO₂ fixation was studied using cells of Rhodospirillumrubrum grown heterotrophically on malate or butyrate. Ratesof CO₂ fixation were higher in the malategrown cells than inthe butyrate-grown bacteria but ribulosebisphosphate (RUP₂)carboxylase/oxygenase activities were higher in the extractsprepared from the butyrate-grown bacteria. The photosyntheticCO₂ fixation in the butyrate-grown R. rubrum cells was inhibitedby KCN, and the inhibitory effect of O₂ on CO₂ fixation wasreversed when cells were returned to an N₂ atmosphere. In themalate-grown cells, photosynthetic CO₂ fixation was insensitiveto KCN and the inhibitory effect exerted by O₂ was practicallyirreversible. ¹⁴CO₂ was not incorporated into glycolate by either malate-or butyrate-grown cells in an N₂ atmosphere, but small amountsof labeled glycolate were found in malate- and butyrate-growncells in air or 100% O₂. Glycolate excreted by these cells in100% O₂ was measured colorimetrically and its identity establishedby mass spectrometry. When the O₂ atmosphere was labeled with¹⁸O₂, only one of the carboxyl oxygens of the excreted glycolatewas labeled, and the enrichment of ¹⁸O in this carboxyl oxygenrelative to the ¹⁸O₂ provided was greater than 80%. These studiesshow that significant glycolate production by R. rubrum onlyoccurs in the presence of O₂ and that in both malateand butyrate-growncells, the glycolate so formed is presumably produced via RuP₂oxygenase. ¹ Paper No. 46 in the series "Structure and Function of ChloroplastProteins", and research supported in part by research grantsfrom the Japanese Ministry of Education (No. 211113), the TorayScience Foundation (Tokyo), and the Nissan Science Foundation(Tokyo). (Received August 19, 1978; )     

Biosynthetic mechanism of glycolate in Chromatium I. Glycolate pathway

The pattern of radioactivity distribution in several amino acidsof Chromatium cells exposed to ¹⁴CO₂ was determined. By transferringthe bacterial cells from an atmosphere of nitrogen to oxygenthere occurred a transient decrease of ¹⁴CO₂ incorporation intoaspartate and glutamate, whereas that into glycine showed aprominent increase. The labeling of both serine and alaninedid not show a marked change under such conditions. The, activitiesof glycolate oxidase and glycolate dehydrogenase in crude extractsof the bacterial cells were very low. The formation of glycolic acid only occurred during the oxidativemetabolism of Chromatium cells grown on bicarbonate as a C source,being negligibly small in bacteria under nitrogen or after growthon malate or acetate. The activities of both ribulose- 1,5-bisphosphateoxygenase and phosphoglycolate phosphatase in the extract preparedfrom the bicarbonate-grown bacterial cells were very low andapparently could not account for the glycolic acid formationthrough these enzymic reactions. Metabolic patterns of glycolicacid in Chromatium are discussed in relation to the photorespiratoryphenomenon. (Received February 24, 1975; )     

Enzymatic Inactivation of Extracellular Carbonic Anhydrase and its Effect on K1/2 (CO2) for Photosynthesis in Chlorella ellipsoidea C-27

The ratio of the extracellular to the intracellular activityof carbonic anhydrase (CA) in cells of Chlorella ellipsoideaC-27, adapted to low levels of CO₂ for 24 h (low-CO₂ cells),was about one to one. Treatment of intact cells with PronaseP inactivated about one-half of the extracellular CA activitywithout affecting photosynthetic activity. The CA activity incell homogenates and in cell-wall ghosts liberated during celldivision was completely inactivated by the same treatment. Pretreatmentwith Glycosidase mix, Chitosanase and Macerozyme enhanced theinactivation of the CA activity in intact cells. These resultssuggest that extracellular CA is evenly distributed throughoutthe whole cell-wall region. The apparent K_1/2 for dissolved inorganic carbon (DIC) in low-CO₂cells doubled when extracellular CA was inactivated by treatmentwith Pronase P, but the K_1/2 obtained was still one-half ofthat in high-CO₂ cells. Photosynthetic ¹⁴CO₂-fixation in low-CO₂cells was enhanced by acetazolamide, whereas H¹⁴CO₃^–-fixationwas suppressed. The results suggest that CO₂ is a dominant substrateutilized by cells and that HCO₃^– is utilized after conversionto CO₂. The present results show that both intracellular andextracellular CA contribute to the increase in affinity forDIC during photosynthesis in low-CO₂ cells of Chlorella ellipsoideaC-27. (Received May 7, 1990; Accepted July 18, 1990)     

Differential Stimulation of PEP-carboxylation in Guard Cells and Mesophyll Cells by Ammonium or Fusicoccin

Dark CO₂-fixation in guard cells of Vicia faba was much moresensitive to ammonium than in mesophyll cells. Addition of ammonium(5.0 mol m^–3; pH₀ 7.6) caused up to a 7-fold increasein dark CO₂-fixation rates in guard cell protoplasts (GCP),whereas in leaf slices, mesophyll cells, and mesophyll protoplaststhe increase was only about 1.4-fold. In both cell or tissuetypes, total CO₂-fixation rates were higher in the light (2–12-foldhigher in GCP and 28-fold in mesophyll); these rates were onlyslightly changed by ammonium treatment. However, separationof ¹⁴C-labelled products after fixation of CO₂ in the lightby GCP revealed a large ammonium-induced shift in carbon flowfrom starch and sugars to typical products of C₄-metabolism(mainly malate and aspartate). In contrast, in mesophyll cellsamino acid and malate labelling was only moderately increasedby ammonium at the expense of sucrose. The data suggest thatin vivo ammonium might facilitate stomatal opening and/or delaystomatal closing through an increased production of organicacids. Key words: PEP-carboxylation, guard cell protoplasts, ammonium, fusicoccin     

Characterization of the Type of Photosynthetic Carbon Dioxide Fixation in Jute (Corchorus olitorius L.)

Activities of photosynthetic and photorespiratory enzymes viz.,ribulose bisphosphate carboxylase, phosphoenol pyruvate carboxylaseand glycolate oxidase from jute (Corchorus olitorius L.; cv.JRO 632) leaves were compared with those from maize (C₄) andsunflower (C₃) leaves. The photosynthetic CO₂ fixation products,the release of ¹⁴CO₂ in light and dark following photosynthesisin ¹⁴CO₂, chlorophyll a: b ratio, gross leaf photosyntheticrate and dry matter production rate were also studied. The resultsshow that jute is a C₃ plant. Key words: Jute, Corchorus olitorius, C₃ photosynthesis     

The Effect of Salinity on Light and Dark CO2-Fixation of Salt-Adapted and Unadapted Cell Cultures of Atriplex and Tomato

The effect of salinity on light and dark CO₂,-fixation was determinedin cells of A triplex portulacoides and tomato (Lycopersiconesculenturn Mill.) grown in culture. CO₂,-fixation of tomatocells was also determined in cultures adapted to mannitol andpolyethylene glycol (PEG). Salinity up to 400 mM NaCI in thecase of A triplex and up to 50 mM in the case of tomato enhancedthe rate of light-induced CO₂,-flxation in unadapted cells.Higher salt concentrations led to a marked decline in CO₂-flxationin both species. In salt-adapted A triplex cells no declinein the rate of light CO²,-flxation was seen even at 500 mM NaCl.Dark CO₂,-fixation was approximately 40% and 80% of the lightfixation in control cell cultures of A triplex and tomato, respectively.No enhancement in dark CO₂,-flxation was seen as salinity wasincreased, but a decline was found at similar salt concentrationsthat decreased fixation in the light. Mannitol-and PEG-adaptedtomato cells fixed CO₂, at somewhat lower rates than the controlcells in the light but not in the dark. Key words: Salinity, CO₂-fixation, cell cultures, Atriplex, tomato     

Effects of growth temperature on photosynthetic carbon metabolism in green plants II. Photsoynthetic 14CO2-incorporation in plants acclimatized to varied temperatures

During photosynthetic ¹⁴CO₂-fixation, leaves of plants suchas wheat, the broad bean and spinach, which had been acclimatizedto high temperature (20–25?C), incorporated much moreradioactivity into sucrose, and less into glycine and serinein comparison with similar plants grown in the cold (mean temperature,5–7?C). Radioactivities incorporated into glycine and serine greatlydescreased on the addition of -hydroxyethylsulfonate or on theremoval of oxygen from the atmosphere, indicating that thesecompounds are synthesized through the glycolate pathway. In leaves of wheat grown under low temperatures, relativelyhigh radioactivity was detected in ribulose 1,5-diphosphateamong the photosynthetic ¹⁴CO₂-fixation products, whereas practicallyno radioactivity was detected in this compound in leaves ofwheat which had been acclimatized to high temperatures. We assumedthat the carboxylation reaction of ribulose 1,5-diphosphateis suppressed in plants acclimatized to low temperatures. It was further inferred that the C-2 and C-2 moiety of ribulose1,5-diphosphate accumulating as a result of suppression of carboxylationis converted to glycine and serine through the glycolate pathway. The possibility was also discussed that during photosyntheticCO₂-fixation in wheat leaves at least a part of the C₆-compoundformed by the carboxylation of ribulose 1,5-diphosphate is directlyconverted to sugar phosphate. ¹Part of this investigation was reported at the 2nd InternationalCongress on Photosynthesis Research at Stresa, Italy, June 1971.This paper is based on a dissertation submitted by S.S. to theFaculty of Science, the University of Tokyo, in partial fulfilmentof the requirements for a Ph.D. degree. ²Present address: Department of Botany, Faculty of Science,University of Tokyo, Tokyo, Japan. (Received July 20, 1973; )     

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; )     

Comparison of carbon dioxide fixation and the fine structure in various assimilatory tissues of Amaranthus retroflexus L

The pattern for primary products of CO₂-fixation and the chloroplaststructure of Amaranthus retrqflexus L., a species which incorporatescarbon dioxide into C₄ dicarboxylic acids as the primary productof photosynthesis, were compared in various chlorophyll containingtissues,i.e., foliage leaves, stems, cotyledons and pale-greencallus induced from stem pith. Despite some morphological differencesin these assimilatory tissues, malate and aspartate were identifiedas the major compounds labelled during a 10 sec fixation of¹⁴CO₂ in all tissues. Whereas, aspartate was the major componentin C₄-dicarboxylic acids formed in foliage leaves, malate predominatedas the primary product in stems, cotyledons and the pale-greencallus. The percentage of ¹⁴C-radioactivity incorporated intoPGA and sugar-P esters increased and ¹⁴C-sucrose was detectedin the prolonged fixation of ¹⁴CO₂ in the light, not only infoliage leaves, but also in stems and cotyledons. ¹ This work was supported by a Grant for Scientific ResearchNo. 58813, from the Ministry of Education, Japan. ² Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo, Japan. ³ Present address: Department of Biochemistry, University ofGeorgia, Athens 30601. Georgia, U. S. A. (Received July 10, 1971; )