Release of carboxylating enzymes from maize and sugar cane leaf tissue during progressive grinding

Summary The release of chlorophyll, chloroplasts, o-diphenols, o-diphenol oxidase activity and carboxylating enzyme activity during the grinding of maize and sugar cane leaf tissue has been correlated with the breakage of different types of cell. Enzymes of the photosynthetic carbon reduction cycle were released in the grinding stage during which the bulk of the mesophyll tissue was disrupted and grana-containing chloroplasts released. Since the largest amount of phenol oxidase activity and of phenols was also released at this stage it is likely that the enzymes were partly inhibited by phenol oxidation products and, therefore, underestimated. PEP carboxylase is released earlier in the grinding process. It is concluded that the photosynthetic carbon reduction cycle enzymes studied are located in mesophyll cell chloroplasts and that the PEP carboxylase resides outside the chloroplasts, either in the cytoplasm of mesophyll cells or in colourless tissue. These results are discussed in relation to current theories regarding the assimilation and shuttling of carbon dioxide in leaves of tropical grasses.     

The occurrence of both C3 and C4 photosynthetic characteristics in a single Zea mays plant

The activities of the carboxylating enzymes ribulose-1,5-biphosphate (RuBP) carboxylase and phosphoenolpyruvate (PEP) carboxylase in leaves of three-week old Zea mays plants grown under phytotron conditions were found to vary according to leaf position. In the lower leaves the activity of PEP carboxylase was lower than that of RuBP carboxylase, while the upper leaves exhibited high levels of PEP carboxylase. Carbon dioxide compensation points and net photosynthetic rates also differed in the lower and upper leaves. Differences in the fine structure of the lowermost and uppermost leaves are shown. The existence of both the C₃ and C₄ photosynthetic pathways in the same plant, in this and other species, is discussed.Abbreviations PEP phosphoenolpyruvate - RuBP ribulose-1,5-biphosphate     

Source of glycolate and cyclic changes in photosynthetic and photorespiratory activity during the development of barley leaves

¹⁴CO₂ assimilation, RuBP earboxylase and PEP carboxylase activities show cyclic changes during the development of barley leaves. Cyclic changes, but in phase opposition with respect to carboxylating enzymes, are shown by RuBP oxygenase, phosphoglycolate phosphatase, glycolate oxidase and nitrate reductase activities. The oxygenase function of RuBP carboxylase appears to be the primary source of glycolate in young leaves, whereas in old ones glycolate could be supplied from some source in addition to RuBP oxygenase activity.     

Carboxylating enzymes and carbonic anhydrase functions were suppressed by zinc deficiency in maize and chickpea plants

Maize and chickpea plants were grown in a controlled environment with 0.5 M Zn or without Zn and various photosynthetic reactions were studied. The chlorophyll level, the rate of photosynthesis and photosystem II activity, the activity of carboxylating enzymes and that of carbonic anhydrase were suppressed by Zn deficiency in both plant species. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was quantified using polyacrylamide gel electrophoresis. Growing plants in a medium without Zn caused a decrease in the total protein level and in the levels of large and small subunits of Rubisco.     

Biochemical characteristics of photosynthetic response to various external salanities in halotolerant and fresh water cyanobacteria

Abstract The effect of various salinities on photosynthetic carbon fixation, activities of carboxylating enzymes, and contents of chlorophyll and protein, was examined in a halotolerant ( Aphanothece halophytica ) and fresh water ( Aphanothece stagnina ) cyanobacterium. These parameters were quantified immediately and after 48 h incubation in altered salinities. Photosynthetic carbon fixation was lower at altered salinities than in controls in both species. A. halophytica with 48 h incubation showed increased carbon fixation with increasing NaCl concentration, whereas A. stagnina did not. Chlorophyll content was drastically reduced for all salinities in A. stagnina but was unchanged in A. halophytica . d -ribulose-1,5-bisphosphate carboxylase (RuBisCO) activity from A. halophytica increased at higher external salinities but decreased in cells of A. stagnina . In vitro, the sensitivity of RuBisCO to NaCl from both the species was similar. It was noted that in A. stagnina NaCl affected chlorophyll content, carbon fixation, and RuBisCO activity in decreasing order.     

Developmental and Varietal Comparisons of Pod Carboxylase Levels in Pisum sativum L.

The activities of phosphoenolpyruvate (PEP) carboxylase andribulose-1, 5-bisphosphate (RuDP) carboxylase have been determinedin the developing pod walls of six genotypes of Pisum sativum.Genotypes were chosen which varied in pod characters such aschlorophyll content and tissue morphology, which it was hopedwould be associated with variation in carboxylase levels. Whilst both enzymes were detected in all genotypes, the levelsof activity varied considerably with pod type and with age.In general RuDP carboxylase activity correlated with chlorophyllconcentration, and yellow podded types had considerably lessactivity than green types. The yellow podded genotypes, however,contained significantly higher levels of PEP carboxylase which,in terms of total carboxylase activity, compensated for thelower RuDP carboxylase levels. The activities of both enzymes were determined within the endocarpand within the mesocarp plus exocarp, using 16-day-old pods.All genotypes showed an enrichment for PEP carboxylase in theendocarp and all but one genotype showed a similar enrichmentfor RuDP carboxylase activity in this layer. The role of the carboxylase enzymes within the pod wall is discussedand it is suggested that their main function may be to maintainan appropriate level of CO₂ within the pod cavity as well asrecycling carbon to the developing seeds. Pisum savitum L., pea, pods, carboxylase levels, genetic variation     

Photorespiration studies in Cycas circinalis L. and Cycas beddomei Dyer

Photosynthetic carboxylating enzymes and the effects of light and temperature on ¹⁴CO₂ efflux in two species of gymnosperm leaves were studied. The activity of RuBP carboxylase was high and that of PEP carboxylase was very low when compared to C₄ plants. The CO₂ compensation point was high. ¹⁴CO₂ efflux was greater in light than in darkness and the ratio (L/D) increased with increase in temperature and light intensity. The inhibitors of glycolate metabolism showed decreased ¹⁴CO₂ evolution in light while dark respiration was unaffected. It is concluded that both Cycas circinalis, L. and Cycas beddomei Dyer are C₃ plants.     

Photosynthetic characterization of photoautotrophic cells cultured in a minimal medium

Photosynthetic properties of photoautotrophic suspensions cultured in a minimal growth medium have been evaluated to determine whether changes have occurred in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, phosphoenol-pyruvate (PEP) carboxylase activity, chlorophyll content, or culture growth. Five photoautotrophic lines Amaranthus powellii, Datura innoxia, Glycine max, Gossypium hirsutum, and a Nicotiana tabacum-Nicotiana glutinosa fusion hybrid were grown in a medium without organic carbon other than phytohormones, and without vitamins. These photoautotrophic lines had total Rubisco activities ranging from 85 to 266 micromoles CO₂ fixed per milligram chlorophyll hour⁻¹, with percent activation of Rubisco ranging from 16 to 53%. Inclusion of protease inhibitors in the homogenization buffer did not result in higher Rubisco activity. PEP carboxylase activity for cells cultured in minimal medium was found to range from 16 to 146 micromoles CO₂ per milligram chlorophyll hour⁻¹, with no higher activity in the C₄Amaranthus cells compared with PEP carboxylase activity in the C₃ species assayed. Rubisco-to-PEP carboxylase ratios ranged from 2.2 to 1 up to 9.4 to 1. Chlorophyll contents increased in all but the Nicotiana cell line, and all of the photoautotrophic culture lines were capable of growth in vitamin-free medium with the exception of SB-P, which requires thiamine.     

Comparison Between Growth Chamber and Field-Grown Zea mays Plants for Photosynthetic Carboxylase Activities and .Other Physiological Characteristics with Respect to Leaf Position

Baldy, P. 1986. Comparison between growth chamber and field-grownZea mays plants for photosynthetic carboxylase activities andother physiological characteristics with respect to leaf position.—J.exp. Bot. 37: 309–314. The lengths and fresh weights of leaves, and the amounts ofchlorophyll and protein per leaf, were higher in maize grownin a controlled environment than for field-grown maize harvestedat a similar stage of growth with the seventh leaf just emerging.However, the fresh weight and chlorophyll per unit leaf areawere not different and more protein was present per Unit leafarea in maize cultivated in the field, particularly in the 4thleaf. Except in the seventh leaf the youngest present, the PEP carboxylaseactivity was 2-fold higher in field-grown maize than in plantsfrom the controlled environment. The RuBP carboxylase activitywas not significantly affected by growth conditions. The maximumactivities of the carboxylases were found in the 5th leaf formaize grown in the controlled environment and the 4th leaf forfield maize; in these two leaves the ratios of the PEP: RuBPcarboxylasc activities were 3·0 and 4·4 respectively. The results are used to justify the choice of the fully expanded4th leaf of l9-d-old plants grown in a controlled environmentfor studies of the enzymes involved in the photosynthetic carbonmetabolism of this C₄ plant. Key words: Growth conditions, PEP carboxylase/RuBP carboxylase activity ratio, Zea mays leaf position     

The influence of temperature on malic Acid metabolism in grape berries: I. Enzyme responses

Phosphoenolpyruvate (PEP) carboxylase activity in immature `Carignane' grape berries (Vitis vinifera L.) had a temperature optimum of about 38 C, whereas malic enzyme activity rose with increasing temperature between 10 and 46 C. In vitro temperature inactivation rates for the PEP carboxylase were markedly greater than for the malic enzyme activity. From the simultaneous action of malic acid-producing enzymes (PEP carboxylase and malic dehydrogenase) and malic acid-degradating enzyme (malic enzyme) systems at different temperatures, the greatest tendency for malic acid accumulation in immature grape berries was at 20 to 25 C. Time-course measurements of enzymic activity from heated, intact berries revealed greater in vivo temperature stability for the malic enzyme activity than for the PEP carboxylase activity.     

果蔗叶片生理生化指标与品质性状的典范相关分析

选用果蔗（Saccharum officinarum L．）14个品种,用典范相关分析法研究不同生育期叶片生理生化指标对果蔗品质性状的影响。结果表明：分蘖期类胡萝卜素含量与蔗茎蔗糖含量,可溶性总糖含量与蔗茎含水率,可溶性总糖含量与蔗汁还原糖含量均呈正相关;伸长初期叶绿素含量、CAT活性与蔗茎蔗糖含量,可溶性蛋白质含量与蔗茎纤维含量均呈正相关;伸长盛期Mg^2＋ -ATP酶活性与蔗茎纤维含量,可溶性总糖含量与蔗茎蔗糖含量呈正相关,C／N与蔗茎含水率呈负相关;成熟期类胡萝卜素含量与蔗茎蔗糖含量呈正相关,可溶性总糖含量与蔗汁还原糖含量呈负相关。     

Growth characteristics of hormone and vitamin independent photoautotrophic cell suspension cultures fromChenopodium rubrum

Summary This communication reports the photoautotrophic growth of hormone and vitamin independent cell suspension cultures ofChenopodium rubrum. The transfer of cells from stationary growth into fresh culture medium results in a high protein formation, followed by an exponential phase of cell division, whereas the onset of rapid chlorophyll formation is delayed for 4 days. At the stage of most rapid cell division there is no net synthesis of starch and sugar. When the cells enter stationary growth, there is a progressive accumulation of chlorophyll, sugar, and starch.Photoautotrophic cell cultures assimilate about 80–90 mol CO₂/mg chlorophyll X hour. Dark CO₂ fixation is about 3.7% to 2.2% of the light values during exponential and stationary growth, respectively. As shown by short-term¹⁴CO₂ fixation, CO₂ is predominantly assimilated through ribulosebisphosphate carboxylase via the Calvin pathway. There is a significant increase in the¹⁴C label of C₄ carboxylic acids in exponentially dividing cells as compared to cells from stationary growth. Thein vitro activity of phosphoenolpyruvate carboxylase and ribulosebisphosphate carboxylase is almost equal during exponential cell division. A decrease in cell division activity is accompanied by a significant change in the specific activities of both carboxylation enzymes. In non dividing cells from stationary growth the activity of ribulosebisphosphate carboxylase is greately enhanced and that of phosphoenolpyruvate carboxylase is reduced, documenting the development of carboxylation capacities typical for C₃-plants.The experimental results provide evidence that phosphoenolpyruvate carboxylase activity might be regulated by ammonia and could be involved in anaplerotic CO₂ fixation which supplies carbon skeletons of the citric acid cycle.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylene-diamine-tetraacetic acid - FDP fructose bisphosphate - F-6-P fructose-6-phosphate - G-6-P glucose-6-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PGA 3-phosphoglyceric acid - PEP phosphoenolpyruvate - RuDP ribulosebisphosphate     

Development of Photosynthetic Activity following Anaerobic Germination in Rice-Mimic Grass (Echinochloa crus-galli var oryzicola)

Shoots of anaerobically germinated Echinochloa crus-galli var oryzicola are nonpigmented whether germinated in light or dark, and chlorophyll synthesis is minimal for the first 12 to 18 hours of greening after exposure to ambient conditions. When chlorophyll development is compared between greening anoxic and etiolated shoots, there is a 100-fold difference in chlorophyll levels at 8 hours, an 8-fold difference at 24 hours, but roughly equal amounts at 60 hours. The chlorophyll a/b ratio approaches 3 earlier in greening anoxic shoots than in greening etiolated shoots, relative to total chlorophyll. The long lag in chlorophyll synthesis can be shortened by giving dark-grown anoxic shoots a 24-hour midtreatment of air before light.

Development of photosynthetic activity in etiolated shoots, determined by CO₂ gas exchange, ¹⁴CO₂ uptake, and activity of carboxylating enzymes closely parallels development of chlorophylls. However, development of photosynthetic capability in greening anoxic shoots does not parallel chlorophyll development; ability to fix carbon lags behind chlorophyll synthesis. A reason for this lag is the very low activity of RuBP carboxylase during the first 36 hours of greening in anoxic shoots. The activity of phosphoenolpyruvate carboxylase is also delayed, but its kinetics more closely match those of chlorophyll development.

     

Changes in Sensitivity to Effectors of Maize Leaf Phosphoenolypyruvate Carboxylase during Light/Dark Transitions

Illumination of previously darkened maize (Zea mays L. cv Golden Cross Bantam T51) leaves had no effect on the concentration of phosphoenolpyruvate (PEP) carboxylase protein, but increased enzyme activity about 2-fold when assayed under suboptimal conditions (pH 7.0 and limiting PEP). In addition, sensitivity to effectors of PEP carboxylase activity was significantly altered; e.g. malate inhibition was reduced and glucose-6-phosphate activation was increased. Consequently, 10- to 20-fold differences in PEP carboxylase activity were observed during dark to light transitions when assayed in the presence of effectors. At pH 7.0 activity of purified PEP carboxylase was not proportional to enzyme concentrations. Below 0.7 microgram PEP carboxylase protein per milliliter, enzyme activity was disproportionately reduced. Including polyethylene glycol plus potassium chloride in the reaction mixture eliminated this discontinuity and substantially increased PEP carboxylase activity and reduced malate inhibition dramatically. Inclusion of polyethylene glycol in the assay mixture specifically increased the activity of PEP carboxylase extracted from dark leaves, and reduced malate inhibition of the enzyme from both light and dark leaves. Collectively, the results suggest that PEP carboxylase in maize leaves is subjected to some type of protein modification that affects both activity and effector sensitivity. We postulate that changes in quaternary structure (dissociation or altered subunit interactions) may be involved.     

Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes.

The synthesis of 10 new phosphoenolpyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP-utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z)- and (E)-3-chloro-PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion-exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the (3)J(H-C) coupling constants between the carboxylate carbons and the vinyl protons. All of these and other known PEP-analogues were tested as reversible and irreversible inhibitors of Mg2+- and Mn2+- activated PEP-utilizing enzymes: enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), pyruvate kinase, PEP carboxylase and enolase. Without exception, the most potent inhibitors were those with substitution of a vinyl proton. Modification of the phosphate and the carboxylate groups resulted in less effective compounds. Enzyme I was the least tolerant to such modifications. Among the carboxylate-modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase. Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg2+, but not with Mn2+. For the irreversible inhibition of these enzymes, (Z)-3-Cl-PEP was found to be a very fast-acting and efficient suicide inhibitor of enzyme I (t(1/2) = 0.7 min).     

Effect of Potassium Deficiency on C3 and C4 Cereals

C₄ cereals (Zea maya L. and Sorghum bicolor L. Moench) and C₃cereals (Triticum aestivum L. and Hordeum vulgare L) were grownin nutrient solutions with constant, interrupted, or absentpotassium supply. The lack of potassium retarded shoot growthand depressed the chlorophyll accumulation in all species ina similar way. After the renewal of potassium, the differencesin the compensation for growth retardation were not correlatedwith the photosynthetic system, but with the recovery of chlorophyllaccumulation in younger leaves. As important for the compensationof shoot growth retardation was a slower senescence of old leavescompared to plants with a constant potassium supply. This wasshown by the chlorophyll content and PEP carboxylase activity.In contrast to C₃ cereals, the C₄ cereals did not react withhigher chlorophyll contents to the same extent after the renewalof the postassium supply. The PEP carboxylase activity, however,was immediately raised higher than in control leaves. Chlorophylland PEP carboxylase activity increased simultaneously only inless aged leaves.     

Purification and characterization of phosphoenolpyruvate carboxylase from a cyanobacterium.

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified 100-fold from the cyanobacterium Coccochloris peniocystis with a yield of 10%. A single isozyme was found at all stages of purification, and activity of other beta-carboxylase enzymes was not detected. The apparent molecular weight of the native enzyme was 560,000. Optimal activity was observed at pH 8.0 and 40 degrees C, yielding a Vmax of 8.84 mumol/mg of protein per min. The enzyme was not protected from heat inactivation by aspartate, malate, or oxalacetate. Michaelis-Menten reaction kinetics were observed for various concentrations of PEP, Mg2+, and HCO3-, yielding Km values of 0.6, 0.27, and 0.8 mM, respectively. Enzyme activity was inhibited by aspartate and tricarboxylic acid cycle intermediates and noncompetitively inhibited by oxalacetate, while activation by any compound was not observed. However, the enzyme was sensitive to metabolic control at subsaturating substrate concentrations at neutral pH. These data indicate that cyanobacterial PEP carboxylase resembles the enzyme isolated from C3 plants (plants which initially incorporate CO2 into C3 sugars) and suggest that PEP carboxylase functions anapleurotically in cyanobacteria.     

Changes of ribulose 1,5-diphosphate carboxylase level during the processes of degeneration and regeneration of chloroplasts in Chlorella protothecoides

RuDP carboxylase was active mainly in chloroplasts and PEP carboxylaseactive principally outside of chloroplasts in Chlorella protothecoides. During the process of chloroplast degeneration in algal cellsinduced by addition of glucose, the activity of RuDP carboxylasesignificantly decreased, whereas the activities of PEP-carboxylaseand -carboxykinase markedly increased. During the process of chloroplast regeneration in "glucose-bleached"algal cells, which contained no detectable amounts of FractionI protein and showed only traces of RuDP carboxylase activity,a light-dependent development of RuDP carboxylase proceededalmost in parallel with the light-induced formation of chlorophyll.The activities of PEP-carboxylase and -carboxykinase, whichwere negligibly low in glucose-bleached cells, developed independentlyof light. Both chloramphenicol and cycloheximide severely inhibited thedevelopment of RuDP carboxylase activity. A relatively low concentrationof glucose also caused a significant suppression. Under theseconditions, chlorophyll formation was inhibited only slightlyby chloramphenicol and very strongly by cycloheximide and glucose. ¹ Deceased, 11 June, 1972. (Received April 25, 1972; )     

Influence of age and illumination on distribution of several calvin cycle enzymes in greening barley leaves

Activities of phosphoriboisomerase, phosphoribulokinase, and ribulose 1,5-diphosphate carboxylase, protein content, and chlorophyll accumulation in dark-grown barley seedlings were measured before and after illumination. Enzymatic activities, levels of soluble protein, and accumulation (upon illumination) of chlorophyll in leaves declined from tips toward the base. In response to increasing time of illumination, chlorophyll accumulation and activities of phosphoribulokinase and ribulose 1,5-diphosphate carboxylase (enzymes located in chloroplasts) increased most in tip portions whereas activity of phosphoriboisomerase and levels of soluble protein (constituents not confined to chloroplasts) increased similarly in all sections of the leaf. Maximum activity of phosphoribulokinase and maximum accumulation of chlorophyll shifted toward median portions of the leaf blade with increased age of seedling before illumination. Maximum activity of ribulose 1,5-diphosphate carboxylase and maximum level of soluble protein occurred in all leaf sections when the seedlings were 7 days of age before illumination.     

CO 2 -fixing enzymes in a marine psychophile

A psychrophilic marine Pseudomonas was found to contain phosphoenolpyruvate (PEP) carboxylase and an adenosine triphosphate-linked PEP carboxykinase. Some properties of these CO(2)-fixing enzymes were compared with those homologous enzymes from the terrestrial mesophile Enterobacter cloacae. The PEP carboxylases from both organisms were activated by acetyl-coenzyme A (CoA) and inhibited by l-aspartate. The enzyme from Pseudomonas was less dependent on the presence of the activator, but maximal activation was attained at acetyl-CoA concentrations much lower (50 mum) than those required to saturate the enzyme from E. cloacae. In both cases the main effect of acetyl-CoA was to decrease the K(m) value for PEP. The activity of PEP carboxylase from Pseudomonas was only slightly inhibited by NaCl, KCl, or NH(4)Cl up to 100 mm, whereas the enzyme from E. cloacae was inhibited by about 70% under similar experimental conditions. Both PEP carboxylase and PEP carboxykinase from Pseudomonas showed considerably lower thermal stability than their counterparts from E. cloacae. Our results suggest that the CO(2)-fixing enzymes from a marine Pseudomonas and E. cloacae are similar in nature and regulation, but they differ in properties related to the peculiar conditions of the marine environment.