Changes in Photosynthetic Capacity in Rice Leaves from Emergence through Senescence. Analysis from Ribulose-1,5-bisphosphate Carboxylase and Leaf Conductance

Changes in the rates of gas exchange and the amount of ribulose1,5-bisphosphate (RuBP) carboxylase protein were determinedin the 12th leaf blades of rice during the reproductive stages.RuBP carboxylase exhibited a large change similar to that inthe assimilation rate at 2% O₂ throughout the leaf's life, butits decrease during senescence was barely faster than the decreasein the assimilation rate. Consequently, the overall relationshipwas slightly curvilinear. By contrast, leaf conductance decreasedmore slowly than the assimilation rate which resulted in theintercellular CO₂ concentration increasing during senescence. In order to determine the maximum activity of RuBP carboxylaseat the intercellular CO₂ concentration, the kinetic parametersand their pH response were determined using purified, and completelyactivated, rice RuBP carboxylase. The maximum carboxylase activityat the intercellular CO₂ concentration was linearly correlatedwith the assimilation rate at 2% O₂ (r=0.989), and was veryclose to that needed to account for the assimilation rate. We conclude that changes in both the amount of RuBP carboxylaseprotein and leaf conductance reflect the change in photosynthesisduring the life span of the leaf. (Received November 26, 1983; Accepted February 20, 1984)     

Polypeptide Composition of Envelope Membranes Isolated from Chloroplasts of C3, C4, and CAM Plants

Chloroplast envelopes were isolated from chloroplasts purifiedfrom Spinacea oleracea L. (C₃), Panicum miliaceum L. (NAD-malicenzyme-type C₁), Digitaria sanguinalis (L.) Scop. (NADP-malicenzyme-type C₄), Kalanchoe daigremontiana Hamet et Perrier (constitutiveCAM), and from Mesembryanthemum crystallinum L. (inducible CAM)performing either C₃ photosynthesis or Crassulacean acid metabolism(CAM). For each species, methods were developed to isolate chloroplastenvelopes free of thylakoid contamination. The polypeptidesof ribulose bisphosphate (RuBP) carboxylase which has been consistentlyreported in envelope preparations of spinach were not foundin envelope preparations of C₄ mesophyll chloroplasts. Silverstaining of envelope polypeptides resolved electrophoreticallyon sodium dodecylsulfate polyacrylamide gradient slab gels produceda more complex profile than did Coomassie staining which haspreviously been used with C₃ envelope preparations, even thoughsilver reacted poorly with polypeptides corresponding to thesubunits of RuBP carboxylase. All of the plants examined possesseda major polypeptide of 27 to 29 kilodaltons (kD) which was previouslysuggested to be the phosphate translocator in spinach. WithC₃ M. crystallinum, the 29 kD polypeptide stained most intensely.After induction of CAM, a 32 kD polypeptide also stained intensely,giving a profile similar to that obtained with the constitutiveCAM species. A 32 kD polypeptide was also prominent in C₄ envelopepreparations, suggesting that a 32 kD polypeptide may be a translocatorprotein which is required in Crassulacean acid metabolism andC4 photosynthesis, but not in C₃ photosynthesis. (Received April 25, 1983; Accepted July 9, 1983)     

Calcium-Dependent and -Independent Phosphoenolpyruvate Carboxylase Kinases in Sorghum Leaves: Further Evidence for the Involvement of the Calcium-Independent Protein Kinase in the in situ Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase

Calcium-dependent phosphoenolpyruvate carboxylase protein kinasewas copurified with C₄ phosphoenolpyruvate carboxylase (C₄ PEPC)from illuminated Sorghum leaves during purification by variousprocedures. Isolated mesophyll cell protoplasts contained bothcalcium-dependent and -independent protein kinases. The latterwas induced by light and weak bases and was found to be themajor protein kinase phosphorylating C₄ PEPC in the mesophyll. (Received July 29, 1997; Accepted November 28, 1997)     

Purification and Storage of Ribulose 1,5-bisphosphate Carboxylase from Rice Leaves

Ribulose 1,5-bisphosphate (RuBP) carboxylase was purified fromrice leaves. By using a buffer containing 12.5% (v/v) glycerolthroughout purification, the enzyme was protected from coldlability and was obtained at a high yield (5.5 mg/g fresh wt).The purified enzyme exhibited different rates of CO₂/Mg²⁺-activationby temperature pretreatment/storage. The purified enzyme was stable for at least one year in phosphatebuffer containing 12.5% (v/v) glycerol at 4°C or 50% (v/v)glycerol at –20°C. (Received March 1, 1983; Accepted June 27, 1983)     

Studies on the Properties of Carboxylating Enzymes in the Epidermis of Commelina communis

A spectrophotometric assay has been used to measure the activityof PEP carboxylase and RuBP carboxylase in the epidermal andmesophyll tissue of Commelina communis. On both a chlorophylland protein basis the PEP carboxylase activity was always greaterin the epidermis than in the mesophyll, whereas RuBP carboxylaseactivity was always highest in the mesophyll. PEP carboxylaseactivity in epidermal extracts was lost very slowly and itspH optimum was a broad one in the range 7·5–8·0.The K_m values for PEP carboxylase in the epidermis and mesophyllobtained from light- and dark-treated plants were not very differentalthough its V_max was much lower in dark-treated tissue. Thesedata are discussed in relation to the possible role of PEP carboxylasein guard cell metabolism.     

Photosynthetic and Photorespiratory Enzymes in Widely Divergent Plant Species with Special Reference to the Moss Ceratodon purpureus: Properties of Ribulose Bisphosphate Carboxylase/ Oxygenase, Phosphoenolpyruvate Carboxylase and Glycolate Oxidase

Rintamäki, E. and Aro, E.-M. 1985. Photosynthetic and photorespiratoryenzymes in widely divergent plant species with special referenceto the moss Ceratodon purpureus: Properties of ribulose bisphosphatecarboxylase/oxygenase, phosphoenolpyruvate carboxylase and glycolateoxidase.—J. exp. Bot. 36: 1677–1684. Km(CO₂) values and maximal velocities of ribulose bisphosphatecarboxylase/oxygenase (E.C. 4.1.1.39 [EC] ) were determined for sixplant species growing in the wild, consisting of a moss, a fernand four angiosperms. The maximum velocities of the RuBP carboxylasesvaried from 0.13 to 0.;62 µmol CO₂ fixed min^–1 mg^–1soluble protein and the Km(CO₂) values from 15 to 22 mmol m^–3CO₂. The highest Km(CO₂) values found were for the moss, Ceratodonpurpureus, and the grass, Deschampsia flexuosa. These plantsalso had the highest ratios of the activities of RuBP carboxylaseto RuBP oxygenase. Glycolate oxidase (E.C. 1.1.3.1 [EC] ) activitieswere slightly lower in D.flexuosa, but not in C. purpureus,than for typical C₃ species. Phosphoenolpyruvate carboxylase(E.C. 4.1.1.31 [EC] ) was not involved in the photosynthetic carboxylationby these two plants. However, another grass, Phragmites australis,was intermediate in PEP carboxylase activity between C₃ andC₄ plants The properties of RuBP carboxylase/oxygenase are discussedin relation to the activities of PEP carboxylase and glycolateoxidase and to the internal CO₂ concentration. Key words: RuBP carboxylase, oxygenase, Km(CO₂), moss     

Ribulose-1,5-bisphosphate carboxylase in the bundle sheath cells of maize leaf

By employing the one-step enzymic digestion of maize leaf tissues,mesophyll protoplasts and bundle sheath strands were separatedwithout cross-contamination. Ribulose-1,5-bisphosphate (RuP₂carboxylase and NADP-malic enzyme were found to be exclusivelylocalized in the bundle sheath cells, whereas phosphoenolpyruvatecarboxylase, the primary carboxylation enzyme in the C₄-photosyntheticpathway, was only present in the mesophyll cells. Immunochemicalprecipitation experiments using the rabbit antisera developedagainst the spinach leaf RuP₂ carboxylase revealed the entireabsence of this enzyme protein and/or immunologically relatedmolecules in the mesophyll cells. The structural relatednessof the maize carboxylase molecule with the spinach enzyme, containingthe large (A) and small (B) subunits was demonstrated, and fromthe quantitative immunoassay it was estimated that the enzymeprotein comprises at least 30% of the total soluble proteinin the bundle sheath cells. ¹ This is paper No.41 in the series "Structure and Functionof Chloroplast Proteins". The research was supported in partby grants from the Ministry of Education (111912, 147106), theToray Science Foundation (Tokyo) and the Nissan Science Foundation(Tokyo). (Received June 23, 1977; )     

Variation in the Specificity Factor of C3 Higher Plant Rubiscos Determined by the Total Consumption of Ribulose-P2

The specificity factors for ribulose 1, 5-bisphosphate carboxylase/oxygenase(rubisco) from six species of photosynthetic organisms are compared.The values were determined by measuring the oxygen uptake duringthe total consumption of ribulose-P₂ in the presence of variousconcentrations of O₂ and CO₂. The specificity factors determinedin this way were similar to values previously published; smallbut significant differences were found between the specificityfactors of rubisco from C₃ higher plant species. Key words: Specificity factor, total consumption, partitioning, carboxylase oxygenase ratios, ribulose bisphosphate carboxylase, rubisco     

Variations in the Contents and Kinetic Properties of Ribulose-1,5-bisphosphate Carboxylases among Rice Species

The K_m(CO₂) ancl V_max of ribulose 1,5-bisphosphate (RuBP) carboxylaseand its protein ratio to total soluble protein from Oryza speciesincluding cultivars (25 varieties) and wild types (11 species,21 strains) were surveyed. Their variabilities among cultivarsof O. sativa were very small. The averages of the K_m(CO₂) andV_max values and the ratio of carboxylase to soluble protein,and their standard errors were 10.2?1.0µM, 1.72?0.13units.mg^–1(pH 8.0 and 25?C) and 52?2%, respectively. However, some differencesseemed to exist based on genome constitution in the Oryza genus.RuBP carboxylases from the species with the A^gA^g genome, O.graberrima and O. breviligulate, exhibited low K_m(CO₂) values(8.0?0.8 µM). High V_max was associated with the CC genome,O. eichingeri and O. officinalis (2.08?0.15 units.mg^–1).A higher ratio of RuBP carboxylase protein to soluble proteinwas found for the AA genome, O. sativa and O. perennis. (Received September 24, 1986; Accepted April 15, 1987)     

Ribulose-1,5-Diphosphate Carboxylase Protein during Flag Leaf Senescence

Ribulose-l,5-diphosphate (RuDP) carboxylase protein and activitywere determined in relation to net photosynthetic rate duringthe senescence of intact flag leaves of wheat on the plant.Initially the decrease in RuDP carboxylase activity was greaterthan the decline in net photosynthesis. The major decrease inRuDP carboxylase activity over this period resulted from a decreasein enzyme specific activity from 11 to 2 µmol CO₂ fixedh^–1 mg^–1 protein. Loss of RuDP carboxylase proteindid not occur until late in senescence by which time chlorophyllconcentration had decreased by more than 50%. Treatment of flagleaves at weekly intervals with either 1000 parts 10^–62-chloro-ethyltrimethylammonium chloride or 100 parts 10^–6gibberellic acid with 1 part 10^–6 kinetin did not significantlyaffect net photosynthetic rate, RuDP carboxylase protein oractivity during senescence.     

Inhibition of Ribulose-P2 Carboxylase/Oxygenase by Fluoride

Fluoride is a potent inhibitor of both reactions of ribulose-P₂carboxylase/oxygenase. Inhibition is almost totally competitivewith respect to CO₂, but uncompetitive with respect to ribulose-P₂Inhibition of photosynthesis after exposure to HF may resultfrom the inhibition of ribulose-P₂ carboxylase by F– accumulatedin the leaves. Key words: Fluoride inhibition, Ribulose P₂ carboxylase, Activation     

Some Properties of Ribulose Bisphosphate Carboxylase Extracted from Tomato Leaves

Ribulose bisphosphate carboxylase (EC 4.1.1.39 [EC] ) activity wasvery low in tomato leaf extracts unless prepared in the presenceof Mg²⁺, and polyclar AT. With young leaves, but not with fully-expanded leaves, the RuBP carboxylase activityextracted was increased by prolonged illumination of the leaves(2 h). The main effect of the light treatment was to increasethe specific activity of the enzyme but there was also a smallincrease in RuBP carboxylase protein. Tomato leaf RuBP carboxylasein extracts had specific activities in the range 0.2–0–6µmol CO₂ min^–1 mg^–-1 total protein extracted,or 0.5–1.2 µmol CO₂ min^–1 mg^–1 RuBPcarboxylase, and an apparent K_m (CO₂) at 20 ?C of 9.3 ? 1.2µM (using a of 6.407). Key words: Tomato leaf, RuBP carboxylase, Properties     

Phosphoenolpyruvate Carboxylase and CarbonEconomy of Apple Seedlings

Seeds of apple cv. Golden Delicious were germinated and cultivatedin the greenhouse until the third leaf emerged. Respirationofgerminating seeds or photosynthesis of the first leaves wasmeasured by infra-red gas analysis and porometry, respectively.To study the role of phosphoenolpyruvate carboxylase (PEPC),the dominant carboxylase in the carbon economy, its CO₂ refixationpotentialwas related to the amount of CO₂ lost in respiration. With arange of 0.2 (dry seeds) to 18 (cotyledons) µmol CO₂ h^–1g^–1 PEPC activity resembled or exceeded the amount ofC0₂ lost in respiration before the third leaf developed. Itis concludedthat PEPC largely contributes to economize the carbonmetabolism of apple seedlings before they become photosyntheticallycompetent. Key words: Apple (Malus pumila Mill.) seedling, carbon economy, phosphoenolpyruvate carboxylase, photosynthesis, respiration     

Ribulose-1, 5-diphosphate carboxylase of Chromatium strain D

RuDP carboxylase isolated from autotrophically grown cells ofphotosynthetic sulfur bacterium, Chromatium strain D, was partiallypurified by (NH₄)₂SO₄ precipitation and Sephadex G-200 gel filtration.The molecular size of the bacterial RuDP carboxylase was foundto be large, analogous to that of the plant enzyme, in agreementwith results of previous workers. Sucrose density gradient centrifugationshowed the S_rel to be approximately 18; the omission of Mg⁺⁺caused no dissociation of the enzyme molecule in its subunits.Chromatium RuDP carboxylase showed similarities to the plantenzyme in some of its kinetic properties; (a) a shift of pHoptimum to the neutral side from the alkaline side on the additionof Mg⁺⁺, (b) deviation of the substrate concentration (NaHCO₃)-activityrelationship from the MICHAELIS formula and (c) a marked stimulativeeffect of Mg⁺⁺. A unique sigmoidal saturation curve of the enzymeto RuDP, which had been detected in Rhodospirillum rubrum andRhodopseudomonas spheroides RuDP carboxylase in the absenceof Mg⁺⁺, was not found. Another characteristic feature of ChromatiumRuDP carboxylase is its partial immunological response to therabbit anti-spinach RuDP carboxylase serum as detected by theinhibition of the carboxylation reaction due to the antibody-antigenreaction. ¹Part X, Structure and Function of Chloroplast Proteins. Supportedin part by research grants from the Ministry of Education ofJapan (No. 8719) and USPHS (AM-10792-03) (Received July 4, 1969; )     

Carbon Isotope Discrimination by Tissues5

The carbon isotope discrimination ratio of the floral parts,leaves, and stems of barley and oat plants were measured todetermine if net CO₂ fixation by PEP carboxylase (describedin these tissues by other workers) makes a significant contributionto total carbon fixation in these tissues. The ¹³C values rangedfrom –26.6 to –29.6% and are within the range normallyexpected for plants with the C₃ pathway of photosynthesis inwhich autotrophic CO₂ fixation proceeds via RuBP carboxylase.We conclude that PEP carboxylase does not make a substantialcontribution to autotrophic CO₂ fixation in the floral partsof these C₃ plants.     

Ribulose Bisphosphate Carboxylase Activity and Photosynthesis during Leaf Development in the Tomato

Besford, R. T., Withers, A. C. and Ludwig, L. J. 1985. Ribulosebisphosphate carboxylase activity and photosynthesis duringleaf development in the tomato.—J. exp Bot. 36: 1530–1541. The carboxylase activity of ribulose-1,5-bisphosphate carboxylase/oxygenaseand of phosphoenolpyruvate carboxylase, and the light saturatedrate of net photosynthesis were measured in the developing 5thleaf of tomato plants. Values for light saturated net photosynthesiswere also calculated from the measured carboxylase activitiesand estimates of internal CO₂ and oxygen concentrations. Thecalculated rate using the activity of ribulose bisphosphatecarboxylase alone for net CO₂ assimilation in 300 mm³ dm^–3CO₂ was greater than the measured rate at 80% and full expansionbut less than the measured rate in younger leaves. When theactivities of both the carboxylases were taken into accountbetter agreement was evident for young leaves but the rate wasfurther overestimated for older leaves The calculated rate forphotosynthesis in 1200 mm³ dm^–3 CO², assuming saturationof ribulose bisphosphate carboxylase with RuBP, was an overestimatefor young leaves but was close to the observed values for leavesnear full expansion. The results are discussed in terms of measuredconductances for CO₂ and the availability of RuBP in the leaf Key words: Tomato, leaf development, photosynthesis, RuBP carboxylase, oxygenase     

Distribution of enzymes related to C3 and C4 pathway of photosynthesis between mesophyll and bundle sheath cells of Panicum hians and Panicum milioides

Enzymes of the C₄, C₃ pathway and photorespiration have beenanalyzed for P. hians and P. milioides, which have chlorenchymatousbundle sheath cells in the leaves. On whole leaf extracts thelevels of PEP carboxylase are relatively low compared to C₄species, RuDP carboxylase is typical of C₃ species, and enzymesof photorespiratory metabolism appear somewhat intermediatebetween C₃ and C₄. Substantial levels of PEP carboxylase, RuDPcarboxylase, and photorespiratory enzymes were found in bothmesophyll and bundle sheath cells. Low levels of C₄-acid decarboxylatingenzymes may limit the capacity for C₄ photosynthesis in P. hiansand P. milioides. The results on enzyme activity and distributionbetween mesophyll and bundle sheath cells are consistent withCO₂ fixation via C₃ pathway in these two species. ¹ This research was supported by the College of Agriculturaland Life Sciences, University of Wisconsin, Madison; and bythe University of Wisconsin Research Committee with funds fromthe Wisconsin Alumni Research Foundation; and by the NationalScience Foundation Grant BMS 74-09611. (Received September 16, 1975; )     

Photosynthetic efficiency of Panicum hians and Panicum milioides in relation to C3 and C4 plants

Panicum hians and Panicum milioides were found to have characteristicsintermediate to those of C₃ and C₄ species with respect to CO₂compensation point, percentage inhibition of photosynthesisby O₂ at various O₂/CO₂ solubility ratios, and water use efficiency.C₄ species have a higher carboxylation efficiency than eitherthe intermediate or C₃ species. During photosynthesis, evenunder 2.5% O₂, C₄ species have a higher affinity for intercellularCO₂ (Km 1.6 µM) apparently due to the initial carboxylationthrough PEP carboxylase. Under low O₂ the intermediate and C₃species had a similar affinity for intercellular CO₂ duringphotosynthesis (Km 5–7 µM) consistent with carboxylationof atmospheric CO₂ through RuDP carboxylase. There were considerablevariation in photosynthesis/unit leaf area at saturating CO₂levels in the species examined which in part is due to differencesin RuDP carboxylase /unit leaf area. The highest rates of photosynthesis/unitleaf area under CO₂-saturating conditions were with the C₃ specieswhich had a correspondingly high level of RuDP carboxylase/unitleaf area. Possibilities for the greater efficiency of P. hiansand P. milioides in comparison to C₃ species in utilizing lowlevels of CO₂ in the presence of atmospheric O₂ are discussed. ¹ This research was supported by the College of Agriculturaland Life Sciences, University of Wisconsin, Madison; and theUniversity of Wisconsin Research Committee with funds from theWisconsin Alumni Research Foundation. (Received June 25, 1977; )     

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.     

Effects of Cyclic AMP on the Activity of Soluble Protein Kinases in Lemna paucicostata

Three protein kinases which phosphorylate histone were isolatedfrom cellular extract of Lemna plants. They were separated byelution from DEAE-Sephacel column and referred to as PI, PITand PHI. The PI protein kinase activity was partially inhibitedby 10µM cyclic AMP, cyclic GMP or cyclic IMP, while thePII enzyme was activated in the presence of these cyclic nucleotides.The PIII enzyme was cAMPindependent, but slightly inhibitedby cyclic CMP and cyclic UMP. The molecular weights of thesethree protein kinases were 165,000, 85,000 and 145,000, respectively,as estimated from Sephacryl S-300 gel filtration. A single cyclicAMP-binding protein was detected in the PII enzyme fractionby using the photoaffinity cAMP-analogue, 8-N₃-cAMP. The proteinwhich specifically bound [³H]-8-N₃-cAMP had an apparent molecularweight of 48,000 as determined by SDS-polyacrylamide gel electrophoresis.The phosphorylation of cellular proteins in Lemna was examinedby SDS-polyacrylamide gel electrophoresis. Four phosphorylatedpolypeptides were detected, the phosphorylations of which werestimulated by cAMP. The molecular weights of these four polypeptideswere 59,000, 19,000, 16,000 and 14,000, respectively. (Received January 26, 1983; Accepted April 13, 1983)