Enzymic Fractionation of the Stable Carbon Isotopes of Carbon Dioxide by Ribulose-1,5-bisphosphate Carboxylase

The enzymic fractionation of the stable carbon isotopes of CO₂ (Δco₂) was determined using a purified preparation of ribulose-1,5-bisphosphate (RuBP) carboxylase isolated from cotton (a C₃ plant) leaves. The bicarbonate concentration in the reaction mixture saturated the enzyme and furnished an infinite pool of ¹²CO₂ and ¹³CO₂ for enzyme fractionation. The RuBP was 96 to 98% pure. The phosphoglycerate synthesized in the reaction mixtures was purified free of RuBP, phosphoglycolate, and other phosphate esters by column chromatography on Dowex 1-Cl⁻ resin. The average Δco₂ value of −27.1% was determined from five separate experiments. A discussion of the isotope fractionation associated with photosynthetic CO₂ fixation in plants shows that the enzymic fractionation of stable carbon isotopes of CO₂ by RuBP carboxylase is of major importance in determining the δ¹³C values of C₃ plants.     

Variation in Concentration of Ribulose-1, 5-bisphosphate Carboxylase in Leaves of Barley, Wheat and Hybrids of Crested Wheatgrasses

Amounts of the enzyme ribulose-1,5-bisphosphate carboxylasewere estimated in seedling leaves of barley (Hordewn vulgareL.) and flag leaves of wheat (Triticum aestitum L.) by radialimmuno diffusion. A fourfold variation among barley varietiesfor amount of RuBPCase at the seedling stage was observed (c.3.5–15mg g^–1 fr. wt). Range in variation for amountof flag leaf RuBPCase among wheat varieties was 6-09-9.39 mgRuBPCase g^–1 fr. wt. F₁ hybrids from interspecific andintergeneric crosses of crested wheatgrasses (Agropyron andElymus spp.) and their amphidiploid analogues were comparedfor amount of RuBPCase in the most recent fully expanded leavesharvested before seed set. Amount of enzyme varied from 3.4to 77.6 mg g^–1 fr. wt among the hybrids. No effect chromosomenumber on enzyme concentration was observed among 13 hybridsand their amphidiploid counterparts. Key words: RuBPCase, wheatgrasses     

The Control of Water and Carbon Dioxide Flux in Water-stressed Sugar Beet

Changes in leaf and canopy water potential of sugar beet growingin soil of decreasing water content depended on soil water potentialand were independent of water flux from the plant when thiswas varied by changing the water vapour content of the air.The calculated hydraulic conductance of the plant increasedas flux increased and decreased as leaf water potential decreasedand as the plant aged. The conductances to water vapour of individualleaves and of the canopy decreased as leaf water potential decreasedand increased with increasing humidity of the air. The lattereffect was independent of changes in leaf water potential. Theconductances were not affected by the rate of evaporation orleaf temperature. The rate of photosynthesis was directly relatedto leaf conductance except in severely stressed, mature leavesin which leaf water potential had a more direct effect on photosynthesis.Stomatal conductances, transpiration, and photosynthesis weregreater in young leaves than mature leaves on the same plantand at the same leaf water potential. These results are discussedin relation to current agricultural irrigation practices usedfor sugar beet.     

Carboxylase Levels and Carbon Dioxide Fixation in Baker''s Yeast

Levels of pyruvate carboxylase (PC), phosphopyruvate carboxylase (PEPC), and malate dehydrogenase (decarboxylating) were compared in wild-type bakers' yeast (I), a cytoplasmic-respiratory mutant (II), a biotin-deficient wild-type yeast (III), and a biotin-deficient respiratory mutant (IV). PC activities were greatly reduced in III and IV, whereas PEPC was reduced in II and IV. Malate dehydrogenase (decarboxylating) could not be detected in any of the yeasts. With yeast I growing on glucose as the sole carbon source, PEPC decreased to negligible levels during the logarithmic phase of growth (glucose repression effect), whereas PC increased. Both enzymes reverted to their original levels during the stationary phase, when glucose in the medium was exhausted. In agreement with the leading role of PC for CO(2) assimilation, the rates of (14)CO(2) fixation in yeasts I and II were approximately equal and were much higher than that in yeast IV. With I and II, most of the (14)C was distributed similarly in oxalacetate derivatives; with yeast IV, most of (14)C appeared in a compound apparently unrelated to CO(2) fixation via C(4)-dicarboxylic acids.     

水稻叶片中过氧化氢与核酮糖-1，5-二磷酸羧化酶/加氧酶降解的关系

甲基紫精(MV)处理水稻植株能快速诱导核酮糖-1,5-二磷酸羧化酶／加氧酶(Rubisco,EC4．1．1．39)及其它可溶性蛋白的降解。MV浓度越高,降解速率越高。MV能诱发叶片内源H     

Simultaneous Measurement of Oxygen, Carbon Dioxide, and Water Vapour Exchange in Intact Plants

A low flow of air is passed through a temperature-controlledplant chamber in order to obtain relatively large (300–500µl-1) differences in [O₂] between influx and efflux streams.These differences are measured with a stabilized O₂ electrodesystem incorporating elements of gas conditioning electroniczero suppression and signal amplification. Changes in [O₂] of400 µl l-1 can be detected at full scale recorder deflectionagainst a background concentration of 21% O₂. The concentrationsof CO₂ and H₂O within the chamber are held constant by con-trolled-flowCO₂-scrubbing and dehumidifying loops. Carbon dioxide, H₂O,and O₂ fluxes are measured and leaf diffusion resistance andinternal [CO₂] are calculated in essentially ‘real time’.     

Activity of Carbon Metabolism Enzymes in Wheat Plants Treated with Kartolin-4 and Exposed to Water Stress

Enzymatic activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC 4.1.1.39), phospho(enol)pyruvate carboxylase (EC 4.1.1.31), NAD malate dehydrogenase (EC 1.1.1.37), and NADP glyceraldehyde phosphate dehydrogenase complex including phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehyde phosphate dehydrogenase (EC 1.2.1.13) were comparatively assayed in wheat seedlings of the cultivar Lyutestsens 758 grown under normal conditions, water deficiency conditions, and subsequent rehydration. Water stress was found to decrease the activity of all enzymes tested, the effect being most pronounced in the case of Rubisco. The content of Rubisco in wheat plants exposed to water deficiency was reduced less significantly than the activity of the enzyme. Pretreatment of plant seeds with kartolin-4 (o-isopropyl-N-2-hydroxyethyl carbamate), a preparation with cytokinin activity, reduced the dehydration-induced inhibition of enzymatic activity. Upon a subsequent rehydration, kartolin-4 facilitated rapid recovery of the photosynthetic activity, the process being based on the kartolin-induced stimulation of reparation reactions. Under conditions of water stress, a partial decrease in the activity of carbon metabolism enzymes in vitrowas accompanied by complete inhibition of photosynthesis in vivo, perhaps, as a result of an abrupt increase in the stomatal resistance.     

Ribulose 1, 5-bisphosphate Carboxylase/ Oxygenase Activities in Leaves of Greenhouse Roses

An enzyme assay was developed to measure the initial and Mg²⁺–CO₂activated forms of Ribulose 1,5-bisphosphate Carboxylase/Oxygenase(Rubisco) in rose leaves. The assay was verified by co-extractionof the leaflets with partially purified spinach Rubisco andthrough correlation with net photosynthetic rates of individualleaflets (r²=0.7324). Changes in activities were measured asa function of depth of leaves in the canopy for two cultivarsof greenhouse hybrid tea roses. Initial Rubisco activity declinedwith increasing canopy depth for both cultivars. The activatedform of the enzyme, however, remained constant with canopy depthfor cv. Red Success; but increased with canopy depth, then declinedafter mid-canopy in the cv. Royalty. Rubisco activities werealso measured in the cv. Red Success grown in CO₂ enriched environments(100 mm³ dm^–3) at three humidity levels. The activitieswere not significantly affected by humidity treatment. However,there was a trend for plants grown at lower humidity to havehigher activated activities. Key words: Humidity, Rubisco, Rosa ? hybrida, Royalty, Red Success     

Relation between Nitrogen and Ribulose-1,5-bisphosphate Carboxylase in Rice Leaves from Emergence through Senescence

The relation between N content and ribulose-l,5-bisphosphate(RuBP) carboxylase protein was examined in the 12th leaf bladeof rice. Plants were grown under different amounts of N afterthe emergence of the 12th leaf blade. RuBP carboxylase proteinincreased with leaf N during leaf expansion. The synthesis ofRuBP carboxylase predominated during this period, and changesin the amounts of carboxylase synthesized until leaf death paralleledchanges in the N influx to the leaves. When the carboxylasereached its maximum content, the proportion of RuBP carboxylaseto leaf N was 27 to 28% irrespective of N treatment. As theleaf senesced, however, this proportion differed significantlywith the treatment. It was higher in the N-deficient leaf thanin the N-sufficient leaf. This was due to different patternsof RuBP carboxylase degradation for the treatments during senescence.RuBP carboxylase was degraded actively during the early stageof senescence in the N-sufficient leaf, whereas its degradationproceeded almost constantly in the N-deficient leaf during senescence. (Received October 17, 1983; Accepted January 27, 1984)     

Regulation of the Activity of Ribulose-1,5-Bisphosphate Carboxylase in Response to Changes in the Photosynthetic Source-Sink Balance in Intact Soybean Plants

Sink-limited conditions cause a reduction in the rate of photosyntheticfixation of CO₂ in single-rooted soybean leaves (Glycine max.Merr.). We suggested previously that this reduction is due tothe deactivation of ribulose-1,5-bisphosphate carboxylase (RuBPcase;EC 4.1.1.39 [EC] ) that is caused by a decrease in the level of freeP_i via a decrease in the rate of conversion of phosphorylatedintermediates to the end-product (sucrose) in sink-limited leaves[Sawada et al. (1989) Plant Cell Physiol. 30: 691, Sawada etal. (1990) Plant Cell Physiol. 31: 697, Sawada et al. (1992)Plant Cell Physiol. 33: 943]. In the present study, we investigatedwhether, in intact soybean plants, sink-limited conditions wouldalso cause a reduction in the rate of photosynthesis and whethersuch a reduction might be due to the deactivation of RuBPcasevia the same regulatory mechanism as that suggested from previousstudies with single-rooted leaves. Continuous removal of flowerbuds from intact plants brought a large decrease in ratio ofthe dry weight of sink organs (stem, roots, pods) to sourceorgan (leaves) as a result of the absence of pod formation.Pods are likely to function as the major sink at the reproductivestage. Upon continuous removal of flower buds, the treated (sink-limited)plants showed a large decrease in the rate of photosyntheticfixation of CO₂ as compared to control plants. RuBPcase in theleaves of treated plants was continuously inactivated with thedecrease in photosynthetic activity. However, the inactivatedenzyme was totally reactivated upon incubation in the presenceof 10 mM NaHCO₃ and 5 mM MgCl₂. The levels of sucrose and ribulose-1,5-bisphosphatein leaves of the treated plants increased significantly. Allthese results coincide exactly with those obtained in previousstudies of single-rooted leaves under the sink-limited conditions. (Received July 14, 1994; Accepted February 21, 1995)     

The Activity and Content of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Wheat Plants as Affected by Water Stress and Kartolin-4

The carboxylating activity and content of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC 4.1.1.39), and other soluble proteins in young seedlings and mature leaves of Lutescens-758, a drought-sensitive cultivar of soft spring wheat Triticum aestivum L., were studied under the conditions of drought and subsequent rehydration. Seedlings and mature plants preliminarily treated with the cytokinin-like compound kartolin-4 were compared to untreated plants. Drought-induced decrease in RuBPCO activity should be attributed not only to proteolytic decomposition of the enzyme protein itself but also to a partial inhibition of its catalytic activity. The decrease in RuBPCO activity was larger than that in RuBPCO content. Water stress induced a marked decrease in the soluble protein content. Kartolin-4 increased the resistance to drought.     

Rehydration of Sugar Beet Plants after Water Stress: Effect of Cytokinins

The possibility to improve the recovery of sugar beet plants after water stress by application of synthetic cytokinins N⁶-benzyladenine (BA) or N⁶-(m-hydroxybenzyl)adenosine (HBA) was tested. Relative water content (RWC), net photosynthetic rate (P_N), transpiration rate (E), stomatal conductance (g_s), chlorophyll (Chl) a and Chl b contents, and photosystem 2 efficiency characterized by variable to maximal fluorescence ratio (F_v/F_m) were measured in control plants, in water-stressed plants, and after rehydration (4, 8, 24, and 48 h). Water stress markedly decreased parameters of gas exchange, but they started to recover soon after irrigation. Application of BA or HBA to the substrate or sprayed on leaves only slightly stimulated recovery of P_N, E, and g_s in rehydrated plants, especially during the first phases of recovery. Chl contents decreased only under severe water stress and F_v/F_m ratio was not significantly affected by water stress applied. Positive effects of BA or HBA application on Chl content and F_v/F_m ratio were mostly not observed.     

Degradation of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves During Dark-induced Senescence

The degradation of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves during dark-induced senescence was studied. An in vivo degradation product of Rubisco large subunit (LSU) with molecular weight of 50 kD was detected by SDS-PAGE and immunoblotting with antibody against tobacco Rubisco. This fragment could also be detected in natural senescence. The result also suggested that the Rubisco holoenzyme had not dissociated when LSU hydrolyzed from 53 kD to 50 kD. And LSU could be fragmented to 50 kD at 30-35 ℃ and at pH 7.5 in crude enzyme extracts of wheat leaves dark-induced for 48 h, which suggested that maybe LSU was degraded to 50 kD by an unknown protease in chloroplast.     

Relationship between the Kinetic Properties and the Small Subunit Composition of Nicotiana Ribulose-1, 5-bisphosphate Carboxylase

Genetic variability in the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in several Nicotiana species has been characterized by isoelectric focusing patterns. This heritable variation provides an opportunity to examine the functional role of each of these subunits. In this study, specifically designed RuBPCase enzymes composed of identical large subunits but different small subunits were constructed in vivo by interspecific hybridization between the species N. sylvestris, N. tabacum, N. glauca, N. glutinosa, N. plumbaginifolia, and N. tomentosiformis. Small subunit polypeptides were combined to form a sequence of one, two, three, and four polypeptides with the large subunit of N. sylvestris. Kinetic properties of these hybrid enzymes were compared. No differences in the specific activity of either carboxylation or oxygenation nor in K_m values for ribulose 1,5-bisphosphate, CO₂, or O₂ were detected among the RuBPCase enzymes from the various interspecific hybrids. Likewise, the ratio of carboxylation to oxygenation was constant.     

Differences between Wheat Genotypes in Specific Activity of Ribulose-1,5-bisphosphate Carboxylase and the Relationship to Photosynthesis

The in vitro ribulose-1,5-bisphosphate (RuBP) carboxylase activity per unit of leaf nitrogen was found to be 30% greater in Triticum aestivum than in T. monococcum. This was due to a higher specific activity of the enzyme from T. aestivum, as the amount of RuBP carboxylase protein per unit of total leaf nitrogen did not differ between the genotypes. The occurrence of higher specific activity of RuBP carboxylase is shown to correlate with possession of the large subunit derived from the B genome of wheat.

Despite the greater RuBP carboxylase activity per unit of leaf nitrogen in T. aestivum, the initial slopes of curves relating rate of CO₂ assimilation to intercellular p(CO₂) are similar in T. aestivum and T. monococcum for the same nitrogen content per unit leaf area. The similarity of the initial slopes is the result of a greater resistance to CO₂ transfer between the intercellular spaces and the site of carboxylation in T. aestivum than in T. monococcum.

     

Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase／Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

Chlorophyll, Ribulose-1,5-diphosphate Carboxylase, and Hill Reaction Activity in Developing Leaves of Populus deltoides

The synthesis of chlorophyll and ribulose diphosphate carboxylase as well as the development of Hill reaction activity were followed in expanding Populus deltoides leaves and related to photosynthetic patterns. Total chlorophyll, which was not correlated with photosynthetic rate in expanding leaves, decreased slightly with age in very young leaves, due to a decrease in chlorophyll b, but then increased linearly. The ratio of chlorophyll a to b, which rose sharply in young leaves, was highly correlated with the onset of net photosynthesis. Hill reaction activity was very low in young leaves and did not increase significantly until leaves were about half expanded. Ribulose diphosphate carboxylase activity increased in a sigmoid fashion with leaf ontogenesis and closely paralleled development of the photosynthetic system. The study demonstrates the importance of chlorophyll a and Calvin cycle enzyme synthesis to photosynthetic development in expanding leaves.     

Induction of the Inactivation and Degradation of Phosphoenolpyruvate Carboxylase and Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in Maize Leaves by Freezing and Thawing

When frozen leaves of 24-day-old maize (Zea mays L.) plant werethawed on moist filter paper at 26°C (freeze-thaw treatment)several enzymes, including phosphoenolpyruvate carboxylase (PEPC)and ribulose-1,5-bisphosphate carboxylase (RuBPC), were rapidlyinactivated and degraded. The kinetics of the inactivation anddegradation were pseudo first-order, and the halftimes for inactivationof PEPC and RuBPC were 3.2 and 2.4 min, respectively. The effectof the freeze-thaw treatment on the inactivation and degradationdiffered among various enzymes: the residual activities of RuBPC,PEPC, hydroxypyruvate reductase, Cyt c oxidase, NADP-malic enzymeand a-mannosidase 10 min after the start of the thawing treatmentwere 7, 16, 54, 64, 97 and 98% of the initial respective levels.Thirty min after the starting of thawing treatment, the amountsof total soluble protein, the large subunit of RuBPC, the smallsubunit of RuBPC, the PEPC subunit and the NADP-malic enzymesubunit had fallen to 61, 2, 16, 8, and 66% of the initial respectiveamounts. The effect of freeze-thaw treatment on PEPC was greater in oldleaves than in young leaves. There was a steady increase ofthe rate of degradation of PEPC by freeze-thaw treatment asplants aged from 6 to 24 days. These results are discussed inthe context of protein degradation in plant cells. (Received August 9, 1993; Accepted January 10, 1994)     

Photosynthesis, Ribulose-1,5-bisphosphate Carboxylase, Electron Transport, and Ribulose 1,5-Bisphosphate of Virescent and Normal Green Wheat Leaves

CO₂ gas exchange, ribulose-1,5-bisphosphate, and electron transport have been measured in leaves of a yellow-green mutant of wheat (Triticum durum var Cappelli) and its wild type strain grown in the field. All these parameters, expressed on leaf area basis, were similar in both genotypes except electron transport which was more than double in the wild type. These results, treated according to a recent photosynthesis model for C₃ plants, seem to indicate that the electron transport rate of mutant leaves is not sufficient to support the carboxylation derived through both the assimilation rate and the in vitro ribulose-1,5-bisphosphate carboxylase activity. It is suggested that under our experimental conditions photosynthetic electron transport is not the sole energy-dependent determinant of ribulose-1,5-bisphosphate regeneration in the mutant.