Participation of Inorganic Orthophosphate in Regulation of the Ribulose-1,5-bisphosphate Carboxylase Activity in Response to Changes in the Photosynthetic Source-Sink Balance

Sink-limited conditions, defined as treatment with continuousillumination, cause a reduction in the rate of photosyntheticfixation of CO₂ in single-rooted leaves of soybean (Glycinemax. Merr. cv. Turunoko). We suggested previously that thisreduction is due to a deactivation of ribulose-1,5-bisphosphatecarboxylase (RuBPcase, E.C. 4.1.1.39 [EC] ) that is caused by a decreasein the level of P_i in the leaves [Sawada et al. (1989) PlantCell Physiol. 30: 691, Sawada et al. (1990) Plant Cell Physiol.31: 697]. In the present study, the mechanism of regulationof RuBPcase activity by P_i was examined. The activity of RuBPcasein the sink-limited leaves, exposed for 6 or 7 d to continuousillumination to alter the source/sink balance, was enhancedwith increasing concentrations of P_i, in a CO₂-free preincubationmedium in the presence of 5 mM MgCl₂ The maximum value [6.3µmole CO₂ (mg Chl)^–1 min^–1] was obtained atapproximately 5 mM P_i after a 5 min incubation, being 3 timesof the activity without the preincubation. The activity of acrude preparation of RuBPcase that had been deactivated by removalof CO₂ and Mg²⁺ ions by the gel filtration was 5.2–9.3nmole CO₂ (mg protein)^–1 min^–1 and was also enhancedby P_i plus Mg²⁺ ions. The maximum value [147–151 nmoleCO₂ (mg protein)^–1 min^–1] was attained at 5 mM P_iafter a 5 min incubation. The cycle of activation and inactivationof deactivated crude RuBPcase was perfectly reversible by additionof P_i to the enzyme and removal of P_i from the enzyme. Levelsof free P_i and of esterified phosphate in the sink-limited leaveswere 69% and 31% of the total phosphate, respectively. By contrast,in the control leaves, these values were 87% and 13%, respectively.These results support our previously stated hypothesis and indicatean important role for free P_i in the regulation of RuBPcaseactivity, in particular in sink-limited plants. (Received February 21, 1992; Accepted July 23, 1992)     

Regulation of Ribulose-l,5-bisphosphate Carboxylase Activity in Response to Changes in the Source/Sink Balance in Single-Rooted Soybean Leaves: The Role of Inorganic Orthophosphate in Activation of the Enzyme

The results of our previous study [Sawada et al. (1989) PlantCell Physiol. 30: 691] implied that, under sink-limited conditions,a decrease in the activity of ribulose-l,5-bisphosphate carboxylase(EC 4.1.1.39 [EC] ) caused a reduction in the rate of photosyntheticfixation of CO₂ in single-rooted leaves of soybean (Glycinemax L. Merr. cv. Tsurunoko). This reduction in the rate of photosynthesisin source leaves seemed to correspond to a decrease in the demandby sink tissues for photoassimilates. In the present study,the activity of RuBPcase in vivo was estimated by measuringthe "initial" activity immediately after extraction from standardleaves (grown under a regime of 10 h of light and 14 h of darkness)and from sink-limited leaves (exposed for 6 or 7 d to continuouslight to alter the source/sink balance). The rate of photosynthesisin the sink-limited leaves decreased to 47% of that in the standardleaves. The "initial" activity of RuBPcase was 4.3 in the standardleaves but only 1.6 µmole CO₂.(mg Chl)^–1.min^–1in the sink-limited leaves. These results appear to indicatethat the reduction in photosynthetic activity under sink-limitedconditions was mostly due to a deactivation of RuBPcase. Theactivity of deactivated RuBPcase in the sink-limited leaveswas restored to 4.1 µmole CO₂.(mg Chl)^–1.min^–1by incubation of the enzyme in a medium that contained onlyNa₂HPO₄. This result suggests that free P_i in chloro-plastsplays an important role in the activation of the enzyme. Thelevel of P_i in the sink-limited leaves was 62% of that in thestandard leaves. On the basis of these various results, it appearsthat the deactivation of RuBPcase in the sink-limited leavesis the result of a decrease in the level of P_i. The role offree P_i in the activation of RuBPcase, in particular at atmosphericconcentrations of CO₂, was also investigated. (Received November 30, 1989; Accepted May 11, 1990)     

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)     

Photosynthesis and Light Activation of Ribulose 1,5-bisphosphate Carboxylase in the Presence of Starch

Limitation of photosynthesis and light activation of ribulose,1,5-bisphosphate carboxylase (RuBPCO) were examined in the 5thleaf of seedlings of red clover (Trifolium pratense L. cv. Renova)for 5 d following an increase in photosynthetic photon fluxdensity (PPFD) from 200 to 550µmol quanta m^–2 s^–1.Net photosynthesis and its stimulation at 2.0 kPa O₂ initialactivity of rapidly extracted RuBPCO, standard activity of RuBPCOafter incubation of the extracts in the presence of CO₂, Mg²⁺,and inorganic phosphate and contents of soluble protein, starch,soluble sugars, and various photosynthetic metabolites weredetermined. Photosynthesis decreased and starch content increased.No decrease in photosynthesis was found if, when PPFD was increased,all leaves except the investigated 5th leaf were removed, suggestingthat the decrease in photosynthesis was due to accumulated carbohydrates.The stimulation of photosynthesis at 2.0 kPa O₂ did not decreaseand the ratio of the total foliar steady-state contents of triosephosphate to 3-phosphoglycerate increased suggesting that thedecrease in photosynthesis was not due to limiting inorganicphosphate in chloroplasts. Intercellular CO₂ partial pressureand RuBP content were not decreased. Nevertheless, the ratioof photosynthesis to initial RuBPCO activity decreased, suggestingthat the catalysis per active RuBPCO site was decreased. Theincrease in PPFD in the growth cabinet and the PPFD at whichleaves were preconditioned for 1 h, affected not only initialactivity but also the standard activity of RuBPCO. The resultssuggest that a varying proportion of RuBPCO was bound to membranesand was contained in the insoluble fraction of the extracts.A comparison of photosynthesis with extracted RuBPCO activitysuggested that membrane bound RuBPCO did not contribute to photosyntheticCO₂ fixation and that the binding and release to and from membranesmodulated actual RuBPCO activity in vivo. Key words: Photosynthesis, ribulose 1,5-bisphosphate carboxylase, starch     

Carbon Dioxide Fixation and Ribulose-1, 5-bisphosphate Carboxylase Activity in Intact Leaves of Sugar Beet Plants Exposed to Salinity and Water Stress

The influence of salinity in the growing media on ribulose-1,5-bisphosphate (RuBP) carboxylase and on CO₂ fixation by intactsugar beet (Beta vulgaris) leaves was investigated. RuBP carboxylase activity was mostly stimulated in young leavesafter exposure of plants for 1 week to 180 mM NaCl in the nutrientsolution. This stimulation was more effective at the higherNaHCO₂ concentrations in the reaction medium. Salinity also enhanced CO₂ fixation in intact leaves mostlyat rate-limiting light intensities. A 60 per cent stimulationin CO₂ fixation rate was obtained by salinity under 450 µEm^–2 s^–1. At quantum flux densities of 150 µEm^–2 s^–1 (400–700 nm) this stimulation was280 per cent. Under high light intensities no stimulation bysalinity was found. In contrast, water stress achieved by directleaf desiccation or by polyethylene glycol inhibited enzymeactivity up to fourfold at –1.2 MPa. Beta vulgaris, sugar beet, ribulose-1, 5-bisphosphate carboxylase, salt stress, water stress, carbon dixoide fixation, salinity     

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     

Light Dependency of Carboxylation Efficiency and Ribulose-1, 5-Bisphosphate Carboxylase Activation in Trifolium subterraneum L. Leaves

The effect of photosynthetic photon flux density (PPFD) on carboxylationefficiency, estimated as the initial slope (IS) of net CO₂ assimilationrate versus intercellular CO₂ partial pressure response curve,as well as on ribulose-1, 5-bisphosphate carboxylase (Rubisco)activation was measured in Trifolium subterraneum L. leavesunder field conditions. The relationship between IS and PPFDfits a logarithmic curve. Rubisco activation accounts for theIS increase only up to a PPFD of 550 µmol photons m^-2s^-1. Further IS increase, between 550 and 1000 µmol photonsm^-2 s^-1, could be related to a higher ribulose fcwphosphate(RuBP) availability. The slow, but sustained IS increase above1000 µmol photons m^-2 s^-1 could be explained by the mesophyllCO₂ diffusion barriers associated with the high chlorophylland protein content in field developed leaves. Key words: Photosynthesis, initial slope, ribulose-1, 5-bissphosphate carboxylase activation, light response, Trifolium subterraneum L     

Activation of Ribulose Bisphosphate Carboxylase Purified from Wheat Leaves

A stable freeze-dried powder was prepared of partly purifiedribulose bisphosphate carboxylase from wheat leaves. As withpreparations from other leaves it is necessary to incubate theenzyme with Mg^2$ and CO₂ to achieve maximum activity. At 25°C this activity was 0.75 IU mg^–1 protein for a preparationactivated at 50 °C for 10 min; the K_m for CO₂ was 15 µM. The time for reactivation of enzyme that had been inactivatedthrough the absence of CO₂ and Mg^2$ was influenced by the lengthof the inactivating treatment. After a short inactivation periodthe enzyme was reactivated within a few minutes, whereas aftera longer period several hours were needed. Enzyme in the latterstate had some properties in common with enzyme inactivatedby lower temperatures but in the presence of CO₂ and Mg^2$. Theenzyme kinetic characteristics are similarly affected by bothkinds of inactivation; the maximum velocity is decreased butthe affinity for CO₂ is not affected. Reactivation following a long inactivating treatment becomesmore dependent on Mg^2$ concentration as the temperature is increasedfrom 0 to 20 °C.     

Photoinhibition under Atmospheric O2, the Activation State of RuBP Carboxylase and the Content of Photosynthetic Intermediates in Soybean and Wheat

Ward, D. A. and Drake, B. G. 1987. Photoinhibition under atmosphericO₂, the activation state of RuBP carboxylase and the contentof photosynthetic intermediates in soybean and wheat.—J.exp. Bot. 38: 1937–1948. Associations between photosynthesis, the activation state ofRuBP carboxylase and the contents of photosynthetic intermediateswere compared in soybean and wheat leaves before and after exposureto photoinhibitory treatments in the presence of atmosphericO₂. Exposing attached leaves to a supra-saturating irradiance(3 800 µmol quanta m^{– 2} s^–1) for 2 h in CO_2-freeair decreased carboxylation efficiency and the light-saturatedphotosynthetic rate in air by approximately 50%. Exposure tothe photoinhibitory treatment for periods in excess of 2 h didnot cause a further decrease of photosynthesis in soybean. Althoughphotosynthesis was reduced, the initial and total (fully-activated)activities of ribulose 1,5-bisphosphate carboxylase (RuBPCase)in leaf extracts were unaltered in each species by the photoinhibitorytreatment. This was true for leaves sampled under both air andat a rate-limiting intercellular CO₂ partial pressure (C_i) of75 µPa Pa^–1. The contents of ribulose l,5-bisphosphate(RuBP) and 3-phosphoglyceric acid (3-PGA) were reduced by thephotoinhibitory treatment in soybean leaves sampled in air andat a rate-limiting C_i, although the RuBP/3-PGA ratio was unaffected.The relative reduction of RuBP content in soybean leaves atrate-limiting C₁ was similar to the corresponding reductionof carboxylation efficiency. For wheat,the relative reductionof RuBP content at rate-limiting C_i (–19%) caused by thephotoinhibitory treatment was considerably less than the correspondingdecrease of carboxylation efficiency (–49%).The RuBP/3-PGAratio of wheat was also increased significantly by the photoinhibitorytreatment The significance of these observations to the regulationof CO₂-limited photosynthesis in leaves experiencing photoinhibitionunder atmospheric oxygen is discussed. Consideration is alsogiven to the previous contention that contemporary measurementsof initial activity in crude extracts may provide a spuriousindication of the amount of the enzyme-CO₂-Mg_{2 +} form of RuBPcarboxylase present in the leaf. Key words: Carboxylation efficiency, RuBP carboxylase, photoinhibition, RuBP, 3-PGA     

Linearity and Functioning Forms in the Carboxylase Reaction of Spinach Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

The reaction of spinach RuBisCO activated with CO₂ and Mg²⁺proceeded in two phases, an initial burst for a few minutesand the subsequent linear phase, in the presence of saturatingconcentrations of CO₂, ribulose 1,5-bisphosphate (RuBP), andMg²⁺. The percentage of the activity in the linear phase tothat in the initial burst was 55% with RuBisCO prepared withpolyethylene glycol, and very close to the value with the enzymereleased immediately from isolated chloro-plasts. RuBisCO preparedwith ammonium sulfate had a much larger decrease of the activityin the linear phase. The Euglena enzyme had a linear courseof reaction with time for up to 20 minutes. The K_m for CO₂ of spinach RuBisCO activated beforehand was 20µM in the initial burst, and 28 µM in the linearphase. In the carboxylase reaction initiated with inactive enzyme,the activity was initially negligible, but in 5 minutes increasedto the level observed in the linear phase of the activated enzyme.The K_m for CO₂ in the linear phase of the pre-inactivated enzymewas 70 µM. The concentration of RuBP was the immediate cause of the two-phasiccourse of the carboxylase reaction of spinach RuBisCO. The curvatureof the time course was not observed below 35 µM RuBP.The enzyme required over 88 µM RuBP for the conventionaltwo-phasic course. Further increase of the concentration ofRuBP increased the extent of the curvature, but did not startthe curvature sooner after the start of the reaction. Even ifspinach RuBisCO was in the linear phase, dilution of RuBP orits consumption by the enzymatic reaction to less than 30 µMcaused the enzyme to show the resumed biphasic reaction courseafter addition of a high concentration of RuBP. ¹This paper is the twenty-fourth in a series on PhotosyntheticCarbon Metabolism in Euglena gracilis. (Received September 19, 1988; Accepted November 25, 1988)     

Effects of dark preincubation and chloramphenicol on blue light-induced CO2 incorporation in Chlorella cells

Chlorella cells incubated in the dark longer than 12 hr showedpronounced blue light-induced ¹⁴CO₂ fixation into aspartate,glutamate, malate and fumarate (blue light effect), whereasthose kept under continuous light showed only a slight bluelight effect, if any. 2) During dark incubation of Chlorellacells, phosphoenolpyruvate carboxylase activity and the capacityfor dark ¹⁴CO₂ fixation decreased significantly, whereas ribulose-1,5-diphosphatecarboxylase activity and the capacity for photosynthetic ¹⁴CO₂fixation (measured under illumination of white light at a highlight intensity) did not decrease. 3) In cells preincubatedin the dark, intracellular levels of phosphoenolpyruvate and3-phosphoglycerate determined during illumination with bluelight were practically equal to levels determined during illuminationwith red light. 4) The blue light effect was not observed incells incubated widi chloramphenicol, indicating that blue light-inducedprotein synthesis is involved in the mechanism of the effect. (Received April 9, 1971; )     

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     

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     

Analysis of the feed-forward effects of sink activity on the photosynthetic source-sink balance in single-rooted sweet potato leaves. I. Activation of RuBPcase through the development of sinks

Single-rooted sweet potato leaves having a petiole with a fragment of stem allocated exceptionally large amounts of photosynthates to tuberous roots, the only major storage organ, throughout an experimental period of 50 d. The increase in photosynthetic activity for CO(2) fixation depended exclusively on the development of sink activity due to the growth of tuberous roots. Thus this model expressed a remarkable feed-forward effect on the photosynthetic source-sink balance. The level of ribulose-1,5-bisphosphate carboxylase (RuBPcase) protein in the leaves increased continuously during the period. The lowered initial as well as total activity of RuBPcase observed at the start of the experiment was raised with the cancellation of the sink-limited state due to the development of tuberous roots. The maximum activity determined after removing some inhibitor(s) from the enzyme by treating the leaf extract with SO(4)(2-) was much greater than the total activity and remained approximately constant throughout the experimental period. The clear decrease in the difference between maximum and total activities with the development of tuberous roots might reflect the reactivation of RuBPcase due to the removal of some inhibitor(s) from the enzyme through the cancellation of the sink-limited state.     

Photosynthesis with single-rooted Amaranthus leaves. II. Regulation of ribuelose-1,5-bisphosphate carboxylase,phosphoenolpyruvate carboxylase,NAD-malic enzyme and NAD-malate dehydrogenase and coordination between PCR and C4 photosynthetic metabolism in response to changes in the source-sink balance

We have studied source-sink relationships with a model consisting of single-rooted leaves without petioles. We previously reported that the rate of photosynthesis decreased when C4 model plants prepared from Amaranthus cruentus leaves were subjected to sink-limited conditions by exposure to continuous light for a few days. It was suggested that the inhibition is due to a coordinated decrease in the activity of ribulose-1,5-bisphosphate carboxylase (RuBPcase) and phosphoenol-pyruvate carboxylase (PEPcase), both essential enzymes for photosynthesis in C4 plants. We further investigated the mechanisms behind the decreased activity of RuBPcase, PEPcase, NAD-malic enzyme and NAD-malate dehydrogenase. The results suggested that (1) the initial activity of RuBPcase is suppressed by a lowering of the P(i) level in chloroplasts, (2) the inhibition of PEPcase is due to dephosphorylation of the enzyme via the inhibition of PEPcase kinase and PEPcase phosphatase, (3) the inhibition of NAD-malic enzyme and NAD-malate dehydrogenase is derived from the oxidation of these enzymes, and (4) some proteinous factor(s) may be involved in the inhibition of the activity of these latter three enzymes. The significance of a coordinated decrease in these enzymes in response to a change in the source-sink balance is discussed.     

Photosynthesis, Ribulose-1,5-bisphosphate Carboxylase and Leaf Characteristics of Nicotiana tabacum L. Genotypes Selected by Survival at Low CO2 Concentrations

The photosynthetic characteristics (responses to CO₂ and light),ribulose-1,5-bisphosphate carboxylase (Rubisco) properties,and the size and number of cells of the mesophyll of Nicotianatabacum L. leaves of genotypes selected for survival at lowatmospheric CO₂ concentrations are described. When grown inthe greenhouse with nutrient solutions, the total dry matterproduction of the selected genotypes was 23% greater than thatof the parent genotype; this increase was related to a greaternumber of mesophyll cells of smaller size in the selected plantscompared to the parent. However, it was not related to changesin the photosynthetic characteristics nor to Rubisco properties.These results suggest that the increased dry matter accumulationof the selected genotypes is not due to a reduction in photorespirationnor an increase in the CO₂ assimilation rates. Rather, the selectionof haploid tobacco plantlets in low CO₂ has resulted in plantswith greater leaf area (shown in previous work), due to theproduction of more cells of smaller size and to lower respirationrates per unit of leaf dry mass (previous work), thus increasinglight capture, reducing the loss of assimilates and increasingtotal plant dry matter production. Key words: Photosynthesis, ribulose-1,5-bisphosphate carboxylase, leaf anatomy, tobacco, genotypes     

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.     

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     

Stimulatory Effect of Chloride Ions on NADP Malic Enzyme from Leaves of two C4 Species of Cyperus

NADP malic enzyme (EC 1.1.1.40 [EC] ) from leaves of two C₄ speciesof Cyperus (C. rotundus and C. brevifolius var leiolepis) exihibiteda low level of activity in an assay mixture that contained lowconcentrations of Cl^–. This low level of activity wasmarkedly enhanced by increases in the concentration of NaClup to 200 mM. Since the activity of NADP malic enzyme was inhibitedby Na₂SO₄ and stimulated by relatively high concentration ofTris-HCl (50–100 mM, pH 7–8), the activation ofthe enzyme by NaCl appears to be due to Cl^–. Variationsin the concentration of Mg²⁺ affected the K_A (the concentrationof activator giving half-maximal activation) for Cl^–,which decreased from 500 mM to 80 mM with increasing concentrationsof Mg²⁺ from 0.5 mM to 7 mM. The K_m for Mg²⁺ was decreased from7.7 mM to 1.3 mM with increases in the concentration of NaClfrom zero to 200 mM, although the increase of V_max was not remarkable.NADP malic enzyme from Cyperus, being similar to that from otherC₄ species, was able to utilize Mn²⁺. The K_m for Mn²⁺ was 5mM, a value similar to that for Mg²⁺. The addition of 91 mMNaCl markedly decreased the K_m for Mn²⁺ to 20 +M. NADP malicenzyme from Setaria glauca, which contains rather less Cl^–than other C₄ species, was inactivated by concentrations ofNaCl above 20 mM, although slight activation of the enzyme wasobserved at low concentrations of NaCl at pH7.6. (Received February 20, 1989; Accepted June 12, 1989)     

Reactions of birch leaves to changes in light during early ontogeny: comparison between in vivo and in vitro techniques to measure carbon uptake

Trends in several photosynthetic parameters and their responseto changed growth light were followed for 15 d in leaves ofyoung birch saplings using a rapid-response gas exchange measuringequipment. These in vivo measurements were compared to biochemicalassays that were made from the same leaves after the gas exchangestudies. The measurements were made on leaves that were selectedprior to the study and were at that time of similar age. Forthe first 7 d the photosynthetic parameters were followed fromthe growth conditions of moderate light (200 µmol m^–2s^–1; referred to as controls later in the text). On day7 some of the saplings were transferred to grow either underhigh (450 µmol m^–2 s^–1; referred to as highlight plants) or low (75 µmol m^–2 s^–1; referredto as low light plants) light and the capability of the preselectedleaves for acclimation was followed for 6 d. For comparison,at the end of the experiment the measurements were made on bothcontrols and on young leaves that had developed under high andlow light. Generally the in vivo measured rate of CO₂ uptake (gross photosynthesis)both at 310 ppm CO₂ and 2000 ppm CO₂ corresponded very wellto the biochemically determined CO₂ fixation capacity in vitroafter rapid extraction (measured as the initial and total activityof Rubisco, respectively). However, if the flux of CO₂ intothe chloroplasts was limited by the closure of the stomata,as was the case of the high light plants, then the in vitromeasured Rubisco activity was greater than the in vivo measuredCO₂ uptake. V_max, calculated from the mesophyll conductanceat 1% O₂, exceeded the initial activity of Rubisco (assayedat saturating RuBP and CO₂) constantly by 60%. The catalyticactivity of Rubisco in birch leaves was overall very low, evenwhen calculated from the total activity of Rubisco (K_cat 0.63–1.18 s^–1), when compared to herbaceous C₃ species. Signs of light acclimation were not observed in most of thephotosynthetic parameters and in chloroplast structure whenmature birch leaves were subjected to changes in growth lightfor 6 d. However, the change of the growth light either to highor low light caused day-to-day fluctuations in most of the measuredphotosynthetic parameters and in the case of the high lightplants signs of photoinhibition and photodestruction were alsoobserved (decrease in the amount of chlorophyll and increasein chlorophyll a/b ratio). As a result of these fluctuationsthese plants achieved a new and lower steady-state conditionbetween the light and dark reactions, as judged from the molarratio of RuBP to Rubisco binding site. Key words: Acclimation, photosynthesis, light, Rubisco, birch