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.     

Ribulose Bisphosphate Carboxylase and Proteolytic Activity in Wheat Leaves from Anthesis through Senescence

Changes in ribulose bisphosphate carboxylase (RuBPCase) and proteolytic activity were followed in the flag leaf and second leaf of field-grown winter wheat (cv. Arthur). These changes were followed in relation to changes in leaf chlorophyll, protein, and photosynthesis, and seed development. Levels of RuBPCase were determined by rocket immunoelectrophoresis as described previously (Wittenbach 1978 Plant Physiol 62: 604-608). RuBPCase constituted 40 to 45% of the total soluble protein in the flag leaf and an even higher percentage of the soluble protein in the second leaf. This ratio remained unchanged until senescence when RuBPCase protein was apparently lost at a faster rate than total soluble protein. No change in the specific activity of RuBPCase on either a milligram protein or RuBPCase basis was observed until senescence. A close correlation existed among the various indices of senescence in the field, namely, the decline in chlorophyll, protein, photosynthesis, and RuBPCase activity. In addition, proteinase activity increased with the onset of senescence. These enzymes readily degraded RuBPCase, exhibiting a pH optimum of 4.8 to 5.0 and a temperature optimum of 50 C. Proteinase activity was modified by sulfydryl reagents suggesting the presence of sulfydryl groups at or near the active sites.     

Regulation of Ribulose Bisphosphate Carboxylase Activity in Intact Wheat Leaves by Light, CO2, and Temperature

The activity of the enzyme ribulose bisphosphate carboxylase(RuBPCase) was estimated after rapidly extracting it from intactwheat leaves pretreated under different light and CO₂ levels.No HCO₃^– was added to the extraction buffer since it isshown to inhibit RuBPCase. The activity increased as light intensityor CO₂ concentration during pretreatment was increased. Enzymeactivity increased as temperature during pretreatment was decreased.Light activation did not affect the affinity of RuBPCase forCO₂. A K_m of 30 µM CO₂ under air level O₂ was determined.CO₂, light and temperature are three main limiting factors ofphotosynthesis. It seems that the activity of RuBPCase is regulatedby these factors according to the requirements for CO₂ fixation.     

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     

Relation between Ribulose Bisphosphate Carboxylase Content and Chloroplast Number in Naturally Senescing Primary Leaves of Wheat

The amounts of ribulose bisphosphate carboxylase protein decreasedrapidly with leaf age to a low content by the middle stage ofsenescence. In contrast, the decrease in chloroplast numberwas slight during the same period. This indicates that the enzymecan be degraded within the chloroplast before the chloroplastsdisintegrate during senescence. (Received September 3, 1983; Accepted November 24, 1983)     

Effects of Light and Elevated Atmospheric CO(2) on the Ribulose Bisphosphate Carboxylase Activity and Ribulose Bisphosphate Level of Soybean Leaves

Soybean (Glycine max L. Merr. cv Bragg) was grown throughout its life cycle at 330, 450, and 800 microliters CO₂ per liter in outdoor controlled-environment chambers under solar irradiance. Leaf ribulose-1,5-bisphosphate carboxylase (RuBPCase) activities and ribulose-1,5-bisphosphate (RuBP) levels were measured at selected times after planting. Growth under the high CO₂ levels reduced the extractable RuBPCase activity by up to 22%, but increased the daytime RuBP levels by up to 20%.

Diurnal measurements of RuBPCase (expressed in micromoles CO₂ per milligram chlorophyll per hour) showed that the enzyme values were low (230) when sampled before sunrise, even when activated in vitro with saturating HCO₃⁻ and Mg²⁺, but increased to 590 during the day as the solar quantum irradiance (photosynthetically active radiation or PAR, in micromoles per square meter per second) rose to 600. The nonactivated RuBPCase values, which averaged 20% lower than the corresponding HCO₃⁻ and Mg²⁺-activated values, increased in a similar manner with increasing solar PAR. The per cent RuBPCase activation (the ratio of nonactivated to maximum-activated values) increased from 40% before dawn to 80% during the day. Leaf RuBP levels (expressed in nanomoles per milligram chlorophyll) were close to zero before sunrise but increased to a maximum of 220 as the solar PAR rose beyond 1200. In a chamber kept dark throughout the morning, leaf RuBPCase activities and RuBP levels remained at the predawn values. Upon removal of the cover at noon, the HCO₃⁻ and Mg²⁺-activated RuBPCase values and the RuBP levels rose to 465 and 122, respectively, after only 5 minutes of leaf exposure to solar PAR at 1500.

These results indicate that, in soybean leaves, light may exert a regulatory effect on extractable RuBPCase in addition to the well-established activation by CO₂ and Mg²⁺.

     

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     

Activation of Ribulose Bisphosphate Carboxylase by Thioredoxin

The activity of ribulose bisphosphate carboxylase (RuBPCase) in the soluble part of ruptured chloroplasts was assayed spectrophotometrically by the oxidation of NADH, using ribose-5-phosphate as substrate. The reaction mixture used in this assay consisted of six enzymes, namely ribose-5-phosphate isomerase, rlbulose-5-phosphate Kinase, RuBPCase, 3-phosphoglyceric acid kinase, glyceraldehyde-3-phosphate dehydrogenase and creatine kinase. By adding exogenous RuBPCaso into the reaction mixture, it was shown that the reaction catalyzed by RuBPCase was rate limiting during the course of assay. The activity of RuBPCase in the soluble part of ruptured chloroplasts was significantly enhanced by the addition of reduced thioredoxin (Td). Because the solution of reduced Td contained DTT which had been used as reductant, it was desirable to ascertain the degree of activation of RuBPCase brought about by DTT alone. Experiments showed Td to be far more effective than DTT in this respect. The results presented in this paper suggests a possible mechanism of the light-activation of RuBPCase, i.e. Td. is first reduced by light through photosystems in chloroplast lamellae, and then the reduced Td activates RuBPCase.     

Ribulose 1, 5-Bisphosphate Carboxylase/Oxygenase Activity and Photorespiration during the Ageing of Flag Leaves of Wheat

Photosynthesis, photorespiration, and ribulose bisphosphatecarboxylase/oxygenase activities were measured in intact flagleaves of wheat during ageing. Photorespiration declined verylittle as the leaves aged, and much less than photosynthesis.These changes could not be explained by changes in the ribulosebisphosphate carboxylase to oxygenase ratio of fraction 1 protein.As the leaves grew older the enzyme activities in extracts ofleaves declined in parallel so the ratio remained constant.     

Breakdown of Ribulose Bisphosphate Carboxylase and Change in Proteolytic Activity during Dark-induced Senescence of Wheat Seedlings

When 8-day-old wheat seedlings (Triticum aestivum L. var. Chris) are placed in the dark the fully expanded primary leaves undergo the normal changes associated with senescence, for example, loss of chlorophyll, soluble protein, and photosynthetic capacity (Wittenbach 1977 Plant Physiol. 59: 1039-1042). Senescence in this leaf is completely reversible when plants are transferred to the light during the first 2 days, but thereafter it becomes an irreversible process. During the reversible stage of senescence the loss of ribulose bisphosphate carboxylase (RuBPCase) quantitated immunochemically, accounted for 80% of the total loss of soluble protein. There was no significant change in RuBPCase activity per milligram of antibody-recognized carboxylase during this stage despite an apparent decline in specific activity on a milligram of soluble protein basis. With the onset of the irreversible stage of senescence there was a rapid decline in activity per milligram of carboxylase, suggesting a loss of active sites. There was no increase in total proteolytic activity during the reversible stage of senescence despite the loss of carboxylase, indicating that this initial loss was not due to an increase in total activity. An 80% increase in proteolytic activity was correlated with the onset of the irreversible stage and the rapid decline in RuBPCase activity per milligram of carboxylase. Delaying senescence with zeatin reduced the rate of loss of carboxylase and delayed both the onset of the irreversible stage and the increase in proteolytic activity to the same degree, suggesting that these events are closely related. The main proteinases present in wheat and responsible for the increase in activity are the thiol proteinases. These proteinases have a high affinity for RuBPCase, exhibiting an apparent K_m at 38 C of 1.8 × 10⁻⁷ m. The K_m for casein was 1.1 × 10⁻⁶ m. If casein is representative of noncarboxylase protein, then the higher affinity for carboxylase may provide an explanation for its apparent preferential loss during the reversible stage of senescence.     

Effect of Nitrogen Fertilizer on Photosynthesis and Ribulose 1,5-Diphosphate Carboxylase Activity in Spring Wheat in the Field

Wheat was grown in the field with different levels of nitrogenousfertilizer, and the rate of photosynthesis and the activityof ribulose 1,5-diphosphate carboxylase in the flag leaves determined.Additional nitrogen increased the dry-weight and leaf area ofthe plants, but did not increase grain yield; the rate of photosynthesisof the flag leaves was unchanged but the activity of ribulose1,5-diphosphate carboxylase increased. The significance of theseobservations to the loss of potential yield of wheat and therelationship between, photosynthesis and carboxylase activityis considered.     

Effect of Carbonic Anhydrase on the Activity of Ribulose Bisphosphate Carboxylase

At concentrations of CO₂ less than saturating, carbonic anhydrase(EC 4.2.1.1 [EC] ) stimulates the carboxylation of ribulose bisphosphatecatalysed by ribulose bisphosphale carboxylase (EC 4.1.1.3 [EC] .9)in vitro. This is not through any beneficial association ofthe two enzymes but is a consequence of the increased rate ofconversion of HCO₃^– ion to CO₂, the substrate for thecarboxylation. Carbonic anhydrase should always be includedin reaction mixtures used to determine the Michaelis constantof ribulose bisphosphate carboxylase for CO₂ where fixationof radioactive CO₂ into phosphoglycerate is the basis of rateestimation. The effect is to decrease the value obtained forthe Michaelis constant.     

水稻叶片在自然衰老过程中1，5-二磷酸核酮糖羧化酶/加氧酶的降解(英文)

１,５二磷酸核酮糖羧化酶／加氧酶（Ｒｕｂｉｓｃｏ）是光合碳同化的关键酶,研究其降解机理对合理调控水稻生长后期光合衰退具有重要意义。前人用人为诱导植物衰老的方法,研究了Ｒｕｂｉｓｃｏ的降解机理,认为该酶降解之前,必需发生亚基间的交联聚合和向类囊体膜转移,这样在结构和空间上有利于水解酶的作用。我们用自然衰老叶片进行研究的结果表明：Ｒｕｂｉｓｃｏ在降解过程中其比活基本保持恒定,意味着未发生酶的失活,也就是说酶结构未发生根本性改变,由此也可初步判断酶未发生亚基间的交联聚合（已证明亚基交联可导致酶失活）。接着用ＳＤＳＰＡＧＥ和蛋白印迹技术证实了上述观点：Ｒｕｂｉｓｃｏ降解之前只有极少量的大亚基聚合体,随后同未聚合大亚基一起很快降解。此外,研究结果进一步表明酶分子在降解之前有少量与叶绿体膜结合,但降解过程中并未见膜结合蛋白增加。根据上述结果我们认为,亚基间交联聚合和向膜转移并非水稻叶片自然衰老时Ｒｕｂｉｓｃｏ降解的必要条件。     

Carboxylation and Detoxification of Xylulose Bisphosphate by Spinach Ribulose Bisphosphate Carboxylase/Oxygenase

The time-dependent, slow inhibition of ribulose bisphosphatecarboxylase (RuBisCO) in the absence of ribulose bisphosphatewas dependent on the concentrations of RuBisCO and xylulosebisphosphate (XuBP). When incubated with excess XuBP, RuBisCOlost its activity gradually with incubation time. When RuBisCOof the concentration of 1.5 µM was incubated with 20 µMXuBP, the activity was inhibited for the initial 10 minutes,after which the activity recovered gradually with time. Therecovery was because XuBP in the incubation mixture was carboxy-latedto form 3-phosphoglycerate. The concentration of XuBP half-saturatingthe XuBP-carb-oxylation reaction of RuBisCO was 12 to 15 µM.The initial inhibition and the subsequent recovery of the activitywere due to the elimination from and re-binding to RuBisCO,respectively, of the activator CO₂. (Received April 20, 1991; Accepted May 21, 1991)     

Photosynthesis and Activation of Ribulose Bisphosphate Carboxylase in Wheat Seedlings : Regulation by CO(2) and O(2)

Photosynthetic carbon assimilation in plants is regulated by activity of the ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase. Although the carboxylase requires CO₂ to activate the enzyme, changes in CO₂ between 100 and 1,400 microliters per liter did not cause changes in activation of the leaf carboxylase in light. With these CO₂ levels and 21% O₂ or 1% or less O₂, the levels of ribulose bisphosphate were high and not limiting for CO₂ fixation. With high leaf ribulose bisphosphate, the K_act(CO₂) of the carboxylase must be lower than in dark, where RuBP is quite low in leaves. When leaves were illuminated in the absence of CO₂ and O₂, activation of the carboxylase dropped to zero while RuBP levels approached the binding site concentration of the carboxylase, probably by forming the inactive enzyme-RuBP complex.

The mechanism for changing activation of the RuBP carboxylase in the light involves not only Mg²⁺ and pH changes in the chloroplast stroma, but also the effects of binding RuBP to the enzyme. In light when RuBP is greater than the binding site concentration of the carboxylase, Mg²⁺ and pH most likely determine the ratio of inactive enzyme-RuBP to active enzyme-CO₂-Mg²⁺-RuBP forms. Higher irradiances favor more optimal Mg²⁺ and pH, with greater activation of the carboxylase and increased photosynthesis.

     

Changes of Anatomical Features, Photosynthesis and Ribulose Bisphosphate Carboxylase-Oxygenase Content of Mango Leaves

Changes in anatomical and physiological features, includingchanges in amount per unit area of anthocyanin and chlorophyll,in leaves of seedling mango (Mangifera indica L. cv. Irwin)trees were determined to understand what controls the rate ofphotosynthesis (Pn) at various stages of development. The youngleaves of seedling trees contained high concentrations of anthocyanin.During enlargement of leaves, the disappearance of anthocyaninand the accumulation of chlorophyll occurred concomitantly;the anthocyanin content began to decrease markedly once theleaf area had reached a maximum. During the early period ofleaf development, the thickness of mesophyll tissue decreasedtemporarily, but when the length of the leaf reached half thatof a mature leaf, the mesophyll began to thicken again. Smallstarch grains appeared in the chloroplasts of the young leavesand chloroplast nucleoids (ct-nuclei) were distributed throughoutthe chloroplasts. When leaves matured, ct-nuclei were displacedto the periphery of chloroplasts because of the accumulationof large starch grains. Compared with young leaves, green andmature leaves contained greater concentrations of ribulose bisphosphatecarboxylase-oxygenase (RuBisCO) protein. The results of immunocytochemicalexamination of RuBisCO under the light microscope reflectedthe results of electrophoresis measurements of RuBisCO. Pn waslow during the chocolate-coloured stage of early leaf development.In green and mature leaves Pn was higher; the average Pn was7·6 mg CO₂ dm^-2 h^-1 under light at intensities above500 µmol m^-2 s^-1.Copyright 1995, 1999 Academic Press Mangifera indica L., mango leaf, chloroplast nucleoids, chloroplast ultrastructure, starch accumulation, anthocyanin, chlorophyll, DAPI staining, SDS-PAGE, immunocytochemical technique     

Influence of Temperature on the Ratio of Ribulose Bisphosphate Carboxylase to Oxygenase Activities and on the Ratio of Photosynthesis to Photorespiration of Leaves

Lehnherr, B., Mächler, F. and Nösberger, J. 1985.Influence of temperature on the ratio of ribulose bisphosphatecarboxylase to oxygenase activities and on the ratio of photosynthesisto photorespiration of leaves.—J. exp. Bot. 36: 1117–1125. Rates of net and gross photosynthesis of intact white cloverleaves were measured by infrared gas analysis and by short termuptake of ¹⁴CO₂ respectively. Ribulose bisphosphate carboxylaseoxygenase (RuBPCO) was purified from young leaves and kineticproperties investigated in combined and separate assays. Theratio of carboxylase to oxygenase activities was compared withthe ratio of photosynthesis to photorespiration at various temperaturesand CO₂ concentrations. The ratio of photosynthesis to photorespiration at 30 Pa p(CO₂)was consistent with the ratio of carboxylase activity to oxygenaseactivity when each was measured above 20 °C. However, theratio of photosynthesis to photorespiration increased with decreasingtemperature, whereas the ratio of carboxylase to oxygenase activitywas independent of temperature. This resulted in a disagreementbetween the measurements on the purified enzyme and intact leafat low temperature. No disagreement between enzyme and leafat low temperature occurred, when the ratio of photosynthesisto photorespiration was determined at increased CO₂ concentrations. The results suggest an effect of low temperature and low CO₂concentration on the ratio of photosynthesis to photorespirationindependent of the enzyme. Key words: Ribulose bisphosphate carboxylase oxygenase, photorespiration, temperature     

Ribulose Bisphosphate Carboxylase Activity in Anther-Derived Plants of Saintpaulia ionantha Wendl. Shag

Plants obtained from anther culture of the African violet, Saintpaulia ionantha Wendl. `Shag' and vegetatively cloned copies of the parent anther donor plant were examined for their ploidy and ribulose-1,5-biphosphate carboxylase (RuBPcase) activity. The cloned parent plants were all diploid and did not vary much in their nuclear DNA, chlorophyll, and RuBPcase activity. Some of the anther-derived plants were similar to the parent plants while others were not. Different levels of ploidy were observed among the androgenetic plants. RuBPcase activities higher than that of the parent plants were found in some anther-derived plants. However, there was no direct correlation between ploidy and RuBPcase activity. Expression of nuclear genes from a single parent in the anther-derived plants and it's diploidization or plastid changes during early stages of microsporogenesis or androgenesis are suggested as possible reasons for the variations observed among them. This could be a useful technique to obtain physiological variants which could be agronomically desirable.