Activation Of RuBPCase Research Articles

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.

Effects of rare earth ions on activity of RuBPcase in tobacco

To investigate the physiological effects of rare earth ions, we have studied the effect of LaCl 3 on the activity of ribulose 1,5-bisphosphate carboxylase (RuBPcase) in tobacco ( Nicotiana tobacum). When treated with 20–80 μmol/l LaCl 3 in Hoagland solution by water culture or by spraying with 400–1600 μmol/l LaCl 3 in pot experiment, the total and initial activities of RuBPcase were raised gradually, but decreased when the concentrations of LaCl 3 were ≥200 μmol/l in water culture and >1600 μmol/l by spraying. It was indicated that LaCl 3 promoted the activation of RuBPcase at low concentrations in vivo. It was also proved that lanthanides (Ln 3+) increased the activity of RuBPcase in vitro. Upon addition of different concentrations of Ln 3+ to replace Mg 2+, the RuBPcase activity exhibited an apparently biphasic response curve. Among the three Ln 3+ tested, the activation effects of La 3+ and Ce 3+ on RuBPcase were greater than that of Gd 3+, but the activation effects of all the three Ln 3+ were lower at high concentrations than that of Mg 2+, showing strongly inhibitory function. The extended X-ray absorption fine structure study of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco)–La complex indicated that La is bound with the oxygen atoms of amino acid residues, with the distance of the LaO bond being 2.51 Å. The mechanism of molecular interaction between Ln 3+ and RuBPcase is discussed.

Characteristics of Five New Photoautotrophic Suspension Cultures Including Two Amaranthus Species and a Cotton Strain Growing on Ambient CO2 Levels

Suspension cultures of cotton (Gossypium hirsutum), Amaranthus cruentus, A. powellii, Datura innoxia, and a Nicotiana tabacum-N. glutinosa fusion hybrid were adapted to grow photoautotrophically under continuous light. The cotton strain grew with an atmosphere of ambient CO(2) (about 0.06 to 0.07% in the culture room) while the other strains required elevated CO(2) levels (5%). Photoautotrophy was indicated by the requirement for CO(2) and for light for growth. The strains grew with doubling times near 14 days and had from 50 to 600 micrograms of chlorophyll per gram of fresh weight. The cells grew in small to moderate sized clumps with cell sizes from 40 to 70 micrometers (diameter). Like most photoautotrophic cultures described so far the ribulose 1,5-bisphosphate carboxylase (RuBPcase) activity levels were well below those of mature leaves. The phosphoenolpyruvate carboxylase levels were not elevated in the C(4)Amaranthus species. The cells showed high dark respiration rates and had lower net CO(2) fixation under high O(2) conditions. Dark CO(2) fixation rates ranged from near 10 to 30% of that in light. Fluorescence emission spectra measurements show that the cell antenna pigments systems of the four strains examined are similar to that of chloroplasts of green plants. The cotton strain which was capable of growth under ambient CO(2) conditions showed the unique properties of a high RuBPcase activation level in ambient CO(2) and a stable ability to show net CO(2) fixation in 21% O(2) conditions.

Water Deficit and Associated Changes in Some Photosynthetic Parameters in Leaves of `Valencia' Orange (Citrus sinensis [L.] Osbeck)

Photosynthetic CO(2) assimilation, transpiration, ribulose-1,5-bisphosphate carboxylase (RuBPCase), and soluble protein were reduced in leaves of water-deficit (stress) ;Valencia' orange (Citrus sinensis [L.] Osbeck). Maximum photosynthetic CO(2) assimilation and transpiration, which occurred before midday for both control and stressed plants, was 58 and 40%, respectively, for the stress (-2.0 megapascals leaf water potential) as compared to the control (-0.6 megapascals leaf water potential). As water deficit became more severe in the afternoon, with water potential of -3.1 megapascals for the stressed leaves vs. -1.1 megapascals for control leaves, stressed-leaf transpiration declined and photosynthetic CO(2) assimilation rapidly dropped to zero. Water deficit decreased both activation and total activity of RuBPCase. Activation of the enzyme was about 62% (of fully activated enzyme in vitro) for the stress, compared to 80% for the control. Water deficit reduced RuBPCase initial activity by 40% and HCO(3) (-)/Mg(2+)-saturated activity by 22%. However, RuBPCase for both stressed and control leaves were similar in K(cat) (25 moles CO(2) per mole enzyme per second) and K(m) for CO(2) (18.9 micromolar). Concentrations of RuBPCase and soluble protein of stressed leaves averaged 80 and 85%, respectively, of control leaves. Thus, reductions in activation and concentration of RuBPCase in Valencia orange leaves contributed to reductions in enzyme activities during water-deficit periods. Declines in leaf photosynthesis, soluble protein, and RuBPCase activation and concentration due to water deficit were, however, recoverable at 5 days after rewatering.

The in-vivo response of the ribulose-1,5-bisphosphate carboxylase activation state and the pool sizes of photosynthetic metabolites to elevated CO2 inPhaseolus vulgaris L.

The short-term, in-vivo response to elevated CO2 of ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39) activity, and the pool sizes of ribulose 1,5-bisphosphate, 3-phosphoglyceric acid, triose phosphates, fructose 1,6-bisphosphate, glucose 6-phosphate and fructose 6-phosphate in bean were studied. Increasing CO2 from an ambient partial pressure of 360-1600 μbar induced a substantial deactivation of RuBPCase at both saturating and subsaturating photon flux densities. Activation of RuBPCase declined for 30 min following the CO2 increase. However, the rate of photosynthesis re-equilibrated within 6 min of the switch to high CO2, indicating that RuBPCase activity did not limit photosynthesis at high CO2. Following a return to low CO2, RuBPCase activation increased to control levels within 10 min. The photosynthetic rate fell immediately after the return to low CO2, and then increased in parallel with the increase in RuBPCase activation to the initial rate observed prior to the CO2 increase. This indicated that RuBPCase activity limited photosynthesis while RuBPCase activation increased. Metabolite pools were temporarily affected during the first 10 min after either a CO2 increase or decrease. However, they returned to their original level as the change in the activation state of RuBPCase neared completion. This result indicates that one role for changes in the activation state of RuBPCase is to regulate the pool sizes of photosynthetic intermediates.

Photosynthetic Characteristics of a Photoautotrophic Cell Suspension Culture of Soybean

A soybean suspension culture (SB-P) which can grow photoautotrophically in 5% CO(2) will not grow in ambient CO(2) levels. This elevated CO(2) requirement seems to be due to the additive effects of a number of factors. The in vivo activity of ribulose-1,5-bisphosphate carboxylase (RuBPcase) is much lower in the SB-P cells, compared to soybean plants. This may be due to the low light intensity used to culture the cells, which has been shown to decrease both the amount and activity in whole plants, resulting in a low rate of net photosynthesis. The RuBPcase activation level is also lowered in air CO(2) levels. The presence of the liquid medium raises the cells CO(2) compensation concentration (the CO(2) concentration reached when the rates of CO(2) fixed by photosynthesis and the CO(2) respired by the cells are equal). These factors, coupled with the high respiratory loss of CO(2) all contribute to reduced net photosynthesis in air, resulting in a photosynthetic capacity that is inadequate for cell survival. Active cell division, low photosynthetic capacity, elevated respiration, and a low ratio of RuBPcase(initial)/phosphoenolpyruvate carboxylase are traits that SB-P cells share with young leaf cells, indicating SB-P cell physiology may be comparable to that of young expanding leaves rather than to that of mature leaves.

Phosphorylation of small subunit plays a crucial role in the regulation of RuBPCase in moss and spinach

RuBPCase has been purified to electrophoretic homogeneity from moss and spinach. On denaturing SDS-polyacrylamide gels the purified enzyme revealed two discrete bands, thereby indicating the presence of large and small subunits. The phosphoprotein nature of RuBPCase was proved by in vivo labelling of enzyme with [ 32P]orthophosphate. Autoradiographic analysis of 32P-labelled RuBPCase on SDS-PAG demonstrated that phosphorylation was restricted to the small subunit. Dephosphorylation of purified RuBPCase with alkaline phosphatase resulted in a dramatic decline (70% decrease) in the biological activity of the enzyme. Fractionation of the dephosphorylated enzyme on denaturing gels revealed only the presence of large subunits of RuBPCase. Thus it became evident that dephosphorylation of RuBPCase brings about the dissociation of small subunits from the catalytic large subunits (octamer). The dephosphorylated small subunits were isolated as dimers. These results clearly indicate that phosphorylation of small subunits is mandatory for the reconstitution of holoenzyme and hence crucial for the activation of RuBPCase.

Variation in photosynthetic electron transport capacity in vivo and its effects on the light modulation of ribulose bisphosphate carboxylase

Photosynthetic electron transport capacity was varied in vivo in sugar beets using iron deficiency, and its effects on the light modulation of ribulose bisphosphate carboxylase (RuBPCase) studied. Three treatment groups corresponding to decreasing amounts of thylakoids per leaf area were examined: iron sufficient (control), moderately iron-stressed, and severely iron-stressed. Reduction in electron transport capacity in vivo was correlated with a substantial decrease in the level of RuBPCase activation, even at saturating irradiances. These results indicate a direct relationship between RuBPCase activation and photosynthetic electron transport. In addition, our data suggest that the activation of RuBPCase could not solely account for the increases in the photosynthetic rate at high irradiances; RuBPCase reached maximal activation at irradiances well below light saturation for net photosynthesis.

Relationships between CO2 Exchange Rates and Activities of Pyruvate,Pi Dikinase and Ribulose Bisphosphate Carboxylase, Chlorophyll Concentration, and Cell Volume in Maize Leaves

The relationships between CO(2)-exchange rate (CER), DNA and chlorophyll (Chl) concentrations, pyruvate,Pi dikinase (PPDK) and ribulose bisphosphate carboxylase (RuBPCase) activities in ten maize (Zea mays L.) genotypes were investigated. The in vivo degrees of activation of PPDK and RuBPCase were estimated to make meaningful comparisons with CER. In leaves at a photosynthetic photon flux density (PPFD) of 720 micromoles per square meter per second, in vivo PPDK degree of activation was 80% of that of PPDK fully activated in vitro, whereas RuBPCase could not be further activated in vitro, suggesting that RuBPCase was fully activated in vivo. CER varied about 50% among the genotypes tested. Significant genetic differences were observed for the average weight of a cell (estimated by gram fresh weight per milligram DNA), but this character was not correlated with CER expressed on a fresh weight basis. CER was correlated with Chl concentration, and with estimates of the in vivo degree of activation of PPDK and RuBPCase. We concluded that in maize, CER is controlled by the metabolic components of photosynthesis rather than by membrane resistances to CO(2). If the latter factor were controlling CER, then smaller cells with higher amounts of exposed cell surface area per unit cell volume would have lower resistance to CO(2) diffusion, and therefore higher CER. When data were expressed on a DNA basis (proportional to a per cell basis), results indicated that larger cells (i.e. those with higher fresh weight per milligram DNA) have a higher content of Chl, and higher PPDK and RuBPCase activities, resulting in higher CER than in smaller cells.

Enzyme Activities and Leaf Constituents in Barley Seedlings at Different Nutrient Levels

Summary The effect of additional fertilization (NPK) on metabolism was studied in the first three leaves of barley. Activities of ribulose-1,5-bisphosphate carboxylase, alkaline fructose-1,6-bisphosphatase, nitrate reductase and protein content were markedly higher at elevated nutrient levels, but content of main non-structural carbohydrates were lower under these conditions. Nutrient limited plants accumulated large amounts of carbohydrates, but their dry weight was reduced, indicating a limited capacity for photosynthesis and utilization of assimilates. The specific activity of RuBPCase increased at low nutrient levels during development of the second leaf, but not at elevated nutrient levels. Therefore, it is likely that RuBPCase protein is degraded less efficiently than other protein. Alternatively, these findings could be interpreted as an altered response to activation of RuBPCase by bicarbonate.

Limiting Factors in Photosynthesis

Although there is now some agreement with the view that the supply of photochemical energy may influence photosynthetic rate (P) at high CO(2) pressures, it is less clear whether this limitation extends to P at low CO(2). This was investigated by measuring P per area as a function of the intercellular CO(2) concentration (C(i)) at different levels of photochemical energy supply. Changes in the latter were obtained experimentally by varying the level of irradiance to normal (Fe-sufficient) leaves of Beta vulgaris L. cv F58-554H1, and by varying photosynthetic electron transport capacity using leaves from Fe-deficient and Fe-sufficient plants. P and C(i) were determined for attached sugar beet leaves using open flow gas exchange. The results suggest that P/area was colimited by the supply of photochemical energy at very low as well as high values of C(i). Using the procedure developed by Perchorowicz et al. (Plant Physiol 1982 69:1165-1168), we investigated the effect of irradiance on ribulose bisphosphate carboxylase (RuBPCase) activation. The ratio of initial extractable activity to total inducible RuBPCase activity increased from 0.25 to 0.90 as leaf irradiance increased from 100 to 1500 microeinsteins photosynthetically active radiation per square meter per second. These data suggest that colimitation by photochemical energy supply at low C(i) may be mediated via effects on RuBPCase activation.

Effects of Light and Elevated Atmospheric CO2 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 CO(2) 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 CO(2) 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 CO(2) per milligram chlorophyll per hour) showed that the enzyme values were low (230) when sampled before sunrise, even when activated in vitro with saturating HCO(3) (-) and Mg(2+), 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 HCO(3) (-) and Mg(2+)-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 HCO(3) (-) and Mg(2+)-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 CO(2) and Mg(2+).

Binding of phosphorylated effectors by active and inactive forms of ribulose 1,5-bisphosphate carboxylase

Activation of ribulose1,5-bisphosphate carboxylase by CO, and Mgz+ is slow and reversible. At subsaturating concentrations of CO, and Mg2+, positive effectors increase and negative effectors decrease the amount of active enzyme at equilibrium. Preequilibrium experiments indicated that both positive and negative effectors inhibit the rates of enzyme activation and deactivation. Greater than 99% inhibition of the activation and deactivation rates was observed at high effector concentrations, indicating that the binding and release of the activators COz and Mg2+ occur only with effector-free enzyme. The deactivation rate Ki values for the negative effector ribose 5-phosphate and the positive effectors inorganic phosphate, fructose 1,6-bisphosphate, and 6-phosphogluconate were smaller than the corresponding activation rate Ki values by factors of 2, 6, 25, and 670, respectively. Thus, phosphorylated effectors impede deactivation more than activation. Equilibrium binding studies indicated that the active and inR i b u l o s e 1,5-bisphosphate carboxylase (EC 4.1.1.39) requires the addition of C 0 2 and a divalent metal ion in forming an activated enzyme complex capable of catalyzing the carboxylation and oxygenation of RuBP1 (Lorimer et al., 1976). The kinetics of this process have been well characterized for several RuBPCase enzymes purified from microbial and plant sources (Badger, 1980; Christeller & Laing, 1978; Laing & Christeller, 1976). The rate-limiting step in C02/Me2+ activation is carbamate formation at a lysine residue (Lys-201) on the 56-kDa catalytic subunit of the enzyme (Lorimer, 1981; Lorimer & Miziorko, 1980), and this is followed by rapid From the Department of Agricultural Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0718 (D.B.J. and R.C.), the Agronomy Department, University of Illinois, Urbana, Illinois 61 801 (D.B.J. and W.L.O.), and the U S . Department of Agriculture, Agricultural Research Service, Urbana, Illinois 61801 (W.L.O.). Received December 22, 1982. Research sponsored in part by Grant 78-59-231 10-1-1 19-1 from the USDA Competitive Research Grants Office (R.C.). Published as Paper No. 7046, Journal Series, Nebraska Agricultural Experiment Station. 0006-2960/83 10422-34 lO$O 1.5010 active enzyme forms have similar affinities for the positive effectors inorganic phosphate (KD = 650 pM) and fructose bisphosphate (KD = 11 pM). The positive effector 3phosphoglycerate was bound with greater affinity by the inactive enzyme (KD = 25 pM) than by the active enzyme (KD = 76 MM). Thus, preferential binding of positive effectors to the active enzyme form is not responsible for the enhancement of enzyme activation at equilibrium. The promotion of activation by positive effectors is mediated by altering the relative rates of activation and deactivation to favor active enzyme. Equilibrium binding studies indicated that the inactive enzyme had a much greater affinity for ribose 5-phosphate (KD = 42 pM) than did the active enzyme (KD = 480 pM). Preferential binding of this negative effector to inactive enzyme exceeds its stabilizing effect on the active enzyme form and thus causes an overall reduction of activation at equilibrium. addition of the metal cofactor as shown in eq 1. E E-C02 E-CO2-Me2+ (1) inactive active Although the mechanisms of activation and inhibition of RuBPCase by sugar phosphates and other anions have received considerable attention, these processes are not well resolved. Because of their potential regulatory role in vivo, chloroplast metabolites such as 6-P-gluconate, FBP, inorganic phosphate, and NADPH have been of primary interest. Several sugar phosphates enhance the activation state of RuBPCase which is partially activated with respect to C 0 2 and Mg2+, while other sugar phosphates lower the activation state of the enzyme ~ ~~~~~~~ ~ ~~~~~ ~~~~~ ~~ ~~ ' Abbreviations: RuBP, ribulose 1,5-bisphosphate; RuBPCase, ribulose1,5-bisphosphate carboxylase; CABP, 2-carboxyarabinitol 1,5-bisphosphate; FBP, fructose 1,6-bisphosphate; Bicine, N,N-bis(2-hydroxyethy1)glycine; P, phosphate; kDa, kilodalton; EDTA, ethylenediaminetetraacetic acid.