Acclimation of Two Tomato Species to High Atmospheric CO(2): II. Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase

Lycopersicon esculentum Mill. cv Vedettos and Lycopersicon chmielewskii Rick, LA 1028, were exposed to two CO₂ concentrations (330 or 900 microliters per liter) for 10 weeks. The elevated CO₂ concentrations increased the initial ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity of both species for the first 5 weeks of treatment but the difference did not persist during the last 5 weeks. The activity of Mg²⁺-CO₂-activated Rubisco was higher in 900 microliters per liter for the first 2 weeks but declined sharply thereafter. After 10 weeks, leaves grown at 330 microliters per liter CO₂ had about twice the Rubisco activity compared with those grown at 900 microliters per liter CO₂. The two species showed the same trend to Rubisco declines under high CO₂ concentrations. The percent activation of Rubisco was always higher under high CO₂. The phosphoenolpyruvate carboxylase (PEPCase) activity measured in tomato leaves averaged 7.9% of the total Rubisco. PEPCase showed a similar trend with time as the initial Rubisco but with no significant difference between nonenriched and CO₂-enriched plants. Long-term exposure of tomato plants to high CO₂ was previously shown to induce a decline of photosynthetic efficiency. Based on the current study and on previous results, we propose that the decline of activated Rubisco is the main cause of the acclimation of tomato plants to high CO₂ concentrations.     

Photosynthetic Acclimation to Elevated CO2 Occurs in Transformed Tobacco with Decreased Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content

Inhibition of net carbon assimilation rates during growth at elevated CO2 was studied in transgenic tobacco (Nicotiana tabacum L.) plants containing zero to two copies of antisense DNA sequences to the small subunit polypeptide (rbcS) gene of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). High- and low-Rubisco tobacco plants were obtained from the selfed progeny of the original line 3 transformant (S.R. Rodermel, M.S. Abbott, L. Bogorad [1988] Cell 55: 673-681). Assimilation rates of high- and low-Rubisco tobacco plants increased 22 and 71%, respectively, when transferred from 35- to 70-Pa CO2 chamber air at 900 [mu]mol m-2 s-1 photon flux density. However, CO2-dependent increases of net carbon assimilation rates of high- and low-Rubisco plants virtually disappeared after 9 d of growth in elevated CO2 chamber air. Total above-ground dry matter production of high- and low-Rubisco plants was 28 and 53% greater, respectively, after 9 d of growth at 70 Pa compared with 35 Pa CO2. Most of this dry weight gain was due to increased specific leaf weight. Rubisco activity, Rubisco protein, and total chlorophyll were lower in both high- and low-Rubisco plants grown in enriched compared with ambient CO2 chamber air. Soluble leaf protein also decreased in response to CO2 enrichment in high- but not in low-Rubisco tobacco plants. Decreased Rubisco activities in CO2-adapted high- and low-Rubisco plants were not attributable to changes in activation state of the enzyme. Carbonic anhydrase activities and subunit levels measured with specific antibodies were similar in high- and low-Rubisco tobacco plants and were unchanged by CO2 enrichment. Collectively, these findings suggested that photosynthetic acclimation to enriched CO2 occurred in tobacco plants either with or without transgenically decreased Rubisco levels and also indicated that the down-regulation of Rubisco in CO2-adapted tobacco plants was related to decreased specific activity of this enzyme.     

Inhibition and Acclimation of Photosynthesis to Heat Stress Is Closely Correlated with Activation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Increasing the leaf temperature of intact cotton (Gossypium hirsutum L.) and wheat (Triticum aestivum L.) plants caused a progressive decline in the light-saturated CO₂-exchange rate (CER). CER was more sensitive to increased leaf temperature in wheat than in cotton, and both species demonstrated photosynthetic acclimation when leaf temperature was increased gradually. Inhibition of CER was not a consequence of stomatal closure, as indicated by a positive relationship between leaf temperature and transpiration. The activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is regulated by Rubisco activase, was closely correlated with temperature-induced changes in CER. Nonphotochemical chlorophyll fluorescence quenching increased with leaf temperature in a manner consistent with inhibited CER and Rubisco activation. Both nonphotochemical fluorescence quenching and Rubisco activation were more sensitive to heat stress than the maximum quantum yield of photochemistry of photosystem II. Heat stress led to decreased 3-phosphoglyceric acid content and increased ribulose-1,5-bisphosphate content, which is indicative of inhibited metabolite flow through Rubisco. We conclude that heat stress inhibited CER primarily by decreasing the activation state of Rubisco via inhibition of Rubisco activase. Although Rubisco activation was more closely correlated with CER than the maximum quantum yield of photochemistry of photosystem II, both processes could be acclimated to heat stress by gradually increasing the leaf temperature.     

Activities of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase, and Oxygen Evolution in Transgenic Tobacco Plants

Three clones of tobacco transformed with the T-DNA of Agrobacterium rhizogenes Ri plasmid A4b cultivated in vitro on a solid agar medium neither showed pronounced morphological diversity nor significantly differed in chlorophyll (Chl) contents from control plants. However, the transformation caused a 27 to 83 % decay in leaf oxygen evolution and in both ribulose-1,5-bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxylase (PEPC) activities. Therefore, the transformation brought about a reduction of active PEPC as well as activable RuBPC amounts in plant tissues. Individual clones did not mutually differ. In tobacco transformed with A. rhizogenes strain TR101 and grown in soil only the mean leaf area tended to reduce. Chl contents, Chl a/b ratio, oxygen evolution, and activities of both RuBPC and PEPC were insignificantly affected by the transformation.     

Effects of O2 and CO2 on Nonsteady-State Photosynthesis (Further Evidence for Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Limitation)

The effects of CO2 and O2 on nonsteady-state photosynthesis following an increase in photosynthetic photon flux density (PPFD) were examined in Spinacia oleracea to investigate the hypotheses that (a) a slow exponential phase (the ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] phase) of nonsteady-state photosynthesis is primarily limited by Rubisco activity and (b) Rubisco activation involves two sequential, light-dependent processes as described in a previous study (I.E. Woodrow, K.A. Mott [1992] Plant Physiol 99: 298-303). Photosynthesis was found to be sensitive to O2 during the Rubisco phase in the approach of photosynthesis to steady state. Analyses of this sensitivity to O2 showed that the control coefficient for Rubisco was approximately equal to 1 during this phase, suggesting that Rubisco was the primary limitation to photosynthesis. O2 had almost no effect on the kinetics (described using a relaxation time, [tau] of the Rubisco phase for leaves starting in darkness or for leaves starting in low PPFD, but [tau] was substantially higher in the former case. CO2 was found to affect both the rate of photosynthesis and the magnitude of [tau] for the Rubisco phase. The [tau] value for the Rubisco phase was found to be negatively correlated with intercellular CO2 concentration (ci), and leaves starting in darkness had higher values of [tau] at any ci than leaves starting in low PPFD. The effects of CO2 and O2 on the Rubisco phase are consistent with the existence of two sequential, light-dependent processes in the activation of Rubisco if neither process is sensitive to O2 and only the second process is sensitive to CO2. The implications of the data for the mechanism of Rubisco activation and for the effects of stomatal conductance on nonsteady-state photosynthesis are discussed.     

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.     

Responses of Ribulose-1,5-Bisphosphate Carboxylase,Protein Content,and Stomatal Conductance to Water Deficit in Maize,Tomato, and Bean

We compared responses of maize, tomato, and bean plants to water stress. Maize reached a severe water deficit (leaf water potential –1.90 MPa) in a longer period of time as compared with tomato and bean plants. Maize stomatal conductance (g _s) decreased at mild water deficit. g _s of tomato and bean decreased gradually and did not reach values as low as in maize. The protein content was maintained in maize and decreased at low water potential (_w); in tomato it fluctuated and also decreased at low _w; in bean it gradually decreased. Ribulose-1,5-bisphosphate carboxylase/oxygenase activity remained high at mild and moderate stress in maize and tomato plants; in bean it remained high only at mild stress.     

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.     

杂交稻核酮糖二磷酸羧化酶的动力学性质

在ｐＨ８．０和３０℃的条件下测定了杂交稻ＲｕＢＰ羧化酶的动力学常数,纯化酶测定值与粗酶快速测定值无明显差异。１２种不同组合的三系杂交稻（Ｆ１）其ＲｕＢＰ羧化酶动力学常数差异不大,Ｋｍ（ＣＯ２）和Ｖｍａｘ的平均值与普通栽培品种也无显著差异。杂交稻汕优６３号ＲｕＢＰ羧化酶的动力学常数和酶蛋白含量与父本恢复系相类似,而母本不育系的Ｖｍａｘ和酶蛋白含量均较高。     

Photosynthesis in Ulva fasciata: V. Evidence for an Inorganic Carbon Concentrating System, and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase CO(2) Kinetics

Evidence of an inorganic carbon concentrating system in a marine macroalga is provided here. Based on an O₂ technique, supported by determinations of inorganic carbon concentrations, of experimental media (as well as compensation points) using infrared gas analysis, it was found that Ulva fasciata maintained intracellular inorganic carbon levels of 2.3 to 6.0 millimolar at bulk medium concentrations ranging from 0.02 to 1.5 millimolar. Bicarbonate seemed to be the preferred carbon form taken up at all inorganic carbon levels. It was found that ribulose-1,5-bisphosphate carboxylase/oxygenase from Ulva had a K_m(CO₂) of 70 micromolar and saturated at about 250 micromolar CO₂. Assuming a cytoplasmic pH of 7.2 (as measured for another Ulva species, P Lundberg et al. [1988] Plant Physiol 89: 1380-1387), and given the high activity of internal carbonic anhydrase (S Beer, A Israel [1990] Plant Cell Environ [in press]) and the here measured internal inorganic carbon level, it was concluded that internal CO₂ in Ulva could, at ambient external inorganic carbon concentrations, be maintained at a high enough level to saturate ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation. It is suggested that this suppresses photorespiration and optimizes net photosynthetic production in an alga representing a large group of marine plants faced with limiting external CO₂ concentrations in nature.     

各种因子对固定化烟草核酮糖1,5-二磷酸羧化酶/加氧酶解离作用的影响

pH,温度、离子强度及效应剂等对固定化烟草RuBP羧化酶在2.5mol/L尿素处理下的解离作用有各种不同的影响。在pH6.0时,仅小亚基从大亚基核(L_8)解离,当pH为中性偏碱时,大亚基核也解离。低温和低离子强度均促进酶的解离,而温度和离子强度对大亚基之间的解离的影响显著大于对大、小亚基之间的影响。这表明酶的亚基之间存在着不同的极性和疏水作用,而大亚基之间的疏水作用比大、小亚基之间的强。6-PG对大、小亚基之间解离的抑制作用表明大亚基上的催化位置与小亚基之间有一定的密切关系。     

Salinity and Nitrogen Effects on Photosynthesis, Ribulose-1,5-Bisphosphate Carboxylase and Metabolite Pool Sizes in Phaseolus vulgaris L

Salinity (100 millimolar NaCl) was found to reduce photosynthetic capacity independent of stomatal closure in Phaseolus vulgaris. This reduction was shown to be a consequence of a reduction in the efficiency of ribulose-1,5-bisphosphate (RuBP) carboxylase (RuBPCase) rather than a reduction in the leaf content of photosynthetic machinery. In control plants, photosynthesis became RuBP-limited at approximately 1.75 moles RuBP per mole 2-carboxyarabinitol bisphosphate binding sites. Salinization caused the RuBP pool size to reach this limiting value for CO₂ fixation at much lower values of intercellular CO₂. Plants grown at low nitrogen and ± NaCl became RuBP limited at similar RuBP pool sizes as the high nitrogen-grown plants. At limiting RuBP pool sizes and equal values of intercellular CO₂ photosynthetic capacity of salt-stressed plants was less than control plants. This effect of salinity on RuBPCase activity could not be explained by deactivation of the enzyme or inhibitor synthesis. Thus, salinity reduced photosynthetic capacity by reducing both the RuBP pool size by an effect on RuBP regeneration capacity and RuBPCase activity by an unknown mechanism when RuBP was limiting.     

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     

Light and CO(2) Response of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Arabidopsis Leaves

The requirements for activation of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) were investigated in leaves of Arabidopsis wild-type and a mutant incapable of light activating rubisco in vivo. Upon illumination with saturating light intensities, the activation state of rubisco increased 2-fold in the wild-type and decreased in the mutant. Activation of fructose 1,6-bisphosphate phosphatase was unaffected by the mutation. Under low light, rubisco deactivated in both the wild-type and the mutant. Deactivation of rubisco in the mutant under high and low light led to the accumulation of high concentrations of ribulose 1,5-bisphosphate. Inhibiting photosynthesis with methyl viologen prevented ribulose 1,5-bisphosphate accumulation but was ineffective in restoring rubisco activation to the mutant. Net photosynthesis and the rubisco activation level were closely correlated and saturated at a lower light intensity in the mutant than in wild-type. At CO₂ concentrations between 100 and 2000 microliters per liter, the activation state was a function of the CO₂ concentration in the dark but was independent of CO₂ concentration in the light. High CO₂ concentration (1%) suppressed activation in the wild-type and deactivation in the mutant. These results support the concept that rubisco activation in vivo is not a spontaneous process but is catalyzed by a specific protein. The absence of this protein, rubisco activase, is responsible for the altered characteristics of rubisco activation in the mutant.     

Exchange Properties of the Activator CO(2) of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

The exchange properties of the activator CO₂ of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO₂ on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO₂. The exchange kinetics of the activator CO₂ were monitored by activation of the enzyme to steady state in the presence of ¹²CO₂, followed by addition of ¹⁴CO₂ and determination of the amount of labeled CO₂ trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (K_obs) for exchange with both the purified enzyme (0.45 min⁻¹) and in illuminated chloroplasts (0.18 min⁻¹) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO₂ was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation.     

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.