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1.
The kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco 总被引:20,自引:0,他引:20
Transgenic tobacco (Nicotiana tabacum L. cv. W38) with an antisense gene directed against the mRNA of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit was used to determine the kinetic properties of Rubisco in vivo. The leaves of these plants contained only 34% as much Rubisco as those of the wild type, but other photosynthetic components were not significantly affected. Consequently, the rate of CO2 assimilation by the antisense plants was limited by Rubisco activity over a wide range of CO2 partial pressures. Unlike in the wild-type leaves, where the rate of regeneration of ribulose bisphosphate limited CO2 assimilation at intercellular partial pressures above 400 ubar, photosynthesis in the leaves of the antisense plants responded hyperbolically to CO2, allowing the kinetic parameters of Rubisco in vivo to be inferred. We calculated a maximal catalytic turnover rate, kcat, of 3.5+0.2 mol CO2·(mol sites)–1·s–1 at 25° C in vivo. By comparison, we measured a value of 2.9 mol CO2·(mol sites)–1·–1 in vitro with leaf extracts. To estimate the Michaelis-Menten constants for CO2 and O2, the rate of CO2 assimilation was measured at 25° C at different intercellular partial pressures of CO2 and O2. These measurements were combined with carbon-isotope analysis (13C/12C) of CO2 in the air passing over the leaf to estimate the conductance for transfer of CO2 from the substomatal cavities to the sites of carboxylation (0.3 mol·m–2·s–1·bar–1) and thus the partial pressure of CO2 at the sites of carboxylation. The calculated Michaelis-Menten constants for CO2 and O2 were 259 ±57 bar (8.6±1.9M) and 179 mbar (226 M), respectively, and the effective Michaelis-Menten constant for CO2 in 200 mbar O2 was 549 bar (18.3 M). From measurements of the photocompensation point (* = 38.6 ubar) we estimated Rubisco's relative specificity for CO2, as opposed to O2 to be 97.5 in vivo. These values were dependent on the size of the estimated CO2-transfer conductance.Abbreviations and Symbols A
CO2-assimilation rate
- gw
conductance for CO2 transfer from the substomatal cavities to the sites of carboxylation
- Kc, Ko
Michaelis-Menten constants for carboxylation, oxygenation of Rubisco
- kcat
Vcmax/[active site]
- O
partial pressure of O2 at the site of carboxylation
- pc
partial pressure of CO2 at the site of carboxylation
- pi
intercellular CO2 partial pressure
- Rd
day respiration (non-photorespiratory CO2 evolution)
- Rubisco
ribulose 1,5-bisphosphate carboxylase/oxygenase
- RuBP
ribulose-1,5-bisphosphate
- Sc/o
relative specificity factor for Rubisco
- SSu
small subunit of Rubisco
- Vcmax, Vomax
maximum rates of Rubisco carboxylation, oxygenation
- *
partial pressure of CO2 in the chloroplast at which photorespiratory CO2 evolution equals the rate of carboxylation 相似文献
2.
Relationship of photosynthetic acclimation to changes of Rubisco activity in field-grown winter wheat and barley during growth in elevated carbon dioxide 总被引:10,自引:0,他引:10
The responses of photosynthesis, Rubisco activity, Rubisco protein, leaf carbohydrates and total soluble protein to three carbon dioxide treatments were studied in winter wheat [Triticum aestivum (L.)] and barley [Hordeum vulgare (L.)]. Barley and wheat plants were grown in small field plots during 1995 and 1996 in clear, acrylic chambers (1.2–2.4 m2) and were provided with continuous carbon dioxide fertilization at concentrations of 350, 525 and 700 mol mol–1. Photosynthetic rates of barley penultimate leaves and wheat flag leaves measured at growth carbon dioxide concentrations decreased with leaf age in all three CO2 treatments during 1995 and 1996. Photosynthetic acclimation to elevated CO2 was observed on seven of eight measurement dates for barley and ten of eleven measurement dates for wheat over both years. Initial Rubisco activity, total soluble protein and Rubisco protein in barley penultimate leaves and wheat flag leaves also decreased with leaf age. Total Rubisco activity was not used because of enzyme degradation. There was a significant CO2 treatment effect on initial Rubisco activity, total soluble protein and Rubisco protein for wheat in 1995 and 1996 and for barley in 1995. Responses of barley penultimate leaf Rubisco activity and leaf protein concentrations to elevated carbon dioxide were nonsignificant in 1996. A significant CO2 treatment effect also was detected when means of Rubisco activity, soluble protein and Rubisco protein for wheat flag leaves were combined over harvests and years. These three flag leaf parameters were not significantly different in the 350 and 525 mol mol–1 CO2 treatments but were decreased during growth in 700 mol mol–1 CO2 relative to the other two CO2 treatments. Ratios of photosynthesis at 700 and 350 mol mol–1 were compared to ratios of Rubisco activity at 700 and 350 mol mol–1 using wheat flag leaf data from 1995 and 1996. Regression analysis of these data were linear [y = 0.586 + 1.103t x (r2 = 0.432)] and were significant at P 0.05. This result indicated that photosynthetic acclimation was positively correlated with changes of initial Rubisco activity in wheat flag leaves in response to CO2 enrichment. Effects of elevated CO2 on wheat leaf proteins during 1995 and 1996 and on barley during 1995 were consistent with an acceleration of senescence. 相似文献
3.
The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803rbc, were detected within 8 days when grown under 5% CO2 in air. These transformants were unable to grow in air (0.03% CO2). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO2 in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803rbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.Abbreviations Cmr
chloramphenicol resistance
- Kmr
kanamycin resistance
- HCR
high CO2 requirer
- Rubisco
ribulose 1,5-bisphosphate carboxylase/oxygenase
- SSC
sodium chloride and sodium citrate
- wt
wild-type 相似文献
4.
Neufeld S Zinchenko V Stephan DP Bader KP Pistorius EK 《Molecular genetics and genomics : MGG》2004,271(4):458-467
Recent investigations have revealed that the cyanobacterial photosystem II complex contains more than 26 polypeptides. The functions of most of the low-molecular-mass polypeptides, including PsbY, have remained elusive. Here we present a comparative characterization of the wild-type Synechocystis sp. strain PCC 6803 and a PsbY-free mutant derived from it. The results show that growth of the PsbY-free mutant was comparable to that of the wild-type when cells were cultivated in complete BG11 medium or under initial manganese or chloride limitation, and when illuminated at 20 or 200 E m–2 s–1. However, while growth rates of both the wild-type and the PsbY-free mutant were reduced when cells were cultivated in BG11 medium in the absence of calcium, the reduction was significantly greater in the case of the PsbY-free mutant. This differential effect on growth of the mutant relative to the wild-type in CaCl2 deficient medium was detected when the cells were illuminated with high-intensity light (200 E m–2 s–1) but not when light levels were lower (20 E m–2 s–1). The differential effect on growth was associated with lower O2 evolving activity in the mutant compared to wild-type cells. The mutant was also found to be more sensitive to photoinhibition, and showed an altered pattern of fluorescence emission at 77 K. In addition, mass spectrometric analysis revealed that PsbY-free cells cultivated in CaCl2 sufficient medium (in which no growth reduction was observed) had a significantly higher O2 evolution from hydrogen peroxide and a lower O2 evolution from water under flash light illumination than wild-type cells. These results imply that photosystem II is slightly impaired in the PsbY-free mutant, and that the mutant is less capable of coping with low levels of Ca2+ than the wild-type.Communicated by R. G. Herrmann 相似文献
5.
Nigel P. Hall Alan C. Kendall Peter J. Lea Janice C. Turner Roger M. Wallsgrove 《Photosynthesis research》1987,11(1):89-96
A barley mutant RPr84/90 has been isolated by selecting for plants which grow poorly in natural air, but normally in air enriched to 0.8% CO2. After 5 minutes of photosynthesis in air containing14CO2 this mutant incorporated 26% of the14C carbon into phosphoglycollate, a compound not normally labelled in wild type (cv. Maris Mink) leaves.The activity of phosphoglycollate phosphatase (EC 3.1.1.18) was 1.2 nkat mg–1 protein at 30°C in RPr 84/90 compared to 19.2 nkat mg–1 protein in the wild-type leaves. Phosphoglycollate phosphatase activity was not detected after protein separation by electrophoresis of leaf extracts from the mutant on polyacrylamide gels; on linear 5% acrylamide gels three bands with enzyme activity were separated from extracts of wild type plants. Gradient gel electrophoresis followed by activity staining showed two bands in Maris Mink tracks of MW 86,000 and 96,000, but no bands in 84/90. This is the first report of isozymes of phosphoglycollate phosphatase in barley which were absent in the mutant extracts. Our results confirm an earlier report of isozymes of this phosphatase in Phaseolus vulgaris [18].The photosynthetic rate of RPr 84/90 in 1% O2, 350 l CO2 l–1 was 9–12 mg CO2 dm–2 h–1 at 20°C, whereas the wild-type rate was 27–29 mg CO2 dm–2 h–1 at 20°C. In 21% O2, 350 l CO2 l–1 the rate was 2–3 mg CO2 dm–2 h–1 in the mutant and 20 mg CO2 dm–2 h–1 in the wild type.Genetic analysis has shown that the mutation segregates as a single recessive nuclear gene. 相似文献
6.
Photosynthetic gas exchange response of poplars to steady-state and dynamic light environments 总被引:3,自引:0,他引:3
The steady-state and dynamic photosynthetic response of two poplar species (Populus tremuloides and P. fremontii) to variations in photon flux density (PFD) were observed with a field portable gas exchange system. These poplars were shown to be very shade intolerant with high light saturation (800 to 1300 mol photons m–2 s–1) and light compensation (70 to 100 mol m–2 s–1) points. Understory poplar leaves showed no physiological acclimation to understory light environments. These plants become photosynthetically induced quickly (10 min). Activation of Rubisco was the primary limitation for induction, with stomatal opening playing only a minor role. Leaves maintained high stomatal conductances and stomata were unresponsive to variations in PFD. Leaves were very efficient at utilizing rapidly fluctuating light environments similar to those naturally occurring in canopies. Post-illumination CO2 fixation contributed proportionally more to the carbon gain of leaves during short frequent lightflecks than longer less frequent ones. The benefits of a more dynamic understory light environment for the carbon economy of these species are discussed. 相似文献
7.
Transgenic tobacco (Nicotiana tabacum L.) plants transformed with antisense rbcS to produce a series of plants with a progressive decrease in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been used to investigate the contribution of Rubsico to the control of photosynthesis at different irradiance, CO2 concentrations and vapour-pressure deficits. Assimilation rates, transpiration, the internal CO2 concentration and chlorophyll fluorescence were measured in each plant. (i) The flux-control coefficient of Rubisco was estimated from the slope of the plot of Rubisco content versus assimilation rate. The flux-control coefficient had a value of 0.8 or more in high irradiance, (1050 mol·m–2·s–1), low-vapour pressure deficit (4 mbar) and ambient CO2 (350 bar). Control was marginal in enhanced CO2 (450 bar) or low light (310 mol·m–2·s–1) and was also decreased at high vapour-pressure deficit (17 mbar). No control was exerted in 5% CO2. (ii) The flux-control coefficients of Rubisco were compared with the fractional demand placed on the calculated available Rubisco capacity. Only a marginal control on photosynthetic flux is exerted by Rubisco until over 50% of the available capacity is being used. Control increases as utilisation rises to 80%, and approaches unity (i.e. strict limitation) when more than 80% of the available capacity is being used. (iii) In low light, plants with reduced Rubisco have very high energy-dependent quenching of chlorophyll fluorescence (qE) and a decreased apparent quantum yield. It is argued that Rubisco still exerts marginal control in these conditions because decreased Rubisco leads to increased thylakoid energisation and high-energy dependent dissipation of light energy, and lower light-harvesting efficiency. (iv) The flux-control coefficient of stomata for photosynthesis was calculated from the flux-control coefficient of Rubisco and the internal CO2 concentration, by applying the connectivity theorem. Control by the stomata varies between zero and about 0.25. It is increased by increased irradiance, decreased CO2 or decreased vapour-pressure deficit. (v) Photosynthetic oscillations in saturating irradiance and CO2 are suppressed in decreased-activity transformants before the steady-state rate of photosynthesis is affected. This provides direct evidence that these oscillations reveal the presence of excess Rubisco. (vi) Comparison of the flux-control coefficients of Rubisco with mechanistic models of photosynthesis provides direct support for the reliability of these models in conditions where Rubisco has a flux-control coefficient approach unity (i.e. limits photosynthesis), but also indicates that these models are less useful in conditions where control is shared between Rubisco and other components of the photosynthetic apparatus.Abbreviations A
assimilation rate
- Ci
intercellular CO2 concentration in the leaf
- CR
flux-control coefficient of Rubisco for photosynthesis
- qE
high-energy-state-dependent quenching of chlorophyll fluorescence
- QA
primary acceptor of PSII
-
rbc S
gene for the nuclear-encoded small subunit of Rubisco
- Rubisco
ribulose-1,5-bisphosphate carboxylase-oxygenase
- Ru1,5bisP
ribulose-1,5-bisphosphate
- VPD
vapour-pressure deficit 相似文献
8.
Linglan M Chao L Chunxiang Q Sitao Y Jie L Fengqing G Fashui H 《Biological trace element research》2008,122(2):168-178
Characterized by a photocatalysis property, nanoanatase is closely related to the photosynthesis of spinach. It could not
only improve light absorbance, transformation from light energy to electron energy, and active chemical energy, but also promote
carbon dioxide (CO2) assimilation of spinach. However, the molecular mechanism of carbon reaction promoted by nanoanatase remains largely unclear.
In this study, we report that the amounts of Rubisco activase (rca) mRNA in the nanoanatase-treated spinach were increased
by about 51%, whereas bulk-TiO2 treatment produced an increase of only 5%. Accordingly, the protein level of Rubisco activase from the nanoanatase-treated
spinach was increased by 42% compared with the control; however, bulk-TiO2 treatment resulted in a 5% improvement. Further analysis indicated that the activity of Rubisco activase in the nanoanatase-treated
spinach was significantly higher than the control by up to 2.75 times, and bulk-TiO2 treatment had no such significant effects. Together, one of the molecular mechanisms of carbon reaction promoted by nanoanatase
is that the nanoanatase treatment results in the enhancement of rca mRNA expressions, protein levels, and activities of Rubisco
activase, thereby leading to the improvement of Rubisco carboxylation and the high rate of photosynthetic carbon reaction. 相似文献
9.
George T. Byrd T. Loboda Clanton C. Black Jr. R. Harold Brown 《Photosynthesis research》1995,44(3):253-260
Onion (Allium cepa L.) plants were examined to determine the photosynthetic role of CO2 that accumulates within their leaf cavities. Leaf cavity CO2 concentrations ranged from 2250 L L–1 near the leaf base to below atmospheric (<350 L L–1) near the leaf tip at midday. There was a daily fluctuation in the leaf cavity CO2 concentrations with minimum values near midday and maximum values at night. Conductance to CO2 from the leaf cavity ranged from 24 to 202 mol m–2 s–1 and was even lower for membranes of bulb scales. The capacity for onion leaves to recycle leaf cavity CO2 was poor, only 0.2 to 2.2% of leaf photosynthesis based either on measured CO2 concentrations and conductance values or as measured directly by 14CO2 labeling experiments. The photosynthetic responses to CO2 and O2 were measured to determine whether onion leaves exhibited a typical C3-type response. A linear increase in CO2 uptake was observed in intact leaves up to 315 L L–1 of external CO2 and, at this external CO2 concentration, uptake was inhibited 35.4±0.9% by 210 mL L–1 O2 compared to 20 mL L–1 O2. Scanning electron micrographs of the leaf cavity wall revealed degenerated tissue covered by a membrane. Onion leaf cavity membranes apparently are highly impermeable to CO2 and greatly restrict the refixation of leaf cavity CO2 by photosynthetic tissue.Abbreviations Ca
external CO2 concentration
- Ci
intercellular CO2 concentration
-
CO2 compensation concentration
- PPFR
photosynthetic photon fluence rate 相似文献
10.
W. P. Quick U. Schurr R. Scheibe E.-D. Schulze S. R. Rodermel L. Bogorad M. Stitt 《Planta》1991,183(4):542-554
Experiments were carried out to determine how decreased expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) affects photosynthetic metabolism in ambient growth conditions. In a series of tobacco (Nicotiana tabacum L.) plants containing progressively smaller amounts of Rubisco the rate of photosynthesis was measured under conditions similar to those in which the plants had been grown (310 mol photons · m–2 · s–1, 350 bar CO2, 22° C). (i) There was only a marginal inhibition (6%) of photosynthesis when Rubisco was decreased to about 60% of the amount in the wildtype. The reduced amount of Rubisco was compensated for by an increase in Rubisco activation (rising from 60 to 100%), with minor contributions from an increase of its substrates (ribulose-1,5-bisphosphate and the internal CO2 concentration) and a decrease of its product (glycerate-3-phosphate). (ii) The decreased amount of Rubisco was accompanied by an increased ATP/ADP ratio that may be causally linked to the increased activation of Rubisco. An increase of highenergy-state chlorophyll fluorescence shows that thylakoid membrane energisation and high-energy-state-dependent energy dissipation at photosystem two had also increased. (iii) A further decrease of Rubisco (in the range of 50–20% of the wildtype level) resulted in a strong and proportional inhibition of CO2 assimilation. This was accompanied by a decrease of fructose-1,6-bisphosphatase activity, coupling-factor 1 (CF1)-ATP-synthase protein, NADP-malate dehydrogenase protein, and chlorophyll. The chlorophyll a/b ratio did not change, and enolase and sucrose-phosphate synthase activity did not decrease. It is argued that other photosynthetic enzymes are also decreased once Rubisco decreases to the point at which it becomes strongly limiting for photosynthesis. (iv) It is proposed that the amount of Rubisco in the wildtype represents a balance between the demands of light, water and nitrogen utilisation. The wildtype overinvests about 15% more protein in Rubisco than is needed to avoid a strict Rubisco limitation of photosynthesis. However, this excess Rubisco allows the wildtype to operate with lower thylakoid energisation, and decreased high-energy-state-dependent energy dissipation, hence increasing light-use efficiency by about 6%. It also allows the wildtype to operate with a lower internal CO2 concentration in the leaf and a lower stomatal conductance at a given rate of photosynthesis, so that instantaneous water-use efficiency is marginally (8%) increased.Abbreviations Ci
CO2 concentration in the air spaces within the leaf
- CF1
coupling factor 1
- Chl
chlorophyll Fru1
- 6bisP
fructose-1,6-bisphosphate
- Fm
fluorescence yield with a saturating pulse in dark-adapted material
- Fo
ground-level of fluorescence obtained using a weak non-actinic modulated beam in the dark
- PGA
glycerate-3-phosphate
-
rbcS
gene for the nuclear-encoded small subunit of Rubisco
- Rubisco
ribulose-1,5-bisphosphate carboxylase-oxygenase
- Ru1, 5bisP
ribulose-1,5-bisphosphate 相似文献
11.
M. Lauerer D. Saftic W. P. Quick C. Labate K. Fichtner E. -D. Schulze S. R. Rodermel L. Bogorad M. Stitt 《Planta》1993,190(3):332-345
Tobacco (Nicotiana tabacum L.) plants transformed with antisense rbcS to decrease the expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) have been used to investigate the contribution of Rubisco to the control of photosynthesis in plants growing at different irradiances. Tobacco plants were grown in controlled-climate chambers under ambient CO2 at 20°C at 100, 300 and 750 mol·m–2·s–1 irradiance, and at 28°C at 100, 300 and 1000 mol·m–2·s–1 irradiance. (i) Measurement of photosynthesis under ambient conditions showed that the flux control coefficient of Rubisco (C
infRubisco
supA
) was very low (0.01–0.03) at low growth irradiance, and still fairly low (0.24–0.27) at higher irradiance. (ii) Short-term changes in the irradiance used to measure photosynthesis showed that C
infRubisco
supA
increases as incident irradiance rises, (iii) When low-light (100 mol·m–2·s–1)-grown plants are exposed to high (750–1000 mol·m–2·s–1) irradiance, Rubisco is almost totally limiting for photosynthesis in wild types. However, when high-light-grown leaves (750–1000 mol·m–2·s–1) are suddenly exposed to high and saturating irradiance (1500–2000 mol·m–2·s–1), C
infRubisco
supA
remained relatively low (0.23–0.33), showing that in saturating light Rubisco only exerts partial control over the light-saturated rate of photosynthesis in sun leaves; apparently additional factors are co-limiting photosynthetic performance, (iv) Growth of plants at high irradiance led to a small decrease in the percentage of total protein found in the insoluble (thylakoid fraction), and a decrease of chlorophyll, relative to protein or structural leaf dry weight. As a consequence of this change, high-irradiance-grown leaves illuminated at growth irradiance avoided an inbalance between the light reactions and Rubisco; this was shown by the low value of C
infRubisco
supA
(see above) and by measurements showing that non-photochemical quenching was low, photochemical quenching high, and NADP-malate dehydrogenase activation was low at the growth irradiance. In contrast, when a leaf adapted to low irradiance was illuminated at a higher irradiance, Rubisco exerted more control, non-photochemical quenching was higher, photochemical quenching was lower, and NADP-malate dehydrogenase activation was higher than in a leaf which had grown at that irradiance. We conclude that changes in leaf composition allow the leaf to avoid a one-sided limitation by Rubisco and, hence, overexcitation and overreduction of the thylakoids in high-irradiance growth conditions, (v) Antisense plants with less Rubisco contained a higher content of insoluble (thylakoid) protein and chlorophyll, compared to total protein or structural leaf dry weight. They also showed a higher rate of photosynthesis than the wild type, when measured at an irradiance below that at which the plant had grown. We propose that N-allocation in low light is not optimal in tobacco and that genetic manipulation to decrease Rubisco may, in some circumstances, increase photosynthetic performance in low light.Abbreviations A
rate of photosynthesis
- C
infRubisco
supA
flux control coefficient of Rubisco for photosynthesis
- ci
internal CO2 concentration
- qE
energy-dependent quenching of chlorophyll fluorescense
- qQ
photochemical quenching of chlorophyll fluorescence
- NADP-MDH
NADP-dependent malate dehydrogenase
- Rubisco
ribulose-1,5-bisphosphate carboxylase-oxygenase
- RuBP
ribulose-1,5-bisphosphate
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137). 相似文献
12.
Nekrasova G. F. Ronzhina D. A. Maleva M. G. P'yankov V. I. 《Russian Journal of Plant Physiology》2003,50(1):57-67
Radioisotope techniques were used to compare photosynthetic CO2 fixation, activities of carboxylating enzymes, and the composition of photosynthates in 42 species of aquatic plants (emergent, floating, and submersed hydrophytes) collected from rivers Sysert' and Iset' in Sverdlovsk oblast (Russia). The submersed leaves, in comparison with the emergent and floating leaves, featured lower rates of potential photosynthesis (by 2.2 mg CO2/(dm2 h) on average), low content of the fraction I protein, and low activity of Rubisco and phosphoenolpyruvate carboxylase (PEPC). The averaged activities of Rubisco and PEPC were diminished in submersed leaves by 10 and 1 mg/(dm2 h), respectively. Different hydrophyte groups showed similar composition of assimilates accumulated after 5-min photosynthesis and did not differ in this respect from terrestrial plants. However, the incorporation of 14C into sucrose and starch in submersed leaves (30 and 9% of total labeling, respectively) was lower than in emergent and floating leaves (45 and 15%, respectively). At the same time, the incorporation of 14C into C4 acids (malate and aspartate) was 1.5 times higher in submersed leaves than in other leaf types. Analysis of leaf differentiation, the Rubisco/PEPC activity ratio, the PEPC activity, and the composition of primary photosynthates in the pulse–chase experiments revealed no evidence of the C4 effect in the submersed hydrophytes examined. The adaptation of hydatophytes to specific conditions of an aquatic environment was structurally manifested in the reduction (by a factor of 3–5) in the number of chloroplasts per 1 cm2 leaf area. This small number of chloroplasts was responsible for low photosynthetic rates in submersed leaves, although metabolic activities of individual chloroplasts were similar for all three hydrophyte groups. 相似文献
13.
Experimental elevation of [CO2] around C3 crops in the field has been shown to increase yields by suppressing the Rubisco oxygenase reaction and, in turn, photorespiration. Bioengineering a cyanobacterial carbon-concentrating mechanism (CCM) into C3 crop species provides a potential means of elevating [CO2] at Rubisco, thereby decreasing photorespiration and increasing photosynthetic efficiency and yield. The cyanobacterial CCM is an attractive alternative relative to other CCMs, because its features do not require anatomical changes to leaf tissue. However, the potential benefits of engineering the entire CCM into a C3 leaf are unexamined. Here, a CO2 and HCO3− diffusion-reaction model is developed to examine how components of the cyanobacterial CCM affect leaf light-saturated CO2 uptake (Asat) and to determine whether a different Rubisco isoform would perform better in a leaf with a cyanobacterial CCM. The results show that the addition of carboxysomes without other CCM components substantially decreases Asat and that the best first step is the addition of HCO3− transporters, as a single HCO3− transporter increased modeled Asat by 9%. Addition of all major CCM components increased Asat from 24 to 38 µmol m−2 s−1. Several Rubisco isoforms were compared in the model, and increasing ribulose bisphosphate regeneration rate will allow for further improvements by using a Rubisco isoform adapted to high [CO2]. Results from field studies that artificially raise [CO2] suggest that this 60% increase in Asat could result in a 36% to 60% increase in yield.C3 species include the major grain crops rice (Oryza sativa) and wheat (Triticum aestivum) and overall accounted for approximately 75% of all primary foodstuff production in 2012 (FAOSTAT, 2013). The yield of many crop species has been substantially improved through breeding and agronomy, but advancement in yield has substantially slowed in many of the major C3 crops in the last decade, suggesting that limits on yield improvement using these techniques are being reached and that other approaches are needed (Long and Ort, 2010; Ray et al., 2012).One largely unexploited approach would be to improve the efficiency of photosynthesis in these species (Zhu et al., 2010). The photosynthetic enzyme Rubisco catalyzes the reaction of CO2 with ribulose bisphosphate (RuBP), which eventually forms carbohydrates. However, Rubisco will also react with oxygen as the first step in photorespiration. This reaction is considered wasteful, since energy is consumed to recover RuBP and CO2 is lost in the process. In C3 plants at 25°C and current atmospheric [CO2], photorespiration results in an approximately 30% decrease in net carbon assimilation (Zhu et al., 2010). Thus, it is a large inefficiency in carbon uptake and a target for improvement.Since CO2 and oxygen act competitively at Rubisco, photorespiration can be decreased by increasing [CO2] around Rubisco. That this will increase yield is demonstrated by the many studies that have artificially increased atmospheric [CO2] around C3 crops growing in the field (Kimball et al., 2002; Long et al., 2006b). Other photosynthetic organisms have evolved mechanisms to internally elevate [CO2] at Rubisco to decrease or eliminate photorespiration. Such carbon-concentrating mechanisms (CCMs) include C4 photosynthesis, as in maize (Zea mays), and the carboxysome and pyrenoid CCMs of single-celled cyanobacteria and algae. The C4 pathway requires the addition of the photosynthetic C4 dicarboxylate cycle and inner photosynthetic cells (i.e. bundle sheath), where Rubisco is localized. Converting a C3 crop to a C4 crop will require a coordination of changes in photosynthetic tissue differentiation and enzyme and transporter localization. In contrast, cyanobacteria achieve the same effect in a single cell by localizing Rubisco to specialized subcellular compartments called carboxysomes, so in theory they would require fewer changes. Carboxysomes are polyhedral bodies with a protein shell that encloses carbonic anhydrase (CA) and Rubisco packed in an ordered or semiordered array (Long et al., 2007; Yeates et al., 2011). Bicarbonate is actively transported from the environment into the cytosol of the cyanobacteria, and CO2 in the cytosol is actively hydrated to HCO3− using NADH. CO2 is also hydrated to HCO3−, which serves to increase the [CO2] gradient between the medium and cytosol, increasing CO2 flux, and also serves to refix CO2 that leaks from the carboxysome. Since the cytosol lacks CA and the plasma membrane has low permeability to HCO3−, a high cytosolic [HCO3−] far from equilibrium with [CO2] is achieved. Bicarbonate diffuses through the protein shell of the carboxysome, and since CA is localized to the inner side of the shell, it is rapidly converted to CO2, given the disequilibrium. The resulting high [CO2] around Rubisco inside the carboxysome accelerates carboxylation and competitively inhibits the oxygenation reaction (for review, see Price et al., 2008, 2011, 2013; Espie and Kimber, 2011).The C4 pathway has been well characterized, and several projects are attempting to engineer it into C3 plants with some success (Slewinski, 2013). However, a primary obstacle has been achieving the necessary two tissue types with the correct localization of the key enzymes (Covshoff and Hibberd, 2012). In this regard, cyanobacterial CCM may provide an attractive parallel approach to eliminating photorespiration in C3 plants for two reasons. First, the biochemical, structural, and genetic components of the CCM are well understood, in that all of the necessary proteins and their genes have been identified. Second, chloroplasts of higher plants evolved from a common ancestor with modern cyanobacteria (Raven and Allen, 2003) and, therefore, are structurally similar. Thus, engineering carboxysomes into the chloroplast could involve introducing an operon containing the genes associated with the cyanobacterial CCM into the chloroplast genome.There are, however, multiple proteins in the cyanobacterial CCM, and simultaneously transforming all of the required genes into an organism may be particularly challenging. Furthermore, some of the proteins appear to have a similar function, and others may be deleterious if introduced without the full apparatus. A random sequence of gene stacking would likely be inefficient, while the addition of some genes may simply be unnecessary. However, the biophysical reactions involved are well understood, and there are some measurements of the kinetics of the enzymatic reactions and transporters. With this information, a mathematical model can be created to simulate the CCM. Such a model will allow deduction of the minimal component set necessary for an improvement of leaf photosynthetic efficiency and identify a logical sequence of gene additions to deliver progressive improvements and avoid any lethal effects. This model will also be used to determine which aspects of the CCM exert the most control over the overall process of photosynthetic CO2 assimilation, information that can then be used to optimize the CCM, help identify the ideal set of components, or identify parameters that need to be more accurately measured in order to improve the effectiveness of the CCM model. Similar kinetic models have been applied to propose systems optimization of the Calvin cycle when plants are grown in elevated [CO2] (Zhu et al., 2007), of the whole C3 photosynthetic system (Zhu et al., 2013), C4 photosynthesis (Yu et al., 2014), and to determine the mechanistic basis of mesophyll conductance (Tholen and Zhu, 2011).This paper develops a kinetic model to determine the potential of the cyanobacterial CCM engineered into C3 crops for improving photosynthesis by determining the necessary components and estimating the potential improvement of CO2 assimilation rate and efficiency. There are four distinct features to the cyanobacterial CCM: (1) a carboxysome with internally localized Rubisco and CA; (2) active transport of HCO3− from the medium into the cyanobacterial cytosol (the equivalent compartment in higher plants is the stroma of the chloroplast); (3) active hydration of CO2 to HCO3− within the cytosol; and (4) the absence of CA in the cyanobacterial cytosol, in contrast to the higher plant stroma, which contains high activities of CA. Cyanobacteria have three HCO3− transporters and two CO2 hydration enzymes that have different kinetics and are induced under different conditions (Price et al., 2011). All of these enzymes were examined, giving a total of seven individual components (listed in Fig. 1A) to the full cyanobacterial CCM model. Because of the common ancestry between cyanobacteria and higher plant chloroplasts, the two have homologous membranes and compartments. Proteins were modeled within a C3 leaf using locations equivalent to those in cyanobacteria. That is, bicarbonate transporters were localized to the inner chloroplast membrane, and carboxysomes were localized to the stroma. The CO2 hydration enzymes are bound to the thylakoid and plasma membranes in cyanobacteria, but the reaction for both occurs within the cyanobacterial cytosol; therefore, in this model, hydration of CO2 by these enzymes was localized to the stroma. This model was used to determine the necessity of each of the features noted above, potential improvements in light-saturated CO2 uptake (Asat; irradiance of 1,800 µmol photons m−2 s−1) that would be achieved on incorporation of each feature in turn, a possible sequence of gene additions that would give an incremental improvement, and the key components of the CCM. The model was also used to test the value of using higher plant isoforms of Rubisco versus prokaryotic isoforms adapted to incorporation within the carboxysome. Sources of uncertainty in the model are also defined.Open in a separate windowFigure 1.Asat of leaf models with sequential addition of components of the cyanobacterial CCM. Each point represents a leaf that contains the component listed for that column and all of the components in the columns to the left of it. The columns represent the sequence of transformations that produce the fastest increase in Asat assuming that it is not possible to simultaneously add carboxysomes and remove stromal CA and that it is not desirable to add CO2 hydration enzymes before removal of stromal CA (A) and assuming that it is possible to simultaneously add carboxysomes and remove stromal CA (B). 相似文献
14.
The photosynthetic properties of a yellow lethal mutant, Oy/oy, and two yellow-green mutants of maize which are allelic (a homozygous recessive oy/oy and a heterozygous dominant Oy/+) were examined. Although Oy/oy had little or no chlorophyll or capacity for CO2 fixation compared to normal siblings, it had 28% as much ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) activity, and from 40% to near normal activities of C4 cycle enzymes.Both yellow-green mutants had only half as much chlorophyll per leaf area as normal green seedlings in greenhouse-grown plants in winter and spring. However, the absorbance of light by the mutants was relatively high, as their transmittance was only 5 to 8% greater than normal leaves. In winter-grown greenhouse plants, the activities of Rubisco and several C4 cycle enzymes in the mutants were unaffected and similar to those of normal seedlings on a leaf area basis. After allowing for small differences in leaf absorbance, the light response curves for photosynthesis in the mutants were similar on a leaf area basis but much higher on a chlorophyll basis than those of the normal seedlings. In spring-grown greenhouse plants the enzyme activities and photosynthesis rates were about 30% lower per leaf area in the yellow-green mutant leaves compared to the wild type. The maximum carboxylation efficiency (measured under low CO2 and 1000 mol quanta m-2 s-1) in the mutants and normal leaves was similar on a Rubisco protein basis. The results indicate that maize can undergo a 50% reduction in chlorophyll content without a corresponding reduction in enzymes of carbon assimilation, and still maintain a high capacity for photosynthesis.Abbreviations Chl
chlorophyll
- PEP
phosphoenolypruvate
- Rubisco
ribulose-1,5-bisphosphate carboxylase oxygenase
This research was supported by CSIRO and by USDA Competitive Grant 86-CRCR-1-2036. 相似文献
15.
Susanne von Caemmerer 《Planta》1989,178(4):463-474
A model of leaf, photosynthesis has been developed for C3–C4 intermediate species found in the generaPanicum, Moricandia, Parthenium andMollugo where no functional C4 pathway has been identified. Model assumptions are a functional C3 cycle in both mesophyll and bundle-sheath cells and that glycine formed in the mesophyll, as a consequence of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco, EC 4.1.1.39), diffuses to the bundle sheath, where most of the photorespiratory CO2 is released. The model describes the observed gas-exchange characteristics of these C3–C4 intermediates, such as low CO2-compensation points () at an O2 pressure of 200 mbar, a curvilinear response of to changing O2 pressures, and typical responses of CO2-assimilation rate to intercellular CO2 pressure. The model predicts that bundle-sheath CO2 concentration is highest at low mesophyll CO2 pressures and decreases as mesophyll CO2 pressure increases. A partitioning of 5–15% of the total leaf Rubisco into the bundle-sheath cells and a bundlesheath conductance similar to that proposed for C4 species best mimics the gas-exchange results. The model predicts C3-like carbon-isotope discrimination for photosynthesis at atmospheric levels of CO2, but at low CO2 pressures it predicts a higher discrimination than is typically found during C3 photosynthesis at lower CO2 pressures.Abbreviations and symbols PEP
phosphoenolpyruvate
- Rubisco
ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39)
- RuBP
ribulose-1,5-bisphosphate
-
p(CO2)
partial pressure of CO2
-
p(O2)
partial pressure of O2. See also p. 471 相似文献
16.
Net O2 evolution, gross CO2 uptake and net HCO
inf3
su–
uptake during steady-state photosynthesis were investigated by a recently developed mass-spectrometric technique for disequilibrium flux analysis with cells of the marine cyanobacterium Synechococcus PCC7002 grown at different CO2 concentrations. Regardless of the CO2 concentration during growth, all cells had the capacity to transport both CO2 and HCO
inf3
su–
; however, the activity of HCO
inf3
su–
transport was more than twofold higher than CO2 transport even in cyanobacteria grown at high concentration of inorganic carbon (Ci = CO2 + HCO
inf3
su–
). In low-Ci cells, the affinities of CO2 and HCO
inf3
su–
transport for their substrates were about 5 (CO2 uptake) and 10 (HCO
inf3
su–
uptake) times higher than in high-Ci cells, while air-grown cells formed an intermediate state. For the same cells, the intracellular accumulated Ci pool reached 18, 32 and 55 mM in high-Ci, air-grown and low-Ci cells, respectively, when measured at 1 mM external Ci. Photosynthetic O2 evolution, maximal CO2 and HCO
inf3
su–
transport activities, and consequently their relative contribution to photosynthesis, were largely unaffected by the CO2 provided during growth. When the cells were adapted to freshwater medium, results similar to those for artificial seawater were obtained for all CO2 concentrations. Transport studies with high-Ci cells revealed that CO2 and HCO
inf3
su–
uptake were equally inhibited when CO2 fixation was reduced by the addition of glycolaldehyde. In contrast, in low-Ci cells steady-state CO2 transport was preferably reduced by the same inhibitor. The inhibitor of carbonic anhydrase ethoxyzolamide inhibited both CO2 and HCO
inf3
su–
uptake as well as O2 evolution in both cell types. In high-Ci cells, the degree of inhibition was similar for HCO
inf3
su–
transport and O2 evolution with 50% inhibition occurring at around 1 mM ethoxyzolamide. However, the uptake of CO2 was much more sensitive to the inhibitor than HCO
inf3
su–
transport, with an apparent I50 value of around 250 M ethoxyzolamide for CO2 uptake. The implications of our results are discussed with respect to Ci utilisation in the marine Synechococcus strain.Abbreviations Chl
chlorophyll
- Ci
inorganic carbon (CO2 + HCO
inf3
su–
)
- CA
carbonic anhydrase
- CCM
CO2-concentrating mechanism
- EZA
ethoxyzolamide
- GA
glycolaldehyde
- K1/2
concentration required for half-maximal response
- Rubisco
ribulose-1,5,-bisphosphate carboxylase-oxygenase
D.S. is a recipient of a research fellowship from the Deutsche Forschungsgemeinschaft (D.F.G.). In addition, we are grateful to Donald A. Bryant, Department of Molecular and Cell Biology and Center of Biomolecular Structure Function, Pennsylvania State University, USA, for sending us the wild-type strain of Synechococcus PCC7002. 相似文献
17.
Summary A mutant strain of Anabaena variabilis, strain SA-1 that supported growth of wheat plants in a hydroponic co-culture in nitrogen (N) free medium also produced enough oxygen (O2) to support root respiration. The steady-state concentration of net O2 in the co-culture was dependent on incident light intensity. At an incident photosynthetic photoflux (PPF) of 1000 mol photons·m–2·s–1, net O2 evolution by the co-culture in the root zone reached a maximum value of about 220 mol O2 evolved·h–1·mg chlorophyll (Chl)–1. The O2 concentration in the rhizosphere of the co-culture stayed above the ambient air level. O2 uptake in the dark by strain SA-1-supplemented wheat roots washed free of cyanobacterium was higher than the root respiration of nitrate-grown plants. Nitrate-grown plants required aeration for maximum growth while the wheat-cyanobacterial co-culture can be cultured without aeration. These results show that O2 produced by strain SA-1 can be used to supply the O2 needs for root respiration of wheat. Respiration reduced net O2 evolution by the mutant SA-1, decreasing the partial pressure of O2 at the sites of cyanobacterial attachment to the roots. This led to an increase in the specific activity of nitrogenase of the co-culture at the high light intensities used to support wheat growth. This activity of about 30 mol ethylene produced·mg Chl–1·h–1 was three-fold higher than the activities obtained with the free-living strain SA-1 assayed at the same light intensity. In the co-culture, ammonia produced by the mutant strain SA-1 was not detectable. The NH
inf4
sup+
produced by strain SA-1 was used by the wheat plants and, under these conditions, the total N content of the plants reached as high as 85% of the total N content of nitrate-grown plants. In the co-culture system the metabolism of wheat and the cyanobacterium complemented each other, leading to higher plant growth in N-free medium.
Offprint requests to: M. Gunasekaran 相似文献
18.
The light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was studied in two species: Phaseolus vulgaris L., which has high levels of the inhibitor of Rubisco activity, carboxyarabinitol 1-phosphate (CA1P), in the dark, and Chenopodium album L., which has little CA1P. In both species, the ratio of initial to fully-activated Rubisco activity declined by 40–50% within 60 min of a reduction in light from high a photosynthetic photon flux density (PPFD; >700 mol · m–2 · s–1) to a low PPFD (65 ± 15 mol · m–2 · s–1) or to darkness, indicating that decarbamylation of Rubisco is substantially involved in the initial regulatory response of Rubisco to a reduction in PPFD, even in species with potentially extensive CA1P inhibition. Total Rubisco activity was unaffected by PPFD in C. album, and prolonged exposure (2–6 h) to low light or darkness was accompanied by a slow decline in the activity ratio of this species. This indicates that the carbamylation state of Rubisco from C. album gradually declines for hours after the large initial drop in the first 60 min following light reduction. In P. vulgaris, the total activity of Rubisco declined by 10–30% within 1 h after a reduction in PPFD to below 100 mol · m–2 · s–1, indicating CA1P-binding contributes significantly to the reduction of Rubisco capacity during this period, but to a lesser extent than decarbamylation. With continued exposure of P. vulgaris leaves to very low PPFDs (< 30 mol · m–2 · s–1), the total activity of Rubisco declined steadily so that after 6–6.5 h of exposure to very low light or darkness, it was only 10–20% of the high-light value. These results indicate that while decarbamylation is more prominent in the initial regulatory response of Rubisco to a reduction in PPFD in P. vulgaris, binding of CA1P increases over time and after a few hours dominates the regulation of Rubisco activity in darkness and at very low PPFDs.Abbreviations CA1P
2-carboxyarabinitol 1-phosphate
- CABP
2-carboxyarabinitol 1,5-bisphosphate
- kcat
substrate-saturated turnover rate of fully carbamylated enzyme
- PPFD
photosynthetically active photon flux density (400–700 nm)
- Rubisco
ribulose-1,5-bisphosphate carboxylase/oxygenase
- RuBP
ribulose-1,5-bisphosphate 相似文献
19.
Shinya Wada Yuji Suzuki Daisuke Takagi Chikahiro Miyake Amane Makino 《Photosynthesis research》2018,137(3):431-441
Under CO2-limited conditions such as during stomatal closure, photorespiration is suggested to act as a sink for excess light energy and protect photosystem I (PSI) by oxidizing its reaction center chlorophyll P700. In this study, this issue was directly examined with rice (Oryza sativa L.) plants via genetic manipulation of the amount of Rubisco, which can be a limiting factor for photorespiration. At low [CO2] of 5 Pa that mimicked stomatal closure condition, the activity of photorespiration in transgenic plants with decreased Rubisco content (RBCS-antisense plants) markedly decreased, whereas the activity in transgenic plants with overproduction of Rubisco (RBCS-sense plants) was similar to that in wild-type plants. Oxidation of P700 was enhanced at [CO2] of 5 Pa in wild-type and RBCS-sense plants. PSI was not damaged by excess light stress induced by repetitive saturated pulse-light (rSP) in the presence of strong steady-state light. On the other hand, P700 was strongly reduced in RBCS-antisense plants at [CO2] of 5 Pa. PSI was also damaged by rSP illumination. These results indicate that oxidation of P700 and the robustness of PSI against excess light stress are hampered by the decreased activity of photorespiration as a result of genetic manipulation of Rubisco content. It is also suggested that overproduction of Rubisco does not enhance photorespiration as well as CO2 assimilation probably due to partial deactivation of Rubisco. 相似文献
20.
Phosphorus-deficient spinach plants were grown by transferring them to nutrient solutions without PO4. Photosynthetic rates were measured at a range of intercellular CO2 partial pressures from 50–500 bar and then the leaves were freeze-clamped in situ to measure ribulose bisphosphate carboxylase (Rubisco) activity and metabolite concentrations. Compared with control leaves, deficient leaves had significantly lower photosynthetic rates, percentage activation of Rubisco, and amounts of ribulose bisphosphate and 3-phosphoglycerate at all CO2 partial pressures. After feeding 10 mM PO4 to the petioles of detached deficient leaves, all these measurements increased within 2 hours. At atmospheric CO2 partial pressure the photosynthetic rate was stimulated in 19 mbar O2 compared with 200 mbar. At higher CO2 partial pressures this stimulation was less but the percentage stimulation in deficient leaves was no different from controls in either CO2 partial pressure. It was concluded that phosphorus deficiency affects both Rubisco activity and the capacity for ribulose bisphosphate regeneration, and possible causes are discussed.Abbreviations A
CO2 assimilation rate
- Ci
intercellular CO2 partial pressure
- PGA
3-phosphoglycerate
- RuP2
ribulose 1,5-bisphosphate
- Rubisco
RuP2 carboxylase/oxygenase 相似文献