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

Photosynthetic CO₂ 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 CO₂ 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 CO₂ 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 HCO₃⁻/Mg²⁺-saturated activity by 22%. However, RuBPCase for both stressed and control leaves were similar in K_cat (25 moles CO₂ per mole enzyme per second) and K_m for CO₂ (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.     

Dark/Light modulation of ribulose bisphosphate carboxylase activity in plants from different photosynthetic categories

Ribulose bisphosphate carboxylase/oxygenase (RuBPCase) from several plants had substantially greater activity in extracts from lightexposed leaves than dark leaves, even when the extracts were incubated in vitro with saturating HCO₃⁻ and Mg²⁺ concentrations. This occurred in Glycine max, Lycopersicon esculentum, Nicotiana tabacum, Panicum bisulcatum, and P. hylaeicum (C₃); P. maximum (C₄ phosphoenolpyruvate carboxykinase); P. milioides (C₃/C₄); and Bromelia pinguin and Ananas comosus (Crassulacean acid metabolism). Little or no difference between light and dark leaf extracts of RuBPCase was observed in Triticum aestivum (C₃); P. miliaceum (C₄ NAD malic enzyme); Zea mays and Sorghum bicolor (C₄ NADP malic enzyme); Moricandia arvensis (C₃/C₄); and Hydrilla verticillata (submersed aquatic macrophyte). It is concluded that, in many plants, especially Crassulacean acid metabolism and C₃ species, a large fraction of ribulose-1,5-bisphosphate carboxylase/oxygenase in the dark is in an inactivatable state that cannot respond to CO₂ and Mg²⁺ activation, but which can be converted to an activatable state upon exposure of the leaf to light.     

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 bar 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.Abbreviations and symbols A net CO₂ assimilation rate - C_a ambient CO₂ partial pressure - C_i intercellular CO₂ partial pressure - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat catalytic turnover rate per RuBPCase molecule - PFD photon flux density (400 to 700 nm on an area basis) - PGA 3-phosphoglyceric acid - P_i orthophosphate - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)     

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

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

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

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

     

Limiting Factors in Photosynthesis: V. Photochemical Energy Supply Colimits Photosynthesis at Low Values of Intercellular CO(2) Concentration

Although there is now some agreement with the view that the supply of photochemical energy may influence photosynthetic rate (P) at high CO₂ pressures, it is less clear whether this limitation extends to P at low CO₂. This was investigated by measuring P per area as a function of the intercellular CO₂ 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.     

Drought Stress and Elevated CO(2) Effects on Soybean Ribulose Bisphosphate Carboxylase Activity and Canopy Photosynthetic Rates

Soybean (Glycine max [L.] cv Bragg) was grown at 330 or 660 microliters CO₂ per liter in outdoor, controlled-environment chambers. When the plants were 50 days old, drought stress was imposed by gradually reducing irrigation each evening so that plants wilted earlier each succeeding day. On the ninth day, as the pots ran out of water CO₂ exchange rate (CER) decreased rapidly to near zero for the remainder of the day. Both CO₂-enrichment and drought stress reduced the total (HCO₃⁻/Mg²⁺-activated) extractable ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity, as expressed on a chlorophyll basis. In addition, drought stress when canopy CER values and leaf water potentials were lowest, reduced the initial (nonactivated) RuBPCase activity by 50% compared to the corresponding unstressed treatments. This suggests that moderate to severe drought stress reduces the in vivo activation state of RuBPCase, as well as lowers the total activity. It is hypothesized that stromal acidification under drought stress causes the lowered initial RuBPCase activities. The K_m(CO₂) values of activated RuBPCase from stressed and unstressed plants were similar; 15.0 and 12.6 micromolar, respectively. RuBP levels were 10 to 30% lower in drought stressed as compared to unstressed treatments. However, RuBP levels increased from near zero at night to around 150 to 200 nanomoles per milligram chlorophyll during the day, even as water potentials and canopy CERs decreased. This suggests that the rapid decline in canopy CER cannot be attributed to drought stress induced limitations in the RuBP regeneration capability. Thus, in soybean leaves, a nonstomatal limitation of leaf photosynthesis under drought stress conditions appears due, in part, to a reduction of the in vivo activity of RuBPCase. Because initial RuBPCase activities were not reduced as much as canopy CER values, this enzymic effect does not explain entirely the response of soybean photosynthesis to drought stress.     

Variation in Quantum Yield for CO(2) Uptake among C(3) and C(4) Plants

The quantum yield for CO₂ uptake was measured on a number of C₃ and C₄ monocot and dicot species. Under normal atmospheric conditions (330 microliters per liter CO₂, 21% O₂) and a leaf temperature of 30°C, the average quantum yields (moles CO₂ per einstein) were as follows: 0.052 for C₃ dicots, 0.053 for C₃ grasses, 0.053 for NAD-malic enzyme type C₄ dicots, 0.060 for NAD-malic enzyme type C₄ grasses, 0.064 for phosphoenolpyruvate carboxykinase type C₄ grasses, 0.061 for NADP-malic enzyme C₄ dicots, and 0.065 for NADP-malic enzyme type C₄ grasses. The quantum yield under normal atmospheric conditions was temperature dependent in C₃ species, but apparently not in C₄ species. Light and temperature conditions during growth appeared not to influence quantum yield. The significance of variation in the quantum yields of C₄ plants was discussed in terms of CO₂ leakage from the bundle sheath cells and suberization of apoplastic regions of the bundle sheath cells.     

Structural, Functional, and Evolutionary Analysis of Ribulose-1,5-Bisphosphate Carboxylase from the Chromophytic Alga Olisthodiscus luteus

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) was purified from the marine chromophyte Olisthodiscus luteus. This study represents the first extensive analysis of RuBPCase from a chromophytic plant species as well as from an organism where both subunits of the enzyme are encoded on the chloroplast genome. The size of the purified holoenzyme (17.9 Svedberg units, 588 kilodaltons) was determined by sedimentation analysis and the size of the subunits (55 kilodaltons, 15 kilodaltons) ascertained by analytical sodium dodecyl sulfate gel electrophoresis. This data predicts either an 8:9 or 8:8 ratio of the large to small subunits in the holoenzyme. Amino acid analyses demonstrate that the O. luteus RuBPCase large subunit is highly conserved and the small subunit much less so when compared with the chlorophytic plant peptides. The catalytic optima of pH and Mg²⁺ have been determined as well as the response of enzyme catalysis to temperature. The requirements of NaHCO₃ and Mg²⁺ for enzyme activation have also been analyzed. The Michaelis constants for the substrates of the carboxylation reaction (CO₂ and ribulose bisphosphate) were shown to be 45 and 48 micromolar, respectively. Competitive inhibition by oxygen of RuBPCase-catalyzed CO₂ fixation was also demonstrated. These data demonstrate that a high degree of RuBPCase conservation occurs among widely divergent photoautotrophs regardless of small subunit coding site.     

Photosynthetic and photorespiratory characteristics of flaveria species

The genus Flaveria shows evidence of evolution in the mechanism of photosynthesis as its 21 species include C₃, C₃-C₄, C₄-like, and C₄ plants. In this study, several physiological and biochemical parameters of photosynthesis and photorespiration were measured in 18 Flaveria species representing all the photosynthetic types. The 10 species classified as C₃-C₄ intermediates showed an inverse continuum in level of photorespiration and development of the C₄ syndrome. This ranges from F. sonorensis with relatively high apparent photorespiration and lacking C₄ photosynthesis to F. Among the intermediates, the photosynthetic CO₂ compensation points at 30°C and 1150 micromoles quanta per square meter per second varied from 9 to 29 microbars. The values for the three C₄-like species varied from 3 to 6 microbars, similar to those measured for the C₄ species. The activities of the photorespiratory enzymes glycolate oxidase, hydroxypyruvate reductase, and serine hydroxymethyltransferase decreased progressively from C₃ to C₃-C₄ to C₄-like and C₄ species. On the other hand, most intermediates had higher levels of phosphenolpyruvate carboxylase and NADP-malic enzyme than C₃ species, but generally lower activities compared to C₄-like and C₄ species. The levels of these C₄ enzymes are correlated with the degree of C₄ photosynthesis, based on the initial products of photosynthesis. Another indication of development of the C₄ syndrome in C₃-C₄ Flaveria species was their intermediate chlorophyll a/b ratios. The chlorophyll a/b ratios of the various Flaveria species are highly correlated with the degree of C₄ photosynthesis suggesting that the photochemical machinery is progressively altered during evolution in order to meet the specific energy requirements for operating the C₄ pathway. In the progression from C₃ to C₄ species in Flaveria, the CO₂ compensation point decreased more rapidly than did the decrease in O₂ inhibition of photosynthesis or the increase in the degree of C₄ photosynthesis. These results suggest that the reduction in photorespiration during evolution occurred initially by refixation of photorespired CO₂ and prior to substantive reduction in O₂ inhibition and development of the C₄ syndrome. However, further reduction in O₂ inhibition in some intermediates and C₄-like species is considered primarily due to the development of the C₄ syndrome. Thus, the evolution of C₃-C₄ intermediate photosynthesis likely occurred in response to environmental conditions which limit the intercellular CO₂ concentration first via refixation of photorespired CO₂, followed by development of the C₄ syndrome.     

Quantification and intracellular distribution of ribulose-1,5-bisphosphate carboxylase in Thiobacillus neapolitanus,as related to possible functions of carboxysomes

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) has been quantified by immunological methods in Thiobacillus neapolitanus cultivated under various growth conditions in the chemostat at a fixed dilution rate of 0.07 h^-1. RuBPCase was a major protein in T. neapolitanus accounting for a maximum of 17% of the total protein during CO₂ limitation and for a minimum of 4% during either ammonium- or thiosulfate limitation in the presence of 5% CO₂ (v/v) in the gasphase. The soluble RuBPCase (i.e. in the cytosol) and the particulate RuBPCase (i.e. in the carboxysomes) were shown to be immunologically identical. The intracellular distribution of RuBPCase protein between carboxysomes and cytosol was quantified by rocket immunoelectrophoresis. The particulate RuBPCase content, which correlated with the volume density of carboxysomes, was minimal during ammonium limitation (1.3% of the total protein) and maximal during CO₂ limitation (6.8% of the total protein). A protein storage function of carboxysomes is doubtful since nitrogen starvation did not result in degradation of particulate RuBPCase within 24 h. Proteolysis of RuBPCase was not detected. Carboxysomes, on the other hand, were degraded rapidly (50% within 1 h) after change-over from CO₂ limitation to thiosulfate limitation with excess CO₂. Particulate RuBPCase protein became soluble during this degradation of carboxysomes, but this did not result in an increase in soluble RuBPCase activity. Modification of RuBPCase resulting in a lower true specific activity was suggested to explain this phenomenon. The true specific activity was very similar for soluble and particulate RuBPCase during various steady state growth conditions (about 700 nmol/min·mg RuBPCase protein), with the exception of CO₂-limited growth when the true specific activity of the soluble RuBPCase was extremely low (260 nmol/min ·mg protein). When chemostat cultures of T. neapolitanus were exposed to different oxygen tensions, neither the intracellular distribution of RuBPCase nor the content of RuBPCase were affected. Short-term labelling experiments showed that during CO₂ limitation, when carboxysomes were most abundant, CO₂ is fixed via the Calvin cycle. The data are assessed in terms of possible functions of carboxysomes.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase - PEP phosphoenolpyruvate - RIE rocket immunoelectrophoresis - CIE crossed immunoelectrophoresis     

Km (CO2) values of ribulose-1,5-bisphosphate carboxylase in grasses of different C4 type

Statistical analysis of K_m (CO₂) values of ribulose-1,5-bisphosphate (RuBP) carboxylase from 35 C₄ grass species shows that the mean value for PEP-carboxykinase (PCK) type C₄ species (41.4±s.e. 2.2 μM CO₂) is significantly different from that of NAD-malic enzyme (NAD-ME) type species (55.3±3.1 μM CO₂) or NADP-malic enzyme (NADP-ME type species (52.5±s.e. 2.0μM CO₂). These C₄ type differences remain detectable within both the eu-panicoid and chloridoid grass subfamilies. By contrast, no between-subfamily differences were found within C₄ types. Variation in K_m (CO₂) values of RuBP carboxylase may be related to in vivo differences in CO₂ concentration at the enzyme site, mediated perhaps by differences in CO₂-leakiness of C₄ leaf ‘photosynthetic carbon reduction’ (PCR or ‘Kranz’) tissue.     

CO(2) Concentrating Mechanism of C(4) Photosynthesis: Permeability of Isolated Bundle Sheath Cells to Inorganic Carbon

Diffusion of inorganic carbon into isolated bundle sheath cells from a variety of C₄ species was characterized by coupling inward diffusion of CO₂ to photosynthetic carbon assimilation. The average permeability coefficient for CO₂ (P_CO2) for five representatives from the three decarboxylation types was approximately 20 micromoles per minute per milligram chlorophyll per millimolar, on a leaf chlorophyll basis. The average value for the NAD-ME species Panicum miliaceum (10 determinations) was 26 with a standard deviation of 6 micromoles per minute per milligram chlorophyll per millimolar, on a leaf chlorophyll basis. A P_CO2 of at least 500 micromoles per minute per milligram chlorophyll per millimolar was determined for cells isolated from the C₃ plant Xanthium strumarium. It is concluded that bundle sheath cells are one to two orders of magnitude less permeable to CO₂ than C₃ photosynthetic cells. These data also suggest that CO₂ diffusion in bundle sheath cells may be made up of two components, one involving an apoplastic path and the other a symplastic (plasmodesmatal) path, each contributing approximately equally.     

Regulation of oxaloacetate, aspartate, and malate formation in mesophyll protoplast extracts of three types of c(4) plants

The use of mesophyll protoplast extracts from various C₄ species has provided an effective method for studying light-and substrate-dependent formation of oxaloacetate, malate, and asparate at rates equivalent to whole leaf C₄ photosynthesis. Conditions regulating the formation of the C₄ acids were studied with protoplast extracts from Digitaria sanguinalis, an NADP-malic enzyme C₄ species, Eleusineindica, an NAD-malic enzyme C₄ species, and Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase C₄ species. Light-dependent induction of CO₂ fixation by the mesophyll extracts of all three species was relatively low without addition of exogenous substrates. Pyruvate, alanine and α-ketoglutarate, or 3-phosphoglycerate induced high rates of CO₂ fixation in the mesophyll extracts with oxaloacetate, malate, and aspartate being the primary products. In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C₄ mesophyll cells. Induction by pyruvate or alanine and α-ketoglutarate was light-dependent, whereas 3-phosphoglycerate-induced CO₂ fixation was not.     

Phosphoenolpyruvate carboxylase in Hydrilla plants with varying CO2 compensation points

Incubation of the submersed aquatic macrophyte, Hydrilla verticillata Royle, for up to 4 weeks in growth chambers under winter-like or summer-like conditions produced high (130 to 150 μl CO₂/1) and low (6 to 8 μl CO₂/l) CO₂ compensation points (Γ), respectively. The activities of both ribulose bisphosphate (RuBP) and phosphoenolpyruvate (PEP) carboxylases increased upon incubation but the major increase was in the activity of PEP carboxylase under the summer-like conditions. This reduced the ratio of RuBP/PEP carboxylases from 2.6 in high Γ plants to 0.2 in low Γ plants. These ratios resemble the values in terrestrial C₃ and C₄ species, respectively. Kinetic measurements of the PEP carboxylase activity in high and low Γ plants indicated the Vmax was up to 3-fold greater in the low Γ plants. The Km (HCO₃ ^?) values were 0.33 and 0.22 mM for the high and low Γ plants, respectively. The Km (PEP) values for the high and low Γ plants were 0.23 and 0.40 mM, respectively; and PEP exhibited cooperative effects. Estimated Km (Mg²⁺) values were 0.10 and 0.22 mM for the high and low Γ plants, respectively. Malate inhibited both PEP carboxylase types similarly. The enzyme from low Γ plants was protected by malate from heat inactivation to a greater extent than the enzyme from high Γ plants. The results indicated that C₄ acid inhibition and protection were not reliable methods to distinguish C₃ and C₄ PEP carboxylases. The PEP carboxylase from low Γ plants was inhibited more by NaCl than that from hight Γ plants. These analyses indicated that Hydrilla PEP carboxylases had intermediate characteristics between those of terrestrial C₃ and C₄ species with the low Γ enzyme being different from the high Γ enzyme, and closer to a C₄ type.     

Phosphoenolpyruvate carboxylase in Hydrilla plants with varying CO2 compensation points

Incubation of the submersed aquatic macrophyte, Hydrilla vertieillata Royle, for up to 4 weeks in growth chambers under winter-like or summer-like conditions produced high (130 to 150 μl CO₂/l) and low (6 to 8 μl CO₂/l) CO₂ compensation points (Γ), respectively. The activities of both ribulose bisphosphate (RuBP) and phosphoenolpyruvate (PEP) carboxylases increased upon incubation but the major increase was in the activity of PEP carboxylase under the summer-like conditions. This reduced the ratio of RuBP/PEP carboxylases from 2.6 in high Γ plants to 0.2 in low Γ plants. These ratios resemble the values in terrestrial C₃ and C₄ species, respectively. Kinetic measurements of the PEP carboxylase activity in high and low Γ plants indicated the Vmax was up to 3-fold greater in the low Γ plants. The Km (HCO₃ ^-) values were 0.33 and 0.22 mM for the high and low Γ plants, respectively. The Km (PEP) values for the high and low Γ plants were 0.23 and 0.40 mM, respectively; and PEP exhibited cooperative effects. Estimated Km (Mg²⁺) values were 0.10 and 0.22 mM for the high and low Γ plants, respectively. Malate inhibited both PEP carboxylase types similarly. The enzyme from low Γ plants was protected by malate from heat inactivation to a greater extent than the enzyme from high Γ plants. The results indicated that C₄ acid inhibition and protection were not reliable methods to distinguish C₃ and C₄ PEP carboxylases. The PEP carboxylase from low Γ plants was inhibited more by NaCl than that from high Γ plants. These analyses indicated that Hydrilla PEP carboxylases had intermediate characteristics between those of terrestrial C₃ and C₄ species with the low Γ enzyme being different from the high Γ enzyme, and closer to a C₄ type.     

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.     

Activity ratios of ribulose-1,5-bisphosphate carboxylase accurately reflect carbamylation ratios

Activity ratios and carbamylation ratios of ribulose-1,5-bisphosphate carboxylase (RuBPCase) were determined for leaves of Phaseolus vulgaris and Spinacia oleracea exposed to a variety of partial pressures of CO₂ and O₂ and photon flux densities (PFD). It was found that activity ratios accurately predicted carbamylation ratios except in extracts from leaves held in low PFD. In particular, it was confirmed that the loss of RuBPCase activity in low partial pressure of O₂ and high PFD results from reduced carbamylation. Activity ratios of RuBPCase were lower than carbamylation ratios for Phaseolus leaves sampled in low PFD, presumably because of the presence of 2-carboxyarabinitol 1-phosphate. Spinacia leaves sampled in darkness also exhibited lower activity ratios than carbamylation ratios indicating that this species may also have an RuBPCase inhibitor even though carboxyarabinitol 1-phosphate has not been detected in this species in the past.     

Environmental Effects on Photorespiration of C(3)-C(4) Species : I. Influence of CO(2) and O(2) during Growth on Photorespiratory Characteristics and Leaf Anatomy

The possibility of altering CO₂ exchange of C₃-C₄ species by growing them under various CO₂ and O₂ concentrations was examined. Growth under CO₂ concentrations of 100, 350, and 750 micromoles per mole had no significant effect on CO₂ exchange characteristics or leaf anatomy of Flaveria pringlei (C₃), Flaveria floridana (C₃-C₄), or Flaveria trinervia (C₄). Carboxylation efficiency and CO₂ compensation concentrations in leaves of F. floridana developed under the different CO₂ concentrations were intermediate to F. pringlei and F. trinervia. When grown for 12 days at an O₂ concentration of 20 millimoles per mole, apparent photosynthesis was strongly inhibited in Panicum milioides (C₃-C₄) and to a lesser degree in Panicum laxum (C₃). In P. milioides, acute starch buildup was observed microscopically in both mesophyll and bundle sheath cells. Even after only 4 days exposure to 20 millimoles per mole O₂, the presence of starch was more pronounced in leaf cross-sections of P. milioides compared to those at 100 and 210 millimoles per mole. Even though this observation suggests that P. milioides has a different response to low O₂ with respect to translocation of photosynthate or sink activity than C₃ species, the concentration of total available carbohydrate increased in shoots of all species by 33% or more when grown at low O₂. This accumulation occurred even though relative growth rates of Festuca arundinacea (C₃) and P. milioides grown for 4 days at 210 millimoles per mole O₂, were inhibited 83 and 37%, respectively, when compared to plants grown at 20 millimoles per mole O₂.     

C(3)-C(4) Intermediate Species in Alternanthera (Amaranthaceae) : Leaf Anatomy, CO(2) Compensation Point, Net CO(2) Exchange and Activities of Photosynthetic Enzymes

Two naturally occurring species of the genus Alternanthera, namely A. ficoides and A. tenella, were identified as C₃-C₄ intermediates based on leaf anatomy, photosynthetic CO₂ compensation point (Γ), O₂ response of г, light intensity response of г, and the activities of key enzymes of photosynthesis. A. ficoides and A. tenella exhibited a less distinct Kranz-like leaf anatomy with substantial accumulation of starch both in mesophyll and bundle sheath cells. Photosynthetic CO₂ compensation points of these two intermediate species at 29°C were much lower than in C₃ plants and ranged from 18 to 22 microliters per liter. Although A. ficoides and A. tenella exhibited similar intermediacy in г, the apparent photorespiratory component of O₂ inhibition in A. ficoides is lower than in A. tenella. The г progressively decreases from 35 microliters per liter at lowest light intensity to 18 microliters per liter at highest light intensity in A. tenella. It was, however, constant in A. ficoides at 20 to 25 microliters per liter between light intensities measured. The rates of net photosynthesis at 21% O₂ and 29°C by A. ficoides and A. tenella were 25 to 28 milligrams CO₂ per square decimeter per hour which are intermediate between values obtained for Tridax procumbens and A. pungens, C₃ and C₄ species, respectively. The activities of key enzymes of C₄ photosynthesis, phosphoenolpyruvate carboxylase, pyruvate Pi dikinase, NAD malic enzyme, NADP malic enzyme and phosphoenolpyruvate carboxykinase in the two intermediates, A. ficoides and A. tenella are very low or insignificant. Results indicated that the relatively low apparent photorespiratory component in these two species is presumably the basis for the C₃-C₄ intermediate photosynthesis.     

Photosynthetic Characteristics of the C(3)-C(4) Intermediate Parthenium hysterophorus

The weedy species Parthenium hysterophorus (Asteraceae) possesses a Kranz-like leaf anatomy. The bundle sheath cells are thick-walled and contain numerous granal chloroplasts, prominent mitochondria, and peroxisomes, all largely arranged in a centripetal position. Both mesophyll and bundle sheath chloroplasts accumulate starch. P. hysterophorus exhibits reduced photorespiration as indicated by a moderately low CO₂ compensation concentration (20-25 microliters per liter at 30°C and 21% O₂) and by a reduced sensitivity of net photosynthesis to 21% O₂. In contrast, the related C₃ species P. incanum and P. argentatum (guayule) lack Kranz anatomy, have higher CO₂ compensation concentrations (about 55 microliters per liter), and show a greater inhibition of photosynthesis by 21% O₂. Furthermore, in P. hysterophorus the CO₂ compensation concentration is relatively less sensitive to changes in O₂ concentrations and shows a biphasic response to changing O₂, with a transition point at about 11% O₂. Based on these results, P. hysterophorus is classified as a C₃-C₄ intermediate. The activities of diagnostic enzymes of C₄ photosynthesis in P. hysterophorus were very low, comparable to those observed in the C₃ species P. incanum (e.g. phosphoenolpyruvate carboxylase activity of 10-29 micromoles per milligram of chlorophyll per hour). Exposures of leaves of each species to ¹⁴CO₂ (for 8 seconds) in the light resulted in 3-phosphoglycerate and sugar phosphates being the predominant initial ¹⁴C products (77-84%), with ≤4% of the ¹⁴C-label in malate plus aspartate. These results indicate that in the C₃-C₄ intermediate P. hysterophorus, the reduction in leaf photorespiration cannot be attributed to C₄ photosynthesis.