The Effect of Leaf Temperature and Photorespiratory Conditions on Export of Sugars during Steady-State Photosynthesis in Salvia splendens.

Export and photosynthesis in leaves of Salvia splendens were measured concurrently during steady-state 14CO2 labeling conditions. Under ambient CO2 and O2 conditions, photosynthesis and export rates were similar at 15 and 25[deg]C, but both declined as leaf temperature was raised from 25 to 40[deg]C. Suppressing photorespiration between 15 and 40[deg]C by manipulating CO2 and O2 levels resulted in higher rates of leaf photosynthesis, total sugar synthesis, and export. There was a linear relationship between the rate of photosynthesis and the rate of export between 15 and 35[deg]C. At these temperatures, 60 to 80% of the carbon fixed was readily exported with sucrose, raffinose, and stachyose, which together constituted over 90% of phloem mobile assimilates. Above 35[deg]C, however, export during photosynthesis was inhibited both in photorespiratory conditions, which inhibited photosynthesis, and in nonphotorespiratory conditions, which did not inhibit photosynthesis. Sucrose and raffinose but not stachyose accumulated in the leaf at 40[deg]C. When leaves were preincubated at 40[deg]C and then cooled to 35[deg]C, export recovered more slowly than photosynthesis. These data are consistent with the view that impairment of export processes, rather than photosynthetic processes associated with light trapping, carbon reduction, and sucrose synthesis, accounted for the marked reduction in export between 35 and 40[deg]C. Taken together, the data indicated that temperature changes between 15 and 40[deg]C had two effects on photosynthesis and concurrent export. At all temperatures, suppressing photorespiration increased both photosynthesis and export, but above 35[deg]C, export processes were more directly inhibited independent of changes in photorespiration and photosynthesis.

Estimating photosynthesis and concurrent export rates in C3 and C4 species at ambient and elevated CO21,2

The ability of 21 C3 and C4 monocot and dicot species to rapidly export newly fixed C in the light at both ambient and enriched CO2 levels was compared. Photosynthesis and concurrent export rates were estimated during isotopic equilibrium of the transport sugars using a steady-state 14CO2-labeling procedure. At ambient CO2 photosynthesis and export rates for C3 species were 5 to 15 and 1 to 10 &mgr;mol C m-2 s-1, respectively, and 20 to 30 and 15 to 22 &mgr;mol C m-2 s-1, respectively, for C4 species. A linear regression plot of export on photosynthesis rate of all species had a correlation coefficient of 0.87. When concurrent export was expressed as a percentage of photosynthesis, several C3 dicots that produced transport sugars other than Suc had high efflux rates relative to photosynthesis, comparable to those of C4 species. At high CO2 photosynthetic and export rates were only slightly altered in C4 species, and photosynthesis increased but export rates did not in all C3 species. The C3 species that had high efflux rates relative to photosynthesis at ambient CO2 exported at rates comparable to those of C4 species on both an absolute basis and as a percentage of photosynthesis. At ambient CO2 there were strong linear relationships between photosynthesis, sugar synthesis, and concurrent export. However, at high CO2 the relationships between photosynthesis and export rate and between sugar synthesis and export rate were not as strong because sugars and starch were accumulated.

Photosynthesis and carbon partitioning in transgenic tobacco plants deficient in leaf cytosolic pyruvate kinase

Whole-plant diurnal C exchange analysis provided a noninvasive estimation of daily net C gain in transgenic tobacco (Nicotiana tabacum L.) plants deficient in leaf cytosolic pyruvate kinase (PKc-). PKc- plants cultivated under a low light intensity (100 &mgr;mol m-2 s-1) were previously shown to exhibit markedly reduced root growth, as well as delayed shoot and flower development when compared with plants having wild-type levels of PKc (PKc+). PKc- and PKc+ source leaves showed a similar net C gain, photosynthesis over a range of light intensities, and a capacity to export newly fixed 14CO2 during photosynthesis. However, during growth under low light the nighttime, export of previously fixed 14CO2 by fully expanded PKc- leaves was 40% lower, whereas concurrent respiratory 14CO2 evolution was 40% higher than that of PKc+ leaves. This provides a rationale for the reduced root growth of the PKc- plants grown at low irradiance. Leaf photosynthetic and export characteristics in PKc- and PKc+ plants raised in a greenhouse during winter months resembled those of plants grown in chambers at low irradiance. The data suggest that PKc in source leaves has a critical role in regulating nighttime respiration particularly when the available pool of photoassimilates for export and leaf respiratory processes are low.

Net Carbon Gain and Growth of Bell Peppers, Capsicum annuum 'Cubico', Following Root Infection by Pythium aphanidermatum.

ABSTRACT The first characterization of alterations in whole-plant photosynthetic rate and carbon assimilation of bell peppers associated with infection by Pythium aphanidermatum is described. Relationships of root disease caused by P. aphanidermatum to whole-plant net carbon exchange rate (NCER), total carbon accumulation, dark respiration rates, water loss, and destructive growth parameters were quantified in vegetative, hydroponically grown pepper plants (Capsicum annuum 'Cubico'). Inoculated plants displayed lower whole-plant NCER. This translated into a loss of 28% in cumulative C gain during 7 days after inoculation and occurred before visible shoot symptoms developed. Leaf area and dry weight of shoots and roots were significantly decreased and the shoot/root ratio was higher in inoculated plants than in noninoculated plants. We propose that reduced NCER in inoculated plants was mainly due to restricted development of leaf area, because no differences in NCER and evapotranspiration were observed between control and inoculated plants when expressed based on leaf area and root dry mass, respectively. These findings indicate that Pythium infection did not affect the photosynthetic apparatus directly and that the reductions in photosynthesis and growth were not caused by inefficient water transport by diseased roots. These results enlarge on the understanding of physiological responses of host plants to early stages of root disease.

Sealed environment chamber for canopy light interception and trace hydrocarbon analyses.

Two sealed chambers were constructed, each measuring approximately 4.5 m x 3 m x 2.5 m (LxWxH). Heat exchangers and air handling components were integrated within the sealed environment. Construction materials were chosen to minimize off-gassing and oxidation. Acceptable materials included stainless steel, Teflon (TM), glass and Heresite (TM) or baked enamel coated metal parts. The glass-topped chambers have externally mounted microwave powered light sources providing minimum PAR at canopy level of 1000 micrometers m-2 s-1. Major gases (CO2, O2) were monitored. Other environmental variables relevant to plant production (humidity, temperature, nutrient solution) were monitored and controlled continuously. Typical environment control capability and system specifications are presented. The facility is described as a venue ideally suited to address specific research objectives in plant canopy light interception, such as the roles of novel microwave powered overhead and inner-canopy light sources for dense plant canopies. In addition, control of recycled hydroponic nutrient solutions and analysis of trace atmospheric hydrocarbons in the context of sealed environment life support can be concurrently monitored.

Plant responses to short- and long-term exposures to high carbon dioxide levels in closed environments.

When higher plants are exposed to elevated levels of CO2 for both short- and long-term periods photosynthetic C-gain and photoassimilate export from leaves are generally increased. Water use efficiency is increased on a leaf area basis. During long-term exposures, photosynthesis rates on leaf and whole plants bases are altered in a species specific manner. The most common pattern in C3 plants is an enhanced rate of whole plant photosynthesis in a well irradiated canopy. Nevertheless, in some herbaceous species prolonged exposure to high CO2 results in remobilization of nitrogenous reserves (i.e., leaf protein degradation) and reduced rates of mature leaf photosynthesis when assayed at ambient CO2 and O2 levels. Both short- and long-term exposures to those CO2 levels (i.e., 100 to 2,000 microliter l-1) which modify photosynthesis and export, also modify both endogenous ethylene gas (C2H4) release, and substrate, 1-aminocyclopropane-1-carboxylic acid (ACC), saturated C2H4 release rates from irradiated leaves. Photosynthetically active canopy leaves contribute most of the C2H4 released from the canopy. Prolonged growth at high CO2 results in a persistent increase in the rate of endogenous C2H4 release from leaves which can, only in part, be attributed to the increase of the endogenous pools of C2H4 pathway intermediates (e.g., methionine, M-ACC, and ACC). The capacity for increasing the rate of C2H4 release in response to short-term exposures to varying CO2 levels does not decline after prolonged growth at high CO2. When leaves, whole plants, and model canopies of tomato plants are exposed to exogenous C2H4 a reduction in the rate of photosynthesis can, in each case, be attributed to the classical effects of C2H4 on plant development and morphology. The effect of C2H4 on CO2 gas exchange of plant canopies is shown to be dependent on the canopy leaf area index.

Regulating plant/insect interactions using CO2 enrichment in model ecosystems.

The greenhouse environment is a challenging artificial ecosystem in which it is possible to study selected plant/insect interaction in a controlled environment. Due to a combination of "direct" and "indirect" effects of CO2 enrichment on plant photosynthesis and plant development, canopy productivity is generally increased. In this paper, we discuss the effects of daytime and nighttime CO2 enrichment protocols on gas exchange of pepper plants (Capsicum annuum L, cv Cubico) grown in controlled environments. In addition, we present the effects of thrips, a common Insect pest, on the photosynthetic and respiratory activity of these plant canopies. Carbon dioxide has diverse effects on the physiology and mortality of insects. However, our data indicate that thrips and whiteflies, at least, are not killed "directly" by CO2 levels used to enhance photosynthesis and plant growth. Together the data suggest that the insect population is affected "indirectly" by CO2 and that the primary effect of CO2 is via its effects on plant metabolism.

A Study of Formate Production and Oxidation in Leaf Peroxisomes during Photorespiration.

When glycolate was metabolized in peroxisomes isolated from leaves of spinach beet (Beta vulgaris L., var. vulgaris) formate was produced. Although the reaction mixture contained glutamate to facilitate conversion of glycolate to glycine, the rate at which H(2)O(2) became "available" during the oxidation of [1-(14)C]glycolate was sufficient to account for the breakdown of the intermediate [1-(14)C]glyoxylate to formate (C(1) unit) and (14)CO(2). Under aerobic conditions formate production closely paralleled (14)CO(2) release from [1-(14)C]glycolate which was optimal between pH 8.0 and pH 9.0 and was increased 3-fold when the temperature was raised from 25 to 35 C, or when the rate of H(2)O(2) production was increased artificially by addition of an active preparation of fungal glucose oxidase.When [(14)C]formate was added to these preparations it was oxidized directly to (14)CO(2) by the peroxidatic action of peroxisomal catalase; however, the breakdown of formate was slow relative to the rate of formate production. For example, when [(14)C]formate was generated from [2-(14)C]glycolate it was not readily oxidized to (14)CO(2) in these organelles. Because the activity of formate-NAD(+) dehydrogenase in cell-free leaf extracts was low compared with that of formyl tetrahydrofolate synthetase it is suggested that most of the formate produced during glycolate oxidation could be metabolized via the one carbon pool and not oxidized directly to CO(2).At 25 C the rate of release of (14)CO(2) from [2-(14)C]glycolate in leaf discs was 40 to 50% of the rate from [1-(14)C]glycolate. Isonicotinyl hydrazide inhibited (14)CO(2) release from both [1-(14)C]- and [2-(14)C]glycolate; but this inhibitor was more effective in blocking (14)CO(2) release from [2-(14)C]glycolate. It is argued that the oxidation of the methylene carbon group of glycolate does not occur as a direct consequence of formate (C(1) unit) breakdown, but is a product of the further metabolism of formate and glycine, possibly, via serine.

Enhancement of Ethylene Release from Leaf Tissue during Glycolate Decarboxylation : A Possible Role for Photorespiration.

When leaf discs of Xanthium strumarium L. and Salvia splendens L. are incubated in sealed flasks in the light, more C(2)H(4) gas is released in the presence of added CO(2) (30-200 millimolar NaHCO(3)) than without CO(2). In Salvia, the maximum rate of C(2)H(4) release occurs when sufficient CO(2) (above 125 millimolar NaHCO(3)) is added to saturate photosynthesis confirming previous studies. The maximum rate of C(2)H(4) release from illuminated discs is similar to the rate in the dark with or without CO(2) in both species. Glycolate enhances a CO(2)-dependent C(2)H(4) evolution from illuminated leaf discs. However, the maximum rate of C(2)H(4) release with glycolate is the same as that observed with saturating CO(2). When photosynthesis is inhibited by darkness or by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, glycolate has no effect.Studies with [2,3-(14)C]-1-aminocyclopropane-1-carboxylic acid (ACC) show that the pattern of C(2)H(4) release and the specific activity of the (14)C(2)H(4) in the presence and absence of glycolate is similar to that described above, indicating that glycolate does not alter uptake of the exogenously supplied precursor (ACC) or stimulate C(2)H(4) release from an endogenous source at appreciable rates. Glycolate oxidase in vitro generates H(2)O(2) which stimulates a slow breakdown of ACC to C(2)H(4), but since exogenous glycolate is oxidized to CO(2) in both the light and the dark it is argued that the glycolate-dependent increase in C(2)H(4) release from illuminated leaf discs is not mediated directly by the action of enzymes of glycolate catabolism. The effects of glycolate and CO(2) are not easily explained by changes in stomatal resistance. The data support the view that glycolate decarboxylation at subsaturating levels of CO(2) in the light stimulates C(2)H(4) release by raising the CO(2) level in the tissue.

Glyoxylate decarboxylation during photorespiration.

At 25° C under aerobic conditions with or without gluamate 10% of the [1-(14)C]glycollate oxidised in spinach leaf peroxisomes was released as (14)CO2. Without glutamate only 5% of the glycollate was converted to glycine, but with it over 80% of the glycollate was metabolised to glycine. CO2 release was probably not due to glycine breakdown in these preparations since glycine decarboxylase activity was not detected. Addition of either unlabelled glycine or isonicotinyl hydrazide (INH) did not reduce (14)CO2 release from either [1-(14)C]glycollate or [1-(14)C]glyoxylate. Furthermore, the amount of "available H2O2" (Grodzinski and Butt, 1976) was sufficient to account for all of the CO2 release by breakdown of glyoxylate. Peroxisomal glycollate metabolism was unaffected by light and isolated leaf chloroplasts alone did not metabolise glycollate. However, in a mixture of peroxisomes and illuminated chloroplasts the rate of glycollate decarboxylation increased three fold while glycine synthesis was reduced by 40%. Although it was not possible to measure "available H2O2" directly, the data are best explained by glyoxylate decarboxylation. Catalase reduced CO2 release and enhanced glycine synthesis. In addition, when a model system in which an active preparation of purified glucose oxidase generating H2O2 at a known rate was used to replace the chloroplasts, similar rates of (14)CO2 release and [(14)C]glycine synthesis from [1-(14)C]glycollate were measured. It is argued that in vivo glyoxylate metabolism in leaf peroxisomes is a key branch point of the glycollate pathway and that a portion of the photorespired CO2 arises during glyoxylate decarboxylation under the action of H2O2. The possibility that peroxisomal catalase exerts a peroxidative function during this process is discussed.

The effect of osmotic stress on the oxidation of glycolate by the blue-green alga Anacystis nidulans.

Anacystis nidulans Richt. was shown to assimilate glycolic acid, and uptake was light-stimulated. In the dark 90% of the glycolate taken up was oxidised to CO2. Both light and dark uptake was completely inhibited by α-hydroxysulphonates but was unaffected by isonicotinyl hydrazide. 3-(3,4-Dichlorophenyl)-1,1-dimethyl urea (DCMU) reduced the rate of light uptake but not to the uptake level of the dark control. Subjecting the algal cells to osmotic stress by incubation in 0.6 M mannitol for 1 h, which reduces the photosynthetic activity of this alga, causes an 80% reduction in both light and dark glycolate uptake although the uptake of glyoxylate, formate, acetate, and glycine is not markedly affected. Osmotic stress had no effect on the uptake and metabolism of glycolate by Anabaena flos-aquae (Lyngbye) Bred. and Oscillatoria sp. The activity of glycolate dehydrogenase in Anacystis was also reduced by osmotic shock while the activity of other enzymes was unaffected.

Effect of Oxygen Concentration on C-Photoassimilate Transport from Leaves of Salvia splendens L.

Partitioning and transport of recently fixed photosynthate was examined following (14)CO(2) pulse-labeling of intact, attached leaves of Salvia splendens L. maintained in an atmosphere of 300 microliters per liter CO(2) and 20, 210, or 500 milliliters per liter O(2). Under conditions of increasing O(2) (210, 500 milliliters per liter), a smaller percentage of the recently fixed (14)C in the leaf was allocated to starch, whereas a greater percentage of the fixed (14)C appeared in amino acids, particularly serine. The increase in (14)C in amino acids was reflected in material exported from source leaves. A higher percentage of (14)C in serine, glycine, and glutamate was recovered in petiole extracts when source leaves were maintained under elevated O(2) levels. Although pool sizes of these amino acids were increased in both the leaves and petioles with increasing photorespiratory activity, no significant changes in either (14)C distribution or concentration of transport sugars (i.e. stachyose, sucrose, verbascose) were observed. The data indicate that, in addition to being recycled intracellularly into Calvin cycle intermediates, amino acids produced during photorespiration may also serve as transport metabolites, allowing the mobilization of both carbon and nitrogen from the leaf under conditions of limited photosynthesis.

Glycolic Acid Oxidase Activity in Cell-free Preparations of Blue-Green Algae.

Glycolic acid oxidase activity was detected in cell-free preparations of Anabaena flos-aquae and Oscillatoria sp. by the reduction of 2,6-dichlorophenolindophenol and by the formation of glyoxylate. Enzyme activity was localized in the 20,000 times gravity supernatant fraction, and optimal activity was obtained at pH 8.0. Activity was lost on storing the preparation at 4 C and could not be restored by addition of flavin mononucleotide.Oxidase activity of the supernatant fraction of Oscillatoria sp. was slightly stimulated by addition of flavin mononucleotide, but that of A. flos-aquae was unaffected by addition of this cofactor. The formation of glyoxylate by supernatant fractions of either alga was markedly inhibited by the addition of alpha-hydroxysulfonates.

Loss of photosynthetic activity in two blue-green algae as a result of osmotic stress.

A progressive loss of photosynthetic activity occurred when cells of Anacystis nidulans and Coccochloris peniocystis were incubated in increasing concentrations of osmotica from 0.2 to 0.7 M, and there was a release of pteridine from the cells proportional to the loss of photosynthetic activity.

Intracellular localization of glycolate dehydrogenase in a blue-green alga.

Glycolate dehydrogenase activity was detected in cell-free extracts of Oscillatoria sp. prepared by osmotic lysis of spheroplasts in 0.05 m potassium phosphate buffer, pH 7.5, containing 0.3 m mannitol. Most of the enzyme activity was found in a particulate fraction and localized in the photosynthetic lamellae after centrifugation in a discontinuous sucrose density gradient. Enzyme activity was detected in this fraction both in the presence and absence of the artificial electron acceptor 2,6-dichlorophenolindophenol (DPIP) and a low rate of O(2) uptake was detected in this lamellar fraction. Activity was lost from the lamellar fraction by repeated washing or by treatment with 0.005% Triton X-100 and the solubilized enzyme activity was DPIP-dependent. The data indicate that both glycolate dehydrogenase and its natural electron acceptor are bound to the photosynthetic lamellae in vivo. In contrast, catalase activity was found in the soluble cytoplasmic fraction.

Regulation of chloroplastic carbonic anhydrase : effect of magnesium.

It was previously reported that magnesium ion inhibited carbonic anhydrase (Bamberger and Avron 1975 Plant Physiol 56: 481-485). Studies with partially purified carbonic anhydrase from spinach (Spinacia oleracea L.) chloroplasts show that the effect was the result of the chloride counterion and not the magnesium ion. Enzyme activity was reduced 50% upon addition of 3 to 10 millimolar MgCl(2) or KCl while all additions of MgSO(4) between 0.3 and 10 millimolar were mildly stimulatory.

Acclimation to High CO(2) in Bean : Carbonic Anhydrase and Ribulose Bisphosphate Carboxylase.

Young bean plants (Phaseolus vulgaris L. cv Seafarer) grew faster in air enriched with CO(2) (1200 microliters per liter) than in ambient CO(2) (330 microliters per liter). However, by 7 days when increases in overall growth (dry weight, leaf area) were visible, there was a significant decline (about 25%) in the leaf mineral content (N, P, K, Ca, Mg) and a drop in the activity of two enzymes of carbon fixation, carbonic anhydrase and ribulose 1,5-bisphosphate (RuBP) carboxylase under high CO(2). Although the activity of neither enzyme was altered in young, expanding leaves during the acclimation period, in mature leaves the activity of carbonic anhydrase was reduced 95% compared with a decline of 50% in ambient CO(2). The drop in RuBP carboxylase was less extreme with 40% of the initial activity retained in the high CO(2) compared with 50% in the ambient atmosphere. While CO(2) enrichment might alter the flow of carbon into the glycolate pathway by modifying the activities of carbonic anhydrase or RuBP carboxylase, there is no early change in the ability of photosynthetic tissue to oxidize glycolate to CO(2).

Effects of Glycolate Pathway Intermediates on Glycine Decarboxylation and Serine Synthesis in Pea (Pisum sativum L.).

Glycine decarboxylation and serine synthesis were studied in pea (Pisum sativum L.) leaf discs, in metabolically active intact chloroplasts, and in mitochondria isolated both partially by differential centrifugation (i.e. ;crude') and by further purification on a Percoll gradient. Glycolate, glyoxylate, and formate reduced glycine decarboxylase activity ((14)CO(2) and NH(3) release) in the crude green-colored mitochondrial fractions, and in the leaf discs without markedly altering serine synthesis from [1-(14)C]glycine. Glycolate acted because it was converted to glyoxylate which behaves as a noncompetitive inhibitor (K(i) = 5.1 +/- 0.5 millimolar) on the mitochondrial glycine decarboxylation reaction in both crude and Percoll-purified mitochondria. In contrast, formate facilitates glycine to serine conversion by a route which does not involve glycine breakdown in the crude mitochondrial fraction and leaf discs. Formate does not alter the conversion of two molecules of glycine to one CO(2), one NH(3), and one serine molecule in the Percoll-purified mitochondria. In chloroplasts which were unable to break glycine down to CO(2) and NH(3), serine was labeled equally from [(14)C]formate and [1-(14)C]glycine. The maximum rate of serine synthesis observed in chloroplasts is similar to that in isolated metabolically active mitochondria. Formate does not appear to be able to substitute for the one-carbon unit produced during mitochondrial glycine breakdown but can facilitate serine synthesis from glycine in a chloroplast reaction which is probably a secondary one in vivo.

Hydrogen peroxide production and the release of carbon dioxide during glycollate oxidation in leaf peroxisomes.

The rate at which H2O2 becomes available during glycollate oxidation for further oxidation reactions, especially that of glyoxylate to formate and CO2, in peroxisomes from spinach-beet (Beta vulgaris L., var. vulgaris) leaves has been determined by measuring O2 uptake in the presence and absence of added catalase. The rates observed under air and pure O2 were sufficient to account for the (14)CO2 released from [l-(14)C]glycollate under these conditions; the two reactions showed similar characteristics. In the course of the reaction, a fall in catalase activity was observed concomitant with an increase in (14)CO2 release. There is no evidence that catalase was disproportionately lost from the peroxisomes during isolation, and it is argued that the CO2 release observed contributes to the photorespiratory CO2 loss in intact leaves.

The effect of temperature on glycollate decarboxylation in leaf peroxisomes.

[1-(14)C]glycollate was oxidised to(14)CO2 by peroxisomes isolated from leaves of spinach beet about 3 times as rapidly at 35°C as at 25°C; the rate was further increased with rise in temperature to a maximum at 55°C. These increases are shown to be mainly due to the increased H2O2 available to oxidise glyoxylate non-enzymically as a result of the higher temperature coefficient of glycollate oxidase activity relative to that of catalase. These results are compared with similar increases in the rate of(14)CO2 release between 25°C and 35°C when [1-(14)C]glycollate was supplied to leaf discs in light or darkness. The role of these reactions in accounting for the temperature effect on the release of photorespiratory CO2 is discussed.