Photorespiratory metabolism and immunogold localization of photorespiratory enzymes in leaves of C3 and C3-C4 intermediate species of Moricandia

Photorespiratory metabolism of the C₃-C₄ intermediate species Moricandia arvensis (L.) DC has been compared with that of the C₃ species, Moricandia moricandioides (Boiss.) Heywood. Assays of glycollate oxidase (EC 1.1.3.1), glyoxylate aminotransferases (EC 2.6.1.4, EC 2.6.1.45) and hydroxypyruvate reductase (EC 1.1.1.29) indicate that the capacity for flux through the photorespiratory cycle is similar in both species. Immunogold labelling with monospecific antibodies was used to investigate the cellular locations of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), glycollate oxidase, and glycine decarboxylase (EC 2.1.2.10) in leaves of the two species. Ribulose 1,5-bisphosphate carboxylase/oxygenase was confined to the stroma of chloroplasts and glycollate oxidase to the peroxisomes of all photosynthetic cells in leaves of both species. However, whereas glycine decarboxylase was present in the mitochondria of all photosynthetic cells in M. moricandioides, it was only found in the mitochondria of bundle-sheath cells in M. arvensis. We suggest that localized decarboxylation of glycine in the leaves of M. arvensis will lead to improved recapture of photorespired CO₂ and hence a lower rate of photorespiration.Abbreviations kDa kilodalton - RuBP ribulose-1,5-bisphosphate     

Incorporation of 14CO2 into C4 acids by leaves of C3-C4 intermediate and C3 species of Moricandia and Panicum at the CO2 compensation concentration

Comparative ¹⁴CO₂ pulse-¹²CO₂ chase studies performed at CO₂ compensation ()-versus air-concentrations of CO₂ demonstrated a four-to eightfold increase in assimilation of ¹⁴CO₂ into the C₄ acids malate and aspartate by leaves of the C₃-C₄ intermediate species Panicum milioides Nees ex Trin., P. decipiens Nees ex Trin., Moricandia arvensis (L.) DC., and M. spinosa Pomel at . Specifically, the distribution of ¹⁴C in malate and aspartate following a 10-s pulse with ¹⁴CO₂ increases from 2% to 17% (P. milioides) and 4% to 16% (M. arvensis) when leaves are illuminated at the CO₂ compensation concentration (20 l CO₂/l, 21% O₂) versus air (340 l CO₂/l, 21% O₂). Chasing recently incorporated ¹⁴C for up to 5 min with ¹²CO₂ failed to show any substantial turnover of label in the C₄ acids or in carbon-4 of malate. The C₄-acid labeling patterns of leaves of the closely related C₃ species, P. laxum Sw. and M. moricandioides (Boiss.) Heywood, were found to be relatively unresponsive to changes in pCO₂ from air to . These data demonstrate that the C₃-C₄ intermediate species of Panicum and Moricandia possess an inherently greater capacity for CO₂ assimilation via phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) at the CO₂ compensation concentration than closely related C₃ species. However, even at , CO₂ fixation by PEP carboxylase is minor compared to that via ribulosebisphosphate carboxylase (EC 4.1.1.39) and the C₃ cycle, and it is, therefore, unlikely to contribute in a major way to the mechanism(s) facilitating reduced photorespiration in the C₃-C₄ intermediate species of Panicum and Moricandia.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - PEP phosphoenolpyruvate - CO₂ compensation concentration - 3PGA 3-phosphoglycerate - SuP sugar monophosphates - SuP₂ sugar bisphosphates Published as Paper No. 8249, Journal Series, Nebraska Agricultural Research Division     

Co-function of C3-and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species

The potential for C₄ photosynthesis was investigated in five C₃-C₄ intermediate species, one C₃ species, and one C₄ species in the genus Flaveria, using ¹⁴CO₂ pulse-¹²CO₂ chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating ¹⁴CO₂ into the C₄ acids malate and aspartate, following an 8-s pulse. The proportion of ¹⁴C label in these C₄ products ranged from 50–55% to 20–26% in the C₃-C₄ intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, ¹⁴CO₂ into aspartate as into malate. Generally, about 5–15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C₄ photosynthesis among the intermediate species based on the apparent rate of conversion of ¹⁴C label from the C₄ cycle to the C₃ cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C₄-cycle products and an increase in percentage label in C₃-cycle products during chase periods with ¹²CO₂, although the rate of change was slower than in the C₄ species, F. palmeri. In these C₃-C₄ intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C₃-C₄ intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C₄-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C₄ and C₃ cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C₃ species, 7–18% of the initial label was in malate+aspartate. However, only 40–50% of this label was in the C-4 position, indicating C₄-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O₂, the absorbed quantum yields for CO₂ uptake (in mol CO₂·[mol quanta]^-1) averaged 0.053 in F. cronquistii (C₃), 0.051 in F. trinervia (Spreng.) Mohr (C₄), 0.052 in F. ramosissima (C₃-C₄), 0.051 in F. anomala (C₃-C₄), 0.050 in F. linearis (C₃-C₄), 0.046 in F. floridana (C₃-C₄), and 0.044 in F. pubescens (C₃-C₄). In 2% O₂ an enhancement of the quantum yield was observed in all of the C₃-C₄ intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O₂ were intermediate in value to the C₃ and C₄ species, indicating a co-function of the C₃ and C₄ cycles in CO₂ assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O₂ presumably reflect an ineffcient transfer of carbon from the C₄ to the C₃ cycle. The response of the quantum yield to four increasing O₂ concentrations (2–35%) showed lower levels of O₂ inhibition in the C₃-C₄ intermediate F. ramosissima, relative to the C₃ species. This indicates that the co-function of the C₃ and C₄ cycles in this intermediate species leads to an increased CO₂ concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O₂.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate     

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.     

Cellular expression pattern of the glycine decarboxylase P protein in leaves of an intergeneric hybrid between the C3-C4 intermediate species Moricandia nitens and the C3 species Brassica napus

 An intergeneric hybrid plant was produced between the C₃-C₄ intermediate species Moricandia nitens and the C₃ species Brassica napus by sexual hybridization and in vitro embryo rescue. The hybrid nature of the plant was apparent in its morphology and flower pigmentation and was confirmed by leaf isozyme patterns. The overall plant morphology and the shape and thickness of leaves of the hybrid plant were intermediate between those of the parent species. However, the bundle-sheath cells of the hybrid resembled those of the C₃ parent and lacked the organelle development of the C₃-C₄ intermediate parent. Immunogold labelling for the presence of the P subunit of the mitochondrial glycine decarboxylase complex revealed a very similar labelling density on mitochondria in bundle-sheath and mesophyll cells in B. napus, while in  M. nitens the P subunit was only detectable in bundle sheath cells. In the hybrid the labelling density on mesophyll cell mitochondria was almost half of that on the bundle-sheath mitochondria. The CO₂ compensation point of the hybrid was significantly less than that of the C₃ parent but was not as low, nor as responsive to changes in light intensity, as for the C₃-C₄ parent. Received: 23 October 1997 / Accepted: 28 November 1997     

Coordination of the cell-specific distribution of the four subunits of glycine decarboxylase and of serine hydroxymethyltransferase in leaves of C3-C4 intermediate species from different genera

The cell-specific distribution of the four subunit proteins (P, L, T and H) of glycine decarboxylase (GDC) and of serine hydroxymethyltransferase (SHMT) has been studied in the leaves of C₃-C₄ intermediate and C₄ species of three genera (Flaveria, Moricandia and Panicum) using immunogold localization. Antibodies raised against these proteins from pea leaf mitochondria were used to probe Western blots of total leaf proteins of F. linearis Lag., M. arvensis (L.) DC and P. milioides Nees ex Trin. (C₃-C₄), and F. trinervia (Spring.) Mohr and P. miliaceum (L.) (C₄). For all species, each antibody recognised specifically a protein of similar molecular weight to that in pea leaves. In leaves of M. arvensis the P protein was present in the mitochondria of the bundle-sheath cells but was undetectable in those of the mesophyll, whereas the L, T and H proteins and SHMT were present in both cell types. The density of immunogold labelling of SHMT on the mitochondria of mesophyll cells was less than that on those of the bundle-sheath cells, which correlates with the relative activities of SHMT in these cell types. These data reveal that the lack of functional GDC in the mesophyll cells of M. arvensis, which is the principal biochemical reason for reduced photorespiration in this species, is due to the loss of a single subunit protein. This lack of coordinate expression of the subunit proteins of GDC within a photosynthetic cell represents a clear difference between M. arvensis and other C₃ and C₃-C₄ species. None of the GDC proteins was detectable in the mesophyll cells of the C₃-C₄ and C₄ Flaveria and Panicum species but all were present in the bundle-sheath cells. The differences in the distribution of the GDC proteins in leaves of the C₃-C₄ species studied are discussed in relation to the evolution of photosynthetic mechanisms.     

Distribution of photorespiratory enzymes between bundle-sheath and mesophyll cells in leaves of the C3−C4 intermediate species Moricandia arvensis (L.) DC

In order to study the location of enzymes of photorespiration in leaves of the C₃–C₄ intermediate species Moricandia arvensis (L.). DC, protoplast fractions enriched in mesophyll or bundlesheath cells have been prepared by a combination of mechanical and enzymic techniques. The activities of the mitochondrial enzymes fumarase (EC 4.2.1.2) and glycine decarboxylase (EC 2.1.2.10) were enriched by 3.0- and 7.5-fold, respectively, in the bundle-sheath relative to the mesophyll fraction. Enrichment of fumarase is consistent with the larger number of mitochondria in bundle-sheath cells relative to mesophyll cells. The greater enrichment of glycine decarboxylase indicates that the activity is considerably higher on a mitochondrial basis in bundle-sheath than in mesophyll cells. Serine hydroxymethyltransferase (EC 2.1.2.1) activity was enriched by 5.3-fold and glutamate-dependent glyoxylate-aminotransferase (EC 2.6.1.4) activity by 2.6-fold in the bundle-sheath relative to the mesophyll fraction. Activities of serine- and alanine-dependent glyoxylate aminotransferase (EC 2.6.1.45 and EC 2.6.1.4), glycollate oxidase (EC 1.1.3.1), hydroxypyruvate reductase (EC 1.1.1.81), glutamine synthetase (EC 6.3.1.2) and phosphoribulokinase (EC 2.7.1.19) were not significantly different in the two fractions. These data provide further independent evidence to complement earlier immunocytochemical studies of the distribution of photorespiratory enzymes in the leaves of this species, and indicate that while mesophyll cells of M. arvensis have the capacity to synthesize glycine during photorespiration, they have only a low capacity to metabolize it. We suggest that glycine produced by photorespiratory metabolism in the mesophyll is decarboxylated predominantly by the mitochondria in the bundle sheath.Abbreviation RuBP ribulose 1,5-bisphosphate     

Intergeneric (intersubtribe) hybridization between Moricandia arvensis and Brassica A and B genome species by ovary culture

Summary Intergeneric hybrids between Moricandia arvensis (C₃–C₄ intermediate species) and Brassica A and B genome species (B. campestris and B. nigra) were produced via ovary culture. When M. arvensis was used as a female parent, the hybrid embryo yield (0.25–0.45 embryo per pollination) was similar between two genomes, regardless of the male parent. The reciprocal hybrid using B. campestris as a female was also obtained, although yield of embryo was lower (0.02 embryo per pollination). On the other hand, no hybrids were obtained without the in vitro technique. As most hybrid embryos could not develop normal shoots, plants were regenerated by inducing shoots on the cultured hypocotyl. The hybrid nature of the regenerated plant was confirmed morphologically and cytogenetically. A certain amount of bivalents (2.52-2.71) in the hybrids indicated the existence of partial chromosome homology between two genera. The present results indicate that ovary culture is an effective technique for overcoming the crossing barrier between M. arvensis and Brassica cultivated species.     

Interspecific somatic hybridization between Brassica napus and Brassica hirta (Sinapis alba L.)

Summary Somatic hybridization between Brassica napus and B. hirta (or Sinapis alba) is described. No cybrid plant with B. napus nucleus exhibiting cytoplasmic male sterility was recovered. Somatic hybrids were identified morphologically and, for some of them, by cytological observations. They were also characterised by Southern hybridization of nuclear rDNA. Chloroplast and mitochondrial DNA restriction analysis showed that 2 plants out of 14 have B. hirta ctDNA, one the B. napus mtDNA and the other a hybrid. Nine possess B. napus ctDNA with a hybrid mtDNA. For six of them, mtDNA patterns present novel bands, suggesting intergenomic recombination during fusion. These hybrids will be included in the breeding program.     

Somatic embryogenesis and plant regeneration from stem explants of Moricandia arvensis

Efficient and reproducible plant regeneration has been established from stem internode explants of Moricandia arvensis, a crucifer of special interest due to its C₃-C₄ intermediate photosynthetic activity. Somatic embryogenesis was induced in one-third of explants cultured on Murashige and Skoog based medium containing 9 mm 2,4-dichlorophenoxyacetic acid. High frequencies of plant regeneration (>90%) resulted when somatic embryos were germinated on medium lacking growth regulators. Regenerated plants were diploid, fertile and morphologically similar to seed-derived plants of M. arvensis. This is the first report of somatic embryogenesis in M. arvensis. This plant regeneration system should facilitate gene identification and localisation studies of C₃-C₄ physiology by insertional mutagenesis, a prerequisite for the isolation and transfer of genes involved in C₃-C₄ metabolism from Moricandia to cultivated brassicas. Received: 28 November 1996 / Revision received: 30 January 1997 / Accepted: 15 February 1997     

C3-C4 intermediate photosynthetic characteristics of cassava (Manihot esculenta Crantz)

Cassava, bean and maize leaves were fed with¹⁴CO₂ in light and the primary products of photosynthesis identified 5 and 10 seconds after assimilation. In maize, approximately three quarters of the labelled carbon was incorporated in C₄ acids, in beans about two thirds in PGA, and in cassava approximately 40–60% in C₄ acids with 30–50% in PGA. These data indicate that cassava possesses the C₄ photosynthetic cycle, however due to the lack of typical Kranz anatomy appreciable carbon assimilation takes place directly through the Calvin-Benson-Bassham cycle.     

Effects of irradiance and temperature on photosynthesis in C3, C4 and C3/C4 Panicum species

Species in the Laxa and Grandia groups of the genus Panicum are adapted to low, wet areas of tropical and subtropical America. Panicum milioides is a species with C₃ photosynthesis and low apparent photorespiration and has been classified as a C₃/C₄ intermediate. Other species in the Laxa group are C₃ with normal photorespiration. Panicum prionitis is a C₄ species in the Grandia group. Since P. milioides has some leaf characteristics intermediate to C₃ and C₄ species, its photosynthetic response to irradiance and temperature was compared to the closely related C₃ species, P. laxum and P. boliviense and to P. prionitis. The response of apparent photosynthesis to irradiance and temperature was similar to that of P. laxum and P. boliviense, with saturation at a photosynthetic photo flux density of about 1 mmol m^-2 s^-1 at 30°C and temperature optimum near 30°C. In contrast, P. prionitis showed no light saturation up to 2 mmol m^-2 s^-1 and an optimum temperature near 40°C. P. milioides exhibited low CO₂ loss into CO₂-free air in the light and this loss was nearly insensitive to temperature. Loss of CO₂ in the light in the C₃ species, P. laxum and P. boliviense, was several-fold higher than in P. milioides and increased 2- to 5-fold with increases in temperature from 10 to 40°C. The level of dark respiration and its response to temperature were similar in all four Panicum species examined. It is concluded that the low apparent photorespiration in P. milioides does not influence its response of apparent photosynthesis to irradiance and temperature in comparison to closely related C₃ Panicum species.Abbreviations AP apparent photosynthesis - I CO₂ compensation point - gl leaf conductance; gm, mesophyll conductance - PPFD photosynthetic photon flux density - PR apparent photorespiration rate - RuBPC sibulose bisphosphate carboxylase     

Production of intertribal somatic hybrids between Brassica napus L. and Lesquerella fendleri (Gray) Wats

Intertribal Brassica napus (+) Lesquerella fendleri hybrids have been produced by polyethylene glycol-induced fusions of B. napus hypocotyl and L. fendleri mesophyll protoplasts. Two series of experiments were performed. In the first, symmetric fusion experiments, protoplasts from the two materials were fused without any pretreatments. In the second, asymmetric fusion experiments, X-ray irradiation at doses of 180 and 200 Gy were used to limit the transfer of the L. fendleri genome to the hybrids. X-ray irradiation of L. fendleri mesophyll protoplasts did not suppress the proliferation rate and callus formation of the fusion products but did significantly decrease growth and differentiation of non-fused L. fendleri protoplasts. In total, 128 regenerated plants were identified as intertribal somatic hybrids on the basis of morphological criteria. Nuclear DNA analysis performed on 80 plants, using species specific sequences, demonstrated that 33 plants from the symmetric fusions and 43 plants from the asymmetric fusions were hybrids. Chloroplast and mitochondrial DNA analysis revealed a biased segregation that favoured B. napus organelles in the hybrids from the symmetric fusion experiments. The bias was even stronger in the hybrids from the asymmetric fusion experiments where no hybrids with L. fendleri organelles were found. X-ray irradiation of L. fendleri protoplasts increased the possibility of obtaining mature somatic hybrid plants with improved fertility. Five plants from the symmetric and 24 plants from the asymmetric fusion experiments were established in the greenhouse. From the symmetric fusions 2 plants could be fertilised and set seeds after cross-pollination with B. napus. From the asymmetric fusions 9 plants could be selfed as well as fertilised when backcrossed with B. napus. Chromosome analysis was performed on all of the plants but 1 that were transferred to the greenhouse. Three plants from the symmetric fusions contained 50 chromosomes, which corresponded to the sum of the parental genomes. From the asymmetric fusions, 11 hybrids contained 38 chromosomes. Among the other asymmetric hybrids, plants with 50 chromosomes and with chromosome numbers higher than the sum of the parental chromosomes were found. When different root squashes of the same plant were analysed, a total of 6 plants were found that had different chromosome numbers.     

Synthesis of high erucic acid rapeseed (Brassica napus L.) somatic hybrids with improved agronomic characters

Novel Brassica napus somatic hybrids have been created through protoplast fusion of B. oleracea var. botrytis and B. rapa var. oleifera genotypes selected for high erucic acid (22:1) content in the seed oil. Fifty amphidiploids (aacc) and one putative hexaploid (aacccc) hybrid were recovered in one fusion experiment. Conversely, only one amphidiploid and numerous regenerates with higher DNA contents were produced in a similar fusion using a different B. rapa partner. Hybridity was confirmed by morphology, isozyme expression, flow cytometry, and DNA hybridization. Analysis of organellar DNA revealed a distinct bias toward the inheritance of chloroplasts from the B. rapa (aa) genome. All amphidiploids set self-pollinated seed. A erucic acid content as high as 57.4% was found in the seed oil of one regenerated plant. Fatty acid composition was stable in the R₁ generation and was coupled with increased female fertility. Other novel agronomic characters in the hybrids recovered include large seed size, lodging resistance, and non-shattering seed pods.     

Production and cytogenetics of intergeneric hybrids between Brassica napus and Orychophragmus violaceus

The intergeneric hybrid between Brassica napus and Orychophragmus violaceus was obtained by means of embryo culture technique with the latter as the pollen parent. The hybrid was morphologically intermediate between its parents, but could produce a lot of seeds when selfed. Somatic separation of the genomes from the two parental species was observed during the mitotic divisions of some of the hybrid cells. Thus, the hybrid became the mixoploid in nature, consisting of haploid and diploid cells of B. napus, and a nuclear — cytoplasmic hybrid, with the cytoplasm of B. napus and the nuclei of O. violaceus, and the hybrid cells. Pollen mother cells with 19, 12 and 6 bivalents, respectively, were produced by the hybrid. From the selfed progeny of the hybrid, mainly two kinds of plants, B. napus and the hybrid, were found. The hybrid plants of the selfed progeny again produced two kinds of plants, B. napus and the hybrid.     

The occurrence of both C3 and C4 photosynthetic characteristics in a single Zea mays plant

The activities of the carboxylating enzymes ribulose-1,5-biphosphate (RuBP) carboxylase and phosphoenolpyruvate (PEP) carboxylase in leaves of three-week old Zea mays plants grown under phytotron conditions were found to vary according to leaf position. In the lower leaves the activity of PEP carboxylase was lower than that of RuBP carboxylase, while the upper leaves exhibited high levels of PEP carboxylase. Carbon dioxide compensation points and net photosynthetic rates also differed in the lower and upper leaves. Differences in the fine structure of the lowermost and uppermost leaves are shown. The existence of both the C₃ and C₄ photosynthetic pathways in the same plant, in this and other species, is discussed.Abbreviations PEP phosphoenolpyruvate - RuBP ribulose-1,5-biphosphate     

Carbon-isotope discrimination by leaves of Flaveria species exhibiting different amounts of C3-and C4-cycle co-function

Carbon-isotope ratios were examined as ¹³C values in several C₃, C₄, and C₃–C₄ Flaveria species, and compared to predicted ¹³C, values generated from theoretical models. The measured ¹³C values were within 4 of those predicted from the models. The models were used to identify factors that contribute to C₃-like ¹³C values in C₃–C₄ species that exhibit considerable C₄-cycle activity. Two of the factors contributing to C₃-like ¹³C values are high CO₂ leakiness from the C₄ pathway and pi/pa values that were higher than C₄ congeners. A marked break occurred in the relationship between the percentage of atmospheric CO₂ assimilated through the C₄ cycle and the ¹³C value. Below 50% C₄-cycle assimialtion there was no significant relationship between the variables, but above 50% the ¹³C values became less negative. These results demonstrate that the level of C₄-cycle expression can increase from, 0 to 50% with little integration of carbon transfer from the C₄ to the C₃ cycle. As expression increaces above 50%, however, increased integration of C₃- and C₄-cycle co-function occurs.Abbreviations and symbols RuBP carboxylase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - PEP carboxylase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - pa atmospheric CO₂ partial pressure - pi intercellular CO₂ partial pressure - isotope ratio - quantum yield for CO₂ uptake     

Light-dependent net CO-evolution by C3 and C4 plants

Light-dependent CO-evolution by the green leaves of C₃ and C₄ plants depends on the CO₂/O₂ ratio in the ambient atmosphere. This and other physiological responses suggest that CO-evolution is a byproduct of photorespiration. At CO₂/O₂ ratios up to 10^-3, the ratio of CO evolved: CO₂ fixed in photosynthesis is significantly higher in C₃ than in C₄ plants. This discrepancy disappears when a correction is made for the CO₂-concentrating mechanism in C₄ photosynthesis, by which CO₂-concentration at the site of ribulose-bis-phosphate carboxylase/oxygenase in the bundle sheaths is raised significantly as compared to the ambient atmosphere. Since the oxygenase function of this enzyme is responsible for glycolate synthesis, i.e., the substrate of photorespiration, this result seems to support the conclusion that CO-evolution is a consequence of photorespiration. CO-evolution may turn out to be a useful and rather straightforward indicator for photorespiration in ecophysiological studies.Abbreviations CAM crassulacean acid metabolism - CO net CO-evolution - CO₂ net CO₂-fixation - PEP-C phosphoenolpyruvate carboxylase - RubP-C ribulose-bisphosphate carboxylase/oxygenase Dedicated to Professor André Pirson on the occasion of his 70th birthday     

Light/dark modulation of phosphoenolpyruvate carboxylase in C3 and C4 species

In this report, the effects of light on the activity and allosteric properties of phosphoenolpyruvate (PEP) carboxylase were examined in newly matured leaves of several C₃ and C₄ species. Illumination of previously darkened leaves increased the enzyme activity 1.1 to 1.3 fold in C₃ species and 1.4 to 2.3 fold in C₄ species, when assayed under suboptimal conditions (pH 7) without allosteric effectors. The sensitivities of PEP carboxylase to the allosteric effectors malate and glucose-6-phosphate were markedly different between C₃ and C₄ species. In the presence of 5 mM malate, the activity of the enzyme extracted from illuminated leaves was 3 to 10 fold higher than that from darkened leaves in C₄ species due to reduced malate inhibition of the enzyme from illuminated leaves, whereas it increased only slightly in C₃ species. The Ki(malate) for the enzyme increased about 3 fold by illumination in C₄ species, but increased only slightly in C₃ species. Also, the addition of the positive effector glucose-6-phosphate provided much greater protection against malate inhibition of the enzyme from C₄ species than C₃ species. Feeding nitrate to excised leaves of nitrogen deficient plants enhanced the degree of light activation of PEP carboxylase in the C₄ species maize, but had little or no effect in the C₃ species wheat. These results suggest that post-translational modification by light affects the activity and allosteric properties of PEP carboxylase to a much greater extend in C₄ than in C₃ species.