Molecular properties of phosphoenolpyruvate carboxylase from C3, C3−C4 intermediate,and C4 Flaveria species

Phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) from Flaveria trinervia Mohr (C₄), F. floridana Johnston (C₃–C₄), and F. cronquistii Powell (C₃) leaves were compared by electrotransfer blotting/enzyme-linked immunoassay (Western-blot analysis), mobility of the native enzyme in polyacrylamide gels and in isoelectric focusing (IEF) gels, peptide mapping, and in-vitro translation of RNA isolated from each plant. The PEPCases from the C₃ and C₃–C₄ plants were very similar to each other in terms of electrophoretic mobilities on gels and isoenzyme patterns on IEF gels, and identical in peptide mapping. Quantitative differences were noted, however, in that the C₃–C₄ intermediate plant contained more PEPCase overall and that the relative activity of individual isoenzymes shifted between the C₃ and C₃–C₄ intermediate PEPCases. The PEPCase from the C₄ plant had a different isoenzyme pattern, a different peptide map, and was far more abundant than the other two enzymes. Western blot analysis demonstrated the cross-reactivity of PEPCases from all three Flaveria species with antibody raised against maize PEPCase. The results provide evidence, at the molecular level, that supports the view of C₃–C₄ intermediate species as C₃-like plants with some C₄-like photosynthetic characteristics, but there are differences from the C₃ plant in the quantity and properties of the PEPCase from the C₃–C₄ intermediate plant.Abbreviations IEF isoelectric focusing - kDa kilodalton - PEPCase phosphoenolpyruvate carboxylase - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase     

In-situ immunofluorescent localization of phosphoenolpyruvate and ribulose 1,5-bisphosphate carboxylases in leaves of C3, C4, and C3−C4 intermediatePanicum species

The in-situ inter- and intracellular localization patterns of phosphoenolpyruvate (PEP) and ribulose 1,5-bisphosphate (RuBP) carboxylases in green leaves of severalPanicum species were investigated using an indirect immunofluorescence technique. Four species were examined and compared:P. miliaceum (C₄),P. bisulcatum (C₃), andP. decipiens andP. milioides (C₃–C₄ intermediates which have Kranz-like leaf anatomy and reduced photorespiration). In the C₄ Panicum, PEP carboxylase was located in the cytosol of the mesophyll cells and RuBP carboxylase was restricted to the bundle-sheath chloroplasts. In contrast, in the C₃ Panicum species, PEP carboxylase was found throughout the leaf chlorenchyma, in both the cytosol and chloroplasts, and RuBP carboxylase was located in the chloroplasts. For the C₃–C₄ intermediate plants, the patterns depended on the species examined. ForP. decipiens, the in-situ localization of both carboxylases was similar to that described forP. bisulcatum and other C₃ plants. However, inP. milioides, PEP carboxylase was found exclusively in the cytosol of the mesophyll cells, as inP. miliaceum and other C₄ species, whereas RuBP carboxylase was distributed in both the mesophyll and bundle-sheath chloroplasts.Abbreviations PEP phosphoenolpyruvate - RuBP ribulose 1,5-bisphosphate     

Immunofluorescent localization of phosphoenolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase/oxygenase proteins in leaves of C3, C4 and C3−C4 intermediate Flaveria species

The concentrations of 17 nucleotides and three nucleosides have been determined in a batch suspension culture of Datura innoxia using a new procedure for extraction, purification and high-performance liquid chromatography separation of these compounds. The nucleotide pools change appreciably in the different phases of growth. These changes indicate the preparation for and initiation of cell proliferation, and reflect metabolic events during cell division, cell elongation and starvation. The main components of the nucleotide pool are uracil nucleotides, with uridine 5-diphosphate sugars as the predominant fraction, and the adenine nucleotides. Although their concentrations vary by a factor of more than 6 the ratio of the uracil to adenine nucleotides is kept fairly constant during growth. The energy charge is maintained at a rather high value. The correlation of these events with nutrient uptake and macromolecular synthesis by the batch culture is presented in the following paper.Abbreviations Glc glucose - GlcNAc 2-acetamido-2-deoxy-d-glucose - HPLC high performance liquid chromatography - UDP uridine 5-diphosphate     

Interspecific variation in assimilation of 14CO2 into C4 acids by leaves of C3, C4 and C3−C4 intermediate Flaveria species near the CO2 compensation concentration

The assimilation of ¹⁴CO₂ into the C₄ acids malate and aspartate by leaves of C₃, C₄ and C₃–C₄ intermediate Flaveria species was investigated near the CO₂ compensation concentration ^* in order to determine the potential role of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) in reducing photorespiration in the intermediates. Relative to air concentrations of CO₂, the proportion of CO₂ fixed by PEP carboxylase at ^* increased in all six C₃–C₄ intermediate species examined. However, F. floridana J.R. Johnston and F. ramosissima Klatt were shown to be markedly less responsive to reduced external CO₂, with only about a 1.6-fold enhancement of CO₂ assimilation by PEP carboxylase, as compared to a 3.0- to 3.7-fold increase for the other C₃–C₄ species examined, namely, F. linearis Lag., F. anomala B.L. Robinson, F. chloraefolia A. Gray and F. pubescens Rydb. The C₃ species F. pringlei Gandoger and F. cronquistii A.M. Powell exhibited a 1.5- and 2.9-fold increase in labeled malate and aspartate, respectively, at ^*. Assimilation of CO₂ by PEP carboxylase in the C₄ species F. trinervia (Spreng.) C. Mohr, F. australasica Hook., and the C₄-like species F. brownii A.M. Powell was relatively insensitive to subatmospheric levels of CO₂. The interspecific variation among the intermediate Flaverias may signify that F. floridana and F. ramosissima possess a more C₄-like compartmentation of PEP carboxylase and ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) between the mesophyll and bundle-sheath cells. Chasing recently labeled malate and aspartate with ¹²CO₂ for 5 min at ^* resulted in an apparent turnover of 25% and 30% of the radiocarbon in these C₄ acids for F. ramosissima and F. floridana, respectively. No substantial turnover was detected for F. linearis, F. anomala, F. chloraefolia or F. pubescens. With the exception of F. floridana and F. ramosissima, it is unlikely that enhanced CO₂ fixation by PEP carboxylase at the CO₂ compensation concentration is a major mechanism for reducing photorespiration in the intermediate Flaveria species. Moreover, these findings support previous related ¹⁴CO₂-labeling studies at air-levels of CO₂ which indicated that F. floridana and F. ramosissima were more C₄-like intermediate species. This is further substantiated by the demonstration that F. floridana PEP carboxylase, like the enzyme in C₄ plants, undergoes a substantial activation (2.2-fold) upon illuminating dark-adapted green leaves. In contrast, light activation was not observed for the enzyme in F. linearis or F. chloraefolia.Abbreviations and symbols PEP phosphoenolpyruvate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - CO₂ compensation concentration - ^* a subatmospheric level of CO₂ approximating Published as Paper No. 8832, Journal Series, Nebraska Agricultural Research Division     

Ecotypic differences in the C3 and C4 photosynthetic activity in Mollugo verticillata,a C3−C4 intermediate

Four populations of Mollugo verticillata L. were compared on the basis of their photosynthetic products, photosynthetic rates, enhancement under low oxygen concentration, and CO₂ compensation points. In addition, pulse-chase labeling experiments were conducted using one of the four populations. Depending on the plant population, C₄ acids ranged from 40% to 11% of the primary products under short-term exposure to ¹⁴CO₂. These compounds were also metabolized during pulse-chase experiments. All four populations had significantly different photosynthetic rates and those rates were correlated with the amounts of labelled C₄ acids produced and C₄-acid turnover. Three populations of M. verticillata had similar compensation points (40 l/l) and degrees of photosynthetic enhancement under low [O₂] (20%), the fourth population was much lower in both characteristics (CO₂ compensation, 25 l/l; low-O₂ enhancement, 12%). The results verify the intermediate nature of photosynthesis in this species, and illustrate populational differences in its photosynthetic and photorespiratory carbon metabolism.Abbreviations PGA 3-phosphoglyceric acid - Kan Kansas - Mass Massachusetts - Mex Mexico     

Purification and properties of glycolate oxidase from plants with different photosynthetic pathways: Distinctness of C4 enzyme from that of a C3 species and a C3–C4 intermediate

Glycolate oxidase (GO; EC 1.1.3.1) was purified from the leaves of three plant species:Amaranthus hypochondriacus L.(NAD-ME type C₄ dicot),Pisum sativum L. (C₃ species) andParthenium hysterophorus L. (C₃–C₄. intermediate). A flavin moiety was present in the enzyme from all the three species. The enzyme from the C₄ plant had a low specific activity, exhibited lower K_M for glycolate, and required a lower pH for maximal activity, compared to the C₃ enzyme. The enzyme from the C₄ species oxidized glyoxylate at <10% of the rate with glycolate, while the GO from the C₃ plant oxidized glyoxylate at a rate of about 35 to 40% of that with glycolate. The sensitivity of GO from C₄ plant to -hydroxypyridinemethane sulfonate, 2-hydroxy-3-butynoate and other inhibitors was less than that of the enzyme from C₃ source. The properties of GO fromParthenium hysterophorus, were similar to those of the enzyme fromPisum sativum. The characteristics of glycolate oxidase from leaves of a C₄ plant,Amaranthus hypochondriacus are different from those of the C₃ species or the C₃–C₄ intermediate.     

Plant regeneration from hypocotyls,cotyledons and stem segments of the C3–C4 intermediate species Moricandia arvensis

Techniques have been developed for the regeneration of Moricandia arvensis from complex explants. Hypocotyl segments and cotyledonary explants regenerated shoots, but the most efficient plant regeneration was from stem sections taken from in vitro grown shoots. Regeneration from these three explant types was tested on a range of concentrations of benzylaminopurine and either naphthylene acetic acid or indole acetic acid. Regeneration from all three explants was much higher on indole acetic acid than on naphthylene acetic acid and the ratio of auxin to cytokinin was also significant in determining the response of explants. Optimum regeneration was on 1mg/l IAA with 1mg/l BAP. Plants could be transferred to soil and grown to flowering in the glasshouse.Abbreviations GDC glycine decarboxylase - BAP benzyl aminopurine - NAA naphthalene acetic acid - IAA indole acetic acid     

Transport of Eicosapentaenoic Acid-Derived PGE3, PGF3α, and TXB3 by ABCC4

Background

Eicosapentaenoic acid-derived prostaglandin (PG) E₃, PGF_3α, and thromboxane (TX) B₃ are bioactive lipid mediators which have anti-cancer and anti-inflammatory effects. To exert their effects, PGE₃, PGF_3α, and TXB₃ must be released to the extracellular space from cells, but the release mechanism has been unclear. We therefore investigated the contribution of ATP-binding cassette transporter C4 (ABCC4), which has been known as a prostanoids efflux transporter, to the release of PGE₃, PGF_3α, and TXB₃.

Materials and Methods

ATP-dependent transport of PGE₃, PGF_3α, and TXB₃ via ABCC4 was investigated by using inside-out membrane vesicles prepared from ABCC4-overexpressing HEK293 cells. To evaluate the contribution of ABCC4 to the release of PGE₃, PGF_3α, and TXB₃, we measured the extracellular and intracellular levels of PGE₃, PGF_3α, and TXB₃ in A549 cells when we used ABCC4 inhibitors (dipyridamole, MK571, and probenecid) or ABCC4 siRNAs. The quantification of PGE₃, PGF_3α, and TXB₃ was performed by using liquid chromatography-tandem mass spectrometry.

Results

The apparent K_m values for ABCC4-mediated transport were 2.9±0.1 µM for PGE₃, 12.1±1.3 µM for PGF_3α, and 11.9±1.4 µM for TXB₃ and the ATP-dependent accumulation of PGE₃, PGF_3α, and TXB₃ into vesicles was decreased by using typical substrates and inhibitors of ABCC4. ABCC4 inhibitors and ABCC4 knockdown showed the reduction of extracellular/intracellular ratio of PGE₃ (40–60% of control) and PGF_3α (60–80% of control) in A549 cells.

Conclusions

Our results suggest that PGE₃, PGF_3α, and TXB₃ are substrates of ABCC4 and ABCC4 partially contributes to the release of PGE₃ and PGF_3α.     

The relative contributions of reduced photorespiration,and improved water-and nitrogen-use efficiencies,to the advantages of C3−C4 intermediate photosynthesis in Flaveria

Summary Analyses of carbon-assimilation patterns in response to intercellular CO₂ concentrations, and the photosynthetic water-and nitrogen-use efficiencies, were conducted for a C₃, a C₄, and three C₃–C₄ species in the genus Flaveria in order to determine some of the advantages and disadvantages of C₃–C₄ intermediate photosynthesis. Operational intercellular CO₂ partial pressures (pi), determined when the atmospheric CO₂ partial pressure (pa) was approximately 330 bar, in the C₃–C₄ species were generally equal to, or greater than, those observed in the C₃ species under well-watered or water-stressed conditions. This reflects equal, or lower, water-use efficiencies (WUEs) in the C₃–C₄ species. The only case in which higher WUEs were observed in the C₃–C₄ species, compared to the C₃ species, was when photosynthesis rates were limited by available nitrogen and were less than 12.5 mol CO₂ m^-2s^-1. At higher photosynthesis rates, the C₃–C₄ species exhibited lower values of photosynthesis rate for equal values of stomatal conductance (lower WUE), compared to the C₃ species. Comparing slopes for the linear regions of the relationship between leaf nitrogen content and net photosynthesis rate (taken as an index of photosynthetic nitrogen-use efficiency, NUE), the C₄ species exhibited the highest NUE, followed by the C₃–C₄ species, F. ramosissima, with the other two C₃–C₄ species and the C₃ species being equal and exhibiting the lowest NUEs. The lack of consistent advantages in NUE and WUE in the C₃–C₄ species F. pubescens and F. floridana suggest that in some C₃–C₄ Flaveria species C₄-like anatomy and biochemistry do not provide the same gas exchange advantages that we typically attribute to the CO₂-concentrating mechanism of fully-expressed C₄ plants.     

Synthesis of 27-Oxo, 27-Hydroxymilbemycins A3 and A4 and Novel 27-Alkoxymilbemycins A3 and A4 from Milbemycins A3 and A4 and…

27-Oxomilbemycins A₃ and A₄ and 27-hydroxymilbemycins A₃ and A₄ were identified as metabolites in soil metabolism studies of milbemycins A₃ and A₄. Chemical derivation methods were developed to synthesize 27-oxomilbemycins A₃ and A₄ and 27-hydroxymilbemycins A₃ and A₄ from milbemycins A₃ and A₄. In addition, 27-alkoxymilbemycin derivatives were also synthesized from the same precursors. Some of the synthesized compounds displayed satisfactory acaricidal activity against the organophosphorus-sensitive two-spotted spider mite (Tetranychus urticae), but did not have superior activity to corresponding milbemycins A₃ and A₄.     

δ13C values of an herbivore and the ratio of C3 to C4 plant carbon in its diet

Summary The ¹³C values of whole body samples of the beetle Tribolium castaneum are closely correlated with the ¹³C values of the plant carbon in its diet. The correlation is always high for diets ranging from 100% C₄ to 100% C₃ plant material. The degree of correlation is independent of the growth rate of the animals.     

Overexpression of myoglobin and assignment of its amide,Cα and Cβ resonances

Summary Sperm whale apomyoglobin was expressed to high levels on minimal media and isotopically labeled with ¹³C and ¹⁵N nuclei. The isotopically labeled apoprotein was purified to homogeneity in a single step by reversed-phase chromatography and reconstituted with hemin and carbon monoxide gas for NMR analysis. Sequence-specific backbone ¹H^N, ¹⁵N and ¹³C as well as side-chain ¹³C resonance assignments have been made for over 90% of the amino acids in the carbon monoxide complex of the protein. Resonance assignments were made by analysis of a series of 3D triple resonance spectra measured on the uniformly labeled sample. These assignments will provide the basis for analyzing the effects of point site mutations on the structure, stability and dynamics of the protein in solution.     

Effects of growth temperature and nitrogen nutrition on expression of C3–C4 intermediate traits in Chenopodium album

Proto-Kranz plants represent an initial phase in the evolution from C₃ to C₃–C₄ intermediate to C₄ plants. The ecological and adaptive aspects of C₃–C₄ plants would provide an important clue to understand the evolution of C₃–C₄ plants. We investigated whether growth temperature and nitrogen (N) nutrition influence the expression of C₃–C₄ traits in Chenopodium album (proto-Kranz) in comparison with Chenopodium quinoa (C₃). Plants were grown during 5 weeks at 20 or 30 °C under standard or low N supply levels (referred to as 20SN, 20LN, 30SN, and 30LN). Net photosynthetic rate and leaf N content were higher in 20SN and 30SN plants than in 20LN and 30LN plants of C. album but did not differ among growth conditions in C. quinoa. The CO₂ compensation point (Γ) of C. album was lowest in 30LN plants (36 µmol mol^–1), highest in 20SN plants (51 µmol mol^–1), and intermediate in 20LN and 30SN plants, whereas Γ of C. quinoa did not differ among the growth conditions (51–52 µmol mol^–1). The anatomical structure of leaves was not considerably affected by growth conditions in either species. However, ultrastructural observations in C. album showed that the number of mitochondria per mesophyll or bundle sheath (BS) cell was lower in 20LN and 30LN plants than in 20SN and 30SN plants. Immunohistochemical observations revealed that lower accumulation level of P-protein of glycine decarboxylase (GDC-P) in mesophyll mitochondria than in BS mitochondria is the major factor causing the decrease in Γ values in C. album plants grown under low N supply and high temperature. These results suggest that high growth temperature and low N supply lead to the expression of C₃–C₄ traits (the reduction of Γ) in the proto-Kranz plants of C. album through the regulation of GDC-P expression.

     

A model of photosynthetic CO2 assimilation and carbon-isotope discrimination in leaves of certain C3−C4 intermediates

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     

Planar π-aromatic C3h B6H 3 + and π-antiaromatic C2h B8H2: boron hydride analogues of D3h C3H 3 + and D2h C4H4

Based upon extensive density functional theory and wave function theory calculations performed in this work, we predict the existence of the perfectly planar triangle C(3h) B(6)H(3)(+) (1, (1)A') and the double-chain stripe C(2h) B(8)H(2) (9, (1)A(g)) which are the ground states of the systems and the inorganic analogues of cyclopropene cation D(3h) C(3)H (3) (+) and cyclobutadiene D(2h) C(4)H(4), respectively. Detailed adaptive natural density partitioning (AdNDP) analyses indicate that C(3h) B(6)H (3) (+) is π plus σ doubly aromatic with two delocalized π-electrons and six delocalized σ-electrons formally conforming to the 4n + 2 aromatic rule, while C(2h) B(8)H(2) is π antiaromatic and σ aromatic with four delocalized π-electrons and ten delocalized σ-electrons. The perfectly planar C(2h) B(8)H(4) (5, (1)A(g)) also proves to be π antiaromatic analogous to D(2h) C(4)H(4), but it appears to be a local minimum about 50 kJ mol(-1) less stable than the three dimensional C(s) B(8)H(4)(6, (1)A'). AdNDP, nucleus independent chemical shifts (NICS) and electron localization function (ELF) analyses indicate that these boron hydride clusters form islands of both σ- and π-aromaticities and are overall aromatic in nature in ELF aromatic criteria.     

Production of somatic hybrids between Brassica oleracea and the C3–C4 intermediate species Moricandia nitens

Protoplast fusion between Brassica oleracea and Moricandia nitens, a C₃–C₄ intermediate wild species, was carried out. Four hundred and twenty five plants were regenerated from 1995 calli. More than 90% of the regenerated plants were verified as true intergeneric hybrids on the basis of morphological observation and molecular-marker analysis. The hybrids were morphologically intermediate between both fusion parents. Variations in flower color and petal number were also observed. The chromosome number and pollen fertility varied across the individual hybrids. Although after self-pollination pollen germinated on the stigma and pollen tubes were visible in the style, the pods did not develop properly without in vitro culture. Measurements of the CO₂ compensation point revealed that six out of eight hybrid plants expressed a gas-exchange character that was intermediate between the C₃–C₄ M. nitens and C₃ B. oleracea parents. Received: 20 January 1999 / Accepted: 16 June 1999