Quantity and Kinetic Properties of Ribulose 1,5-Bisphosphate Carboxylase in C3, C4, and C3-C4 Intermediate Species of Flaveria (Asteraceae)

The kinetic properties of ribulose 1,5-bisphosphate carboxylase(RuBPC) appear to have been modified during evolution of photosynthesisto adjust to changes in substrate availability. C₄ plants areconsidered to have a higher concentration of CO₂ available toRuBPC than C₃plants. In this study, the K_m(CO₂ and catalyticcapacity (k_cat) of RuBPC and the ratio of RuBPC protein to totalsoluble protein from several Flaveria species, including C₃,C₃-C₄ intermediate, and C₄ species, were determined. The C₃and intermediate species had similar K_m(CO₂) values while theC₄ species on average had higher K_m(CO₂) values. The mean ratioof K_cat/K_m for species of each group was similar, supportingthe hypothesis that changes in K_m and K_cat, are linked. Theallocation of total soluble protein to RuBPC was lowest in theC₄ Flaveria species, intermediate in the C₃-C₄ species, andhighest in the C₃ species. The results suggest that during evolutionof C₄ photosynthesis adjustments may occur in the quantity ofRuBPC prior to changes in its kinetic properties. (Received January 4, 1989; Accepted April 11, 1989)     

Thermal Characteristics of C4 Photosynthetic Enzymes from Leaves of Three Sugarcane Species Differing in Cold Sensitivity

Phosphoenolpyruvate carboxylase, NADP-malate dehydrogenase andNADP-malic enzyme in desalted extracts from the leaves of threesugarcane species differing in cold sensitivity were relativelystable at cold temperatures, and their Arrhenius plots appearedas straight lines. Pyruvate,P_i dikinase (PPDK) from the threespecies was cold-inactivated, and its Arrhenius plots exhibiteda clear break-point around 10.6°C. Analysis of cold labilityof PPDK using deuterium oxide and Triton X-100 showed that theinteractions between the subunits possibly involve hydro-phobicbonds which would lead to cold lability. There were no apparentdifferences among the three sugarcane species in the thermalproperties of the four C₄ photosynthetic enzymes. The resultssuggest that the differences in cold sensitivity of sugarcanephotosynthesis may not relate to the thermal properties of C₄photosynthetic enzymes per se. ¹ Present address: Department of Biochemistry, University ofNebraska, Lincoln, NE 68588-0664, U.S.A.     

Cold stability of pyruvate,orthophosphate dikinase of Flaveria brownii

The nucleotide sequences of the complementary DNA of pyruvate, P_i dikinase (PPDK) from Flaveria bidentis, a C₄ plant which possesses a cold-sensitive form of PPDK, and Flaveria brownii, a C₄-like plant which possesses a cold-tolerant form of PPDK, were determined. PPDK was isolated from the leaves of both Flaveria species and purified and the N-terminal amino acid sequences characterised. Together with a maize PPDK cDNA, cDNA inserts which code for the mature form of PPDK of F. bidentis and of F. brownii were expressed in bacteria and the cold sensitivity of the expressed PPDK studied. The cold sensitivity of the PPDK expressed in bacteria mimics the cold sensitivity of PPDK found in vivo in all three plant species. This study indicates that the cold sensitivity of plant PPDK is controlled by the primary structure of the enzyme.     

Photosynthetic Characteristics of C3-C4 Intermediate Flaveria Species II. Kinetic Properties of Phosphoenolpyruvate Carboxylase from C3, C4 and C3-C4 Intermediate Species

The kinetic properties of phosphoenolpyruvate (PEP) carboxylasehave been studied among several Flaveria species: the C₃ speciesF. cronquistii, the C₃–C₄ species F. pubescens and F.linearis, and the C₄ species F. trinervia. At either pH 7 or8, the maximum activities (in µmol.mg Chl^–1.h^–1)for F. pubescens and linearis (187–513) were intermediateto those of the C₃ species (12–19) and the C₄ species(2,182–2,627). The response curves of velocity versusPEP concentration were hyperbolic for the C₃ and C₃–C₄species at either pH 7 or 8 while they were sigmoidal for theC₄ species at pH 7 and hyperbolic at pH 8. The K_m values forPEP determined from reciprocal plots were lowest in the C₃ species,and of intermediate value in the C₃–C₄ species comparedto the K' values of the C₄ species determined from Hill plotsat either pH 7 or 8. Glucose-6-phosphate (G6P) decreased theK_m values for PEP at both pH 7 and 8 in the C₃ and C₃–C₄species. In the C₄ species, G6P decreased the K' values at pH8 but increased the K' values at pH 7. In all cases, G6P hadits effect by influencing the activity at limiting PEP concentrationswith little or no effect on the maximum activity. At pH 8 andlimiting concentrations of PEP the degree of stimulation ofthe activity by G6P was greatest in the C₄ species, intermediatein F. linearis, a C₃–C₄ species, and lowest in the C₃species. In several respects, the PEP carboxylases of the C₃–C₄Flaveria species have properties intermediate to those of theC₃ and C₄ species. (Received April 30, 1983; Accepted August 22, 1983)     

Comparative Studies of NAD-Malic Enzyme from Leaves of Various C4 Plants

Using butyl-TSK-gel chromatography, we purified NAD-malic enzyme(ME) (EC 1.1.1.39 [EC] ), which is involved in C₄ photosynthesis,to electrophoretic homogeneity, from leaves of Amaran-thus tricolor.Molecular weights of the native and SDS-denatured enzyme fromA. tricolor were 490 kDa and 61 kDa, respectively. During assayof the enzyme there was a slow reaction transient in the formof a lag before a steady-state rate was reached. The durationof this lag was inversely proportional to the concentrationof each substrate and the activator, fructose- 1,6-bis-phosphate(FBP). The optimal pH of the reaction fell with decreasing concentrationsof either malate or FBP. High pH prolonged the lag in reaction. Double reciprocal plots of the enzymatic activity as a functionof the concentration of malate yielded straight lines and didnot show any cooperativity for binding of malate. The enzymefrom A. tricolor was not inhibited by either HCO^–₃ orCO₂. At different concentrations of malate, the nature of theactivating effect of FBP was compared among the purified enzymesfrom A. tricolor and the C₄ monocots Eleusine coracana and Panicumdichotomiflorum. At low levels of malate, FBP markedly stimulatedthe enzyme from each species. In contrast, at saturating levelsof malate, the response of enzymes to increasing concentrationsof FBP was different and depended on the source of enzyme. The immunochemical properties of the enzymes from the threespecies were compared using an enzyme-linked immunoadsorbentassay with antisera raised against the purified enzymes fromthe three species. Different cross-reactivities were observedamong the enzymes from different sources. The N-terminal aminoacid sequences of NAD-MEs from the three species were determinedand some differences were found among the three enzymes. ²Permanent address; Tohoku National Agricultural ExperimentStation, Morioka, 020-01 Japan. ³Permanent address; National Grassland Research Institute, Nishinasuno,Tochigi, 329-27 Japan. (Received December 12, 1988; Accepted February 17, 1989)     

The Gene for Pyruvate,Orthophosphate Dikinase in C4 Plants: Structure, Regulation and Evolution

Pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1 [EC] ) is a keyenzyme in photosynthesis in plants that exploit the C4 photosyntheticpathway for the fixation of CO₂. This review focuses on thestructure, regulation and evolution of the C4-type ppdk genein the maize genome. The C4-ppdk gene in maize consists of 19exons spanning about 12 kbp. The gene is transcribed from twodifferent initiation sites under the control of two promotersto produce two mRNAs of different sizes. The larger one containsthe exon 1 sequence that encodes the chloroplast transit peptideand its product acts as C4-PPDK in chloroplasts, while the smallerone does not contain the sequence and its product may functionas a C3-enzyme in the cytosol. This unusual dual promoter systemis not unique to the maize C4-type ppdk gene since the sameorganization is also observed in the rice (C3 plant) ppdk geneand in Flaveria. Thus, the two-promoter system is common toplant ppdk genes from C3 and C4, monocot and dicot plants. Adiscussion is also presented of the generation of a system forregulation of the expression of the C4-type ppdk gene. A chimericgene consisting of a reporter gene under the control of thepromoter of maize CA-ppdk is exclusively expressed in photosynthetictissues and not in roots or stems of transgenic rice. The expressionof the introduced gene is also regulated by light: it is lowin etiolated leaves and is enhanced by illumination. These resultsindicate that the regulatory system that controls ppdk expressionin maize is not unique to C4 plants. ¹Recipient of the JSPP Young Investigator Award, 1995.     

Multiple Forms of Acid Phosphatase in Cotyledons of Vigna mungo Seedlings: Immunological Detection and Quantitation

Using a combination of column chromatography and gel electrophoresis,we have found that acid phosphatase in cotyledons of Vigna mungoseedlings is composed of at least six forms (Ia₁, Ia₂, Ib₁,Ib₂, IIa and IIb). We purified one of the major forms, Ia₁,as a polypeptide of 53 kDa. Using an antiserum raised againstthe enzyme Ia₁, we examined the immunological relationshipsbetween the multiple forms from cotyledons and the distributionof the enzyme in organs of maturing and germinating seeds. (Received December 25, 1989; Accepted July 11, 1990)     

Stimulatory Effect of Chloride Ions on NADP Malic Enzyme from Leaves of two C4 Species of Cyperus

NADP malic enzyme (EC 1.1.1.40 [EC] ) from leaves of two C₄ speciesof Cyperus (C. rotundus and C. brevifolius var leiolepis) exihibiteda low level of activity in an assay mixture that contained lowconcentrations of Cl^–. This low level of activity wasmarkedly enhanced by increases in the concentration of NaClup to 200 mM. Since the activity of NADP malic enzyme was inhibitedby Na₂SO₄ and stimulated by relatively high concentration ofTris-HCl (50–100 mM, pH 7–8), the activation ofthe enzyme by NaCl appears to be due to Cl^–. Variationsin the concentration of Mg²⁺ affected the K_A (the concentrationof activator giving half-maximal activation) for Cl^–,which decreased from 500 mM to 80 mM with increasing concentrationsof Mg²⁺ from 0.5 mM to 7 mM. The K_m for Mg²⁺ was decreased from7.7 mM to 1.3 mM with increases in the concentration of NaClfrom zero to 200 mM, although the increase of V_max was not remarkable.NADP malic enzyme from Cyperus, being similar to that from otherC₄ species, was able to utilize Mn²⁺. The K_m for Mn²⁺ was 5mM, a value similar to that for Mg²⁺. The addition of 91 mMNaCl markedly decreased the K_m for Mn²⁺ to 20 +M. NADP malicenzyme from Setaria glauca, which contains rather less Cl^–than other C₄ species, was inactivated by concentrations ofNaCl above 20 mM, although slight activation of the enzyme wasobserved at low concentrations of NaCl at pH7.6. (Received February 20, 1989; Accepted June 12, 1989)     

Purification, Properties and Phosphorylation of Anaerobically Induced Enolase in Echinochloa phyllopogon and E. crus-pavonis

Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11 [EC] ) activityis differentially induced by anoxia in the flood-tolerant speciesE. phyllopogon (Stev.) Koss and the flood-intolerant speciesE. crus-pavonis (H.B.K.) Schult. To examine the regulation ofenolase at the protein level, we purified the enzyme from bothspecies to near homogeneity and compared their physico-chemicaland catalytic properties. Enolase purified from E. phyllopogonexhibits optimal activity at pH 7.0, a K_m of 80 µM for2-PGA, a Q₁₀ of 1.97 and an E_a of 12.3 kcal mol^-1. Similarly,enolase from E. crus-pavonis exhibits optimal activity at pH7.0, a K_m of 50 µM for 2-PGA, a Q₁₀ of 2.04 and an E_aof 12.9 kcal mol^-1. The enzyme from both species is thermostable(100% active after 15 min, 50°C) and is a homodimer of 52.5kDa subunits as resolved by SDS-PAGE and immunoblotting. E.phyllopogon enolase was phosphorylated in vitro using either[     

Immunological Studies on Pyruvate Orthophosphate Dikinase in C3 Plants

Pyruvate orthophosphate dikinase (PPDK) was detected in someC₃ plants, wheat, barley, rice and tobacco, by protein blottingusing an antibody against maize PPDK, although the amounts weremuch lesser than those of C₄ plants. The PPDK activity in immaturegrains of rice was specifically immunoprecipitated by the anti-(maize)PPDK antibody. The molecular weight of the subunit of PPDK inall tested C₃ plants was similar (ca. 95 kD) to that of maizePPDK, and the fragment patterns of the C₃ PPDKs in peptide mappingwere also similar to that of maize PPDK. These results suggestthat C₃ PPDKs have a primary structure similar to that of maizePPDK. In order to obtain information about the expression of PPDKin C₃ plants, changes in the enzyme activity and in the amountof PPDK protein were investigated during the greening of riceseedlings. PPDK, which was found in the etiolated seedlings,decreased temporarily in an early stage of greening and thenincreased. The mechanism of this variation is discussed. ¹ To whom correspondence should be addressed. (Received December 9, 1986; Accepted March 12, 1987)     

Purification of NADP-Malic Enzyme and Phosphoenolpyruvate Carboxylase from Sugar Cane Leaves

A procedure is described for the purification of phosphoenolpyruvatecarboxylase (EC 4.1.1.31 [EC] ) and NADP-dependent malic enzyme (EC1.1.1.40 [EC] ) from sugar cane leaves. Each enzyme was purified tohomogeneity as judged by sodium dodecyl sulfate-polyacrylamidegel electro-phoresis, with about 30% yield. Phosphoenolpyruvatecarboxylase was purified 54-fold. A molecular weight of 400,000and a homotetrameric structure were determined for the nativeenzyme. The purified carboxylase had a specific activity of20.0 {diaeresis}mol (mg protein)_–1 min_–1, and wasactivated by glucose-6-phosphate and inhibited by L-malate.K_m values at pH 8.0 for phosphoenolpyruvate and bicarbonatewere 0.25 and O.l0 mM, respectively. NADP-malic enzyme, 356-foldpurified, exhibited a specific activity of 71.2 {diaeresis}mol(mg protein)_–1 min_–1 and was characterized as ahomotetramer with native molecular weight of 250,000. Purifiedmalic enzyme showed an absolute specificity for NADP⁺ and requireda divalent metal ion for activity. K_m values of 0.33 and 0.008mM for L-malate and NADP⁺, respectively, were determined. Thisenzyme was inhibited by several organic acids, including ketoand amino acids; while succinate and citrate increased the enzymeactivity when assayed with 10{diaeresis}M L-malate. The effectsshown by amino acids and by citrate were dependent on pH, beinghigher at pH 8.0 than at pH 7.0. (Received October 26, 1988; Accepted February 3, 1989)     

Isolation and Purification of a Ubiquinone-Cytochrome b-c1 Complex from a Photosynthetic Bacterium, Rhodopseudomonas sphaeroides

A ubiquinone-cytochrome b-c₁ complex was removed from chromatophoremembranes of a Rhodopseudomonas sphaeroides green mutant bydeoxycholate-cholate treatment of the chromatophores. The complexwas purified by ammonium sulfate fractionation and gel filtration. The molecular weight of the purified complex was 240,000 (240kD) and it was composed of seven subunits with molecular weightsof 47 kD, 42 kD, 38 kD, 32 kD, 30 kD, 24 kD and 16 kD. The complexcontained 1.54 and 3.42 nmol of cytochrome c₁ and two differentcytochrome b species per mg protein, respectively. It also contained7.07 nmol of ubiquinone, 6.37 nmol of non-heme iron and about3 nmol of carotenoids per mg protein. No flavins were detected.Heme staining indicated that the 32 kD-and 24 kD-subunits werecytochromes. The midpoint potential of cytochrome c₁ was 245 mV, and thevalues for the cytochromes b were 60 mV and –75 mV atpH 7.2. The peak of the -band of the reduced-minus-oxidizeddifference spectrum of cytochrome c₁ was located at 552.5 nm,arid peaks of the b-type cytochromes with higher and lower midpointpotentials were located at 562 nm and 563 nm. The chemical and the subunit compositions of the purified complexreported here were similar to those obtained for the inner membranesof mitochondria of various organisms. (Received April 5, 1982; Accepted June 14, 1982)     

Purification and characterization of CMP-N-acetylneuraminic acid hydroxylase from pig submandibular glands

N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-giycosideby the action of CMPN-acetylneuramlnic acid (CMP-Neu5Ac) hydroxylase.This enzyme is a soluble cytochrome bs-dependent monooxygenaseand has been purified to apparent homogeneity from pig submandibularglands by precipitation with N-cetyN,N,N-trimethylam-moniumbromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose,Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12.This procedure resulted in an 8960-fold purification of thehydroxylase with a recovery of 0.8%. The molecular mass of thisprotein was shown to be 65 kDa on SDS-PAGE and 60 kDa as determinedby gel filtration on Superose S.12, which suggests that theenzyme is a monomer. The purified CMP-Neu5Ac hydroxylase isactivated by FeSO₄ and inhibited by iron-binding reagents suchas o-phenanthroline, KCN, Tiron and ferro-zine. An apparentKm of 11 µM was determined for the substrate CMP-Neu5Acusing purified hydroxylase in the presence of Triton X-100-solubilizedmicrosomes. In a reconstituted system consisting of purifiedhydroxylase, cytochrome b₅, cytochrome b₅ reductase and catalase,an apparent K_m of 3 µM was measured. The apparent K_mforcytochrome b₅ in this system was 0.24 µM. Immunizationof a rabbit with enriched and purified hydroxylase led to anantiserum that inhibited CMP-Neu5Ac hydroxylase activity andreacted with the purified 65 kDa protein on a Western blot afterSDS-PAGE. Antibodies specific for this 65 kDa protein were isolatedand showed a strong reaction with the purified CMP-Neu5Ac hydroxylasefrom mouse liver after immunoblotting. Initial experiments withthis monospecific antibody suggest that the activity of thehydroxylase in a particular tissue correlates with the amountof immuno-reactive protein. cytochrome b₅ N-glcoloylneuraminic acid hydroxylase pig submandibular gland mucin sialic acid     

Betacyanin-Decolorizing Enzymes from Phytolacca americana

Betacyanin-decolorizing enzymes (BDEs) in Phytolacca americanawere partially purified and characterized. Acidic enzymes, BDE₁and BDE₂, and basic enzymes, BDE₃ and BDE₄, were partially purifiedby column chromatography on DEAE-Sepharose CL-6B and CM-SepharoseCL-6B. These enzymes degraded betanin to its aglycon (betanidin)and then to a product with a peak of absorbance at 485 nm. Theoptimum pH values were 5.0 for BDE₁, 3.5 for BDE₂, and 5.5 forBDE₃ and BDE₄. BDE activity was detected in extracts of theleaf, stem, seed, and root. The highest specific activity ofBDE₂ was found in the red epidermis of the stem. The activityof BDE₂ was inhibited by NH₄NO₃, KNO₃, KC1, and NaCl, but theactivities of the other BDEs were not inhibited. These resultssuggest that BDEs other than BDE₂ control the degradation ofbetacyanin in the intracellular space. (Received March 27, 1989; Accepted December 8, 1989)     

Purification and Characterization of a Cytochrome P450 (trans-Cinnamic Acid 4-Hydroxylase) from Etiolated Mung Bean Seedlings

A Cyt P450 (P450_C4H) possessing trans-cinnamate 4-hydroxylase(C4H) activity was purified to apparent homogeneity from microsomesof etiolated mung bean seedlings. Upon SDS-polyacrylamide gelelectrophoresis, the purified preparation gave a single proteinband with a molecular mass of 58-kDa. Its specific P450 contentwas 12.6 nmol (mg protein)^–1. Using NADPH as electrondonor, purified P450_C4H aerobically converted trans-cinnamicacid to p-coumaric acid with a specific activity of 68 nmolmin^–1 nmol^–1 P450 in a reconstituted system containingNADPH-Cyt P450 reductase purified from the seedlings or rabbitliver microsomes, dilauroyl phosphatidylcholine, and cholate.This specific activity is by far the highest for reconstitutedC4H systems so far reported and provides direct evidence thatC4H activity is actually associated with a P450 protein. Inthe oxidized state P450_C4H showed a typical low-spin type absorptionspectrum with a Soret peak at 419 nm. A partial spectral shiftto the high spin state was observed when trans-cinnamic acidwas added to oxidized P450_C4H. By spectral titration, the dissociationconstant of the cinnamic acid-P450_C4H complex was determinedto be 2.8 µM. This value is similar to the K_m value (1.8µM) for trans-cinnamic acid determined in the reconstitutedsystem. (Received November 20, 1992; Accepted February 17, 1993)     

Differential Cellular Isotope Effects of Deuterium on Photosynthetic Metabolism of Carbon in Chlorella ellipsoidea

Isotope effects of deuterium on photosynthetic metabolism ofcarbon in Chlorella ellipsoidea were investigated. Photosyntheticfixation of ¹⁴C in D₂O was about a half of that in H₂O. Eachstep in the photosynthetic metabolism of carbon was affecteddifferently by D₂O in the medium and constitutive D. (Received June 15, 1989; Accepted October 23, 1989)     

Quantitative Analysis of Endogenous Gibberellins in Normal and Dwarf Cultivars of Rice

Endogenous levels of gibberellins in shoots and ears of twodwarf rice (Oryza sativa L.) cultivars, Tan-ginbozu (dx mutant)and Waito-C (dy mutant), were analyzed and compared with thoseof normal rice cultivar, Nihonbare. The endogenous levels of13-hydroxylated gibberellins in Tan-ginbozu were much lowerthan those in Nihonbare. In Waito-C, the levels of GA₁₉ andGA₂₀ in the shoots were higher but that of GA₁ was lower thanthe levels of these gibberellins in Nihonbare. These resultssupport the hypothesis that the dy gene controls the 3ß-hydroxylationof GA₂₀ to GA₁ while the dx gene controls a much earlier stepin the gibberellin biosynthesis. Our results indicate that GA₁is the active gibberellin that regulates the vegetative growthof rice. The endogenous levels of GA₄ in the ears of the twodwarf cultivars of rice were higher than the level of GA₄ inthe ears of the normal cultivar, Nihonbare suggesting that thebiosynthesis of gibberellin is not blocked in the anthers ofthe dwarf rice. (Received April 27, 1989; Accepted July 12, 1989)     

A Comparison of the Growth, Photosynthesis, Stomatal Conductance and Water Use Efficiency of Moricandia and Brassica Species

When grown in pots and well-watered, the relative growth ratesof the above ground parts of two species of Moricandia (M. arvensis,an intermediate C₃–C₄ species, and M. moricandioides,a C₃ species) were inferior to those of two cultivated Brassicaspecies (B. campestris and B. napus). The Moricandia specieshad thicker leaves (greater d.wt per unit leaf area) with morechlorophyll than the Brassica species and had slightly greaterrates of photosynthesis per unit leaf area at an irradiance(400–700 nm) of 2000 µmol quanta m^–2 s ^–1.Leaves of M. arvensis, known to have a CO₂ compensation pointbetween that of C₃ and C₄ species, had a lower ratio of theintercellular to atmospheric partial pressure of CO₂ (C₁/C_a)and a greater instantaneous water use efficiency (WUE) thanthose of M. moricandioides and the Brassica species. Carbon isotope discrimination (     

Intracellular Localization of Phosphoenolpyruvate Carboxykinase in Bundle Sheath Cells of C4 Plants

Bundle sheath protoplasts (BSP) were isolated and purified fromfour C₄ species of the phosphoenolpyruvate (PEP) carboxykinasetype (Panicum maximum, P. texanum, Chloris gayana and Eriochloaborumensis), and cell organellses were separated from the BSPextract by differential centrifugation or sucrose density gradientcentrifugation. Separation of the organelles was ascertainedby the distribution of marker enzymes for chloroplasts, mitochondria,peroxisomes and cytoplasm. Contrary to the previous report [Rathnamand Edwards (1975) Arch. Biochem. Biophys. 171: 214], the distributionof PEP carboxykinase in BSP of P. maximum was the same as thatof UDP-glucose pyrophosphorylase, a marker for cytoplasm, andPEP carboxykinase activity was not recovered in the intact chloroplasts.The same results were obtained with P. texanum, C. gayana andE. borumensis. Therefore, we conclude that PEP carboxykinase is exclusivelylocalized in the cytoplasm of bundle sheath cells of C₄ plants. (Received July 23, 1983; Accepted October 17, 1983)