Photosynthesis in Panicum milioides, a Species with Reduced Photorespiration

The capacity for C₄ photosynthesis in Panicum milioides, a specieshaving reduced levels of photorespiration, was investigatedby examining the activity of certain key enzymes of the C₄ pathwayand by pulse-chase experiments with ¹⁴CO₂. The ATP$P₁ dependentactivity of pyruvate,P₁ dikinase in the species was extremelylow (0.14–0.18 µmol mg chlorophyll^–1 min^–1).Low activity of the enzyme was also found in Panicum decipiensand Panicum hians (related species with reduced photorespiration)and in Panicum laxum (a C₃ species). The antibody to pyruvate,P₁dikinase caused about 70% inhibition of the ATP$P₁ dependentactivity of the enzyme in P. milioides. The activity of NAD-malicenzyme and NADP-malic enzyme in ^P. ^milioides was equally low(approximately 0.1–0.2 µmol mg chlorophyll^–1min^–1) and similar to the activity in P. decipiens, P.hians and P. laxum. Photosynthetic pulse-chase experiments underatmospheric conditions showed a typical C₃-like pattern of carbonassimilation including the labelling of glycine and serine asexpected during photorespiration. During the pulse with ¹⁴CO₂only about 1% of the labelled products appeared in malate and2–3% in aspartate. During a chase in atmospheric levelsof CO₂ for up to 6 min there was a slight increase in labellingin the C₄ acids. The amount of label in carbon 4 of aspartatedid not change during the chase, indicating little or no turnoverof the C₄ acid via decarboxylation. The results indicate thatunder atmospheric conditions P. milioides assimilates carbondirectly through the C₃ pathway. Photorespiration as indicatedby the CO₂ compensation point may be repressed in the speciesby a more efficient recycling of photorespired CO₂. (Received June 8, 1982; Accepted July 22, 1982)     

PEP Carboxylases from Two C4 Species of Panicum with Markedly Different Susceptibilities to Cold Inactivation

Phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31 [EC] ) assayedin extracts of Panicum maximum Jacq. loses up to 50% of itsactivity after incubation for 60 minutes at 0C while the enzymefrom P. miliaceum L. is completely stable under these conditions.Following dilution at room temperature the enzyme from P. maximumis labile, while that from P. miliaceum is stable. The P. maximumenzyme can be largely stabilized against dilution and againstcold-inactivation by D₂O which stabilizes hydrophobic bondsand the compatible solutes proline, betaine and trimethylamine-N-oxide.Mineral ions, previously demonstrated to be protective againstcold inactivation of pyruvate, P_i dikinase from maize, provideno protection of P. maximum PEPC against either cold or dilution.The chaotropic ion SCN^- causes partial inactivation of the enzymefrom P. miliaceum in the cold. The possible interrelationshipbetween inactivation by dilution and inactivation by cold isdiscussed. The enzyme from both species, when assayed withoutpreincubation at low temperature, exhibits similar, slightlycurvilinear Arrhenius plots; and no differences were found betweenthe two species in the temperature dependence of photosynthesis. ¹Present address: Botany Dept., University of California, DavisCA 95616 U.S.A.     

Glycolate synthesis in a C3 C4 and intermediate photosynthetic plant type

Excised leaves of a C₃-photosynthetic type, Hordeum vulgare,a C₄-type, Panicum miliaceum, and an intermediate-type, Panicummilioides, were allowed to take up through their cut ends a1 mM solution of butyl hydroxybutynoate (BHB), an irreversibleinactivator of glycolate oxidase. After 30 to 60 min in BHB,extractable glycolate oxidase activity could not be detectedin the distal quarter of the leaf blades. Following this pretreatment,recovery of ¹⁴C-glycolate from ¹⁴CO₂ incorporated in a 10 minperiod was nearly maximal for each of the three plant types.Labeled glycolate was 51% of the total ¹⁴CO₂ incorporated forthe C₃-species, 36% for the intermediate-species, and 27% forthe C₄-species Increased labeling of glycolate was compensatedfor primarily by decreased labeling of the neutral and basicfractions for the C₃ and intermediate-type species. In the C₄-type,label decreased primarily in the neutral and insoluble fractions,but increased in the basic fraction. A lower rate of glycolatesynthesis is indicative of a lower rate of photorespirationand consistent with a lower O₂/CO₂ ratio present in the bundle-sheathcells of C₄-plants. We conclude that both decreased glycolatesynthesis and the refixation of photorespiratory-released CO₂are important in maintaining a lower rate of photorespirationin C₄-plants compared to C₃ plants. Intermediate glycolate synthesisin Panicum milioldes is consistent with its intermediate levelof O₂ inhibition of photosynthesis and intermediate rate ofphotorespiration. (Received May 6, 1978; )     

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.     

Proton/Pyruvate Cotransport into Mesophyll Chloroplasts of C4 Plants

Light-enhanced active pyruvate uptake into mesophyll chloroplastsof C₄ plants was reported to be mimicked by either of the twotypes of cation jump: H⁺-jump in maize and phylogenically relatedspecies (H⁺-type) and Na⁺-jump in all the other C₄ species tested(Na⁺-type) [Aoki, N., Ohnishi, J. and Kanai, R. (1992) PlantCell Physiol. 33: 805]. In this study, medium and stromal pH was monitored in the suspensionof C₄ mesophyll chloroplasts. Medium alkalization lasting for5 to 10 seconds after pyruvate addition was detected by a pHelectrode and observed only in the light and only in mesophyllchloroplasts from H⁺-type species, Zea mays L. and Coix lacryma-jobiL., but not in those from Na⁺-type species Panicum miliaceumL., Setaria italica (L.) Beauv. and Panicum maximum Jacq. Theinitial rate of H⁺ consumption showed good correlation with[¹⁴C]pyruvate uptake measured by silicone oil filtering centrifugation,both being inhibited by N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole to the same degree. The ratio of the rate of H⁺ uptaketo that of pyruvate uptake was always about 1. Pyruvate-inducedacidification of the stroma was observed in maize mesophyllchloroplasts. These results show one to one cotransport of H⁺and pyruvate anion into mesophyll chloroplasts of H⁺-type C₄species in the light. (Received January 5, 1994; Accepted May 6, 1994)     

Effect of Low Relative Humidity on {delta}13C Value in Two C3 Grasses and in Panicum milioides, a C3-C4 Intermediate Species

When grown under conditions of low relative humidity, the C₃–C₄intermediate Panicum milioides, as well as the C₃ grasses Triticumaestivum and Poa pratense, exhibited ¹³C values which were upto 2–7%o less negative than the ¹³C values of the correspondingplants grown at high relative humidity. At both humidity levels,there was no evidence of a substantial contribution of phosphoenolpyruvatecarboxylase to carbon gain in Panicum milioides     

Sodium Requirement of Monocotyledonous C4 Plants for Growth and Nitrate Reductase Activity

The effects of Na application on growth and nitrate reductaseactivity of seven C₄ plant species, Zea mays, Echinochloa crus-galli,Panicum miliaceum, Panicum coloratum, Panicum dichotomiflorum,Panicum maximum and Chloris gayana were studied. Except forZ. mays and P. miliaceum, Na application enhanced growth significantly,and concurrent increases in nitrate reductase activities weredetected in Panicum coloratum, Panicum dichotomiflorum, Panicummaximum and Chloris gayana. ¹Present address: International Research Institute, Ciba GeigyJapan Ltd., Takarazuka, Hyogo 665, Japan. ²Present address: Photobiology Lab., Research Institute forFood Science, Kyoto Univ., Uji, Kyoto 611, Japan. (Received May 2, 1988; Accepted August 22, 1988)     

Protection of Pyruvate,Pi Dikinase from Maize against Cold Lability by Compatible Solutes

Most C₄ species are chilling sensitive and certain enzymes like pyruvate,Pi dikinase of the C₄ pathway are also cold labile. The ability of cations and compatible solutes to protect maize (Zea mays) dikinase against cold lability was examined. The enzyme in desalted extracts at pH 8 from preilluminated leaves could be protected against cold lability (at 0°C) by the divalent cations Mn²⁺, Mg²⁺, and Ca²⁺. There was substantial protection by sulfate based salts but little protection by chloride based salts of potassium or ammonium (concentration 250 millimolar). The degree of protection against cold lability under limiting MgCl₂ (5 millimolar) was pH sensitive (maximum protection at pH 8), but independent of ionic strength (up to 250 millimolar by addition of KCl). In catalysis Mg²⁺ is required and Mn²⁺ could not substitute as a cofactor. Several compatible solutes reduced or prevented the cold inactivation of dikinase (in desalted extracts and the partially purified enzyme), including glycerol, proline, glycinebetaine and trimethylamine-N-oxide (TMAO). TMAO and Mg²⁺ had an additive effect in protecting dikinase against cold inactivation. TMAO could largely substitute for the divalent cation and addition of TMAO during cold treatment prevented further inactivation. Cold inactivation was partially reversed by incubation at room temperature; with addition of TMAO reversal was complete. The temperature dependence of inactivation at pH 8 and 3 millimolar MgCl₂ was evaluated by incubation at 2 to 17°C for 45 minutes, followed by assay at room temperature. At preincubation temperatures below 11°C there was a progressive inactivation which could be prevented by TMAO (450 millimolar). The results are discussed relative to possible effects of the solutes on the quaternary structure of this enzyme, which is known to dissociate at low temperatures.     

(Na+-K+)-stimulated ATPase in Leaves of C4 Plants: Possible Involvement in Active Transport of C4 Acids

Leaves of three C₄ plants, Setaria italica, Pennisetum typhoides,and Amaranthus paniculatus possessed five- to ten-fold higheractivities of a (Na⁺-K⁺)-dependent ATPase than those of twoC₃ plants, Oryza sativa and Rumex vesicarius. Na⁺-K⁺ ATPasefrom leaves of Amarathus exhibited an optimal pH of 7?5 andan optimal temperature of 35 ?C. It required 40 mM K⁺ and 80mM Na⁺ for maximal activity. Ouabain partially inhibited (Na⁺-K⁺)-dependentATPase activity in leaves of C₄ plants. Ouabain also blockedthe movement of label from initially formed C₄ acids into endproducts in leaves of only C₄ plants, Setaria and Amaranthusbut not in a C₃ plant, Rumex. We propose that Na⁺-K⁺ ATPasemay mediate transfer of energy during active transport of C₄acids from mesophyll into the bundle sheath.     

Panicum milioides, a Gramineae plant having Kranz leaf anatomy without C4-photosynthesis

Light and electron microscopic observations of the leaf tissueof Panicum milioides showed that the bundle sheath cells containeda substantial number of chloroplasts and other organelles. Theradial arrangement of chlorenchymatous bundle sheath cells,designated as Kranz leaf anatomy, has been considered to bespecific to C₄ plants. However, photosynthetic ¹⁴CO₂ fixationand ¹⁴CO₂ pulse-and-chase experiments revealed that the reductivepentosephosphate pathway was the main route operating in leavesof P. milioides. The interveinal distance of the leaves wasintermediate between C₃and C₄Gramineae species. These resultsindicate that P. milioides is a natural plant species havingchracteristics intermediate between C₃ and C₄ types. (Received March 6, 1975; )     

Characterization of membrane-bound Mg++-activated ATPase isolated from the lower epidermis of tobacco leaves

Membrane-bound Mg⁺⁺-activated ATPase was separated from thelower epidermis of tobacco leaves (Nicotiand tabacum L. SamsunNN) on stepwise sucrose density gradient centrifugation. Membrane-bound epidermal ATPase was localized in the interfaceof densities in sucrose of 1.12 to 1.16 in the sedimentary fractionbetween 1,500?g to 10,000?g from the homogenate of the lowerepidermis. The epidermal ATPase activity was activated by divalentcations (Mg⁺⁺>Mn⁺⁺Co⁺⁺>Fe⁺⁺>Zn⁺⁺>Ca⁺⁺) and furtherstimulated by KCl by ca. 20%. The pH optimum for Mg⁺⁺-activationof the epidermal ATPase was ca. 6.0. The enzyme hydrolyzed ATPmore rapidly than other nucleoside triphosphates. The optimumtemperature for activation of the epidermal ATPase activitywas ca. 40?C. 50% of the epidermal ATPase activity was lostin 18 min at 55?C and in 2.5 days at 2.5?C. The apparent Kmvalue of the epidermal ATPase was 4.7?10^–4 M and Vmaxwas 65.4 nmoles Pi/mg protein/min. The epidermal ATPase wasstrongly inhibited by N, N'-dicyclohexylcarbodiimide (DCCD)in vitro whereas oligomycin, carbonyl cyanide m-chlorophenylhydrazone(CCGP), indoleacetic acid (IAA) and abscisic acid (ABA) wereinsensitive to the epidermal ATPase activity. (Received May 23, 1978; )     

The Occurrence of 1,3-{beta}-Glucanase in Healthy and Verticillium-infected, Resistant and Susceptible Tomato Plants

Leaf, stem, and root extracts of near-isogenic tomato plantscv. Craigella, resistant and susceptible to Verticillium albo-atrum,showed constitutive 1,3-ß-glucanase activity whichincreased following inoculation with the pathogen. Partiallypurified enzyme extracts were obtained by dialysing a 30–80%ammonium sulphate fraction of the tissue brei. The enzyme hadpH and temperature optima of 5?5 and 44 ?C respectively, withhigh activity between 50 and 60 ?C. The response to laminarinconcentration was linear between 1?2 and 7?5 mg ml^–1.Root inoculation of susceptible plants with 10⁶ propagules ml^–1V. albo-atrum led to a umform 300 per cent increase in all steminternodes except the terminal one, which was 500 per cent ofthe controls. No spatial relationship of enzyme activity tothe localization of fungus within the stem was apparent. Petioles,leaves, and roots of susceptible infected plants similarly showedan increase in activity but less than that in stems. Changedlevels of stern enzyme activity at different times after inoculationwere associated with reductions in the number of vessels containinghyphae. Extracts of plants of the resistant isoline showed increasedglucanase activity over controls, but this was substantiallylower than that in susceptible plants and was associated withthe greatly reduced mycelial colonization in resistant plants. It is concluded that single gene resistance in tomato to Verticilliumis not associated with innately higher levels of 1,3-ß-glucanasein healthy plants. The increased activity in infected plantsis proportional to the overall quantity of pathogen in the plantor of pathogenic metabolites.     

Pyruvate Uptake by Mesophyll and Bundle Sheath Chloroplasts of a C4 Plant, Panicum miliaceum L.

Intact chloroplasts were isolated from mesophyll and bundlesheath protoplasts of a C₄ plant, Panicum miliaceum L., to measurethe uptake of [1-¹⁴C]pyruvate into their sorbitol-impermeablespaces at 4?C by the silicone oil filtering centrifugation method.When incubated in the dark, both chloroplasts showed similarslow kinetics of pyruvate uptake, and the equilibrium internalconcentrations were almost equal to the external levels. Whenincubated in the light, only mesophyll chloroplasts showed remarkableenhancement of the uptake, the internal concentration reaching10–30 times of the external level after 5 min incubation.The initial uptake rate of the mesophyll chloroplasts was enhancedabout ten fold by light and was saturated with increasing pyruvateconcentration; K_m and V_max were 0.2–0.4 mM and 20–40µmol(mg Chl)^–1 h^–1, respectively. The lightenhancement was abolished by DCMU and uncoupling reagents suchas carbonylcyanide-m-chlorophenylhydrazone and nigericin. Theseresults indicate the existence of a light-dependent pyruvatetransport system in the envelope of mesophyll chloroplasts ofP. miliaceum. The uptake activity of mesophyll chloroplastsboth in the light and the dark was inhibited by sulfhydryl reagentssuch as mersalyl and p-chloromercuriphenylsulfonate, but thebundle sheath activity was insensitive to the reagents. Thesefindings are further evidence for the differentiation of mesophylland bundle sheath chloroplasts of a C₄ plant with respect tometabolite transport. (Received July 3, 1986; Accepted October 8, 1986)     

Humidity Pretreatment Affects the Responses of Stomata and CO2 Assimilation to Vapor Pressure Difference in C3 and C4 Plants

Experiments were carried out to investigate the long-term influenceof humidity on the short-term responses of stomata and CO₂ assimilationto vapor pressure difference in Oryza sativa (rice, C₃ species)and Panicum maximum (green panic, C₄ species). Plants were grownfor four weeks in growth chambers set at 35% and 85% relativehumidity at 25C air temperature, 38+2 Pa CO₂ partial pressureand 1,700µmol m^-2s^-1 photon flux density. Soil was saturatedwith water in both humidity treatments. Low humidity pretreatmentscaused low leaf conductance and low rates of transpiration andCO₂ assimilation in O. sativa, but small changes in stomatalresponses to humidity and in CO₂ assimilation were found inP. maximum. From the short-term gas exchange experiments, itwas noted that the responsiveness of leaf conductance to vaporpressure difference were affected by humidity pretreatmentsin O. sativa, whereas unaffected in P. maximum. In O. sativameasurements of CO₂ assimilation as a function of internal CO₂partial pressure (A-Ci curve) indicated that low humidity pretreatmentsreduced the CO₂ assimilation at high internal CO₂ partial pressure,but the initial slope of the A-Ci curve was unaffected. Furthermore,plant characteristics such as total dry weight and leaf areaof plants subjected to low umidity were lower than plants subjectedto high humidity. The reductions in O. sativa, however, werelarger than in P. maximum. Stomatal frequency from low humiditygrown plant was higher than that from high humidity grown plantsin both species although there is no significant difference.The data indicated that if the short term inhibition of netCO₂ assimilation at a high vapor pressure difference was imposedduring vegetative growth, the photosynthetic biochemistry andthe resultant plant growth were largely depressed in O. sativa,a C₃ species. (Received May 26, 1992; Accepted November 2, 1992)     

Purification and some properties of L-tyrosine carboxy-lyase from barley roots

L-Tyrosine carboxy-lyase (E. C. 4. 1. 1. 25) was extracted fromthe roots of barley seedlings and purified approximately 25fold. Optimum pH for the enzyme activity was found to be 7.3.The Km value for L-tyrosine was calulated as 4.5?10^–4M.D-Isomer did not react with the enzyme. L-DOPA, m-tyrosine ando-tyrosine were decarboxylated to some extent. Pyridoxal phosphateactivated the enzyme 4 fold. Caffeic acid and p-coumaric acidare competitive inhibitors. Ki values were 4.5?10^–5M forcaffeic acid and 1.6?10^–4M for p-coumaric acid. L-DOPAand m-tyrosine had an inhibitory effect on the decarboxylationof L-tyrosine. Hydroxylamine, semicarbazide, p-CMB, Fe⁺⁺, Cu⁺⁺,and Hg⁺⁺ inhibited the decarboxylation of tyrosine. Enzyme activitywas also found in extracts from Triticum aestivum, Zea maysand Cytisus scoparius. (Received November 30, 1973; )     

Differentiation of Photorespiratory Activity between Mesophyll and Bundle Sheath Cells of C4 Plants I. Glycine Oxidation by Mitochondria

Mitochondria were isolated from mesophyll protoplasts and bundlesheath protoplasts or strands which were obtained by enzymaticdigestion of six C₄ species: Zea mays, Sorghum bicolor, Panicummiliaceum, Panicum capillare, Panicum maximum and Chloris gayana,representative of three C₄ types. Photorespiratory glycine oxidationand related enzyme activities of mesophyll and bundle sheathmitochondria were compared. Mesophyll mitochondria showed good P/O ratios with malate andsuccinate as substrate but lacked the ability to oxidize glycine.On the other hand, mitochondria isolated from bundle sheathprotoplasts of P. miliaceum and bundle sheath strands of Z.mays possessed glycine oxidation activity similar to that ofmitochondria from C₃ plant leaves. The two enzymes involvedin glycine metabolism in mitochondria, serine hydroxymethyltransferaseand glycine decarboxylase, were also assayed in the mitochondriaof the two cell types. The activities of the two enzymes inbundle sheath mitochondria were in the range found in C₃ mitochondria.In contrast, the activities in mesophyll mitochondria were eithernot detectable or far lower than those in bundle sheath mitochondriaand ascribed to contaminating bundle sheath mitochondria. The present results indicate the deficiency of a complete glycineoxidation system in mesophyll mitochondria and also a differentiationbetween mesophyll and bundle sheath cells of C₄ plants withrespect to the photorespiratory activities of the mitochondria. (Received June 8, 1983; Accepted August 29, 1983)     

Carbon isotopic ratios of chemical constituents of Panicum maximum L.

Panicum maximum L. was not intermediate in ¹³C/¹²C ratios betweenC₃ and C₄-plants. Nor were starch, organic acid, and amino acidfractions significantly different from one another. (Received March 11, 1974; )     

Use of inhibitors to distinguish between C4 acid decarboxylation mechanisms in bundle sheath cells of C4 plants

Both malate and aspartate were decarboxylated at the 4-carbonposition by isolated bundle sheath strands of C₄ plants butto different extents depending upon the species. In Digitariasanguinalis, an NADP-malic enzyme (NADP-ME) species, 100 µMoxalic acid blocked malate decarboxylation through NADP-ME withoutaffecting aspartate decarboxylation which apparently occursthrough NAD-ME. In several phosphoenolpyruvate carboxykinase(PEP-CK) type C₄ species, 200 µM 3-mercaptopicolinic acid(3-MPA), an inhibitor of PEP-CK, specifically inhibited themalate decarboxylation and partially inhibited aspartate decarboxylation.The aspartate decarboxylation insensitive to 3-MPA may occurthrough NAD-ME. Neither inhibitor prevented C₄ acid decarboxylationin bundle sheath cells of NAD-ME species. The inhibitors thusserved to differentiate between the decarboxylation of C₄ acidsin PEP-CK and NADP-ME type C₄ species through their major decarboxylasefrom that of their less active decarboxylation through NAD-ME. ¹ Present address: Department of Biochemistry and Microbiology,Rutgers University, New Brunswick, NJ 08903, U. S. A. (Received January 28, 1977; )     

The Responses of Net Photosysthesis to Light and Temperature in Spartina townsendii(sensu lato), a C4 Species from a Cool Temperate Climate

Carbon dioxide and water vapour exchanges for single attachedleaves of the temperate C₄ grass Spartina townsendii were measuredunder controlled environment conditions in an open gas-exchangesystem. The responses of net photosynthesis, stomatal resistance,and residual resistance to leaf temperature and photon fluxdensity are described. The light and temperature responses ofnet photosynthesis in S. townsendii are compared to informationon these responses in both temperate C₃ grasses and sub-tropicalC₄ grasses. Adaptation of photosynthesis in this C₄ speciesto a cool temperate climate is indicated both by the light andtemperature responses of net photo-synthesis. Unlike the C₄grasses examined previously, significant rates of net photosynthesiscan be detected at leaf temperatures below 10?C. Rates of netphotosynthesis equal or exceed those reported for temperateC₃ grasses at all of the temperature (5–40?C) and photonflax density (13–2500µmol m^–2 s^–1) conditionsexamined. Maximum rates of net photosynthesis in S. townsendiiare almost double those reported for C₃ herbage grasses. Unliketemperate C₃ grasses, the major limitation to net photosynthesisat low leaf temperatures (10?C and below) is the stomatal resistance,showing that the low residual resistance characteristic of C₄species is maintained in S. townsendii even at low leaf temperatures.     

Photosynthetic Carbon Metabolism in an Amphibious Sedge, Eleocharis baldwinii (Torr.) Chapman: Modified Expression of C4 Characteristics under Submerged Aquatic Conditions

The photosynthetic characteristics of Eleocharis baldwinii (Torr.)Chapman, an amphibious leafless plant in the Cyperaceae, wereinvestigated in both the terrestrial form and the submergedform of the plant. Anatomical observation of the culm, whichis the photosynthetic organ in this plant, revealed that theterrestrial form has the Kranz type of anatomy, whereas thesubmerged form has an inner structure that is similar to thatof submerged aquatic plants, with a reduction in both the numberand the size of bundle sheath cells and vascular bundles andrelatively well developed mesophyll cells. In ¹⁴C-pulse ¹²C-chaseexperiments with the terrestrial form, 80% of the total fixed¹⁴C was incorporated into C₄ dicarboxylic acids after a 10-spulse. The radioactivity in the C₄ acids decreased rapidly,while that in sucrose increased to 36% during a 120-s chase.In the submerged form, 64% and 30% of the total fixed ¹⁴C wasincorporated into C₄ acids and phosphate esters, respectively,after a 10-s pulse. The radioactivity of these compounds decreasedrelatively slowly during a 120-s chase. The specific activitieson a chlorophyll basis of C₄ photosynthetic enzymes that areinvolved in the NAD-ME subtype were high in the terrestrialform, while they were intermediate between those of C₃ and C₄plants in the submerged form. The activity of ribulose 1,5-bisphosphatecarboxylase was 1.5 times higher in the submerged form thanin the terrestrial form. By contrast, the activity of carbonicanhydrase exhibited the reverse tendency. Western blot analysisof soluble proteins extracted from the mesophyll cells and thebundle sheath strands of the terrestrial form demonstrated thatribulose 1,5-bisphosphate carboxylase/oxygenase protein waspresent in the mesophyll cells as well as in the bundle sheathcells, with a higher level in the latter, although phosphoenolpyruvatecarboxylase and pyruvate, P_i dikinase proteins were restrictedto the mesophyll cells. In the submerged form, diurnal fluctuationsin levels of malate were observed with significant fixationof CO₂ at night. However, the diurnal changes of malate weresmaller than those reported for CAM plants. These data indicatethat the terrestrial form of Eleocharis baldwinii fixes atmosphericCO₂ essentially via the C₄ pathway, while the submerged formfixes inorganic carbon via a complex metabolic system that resemblesan intermediate between C₃ and C₄ metabolism in associationwith a CAM-like profile. (Received September 12, 1994; Accepted November 21, 1994)