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; )     

Salsola arbusculiformis, a C3-C4Intermediate in Salsoleae (Chenopodiaceae)

Salsola arbusculiformis is identified as a C₃–C₄intermediatespecies based on anatomical, biochemical and physiological characteristics.This is the first report of a naturally occurring intermediatespecies in the Chenopodiaceae, the family with the largest numberof C₄species amongst the dicots. In the genus Salsola, mostspecies have Salsoloid anatomy with Kranz type bundle sheathcells and C₄photosynthesis, while a few species have Sympegmoidanatomy and were found to have non-Kranz type bundle sheathcells and C₃photosynthesis. In the cylindrical leaves of C₄Salsolawith Salsoloid type anatomy, there is a continuous layer ofdistinct, chlorenchymatous Kranz type bundle sheath cells surroundedby a single layer of mesophyll cells; whereas species with Sympegmoidtype anatomy have an indistinct bundle sheath with few chloroplastsand multiple layers of chlorenchymatous mesophyll cells. However,S. arbusculiformis has intermediate anatomical features. Whileit has two-to-three layers of mesophyll cells, characteristicof Sympegmoid anatomy, it has distinctive, Kranz-like bundlesheath cells with numerous chloroplasts and mitochondria. Measurementsof its CO₂compensation point and CO₂response of photosynthesisshow S. arbusculiformis functions as an intermediate specieswith reduced levels of photorespiration. The primary means ofreducing photorespiration is suggested to be by refixing photorespiredCO₂in bundle sheath cells, since analysis of photosyntheticenzymes (activity and immunolocalization) and¹⁴CO₂labellingof initial fixation products suggests minimal operation of aC₄cycle. Copyright 2001 Annals of Botany Company Immunolocalization, photosynthetic enzymes, C₃–C₄intermediate, C₄-plants, leaf anatomy, Chenopodiaceae, Salsola arbusculiformis     

Distribution of enzymes related to C3 and C4 pathway of photosynthesis between mesophyll and bundle sheath cells of Panicum hians and Panicum milioides

Enzymes of the C₄, C₃ pathway and photorespiration have beenanalyzed for P. hians and P. milioides, which have chlorenchymatousbundle sheath cells in the leaves. On whole leaf extracts thelevels of PEP carboxylase are relatively low compared to C₄species, RuDP carboxylase is typical of C₃ species, and enzymesof photorespiratory metabolism appear somewhat intermediatebetween C₃ and C₄. Substantial levels of PEP carboxylase, RuDPcarboxylase, and photorespiratory enzymes were found in bothmesophyll and bundle sheath cells. Low levels of C₄-acid decarboxylatingenzymes may limit the capacity for C₄ photosynthesis in P. hiansand P. milioides. The results on enzyme activity and distributionbetween mesophyll and bundle sheath cells are consistent withCO₂ fixation via C₃ pathway in these two species. ¹ This research was supported by the College of Agriculturaland Life Sciences, University of Wisconsin, Madison; and bythe University of Wisconsin Research Committee with funds fromthe Wisconsin Alumni Research Foundation; and by the NationalScience Foundation Grant BMS 74-09611. (Received September 16, 1975; )     

Effects of Light Quality on Photosynthetic Carbon Metabolism in C4 and C3 Plants: Rapid Movements of Photosynthetic Intermediates Between Mesophyll and Bundle Sheath Cells

The influence of varying light intensity and quality on thecarbon labelling patterns in Rumex vesicarius (a C₃ plant),Setaria italica (a malate-formingC₄ plant), and Amaranthus paniculatus(an aspartate-forming C₄ plant) was studied. In A. paniculatusand B. vesicarius blue light decreased the transfer of radioactivityto sugars and starch but in S. italica only slightly decreasedradioactivity in sugar phosphates, sucrose, and insolubles.Negligible transfer was observed from the C₄ acids to sugarphosphates, sucrose, and starch under dim blue-green and blue-yellowlights in S. italica and A. paniculatus. Blue light favouredthe formation of malate, aspartate, and alanine in all threeplants. The differential effect of blue and red light suggesteda variation in the mechanisms of C₄-photosynthesis in Setariaand Amaranthus. Leaves of S. italica and A. paniculatus were allowed to photosynthesizein ¹⁴CO₂ for 5 s and then the distribution of the labelled productsbetween the mesophyll and the bundle sheath cells was determinedduring subsequent photosynthesis in ¹²CO₂. Malate and aspartatewhich appeared initially in the mesophyll layer moved rapidlyinto the bundle sheath cells. Phosphoglyceric acid originatingin the bundle sheath moved swiftly to the mesophyll layer. Sugarphosphates were recovered from both the mesophyll and the bundlesheath cells. Most of the starch was found in the bundle sheathcells while sucrose and alanine were localized in the mesophyllcells.     

Photosynthesis in Cell Suspension Culture of a C4 Plant, Gisekia pharnaceoides L.

Photomixotrophic cell suspension culture was established fromthe leaf derived callus cells of Gisekia pharnaceoides L., aC₄ dicotyledonous weed. The late log phase cells possessed shade-typecharacters such as low chlorophyll a/b ratio, less pronouncedO₂ evolution and CO₂ fixation, saturation of photosyntheticCO₂ fixation at low intensity. The chloroplasts from these cellscontained granal stacking with high degree of a very few granawhich are characterized by their wide and high degree of stackings. The predominant labelling of 3-phosphoglyceric acid and sugarphosphates (40% of the total ¹⁴C incorporated) during 5 s exposureto ¹⁴CO₂ in light and subsequent decrease in percentage of ¹⁴Cin these compounds with increase in exposure time indicatedthe operation of the C₃ pathway in these cells. The simultaneoussynthesis of malate (23% of the total ¹⁴C incorporated) is relatedto the much pronounced glycolytic and tricarboxylic acid cycleactivities in these cells. The initial proliferation of callimainly from the zones of vascular supplies in the leaf, highstarch content of the cells, presence of large starch grainsin all the chloroplasts, activities of Calvin cycle enzymes,heavy labelling of C₃ type intermediates and less labellingof aspartate as early photosynthates and rapid accumulationof radioactivity into starch during ¹⁴CO₂ assimilation indicatedthat most of the cells in photomixotrophic culture were derivedfrom bundle sheath cells or the leaf cells of Gisekia changetheir function under culture conditions. ¹Present address: Tropical Botanic Garden and Research Institute,Navaranga Road, Trivandrum 695 011, India. (Received January 29, 1982; Accepted June 4, 1983)     

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; )     

Effects of Anoxia on Phloem Loading in C3 and C4 Species

Changes in phloem loading rate were inferred from observationsof ¹¹C export from attached leaves of C₃ and C₄ monocots anddicots. Loading decreased under anoxia in C₃ leaves, but notin general in the leaves of either C₄ monocots or dicots inthe light. However, loading rate in the C₄ leaves did reduceif the leaf was also darkened or received no CO₂. We suggestthat insensitivity to anoxia in C₄ leaves is due to oxygen fromphotosynthesis which is retained in the bundle sheath at a concentrationsufficient to energize a phloem loading system. There may alsobe another system which is insensitive to anoxia since the effectsof shade and CO₂ deprivation were not always seen, and loadingwas not completely stopped by these treatments. Key words: Phloem loading, C₃, C₄     

(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.     

Photosynthetic Characteristics of Cassava (Manihot esculenta Crantz), a C3 Species with Chlorenchymatous Bundle Sheath Cells

Cultivars of cassava, Manihot esculenta Crantz, were studiedto determine the mechanism of photosynthetic carbon assimilationin this species. The results, contrary to recent reports, indicatethat cassava is a C₃ plant based on a number of physiologicaland biochemical photosynthetic characteristics. The CO₂ compensationpoints among 10 cassava cultivars ranged from 55 to 62 µlliter^–1, which was typical for C₃ plants including castorbean, a member of the same family (Euphorbiaceae). The initialproducts of photosynthesis in cassava are C₃-like; the activitiesof several key C₄ enzymes in cassava are low and similar tothose of C₃ plants. Data on the rates of photosynthesis perunit of leaf area and the photosynthetic response of cassavato CO₂ is also consistent with C₃ photosynthesis. Cassava hasa distinctive chlorenchymatous vascular bundle sheath locatedbelow a single layer of palisade cells. Unlike C₃-C₄ intermediatesand C₄ species, the bundle sheaths of cassava are not surroundedby mesophyll cells. The bundle sheath cells which occur at highfrequency in cassava may function in both photosynthesis andtransport of photosynthates in the leaf. (Received July 31, 1990; Accepted September 25, 1990)     

The Influence of Leaf Age on C(4) Photosynthesis and the Accumulation of Inorganic Carbon in Flaveria trinervia, a C(4) Dicot

Characteristics of C₄ photosynthesis were examined in young, mid-age, and mature leaves of Flaveria trinervia (an NADP-malic enzyme-type C₄ dicot). The turnover of [4-¹⁴C] (malate plus aspartate) following a pulse with ¹⁴CO₂ was similar in leaves of different ages (apparent half-time of 18-25 seconds). However, the rate of ¹⁴CO₂ incorporation in mid-age leaves was about 1.5-fold higher than in young leaves, and about 2.5-fold higher than in mature leaves. The rate of ¹⁴CO₂ fixation was proportional to the total active pool of malate plus aspartate but was not correlated with the total photosynthetically derived inorganic carbon pool. The leaf's ability to concentrate inorganic carbon photosynthetically declined during leaf expansion, from 29 down to 7 nanomoles per milligram chlorophyll. Similarly, the active aspartate pool also declined during leaf expansion, from about 123 down to 20 nanomoles per milligram chlorophyll. Enhanced metabolism of aspartate to CO₂ and pyruvate in young leaves is suggested to facilitate the maintenance of high CO₂ levels in bundle sheath cells which are thought to have a higher conductance to CO₂.     

Striga hermonthica decreases photosynthesis in Zea mays through effects on leaf cell structure

Maize seedlings were grown in pots either with or without preconditionedseeds of the parasitic weed, Striga hermonthica. After between4 and 8 weeks, net photosynthesis in the leaves of maize plantsinfected with Striga decreased compared to leaves of uninfectedcontrol plants. The activities of four enzymes of photosyntheticmetabolism were, however, little affected by infection. A pulse-chaseexperiment using ¹⁴CO₂ showed that C₄ acids were the main earlyproducts of assimilation even when the rate of photosynthesiswas much decreased by infection, but more radio-activity appearedin glycine and serine than in leaves of healthy maize plants.Leaves of infected maize required longer to reach a steady rateof photosynthesis upon enclosure in a leaf chamber than leavesof uninfected plants after similar treatment. Electron microscopy of transverse sections of the leaves ofinfected maize indicated that the cell walls in the bundle sheathand vascular tissue were less robust than in leaves of healthyplants. The results suggest that infection with Striga causesan increase in the permeability of cell walls in the bundlesheath, leakage of CO₂ from the bundle sheath cells and decreasedeffectiveness of C₄ photosynthesis in host leaves. Key words: Zea mays, Striga hermonthica, photosynthesis, photorespiration, enzyme activity     

Maximum Quantum Yields of O2 Evolution in C4 Plants under High CO2

The quantum yields of photosynthetic O₂ evolution were measuredin 15 species of C₄ plants belonging to three different decarboxylationtypes (NADP-ME type, NAD-ME type and PEP-CK type) and 5 speciesof C₃ plants and evaluated relative to the maximum theoreticalvalue of 0.125 mol oxygen quanta^-1. At 25°C and 1% CO₂,the quantum yield in C₄ plants averaged 0.079 (differences betweensubgroups not significant) which was significantly lower thanthe quantum yield in C₃ plants (average of 0.105 for 5 species).This lower quantum yield in C₄ plants is thought to reflectthe requirement of energy in the C₄ cycle. For the C₄ NADP-MEtype plant Z. mays and NAD-ME type plant P. miliaceum, quantumyields were also measured over a range of CO₂ levels between1 and 20%. In both species maximum quantum yields were obtainedunder 10% CO₂ (0.105 O₂ quanta_-1 in Z. mays and 0.097 O₂ quanta_-1in P. miliaceum) indicating that at this CO₂ concentration thequantum yields are similar to those obtained in C₃ plants underCO₂ saturation. The high quantum yield values in C₄ plants undervery high CO₂ may be accomplished by direct diffusion of atmosphericCO₂ to bundle sheath cells, its fixation in the C₃ pathway,and feedback inhibition of the C₄ cycle by inorganic carbon. (Received June 6, 1995; Accepted August 15, 1995)     

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; )     

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)     

Gas Exchange and Carbohydrate and Nitrogen Concentrations in Leaves of Pascopyrum smithii (C3) and Bouteloua gracilis (C4) at Different Carbon Dioxide Concentrations and Temperatures

Pascopyrum smithii (C₃) andBouteloua gracilis (C₄) are importantforage grasses native to the Colorado shortgrass steppe. Thisstudy investigated photosynthetic responses of these grassesto long-term CO₂enrichment and temperature in relation to leafnonstructural carbohydrate (TNC) and [N]. Glasshouse-grown seedlingswere transferred to growth chambers and grown for 49 d at twoCO₂concentrations (380 and 750 µmol mol^-1) at 20 and 35°C, and two additional temperatures (25 and 30 °C) at750 µmol mol^-1CO₂. Leaf CO₂exchange rate (CER) was measuredat a plant's respective growth temperature and at two CO₂concentrationsof approx. 380 and 700 µmol mol^-1. Long-term CO₂enrichmentstimulated CER in both species, although the response was greaterin the C₃,P. smithii . Doubling the [CO₂] from 380 to 750 µmolmol^-1stimulated CER ofP. smithii slightly more in plants grownand measured at 30 °C compared to plants grown at 20, 25or 35 °C. CO₂-enriched plants sometimes exhibited lowerCER when compared to ambient-grown controls measured at thesame [CO₂], indicating photosynthetic acclimation to CO₂growthregime. InP. smithii , such reductions in CER were associatedwith increases in TNC and specific leaf mass, reductions inleaf [N] and, in one instance, a reduction in leaf conductancecompared to controls. InB. gracilis , photosynthetic acclimationwas observed more often, but significant changes in leaf metabolitelevels from growth at different [CO₂] were generally less evident.Temperatures considered optimal for growth (C₃: 20 °C; C₄:35 °C) sometimes led to CO₂-induced accumulations of TNCin both species, with starch accumulating in the leaves of bothspecies, and fructans accumulating only inP. smithii. Photosynthesisof both species is likely to be enhanced in future CO₂-enrichedand warmer environments, although responses will sometimes beattenuated by acclimation. Acclimation; blue grama (Bouteloua gracilis (H.B.K.) Lag ex Steud.); leaf nitrogen concentration; nonstructural carbohydrates; photosynthesis; western wheatgrass (Pascopyrum smithii (Rydb.) Love)     

Carbon dioxide assimilation and photosystem II deficiency in bundle sheath strands isolated from C4 plants

The activities of Hill reaction and photosynthetic ¹⁴CO₂ fixationin bundle sheath strands enzymatically isolated from millet(Panicum miliaceum) were 3–15 times as high as those observedin corn (Zea mays). In both preparations, 3-phosphoglyceratewas the initial ¹⁴CO₂ fixation product and the radioactivitywas incorporated into sucrose and insoluble compounds (glucose-polymers)during the later period. After 20 sec of photosynthetic ¹⁴CO₂fixation, the percent of ¹⁴C incorporated into sugar phosphatesin millet was about 3 times as high as that in corn, while incorn, the percent of ¹⁴C in 3-phosphoglycerate was higher thanthat observed in millet throughout the experimental period.When ¹⁴C-phosphoglycerate was added to the isolated bundle sheathstrands, the rates of transfer of the radioactivity to dihydroxyacetonephosphate and sugar diphosphates in millet were significantlyhigher than those in corn. These results indicate that in thebundle sheath strands isolated from corn in which photosystemII activity is deficient, the reductive pentose cycle is impairedat the reduction step of 3-phosphoglycerate to glyceraldehydephosphate due to the limited supply of NADPH through the photoelectrontransport system. In contrast, the bundle sheath strands isolatedfrom millet which have adequate photosystem II activity cancarry out normal photosynthetic CO₂ fixation. (Received January 23, 1975; )     

Leaf anatomy, post-illumination CO2 burst and NAD-malic enzyme activity of Panicum dichotomiflorum

Light microscopic observation of leaf blades of Panicum dichotomiflorumshowed that a mestome sheath was present and chloroplasts inbundle sheath cells were in the centrifugal position. However,a sharp pattern of post-illumination CO₂ burst was observedin less than 30 sec after the extinction of light. Among threeC₄-acid decarboxylating enzymes, only the activity of NAD-malicenzyme was high. These results indicate that P. dichotomiflorumis a NAD-malic enzyme type species having centrifugal chloroplastsin bundle sheath cells and the sharp pattern of post-illuminationCO₂ burst is closely correlated with the C₄-acid decarboxylationsystem through NAD-malic enzyme ¹This research was supported by a grant from the Ministry ofAgriculture, Forestry and Fishery (GEP55-II-1-7). (Received August 18, 1980; )     

Leaf anatomy, post-illumination CO2 burst and NAD-malic enzyme activity of Panicum dichotomiflorum

Light microscopic observation of leaf blades of Panicum dichotomiflorumshowed that a mestome sheath was present and chloroplasts inbundle sheath cells were in the centrifugal position. However,a sharp pattern of post-illumination CO₂ burst was observedin less than 30 sec after the extinction of light. Among threeC₄-acid decarboxylating enzymes, only the activity of NAD-malicenzyme was high. These results indicate that P. dichotomiflorumis a NAD-malic enzyme type species having centrifugal chloroplastsin bundle sheath cells and the sharp pattern of post-illuminationCO₂ burst is closely correlated with the C₄-acid decarboxylationsystem through NAD-malic enzyme ¹This research was supported by a grant from the Ministry ofAgriculture, Forestry and Fishery (GEP55-II-1-7). (Received August 18, 1980; )     

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)     

Effects of Prolonged Darkness on the Sensitivity of Leaf Respiration to Carbon Dioxide Concentration in C3and C4Species

Predicting responses of plant and global carbon balance to theincreasing concentration of carbon dioxide in the atmosphererequires an understanding of the response of plant respirationto carbon dioxide concentration ([CO₂]). Direct effects of thecarbon dioxide concentration at which rates of respiration ofplant tissue are measured are quite variable and their effectsremain controversial. One possible source of variation in responsivenessis the energy status of the tissue, which could influence thecontrol coefficients of enzymes, such as cytochrome-c oxidase,whose activity is sensitive to [CO₂]. In this study we comparedresponses of respiration rate to [CO₂] over the range of 60to 1000 µmol mol^-1in fully expanded leaves of four C₃andfour C₄herbaceous species. Responses were measured near themiddle of the normal 10 h dark period, and also after another24 h of darkness. On average, rates of respiration were reducedabout 70% by the prolonged dark period, and leaf dry mass perunit area decreased about 30%. In all species studied, the relativedecrease in respiration rate with increasing [CO₂] was largerafter prolonged darkness. In the C₃species, rates measured at1000 µmol mol^-1CO₂averaged 0.89 of those measured at 60µmol mol^-1in the middle of the normal dark period, and0.70-times when measured after prolonged darkness. In the C₄species,rates measured at 1000 µmol mol^-1CO₂averaged 0.79 of thoseat 60 µmol mol^-1CO₂in the middle of the normal dark period,and 0.51-times when measured after prolonged darkness. In threeof the C₃species and one of the C₄species, the decrease in theabsolute respiration rate between 60 and 1000 µmol mol^-1CO₂wasessentially the same in the middle of the normal night periodand after prolonged darkness. In the other species, the decreasein the absolute rate of respiration with increase in [CO₂] wassubstantially less after prolonged darkness than in the middleof the normal night period. These results indicated that increasingthe [CO₂] at the time of measurement decreased respiration inall species examined, and that this effect was relatively largerin tissues in which the respiration rate was substrate-limited.The larger relative effect of [CO₂] on respiration in tissuesafter prolonged darkness is evidence against a controlling roleof cytochrome-c oxidase in the direct effects of [CO₂] on respiration.Copyright 2001 Annals of Botany Company Carbon dioxide, respiration, Abutilon theophrasti(L.), Amaranthus retroflexus(L.),Amaranthus hypochondriacus (L.), Datura stramonium(L.), Helianthus annuus(L.), Solanum melongena(L.), Sorghum bicolor(L. Moench), Zea mays