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)     

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; )     

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)     

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; )     

Influence of Organic-Phosphates on 3-Phosphoglycerate Dependent O2 Evolution in C3 and C4 Mesophyll Chloroplasts

In C₄ plants phosphoenolpyruvate (PEP) of the C₄ cycle may betransported on a chloroplast transporter which also transports3-phosphoglycerate (3-PGA) and triosephosphates. In C₃ plantsPEP is not considered to be effectively transported on the chloroplastphosphate translocator. The influences of certain organic phosphates,having a similar structure to either PEP or triose-phosphates,on 3-PGA dependent O₂ evolution by C₄ (Digitaria sanquinalisL. Scop.) and C₃ (Hordeum vulgare L.) mesophyll chloroplastswere investigated. In the C₄ mesophyll chloroplasts phosphoglycolatewas a competitive inhibitor (K_i = 2.1 mM) of 3-PGA dependentO₂ evolution, and was as effective as previously reported forPEP. 2-Phosphoglycerate was also a competitive inhibitor (K_t= 8.6 mM) of O₂ evolution in the C₄ mesophyll chloroplasts with3-PGA as substrate, while phospholactate was a weak inhibitorand glyphosate had no effect. Neither PEP, phosphoglycolatenor 2-phosphoglycerate were effective inhibitors of 3- PGA dependentO₂ evolution in the C₃ chloroplasts. Phosphohydroxypyruvatewas a competitive inhibitor of 3-PGA dependent O₂2 evolutionin both chloroplast types. The selectivity in inhibition ofO₂ evolution with 3-PGA as substrate suggests that the C₄ mesophyllchloroplasts can recognize certain organic phosphates with thephosphate in the C-2 or C-3 position but that the C₄ mesophyllchloroplasts can only effectively recognize certain organicphosphates with the phosphate in the C-3 position. The resultsalso support the view that 3-PGA and PEP are transported onthe same phosphate translocator in C₄ mesophyll chloroplasts. ¹ Current address: Department of Horticulture, 2001 Fyffe Court,The Ohio State University, Columbus, Ohio 43210-1096. (Received March 24, 1987; Accepted April 16, 1987)     

Intracellular localization of certain photosynthetic enzymes in bundle sheath cells of plants possessing the C4 pathway of photosynthesis.

Bundle sheath cells were enzymatically isolated from representatives of three groups of C₄ plants: Zea mays (NADP malic enzyme type), Panicum miliaceum (NAD malic enzyme type), and Panicum maximum (phosphoenolpyruvate (PEP) carboxykinase type). Cellular organelles from bundle sheath homogenates were partially resolved by differential centrifugation and on isopycnic sucrose density gradients in order to study compartmentation of photosynthetic enzymes. A 48-h-dark pretreatment of the leaves allowed the isolation of relatively intact chloroplasts. Enzymes that decarboxylate C₄ acids and furnish CO₂ to the Calvin cycle are localized as follows: NADP malic enzyme, chloroplastic in Z. mays; NAD malic enzyme, mitochondrial in all three species; PEP carboxykinase, chloroplastic in P. maximum. The activity of NAD malic enzyme in the three species was in the order of P. miliaceum > P. maximum > Z. mays. There were high levels of aspartate and alanine aminotransferases in bundle sheath extracts of P. miliaceum and P. maximum and substantial activity in Z. mays. In all three species, aspartate aminotransferase was mitochondrial whereas alanine aminotransferase was cytoplasmic. Based on the activity and localization of certain enzymes, the concept for aspartate and malate as transport metabolites from mesophyll to bundle sheath cells in C₄ species of the three C₄ groups is discussed.     

Some Factors Concerning the Centripetal Disposition of Bundle Sheath Chloroplasts during the Leaf Development of Eleusine coracana

Factors concerning the chloroplast disposition in bundle sheathcells were investigated in finger millet (Eleusine coracanaGaertn.), and NAD malic enzyme type C₄ plant with the centripetalarrangement of bundle sheath chloroplasts. Segments were cutfrom immature regions of emerging leaves in which the centripetalarrangement of bundle sheath chloroplasts had not yet been established.The leaf segments were floated on solutions with or withoutreagents. Sections were made of the segments at time intervalsand the distribution of bundle sheath chloroplasts was observedby light microscopy. The bundle sheath chloroplasts migratedto the vascular bundle and established a centripetal arrangementby 12-16 h in control solutions. Auxins, cycloheximide and cytochalasinB inhibited the disposition of bundle sheath chloroplasts whilechloramphenicol and colchicine had no effect. The inhibitoryeffect of auxins appeared only at early stages of chloroplastmigration while cycloheximide and cytochalasin B were effectiveeven at later stages. Cessation of elongation growth, cytoplasmicprotein synthesis and microfilaments seemed to be associatedwith the centripetal disposition of bundle sheath chloroplasts.Copyright1993, 1999 Academic Press Bundle sheath chloroplast, C₄ plant, chloroplast orientation, Eleusine coracana, finger millet     

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)     

Induction of Glucose 6-Phosphate Transport Activity in Chloroplasts of Mesembryanthemum crystallinum by the C3-CAM Transition

To study possible changes in the transport metabolites betweenchloroplasts and cytoplasm during CAM induction of Mesembryanthemumcrystallinum, we compared substrate specificity of P1¹ translocator(s)in isolated chloroplasts from the C₃ and CAM-induced plants.The [¹⁴C]glu-cose 6-phosphate (G6P) transport activity was significantonly in the chloroplasts of CAM-mode plants and not detectablein those of C₃-mode, while a similar high rate of [³²P]P_i uptakewas observed with both types of chloroplasts. Kinetic analysisof G6P uptake in the CAM chloroplasts showed a high V_max [10.6µmol (mg Chl)^–1 h^–1] and a comparatively lowK_m value (0.41 mM); the latter was similar to K_i values of P_i,3-phosphoglycerate and phospho-enolpyruvate, 0.30, 0.34 and0.47 mM, respectively. On the other hand, [32P]Pi uptake inthe CAM chloroplasts was inhibited competitively by G6P witha K_i value (8.4 mM) 20-fold higher than the K_m value for G6Puptake, while that in C₃ chloroplasts was not inhibited at all.These results suggest that a new G6P/P_i, counterexchange mechanismis induced in the chloroplast envelope of CAM-induced M. crystallinumin addition to the ordinary type of P, translocator, that cannottransport G6P, already present in the C₃-type chloroplasts. (Received March 17, 1997; Accepted May 10, 1997)     

Characterization of C4 Type Leaf Anatomy in Grasses (Poaceae). Mesophyll: Bundle Sheath Area Ratios

The cross-sectional area of ‘primary carbon assimilation’(PCA) (or mesophyll) tissue and of ‘photosynthetic carbonreduction’ (PCR) (or parenchymatous bundle sheath, PBS)tissue associated with each vein has been measured in transversesections of leaf blades of 124 grass species (Poaceae). Thespecies sample is representative of all major grass taxa, andof all photosynthetic types found in this family, viz. C₃, C₃/C₄intermediate, C₄ NADP-malic enzyme type (NADP-ME), C₄ NAD-malicenzyme type (NAD-ME) and PEP carboxykinase type (PCK). MeanPCA (or mesophyll) area per vein varies between photosynthetictypes in the order C₃ > NAD-ME > PCK = NADP-ME, mean PCR(or PBS) area per vein in the order NAD-ME > PCK = C₃ >NADP-ME, and mean PCA/PCR (or mesophyll/PBS) area ratio in theorder C₃ > NADP-ME > NAD-ME > PCK. Since grass leaveshave parallel venation, tissue areas and area ratios are directlyproportional to tissue volumes and volume ratios. Regressionanalyses of plots of PCA (or mesophyll) area per vein againstPCR (or PBS) area per vein yield characteristic slopes for photosynthetictypes. Differences between types in all these parameters arenearly always statistically significant, even within high leveltaxonomic groups (Eupanicoids and Chloridoids). However, differencesbetween major taxa (Eupanicoids, Andropogonoids, Chloridoids),within a photosynthetic type, are frequently not significant.This histometric characterization of photosynthetic types isdiscussed in relation to the co-operation of PCA and PCR tissuesin C₄ photosynthesis, to possible differences between C₄ typesin PCR spatial requirements and to the developmental originof PCR tissue. Grasses, Poaceae, C₄ photosynthesis, C₄ leaf blade anatomy, ‘Kranz’, NADP-malic enzyme, NAD-malic enzyme, PEP carboxykinase, PCA tissue, PCR tissue, taxonomy     

Centripetal Disposition of Bundle Sheath Chloroplasts During the Leaf Development of Eleusine coracana

Distribution of chloroplasts in bundle sheath cells was examinedby light and electron microscopy during the leaf developmentof finger millet (Eleusine coracana Gaertn.), an NAD malic enzymetype C₄ plant with centripetal arrangement of bundle sheathchloroplasts. Young chloroplasts are almost evenly distributedalong the cell walls in bundle sheath cells of folded immatureleaves. In elongating leaves and above the elongation zone thebundle sheath chloroplasts tend to lie along the radial wallsand the walls adjacent to the vascular bundle. They furthermigrate near to the vascular bundle and finally establish acentripetal arrangement. Mitochondria, microbodies and nucleusmigrate along with the chloroplasts. Etioplasts and other organellesare centripetally located in the bundle sheath cells of etiolatedseedlings grown in the dark. Bundle sheath chloroplast, C₄ plant, chloroplast, chloroplast orientation, Eleusine coracana, finger millet     

Polypeptide Composition of Envelope Membranes Isolated from Chloroplasts of C3, C4, and CAM Plants

Chloroplast envelopes were isolated from chloroplasts purifiedfrom Spinacea oleracea L. (C₃), Panicum miliaceum L. (NAD-malicenzyme-type C₁), Digitaria sanguinalis (L.) Scop. (NADP-malicenzyme-type C₄), Kalanchoe daigremontiana Hamet et Perrier (constitutiveCAM), and from Mesembryanthemum crystallinum L. (inducible CAM)performing either C₃ photosynthesis or Crassulacean acid metabolism(CAM). For each species, methods were developed to isolate chloroplastenvelopes free of thylakoid contamination. The polypeptidesof ribulose bisphosphate (RuBP) carboxylase which has been consistentlyreported in envelope preparations of spinach were not foundin envelope preparations of C₄ mesophyll chloroplasts. Silverstaining of envelope polypeptides resolved electrophoreticallyon sodium dodecylsulfate polyacrylamide gradient slab gels produceda more complex profile than did Coomassie staining which haspreviously been used with C₃ envelope preparations, even thoughsilver reacted poorly with polypeptides corresponding to thesubunits of RuBP carboxylase. All of the plants examined possesseda major polypeptide of 27 to 29 kilodaltons (kD) which was previouslysuggested to be the phosphate translocator in spinach. WithC₃ M. crystallinum, the 29 kD polypeptide stained most intensely.After induction of CAM, a 32 kD polypeptide also stained intensely,giving a profile similar to that obtained with the constitutiveCAM species. A 32 kD polypeptide was also prominent in C₄ envelopepreparations, suggesting that a 32 kD polypeptide may be a translocatorprotein which is required in Crassulacean acid metabolism andC4 photosynthesis, but not in C₃ photosynthesis. (Received April 25, 1983; Accepted July 9, 1983)     

Differential Positioning of C4 Mesophyll and Bundle Sheath Chloroplasts: Recovery of Chloroplast Positioning Requires the Actomyosin System

In C₄ plants, bundle sheath (BS) chloroplasts are arranged inthe centripetal position or in the centrifugal position, althoughmesophyll (M) chloroplasts are evenly distributed along cellmembranes. To examine the molecular mechanism for the intracellulardisposition of these chloroplasts, we observed the distributionof actin filaments in BS and M cells of the C₄ plants fingermillet (Eleusine coracana) and maize (Zea mays) using immunofluorescence.Fine actin filaments encircled chloroplasts in both cell types,and an actin network was observed adjacent to plasma membranes.The intracellular disposition of both chloroplasts in fingermillet was disrupted by centrifugal force but recovered within2 h in the dark. Actin filaments remained associated with chloroplastsduring recovery. We also examined the effects of inhibitorson the rearrangement of chloroplasts. Inhibitors of actin polymerization,myosin-based activities and cytosolic protein synthesis blockedmigration of chloroplasts. In contrast, a microtubule-depolymerizingdrug had no effect. These results show that C₄ plants possessa mechanism for keeping chloroplasts in the home position whichis dependent on the actomyosin system and cytosolic proteinsynthesis but not tubulin or light.     

Variations in the Leaf Anatomy among some C4 Panicum Species

The Dichotomiflora group of Panicum contains NAD-malic enzyme(ME) species with centrifugal chloroplasts in Kranz cells, NAD-ME(F)species as well as NAD-ME species with centripetal chloroplastsin Kranz cells, NAD-ME (P) species. Many attributes of leafanatomy of 22 C₄ Panicum species were investigated to identifydifferences among four different C₄ subtypes, i.e. NADP-ME,NAD-ME(F), NAD-ME(P) and PEP-CK species grouped by the C₄-aciddecarboxylating enzymes and chloroplast location in Kranz cellsin combination. Differences were found in the number of Kranzcells surrounding a large vein, and the number surrounding asmall vein, the interveinal distances, the proportion of leafcross sectional area occupied by epidermis plus sclerenchyma,by mesophyll cells, by Kranz cells, and by vascular bundles.There were also differences in the ratios of the area of thedifferent cell types. The number of the characters significantlydifferent between a respective pair of C₄ subtypes was the largestbetween NAD-ME(F) and NAD-ME(P) species. In principal componentanalysis applied to 11 leaf anatomical characters, the differentC₄ subtypes clustered into small groups, although the rangeof variations of PEP-CK species and those of NAD-ME(F) speciesoverlapped. The results were discussed in relation to taxonomyand ecological adaptation of Panicum species in the differentC₄ subtypes. C₄ photosynthesis, NADP-malic enzyme, NAD-malic enzyme, Phosphoenolpyruvate carboxykinase, C₄ leaf anatomy, Panicum, Kranz, Dichotomiflora group     

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; )     

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; )     

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)     

Quantitative Determination of RuBP Carboxylase-Oxygenase Protein in Leaves of Several C3 and C4 Plants

RuBP carboxylase-oxygenase protein in three C₃ species (Nicotianatabacum L., Solanum tuberosum L., Triticum aestivum L.) andthree C₄ species (Panicum miliaceum L., Panicum texanum Buckl.,Zea mays L.) was quantitatively determined by sucrose densitygradient centrifugation and by immunochemical assay using antibodyraised to crystallized tobacco leaf RuBP carboxylase-oxygenase.The C₃ species had 3- to 6-fold higher concentrations of RuBPcarboxylase-oxygenase than the C₄ species when expressed oneither a chlorophyll or a leaf area basis. The C₃ species alsoallocated a higher fraction of their total soluble protein tothis enzyme (from 25 to 60% for the C₃ species compared to 8to 23% for the C₄ species). There was no RuBP carboxylase-oxygenaseprotein or activity in the C₄ mesophyll cells, while the enzymeconstituted from 20 to 40% of the total soluble protein in theC₄ bundle sheath cells. A close correlation (r = +0·91)was found between catalytic activity and level of the enzymeprotein in the species examined.     

Leaf Anatomy and Carbon Discrimination in NAD-malic Enzyme Panicum Species and their Hybrids Differing in Bundle Sheath Cell Ultrastructure

The relationship between leaf anatomy, ultrastructure and carbondiscrimination was investigated in leaves of two F₁hybrids (F₁-1and F₁-2) between two different types of the grassPanicum [anNAD-malic enzyme (ME) C₄species], which differ in bundle sheathultrastructure. The female parent was Kabulabula grass, whichhas centrifugal chloroplasts in bundle sheath cells and is designatedan NAD-ME(F) species, while the male parent was Makarikari grass,which has centripetal chloroplasts in the bundle sheath cellsand is designated an NAD-ME(P) species. Suberin lamellae arepresent in Kabulabula grass but are lacking in Makarikari grass.Both F₁hybrids had the same chromosome number (2n =36) as theparents but exhibited both univalent (about 45%) and bivalent(about 55%) chromosome pairing which was the major basis forthe identification of F₁hybrids. In F₁-1, elongated bundle sheathcell chloroplasts are arranged mainly in a centripetal position,similar to those in the male parent, Makarikari grass. In contrast,most of the bundle sheath cells in F₁-2 are packed with starch-containingchloroplasts, although in some cells chloroplasts tended tobe centripetally arranged. In both F₁hybrids, suberin lamellaewere found in the bundle sheath cell walls, similar to the femaleparent, Kabulabula grass. The ¹³C values of both F₁hybrids were-11.4 to -11.7, almost the same as those of Kabulabula grass(-11.4), but significantly higher than those of Makarikari grass(-12.7). These results indicate that the chloroplast orientationin the bundle sheath cells and the presence of suberin lamellaeare not obligatorily linked in their expression and suggestthat suberin lamellae may play an important role in discriminationagainst¹³C. Panicum ; NAD-malic enzyme species; hybrid; chloroplast position; ¹³C discrimination; suberin lamellae