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; )     

Electron Microscopic Observations of the Structure of Sieve-connexions in the Phloem of Angiosperms and Gymnosperms

The sieve elements of Pinus silvestris L., Sorbus aucupariaL., Vitis vinifera L., and Cucurbita pepo L. have been examinedelectron microscopically in ultra-thin section, and the structuresof the corresponding sieve areas or sieve plates have been describedand compared. In Pinus the sieve areas contain groups of connectingstrands which enter the wall from the lumen side as individualsbut coalesce within it in the median cavity. This cavity hasdeveloped by wall breakdown in the middle lamella and primarywall region and corresponds to the median nodule visible undera light microscope. Neither in this nor in the other speciesobserved is there any visible closing membrane. Structural differences between the four species are shown tosuggest that the major evolutionary trend in the evolution ofspecialized conducting strands has been the enlargement of theconnecting strands from groups of small separate strands toa smaller number of larger strands as the median cavity becomesenlarged to form a canal through the wall. The connecting strands appear invariably to be dense, highlyosmiophilic and continuous with the cytoplasmic surface of thecell. No signs of micropores or of other tubular structure inthe strands have been found. The structures thus revealed aremore nearly compatible with active transport of materials acrossthe sieve plate than they are with any purely physical mechanism.It is suggested that they are incompatible with any mass flowhypothesis.     

Structures in Sieve Elements Cut with a Cryostat Following Different Rates of Freezing

Vascular bundles of the internodes of squash (Cucurbita pepo)were frozen while still attached to the stem by their ends.Four different methods of freezing were employed. With slowercooling rates the sieve tube contents showed distinct evidenceof damage due to ice-crystal formation while tissues were wellpreserved when rapidly frozen. The sieve tubes contained longitudinallyorientated structures which, in rapidly frozen tissues, apparentlyconsisted of discrete strands which appeared to pass into thesieve plate pores. It is concluded that the method of freezing permits the cuttingof sections which represent the in vivo structure of phloemand the results support the concept of translocation by meansof transcellular strands in sieve tubes.     

Transcellular Strands in Sieve Tubes; What Are They?

We show that sieve elements of Nymphoides peltata (S. G. Gmel.)O. Kuntze contain strands which are bundles of P-protein filaments.We observe the strands under the light microscope (differential-interferencecontrast), and in the scanning electron microscope which showssome of them to be arranged as a parietal network. We find bundlesof filaments which correspond to these strands in sections ofembedded sieve elements in the transmission electron microscope,and also in freeze-fracture replicas of sieve elements in vascularbundles frozen intact while translocating carbon-14. Not allthe strands are necessarily transcellular; some may end in theparietal layer just to the inside of the plasmalemma where theyappear to come in contact with membranes, possibly of endoplasmicreticulum. The filaments in the strands have the same bandedappearance as filaments in the sieve pores. We are unable tofind any membrane or other special boundary round the strands;we propose they should be called ‘filamentous strands’.We suggest that the filaments are aggregated into strands bythe Bernoulli effect when fluid flows through sieve elements.We suggest that the strands may be formed by flow during translocationas well as by flow due to injury.     

Colchicine-induced Paracrystals in Root Cells of Wheat (Triticum aestivum L.)

Tubulin conformations other than microtubules in the meristematiccells of wheat roots grown in the presence of 2 mM colchicinesolution were investigated by immunofluorescence and electronmicroscopy. In the affected cells microtubules disappeared andwere replaced by tubulin fluorescent strands that occurred inthe cortical cytoplasm. With increasing time of exposure tocolchicine the tubulin strands became better organized and occurredalso in the subcortical cytoplasm and finally they were restrictedto the area around the nucleus. In prophase and preprophasecells thick strands occupied the cortical cytoplasmic zone wherein normal cells a preprophase microtubule band (PMB) was expectedto be assembled. In the colchicine-treated cells electron microscopy revealedan accumulation of paracrystalline aggregates, which initiallyoccurred along the cell wall and later deeper in the cytoplasm,in the perinuclear regions and the cytoplasmic invaginationsof the nucleus. In transverse planes the paracrystalline strandsappear to consist of hexagonal subunits in a 'honeycomb' arrangement,while in longitudinal and oblique sections they exhibit variableimages. Since their distribution coincides with that of thetubulin strands visualized by immunofluorescence, they are consideredto be the same structure. Therefore, the paracrystals consistof, or at least contain, tubulin. They are most likely to bepolymers of tubulin-colchicine complexes.Copyright 1995, 1999Academic Press Wheat roots, colchicine, immunofluorescence, electron microscopy, tubulin paracrystals, Triticum aestivum L     

FURTHER ELECTRON MICROSCOPE STUDIES ON FIBRILLAR ORGANIZATION OF THE GROUND CYTOPLASM OF CHAOS CHAOS

Further evidence for fibrillar organization of the ground cytoplasm of Chaos chaos is presented. Fixations with osmium tetroxide at pH 6 or 8 and with glutaraldehyde at pH 6 or 7 were used on two preparations: (a) single actively streaming cells; (b) prechilled cells treated with 0.05% Alcian blue in the cold and returned to room temperature for 5–10 min. In addition, a 50,000 g pellet of homogenized cells was examined after fixation with glutaraldehyde-formaldehyde alone. In sections from actively streaming cells considerable numbers of filaments were observed in the uroid regions after glutaraldehyde fixation, whereas only traces of filaments were seen after osmium tetroxide fixation at either pH 6 or 8. Microtubules were not seen. In sections from dye-treated cells, filaments (4–6 mµ) and fibrils (12–15 mµ) were found with all three fixatives. The 50,000 g pellet was heterogeneous but contained both clumps of fibrils and single thick fibrils like those seen in the cytoplasm of dye-treated cells. Many fibrils of the same dimensions (12–15 mµ wide, 0.5 µ long) were also seen in the supernatant above the pellet. Negative staining showed that some fibrils separated into at least three strands of 4–6 mµ filaments.     

Antagonism Between Malate and Ethylene in the Regulation of Avena sativa Coleoptile Elongation

Etiolated Avena sativa L. coleoptile sections were used to determinethe influence of C₂H₄ on in vivo and in vitro rates of CO₂ fixation,and to measure the influence of various permutations of C₂H₄,CO₂, and malate on growth. Whereas 1 mM malate or 320 µI^-1 CO₂ stimulated growth by approximately 100 per cent, inhibitionof growth by 10-8 µ I_-1 C₂H₄ was substantial only in thepresence of malate or CO₂ The increase in growth rate in responseto these two agents was eliminated by the simultaneous applicationof C₂H₄. The in vivo rate of dark [¹⁴C]bicarbonate fixationand in vitro enzymic assays of fixation were not measurablyinhibited by C₂H₄. These results are discussed in the lightof evidence which indicates that CO₂-stimulated growth is mediatedby dark fixation. The data do not support the view that C₂H₄inhibition of growth results from an inhibition of fixation,but suggests that C₂H₄ may inhibit some step in the processby which malate stimulates growth.     

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i) to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.     

The Mechanics of the Flower Stem of the Sedge Carex acutiformis

The mechanics of the triangular stems of Carex acutiformis wasinvestigated by subjecting sections to bending and torsionaltests. The stem was rigid in bending, being stiffened peripherallyby lignified material around the vascular bundles, but becauseof its triangular shape it was vulnerable to local buckling.Despite being and narrow the stem was able to support the seedhead, though it sagged appreciably towards the tip. In contrastthe stem had very low torsional rigidity, both because of itstriangular shape because the strands of lignified material wereisolated from each other. In its lowland habitat this allowsthe drooping stem to twist away from the light winds, so reducingdrag and the chances of self-fertilization. This method of reconfiguringis not possible in the shorter, stiffer mountain sedges whichmust withstand higher winds; many therefore have more circularstems which will be more efficient at resisting bending.Copyright1993, 1999 Academic Press Carex acutiformis, sedge, mechanics, bending, torsion     

Identification and Localization of Frankia Strains in Alnus Nodules by In Situ Hybridization of nif H mRNA with Strain-Specific Oligonucleotide Probes

In situ hybridization of Frankia mRNA with specific probes wasused to localize the strains Arl3 and AcoN24d in Alnus nodulesobtained after inoculation with one or both strains. The probesconsisted of 18-mer oligonucleotides, complementary to strain-specificsequences located within the nif H gene. Sections of nodulesinoculated with only one strain revealed a specific hybridizationbetween the probe and the corresponding Frankia strain mRNA.In sections of dually-inoculated nodules the presence of thestrain AcoN24d in the nodule was clearly shown whereas thoseof the strain Arl3 could not be detected. This suggests thatthe strain Arl3 is less infective than the strain AcoN24d andis not present within the nodule. Key words: Nitrogen fixation, actinorhizae, autoradiography, histochemistry     

Vegetative Anatomy of Uncinia (Cyperaceae)

The vegetative anatomy of 18 (19) Uncinia spp. (Cyperaceae-Cariceae)including representatives of most taxonomic subdivisions wasstudied to determine the range of variation in certain anatomicaland morphological characters of the vegetative organs withinthe genus. The two South American species U. erinacea and U. kingii differfrom all others, the former in having a closed cylinder of sclerenchymain the culm, and the latter a grooved culm. The three tall SouthAmerican spp., U. brevicaulis, U. hamata, and U. phleoides var.trichocarpa, are characterized by adaxial intercostal fibrestrands in the leaves, and in this respect show affinities withtall New Zealand species, U. sinclairii and U. uncinata. Thelast species does not have the intercostal strands. The muchsmaller U. tenuis from S. America resembles, in the size andshape of transverse sections of leaf and culm, a group of speciesfrom New Zealand comprising U. angustifolia, U. egmontiana,and U. rupestris. To these could be added U. banksii, U. hookeri,and perhaps also U. tenella, although the last two spp. exhibitsome distinctive characters in the transverse section of theleaf and also in the leaf epidermis in surface view. No exactcounterpart to the Australian and New Zealand spp. U. divaricata,U. riparia, U. rubra and U. scabra was found amongst the S.American material. This group of spp. is distinctive, for example,because of the triangular or irregularly triangular shape oftransverse sections of the culm and the large amount of sclerenchymain transverse sections of the leaf. The range of structural variation appears to be particularlywide in the S. American spp., which represent two extreme typesin the shape of the leaf in transverse section. The other talland small species have their respective counterparts in eacharea, although they are more profusely represented in New Zealandthan in America, and therefore show a correspondingly greaterrange of structure.     

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)     

Can the 15N Dilution Technique be used to Study N2 Fixation in Tropical Tree Symbioses as Affected by Water Deficit?

Three methods were used to study N₂ fixation and effects ofwater deficit on N₂ fixation: C₂H₂ reduction assay (ARA), ¹⁵Ndilution technique and accumulated N content. In addition, ¹⁵Ndilution was calculated both in a traditional way and in a modifiedway, which takes into consideration N and ¹⁵N content for theplants before the experiment started. The three methods wereapplied on the following Rhizobium-symbioses: Acacia albidaDel (Faidherbia albida (Del) A. Chev.) and Leucaena leucocephala(Lam) de Wit., and the Frankia-symbiosis Casuarina equisetifoliaL. The plants wereabout 4-months-old when they were harvested. Nitrogen derived from N₂ fixation in control plants of Acaciaalbida was 54·2 mg as measured with ARA, while it was28·5 mg as measured with the ¹⁵N dilution technique,compared to 30·7 mg calculated as accumulated N. In comparison,L. leucocephala fixed 41·6 mg N (ARA), 53·5 mgN(15N dilution technique) and 56·3 mg N (accumulatedN). The Frankia-symbiosis had fixed 27·4 mg N as measuredby ARA, 8·1 mg N as measured by ¹⁵N dilution techniqueand 12·3 mg N as accumulated N. There were no differencesbetween the estimates based ontraditional and modified waysof calculating ¹⁵N dilution. The immediate effect of water deficit treatment on N₂ fixationwas continuously measured inall species with ARA, which startedto decrease approximately 10 d after the initiation of the treatment,and declined to less than 5% of the initial level after 21–28d. The decrease in the amount of N derived from N₂ fixation wasstudied in L. leucocephala during the period of treatment. Therewas a 26% decrease in amount of N derived from N₂ fixation asresult of water deficit (as measured with ARA), while the decreasewas 23% when measured withboth the ¹⁵N dilution method and asaccumulated N. The three different methods for measuring N₂ fixation and effectsof water deficit on N₂ fixation are discussed. Key words: Acacia albida, ARA, Casuarina equisetifolia, Leucaena leucocephala, ¹⁵N dilution, N₂N fixation, water deficit     

The Vascular Pattern of a Rhizomatous Ginger (Alpinia speciosa L. Zingiberaceae). 2. The Rhizome

The vascular system in the underground rhizome of Alpinia speciosaL. (Zingiberaceae) is seen to be arranged in three distinctzones. (1) An inner system of ‘scattered’ vascularbundles which serial cinematography reveals to have an axialpattern conforming to the basic ‘palm’ configuration(a system of upwardly branching leaf traces with interconnections).(2) An intermediate zone comprising a thin perforated cylinderof anastomosing vascular strands having direct contact withboth roots and inner system bundles. (3) An outer system offreely-anastomosing vascular bundles. Connexion of outer andinner system occurs in the form of extensive bridging from innersystem leaf traces as they depart obliquely between the outersystem network. The interrelation of the three systems, plus root and branchinsertion, is illustrated by means of diagrammatic three-dimensionalreconstructions. The intermediate zone is intimately associatedwith root insertions and with the inner system, and is shownto obliviate potential bottlenecks at the point of lateral branchinsertion in this sympodial rhizome system. A comparison ismade with other monocotyle-donous vascular systems. Alpinia speciosa L., shell ginger, rhizome, vascular anatomy     

Initiation of Procambial Strands in Axillary Buds of Dactylis glomerata L., Secale cereale L., and Lolium perenne L.

In axillary buds of Dactylis glomerata L., Secale cereale L.,and Lolium perenne L., the first two procambial strands of theprophyll and the median strand of the first normal leaf areinitiated in the bud in isolation from the vascular system ofthe parent axis. They rapidly form connections with the vascularsystem of the parent axis, presumably by downward extension,as is the case of the strands of leaf primordia on the mainaxis.     

PLANT MICROTECHNIQUE: SOME PRINCIPLES AND NEW METHODS

Some easily seen structural features of living plant cells are destroyed or badly distorted by most of the common fixatives and embedding media used in plant histology. In stained sections of plant tissues fixed in FAA (formalin-acetic acid-alcohol mixtures) and embedded in paraffin wax, for example, mitochondria and fine transvacuolar strands of cytoplasm are usually not visible. Many structural features such as these can be preserved, however, with suitable fixatives and embedding media. Specifically we recommend fixation in non-coagulant fixatives (e.g., osmium tetroxide, acrolein, glutaraldehyde, formaldehyde) and the use of plastics as embedding media, and we describe in detail a method of fixation in acrolein and embedding in glycol methacrylate polymer. In a wide range of plant specimens prepared in this way, stained sections 1–3 microns thick showed excellent preservation of tissue and cell structures.     

Preservation of the Tonoplast in Metaphloem Sieve Elements of Embryonic Roots of Zea mays L.

Decortication of embryonic roots of 4- to 5-day-old Zea seedlingsand subsequent chemical fixation permitted comparison of cutand uncut developing sieve elements In a decorticated root wheresieve tubes are not severed, metaphloem sieve elements in latestates of development and some mature sieve elements exhibita highly vacuolate condition When roots are cut or diced inthe course of fixation intact vacuoles are not observed in latestages of sieve-element ontogeny The degree of callose formationat sites of developing sieve-plate pores and in the pores ofmature sieve elements varies greatly with both decorticationand non-decortication treatments Nuclei were not observed insieve elements at the electron microscope level, but they wereseen at the light microscope level in serial sections of sieveelements in the late to mature developmental stages representedAlthough the occurrence and distribution of plastids, mitochondria,endoplasmic reticulum, dictyosomes and nbosomes also vanes insieve elements of decorticated roots, disruption or surgingof sieve-element contents is greater for sieve tubes that aresevered during fixation treatment A discussion is presentedrelating effects of trauma on observed developmental stagesand sieve-element structure Zea mays L, maize, corn, phloem, Sieve elements, tonoplast, ultrastructure     

Contributions to the Morphology of Some Species of Microsorium

Morphology of eleven epiphytic and rupicaulous species of Microsoriumis described. The paleae in the genus are either peltate withthe basal region developing secondarily as a hood over the stalk,or basally attached with auricles on either side of the stalk.The auricles in M. hancockii and M. pteropus develop from singleinitial cells adjacent to the stalk, while in the others nospecialized initial cells occur. Marginal and terminal glandularhairs occur on the paleae, except in M. hancockii and M. pteropusin which marginal hairs are absent. Slender sclerenchyma strands are scattered profusely in theground tissue of the rhizome. The stelar cylinder is dictyostelicand is dissected by lacunae into cylindrical vascular bundles.Leaf traces are multiple strands originating as branches fromthe dorsal median vascular bundle of the stelar cylinder andthe one next to it on each side. Two or three closely placedvascular bundles of the rhizome constitute the vascular connexionto each branch of the rhizome. Venation of the leaf lamina is reticulate with most of the free-endingveinlets entering foliar hydathodes. The juvenile leaves bearhairs similar to the pro-thallial hairs and are spatulate witha medianly placed forked vein. Sori are generally punctiform,but in M. hancockii and M. pteropus spread slightly over theveins, often forming elongated coenosori. Uniseriate (multiseriatein M. rubidum), multicellular paraphyses occur in all speciesexcept M. scolopendria. The spores are monolete and either psilateor granulate. The prothalli develop from 3-5 cells long germ filaments inwhich the anterior cells divide longitudinally and form an ameristicprothallial plate. An apical meristematic cell is formed later,and a cordate prothallus is developed, except in M. hancockiiand M. pteropus in which a definite meristem is never formedand the prothalli are ribbon-shaped and branched. The cordateprothalli possess polypodiaceous hairs: the ribbon-shaped onesare more or less naked and devoid of any midrib. The chromosome number in the species is n = 36 (zn = 72).     

Interspecific Pollen-Pistil Interaction in Eucalyptus L'Her. (Myrtaceae): The Effect of Taxonomic Distance

The pollen-pistil interaction was investigated in three intraspecific,57 interspecific and six intergeneric crosses using three speciesof Eucalyptus (Myrtaceae) subgenus Symphyomyrtus, section Bisectariaas female parents Interspecific prefertilization isolation occursin the pistil and manifests as a number of pollen-tube abnormalitiesin the style and ovary associated with a lowered probabilityof ovule penetration The major selection points in the pistilare the upper style and the ovary The seventy of abnormalitiesand the probability of pollen-tube arrest in the pistil wasproportional to the taxonomic distance between parent speciesOvule penetrations were seen mainly in crosses within the sectionBisectaria or between the sections Bisectaria and Adnataria Pollen storage, style length and mean maximum temperature duringthe flowering period of the male parent had no significant effecton pollen-tube growth in the crosses used Mechanisms of reproductiveisolation are discussed in relation to evolutionary relationshipsand the implications for taxonomic groupings Eucalyptus L'Hér, pollen-pistil interaction, incongruity, interspecific hybridization, pollen-tube growth, breeding system, taxonomy     

Metabolism of epidermal tissues, mesophyll cells, and bundle sheath strands resolved from mature nutsedge leaves

Mesophyll cells and bundle sheath strands were isolated from Cyperus rotundus L. leaf sections infiltrated with a mixture of cellulase and pectinase followed by a gentle mortar and pestle grind. The leaf suspension was filtered through a filter assembly and mesophyll cells and bundle sheath strands were collected on 20-μm and 80-μm nylon nets, respectively. For the isolation of leaf epidermal strips longer leaf cross sections were incubated with the enzymes and gently ground as above. Loosely attached epidermal strips were peeled off with forceps. The upper epidermis, which lacks stomata, could be clearly distinguished from the lower epidermis which contains stomata. Microscopic evidence for identification and assessment of purity is provided for each isolated tissue.Enzymes related to the C₄-dicarboxylic acid cycle such as phosphoenolpyruvate carboxylase, malate dehydrogenase (NADP⁺), pyruvate, P_i dikinase were found to be localized, ≥98%, in mesophyll cells. Enzymes related to operating the reductive pentose phosphate cycle such as RuDP carboxylase, phosphoribulose kinase, and malic enzyme are distributed, ≥99%, in bundle sheath strands. Other photosynthetic enzymes such as aspartate aminotransferase, pyrophosphatase, adenylate kinase, and glyceraldehyde 3-P dehydrogenase (NADP⁺) are quite active in both mesophyll and bundle sheath tissues.Enzymes involved in photorespiration such as RuDP oxygenase, catalase, glycolate oxidase, hydroxypyruvate reductase (NAD⁺), and phosphoglycolate phosphatase are preferentially localized, ≥84%, in bundle sheath strands.Nitrate and nitrite reductase can be found only in mesophyll cells, while glutamate dehydrogenase is present, ≥96%, in bundle sheath strands.Starch- and sucrose-synthesizing enzymes are about equally distributed between the mesophyll and bundle sheath tissues, except that the less active phosphorylase was found mainly in bundle sheath strands. Fructose-1,6-diP aldolase, which is a key enzyme in photosynthesis and glycolysis leading to sucrose and starch synthesis, is localized, ≥90%, in bundle sheath strands. The glycolytic enzymes, phosphoglyceromutase and enolase, have the highest activity in mesophyll cells, while the mitochondrial enzyme, cytochrome c oxidase, is more active in bundle sheath strands.The distribution of total nutsedge leaf chlorophyll, protein, and PEP carboxylase activity, using the resolved leaf components, is presented. ¹⁴CO₂ Fixation experiments with the intact nutsedge leaves and isolated mesophyll and bundle sheath tissues show that complete C₄ photosynthesis is compartmentalized into mesophyll CO₂ fixation via PEP carboxylase and bundle sheath CO₂ fixation via RuDP carboxylase. These results were used to support the proposed pathway of carbon assimilation in C₄-dicarboxylic acid photosynthesis and to discuss the individual metabolic characteristics of intact mesophyll cells, bundle sheath cells, and epidermal tissues.