Ruminococcus flavefaciens Cell Coat and Adhesion to Cotton Cellulose and to Cell Walls in Leaves of Perennial Ryegrass (Lolium perenne)

Ruminococcus flavefaciens was shown to possess a prominent glycoprotein coat, which contained rhamnose, glucose, and galactose as its principal carbohydrates. Periodate-reactive carbohydrate occurred as a surface layer of the coat. The ruminococci adhered strongly by means of this coat to cotton cellulose and to cell walls in leaf sections of Lolium perenne L. (perennial ryegrass). The coat was diffuse at the point of contact so that the bacterial cell wall was in close contact with the substrate. Adhesion was influenced by the availability of damaged plant cell walls and by the cell wall type and occurred most rapidly to cell walls of the epidermis and sclerenchyma, followed by the phloem and mesophyll. Plaques of bacteria with filamentous coat extensions developed on all these tissues. The bacteria did not readily adhere to the walls of the bundle sheath cells or metaxylem or protoxylem vessels and did not adhere to the cuticle or chloroplasts. The epidermal and phloem cell walls were more rapidly digested than the walls of other cell types.     

Role of free space in translocation in sugar beet

The involvement of the free space in phloem loading of sucrose was studied in sugar beet source leaves (Beta vulgaris, L.). Sucrose, supplied exogenously to the abraded upper surface of leaves at a concentration of 20 mm, was available for translocation at rates similar to those obtained with photosynthesis. The exogenous sucrose substituted as a source of translocate for assimilate derived from photosynthesis when the latter process was disrupted by plasmolysis of the leaf with 0.8 M mannitol. The mesophyll symplast was not completely disrupted by this treatment, however. Data from the sugar uptake experiments indicate that phloem loading can occur from the free space.     

Phloem Unloading in Developing Leaves of Sugar Beet : I. Evidence for Pathway through the Symplast

Physiological and transport data are presented in support of a symplastic pathway of phloem unloading in importing leaves of Beta vulgaris L. (`Klein E multigerm'). The sulfhydryl reagent p-chloromercuribenzene sulfonic acid (PCMBS) at concentration of 10 millimolar inhibited uptake of exogenous [¹⁴C]sucrose by sink leaf tissue over sucrose concentrations of 0.1 to 5.0 millimolar. Inhibited uptake was 24% of controls. The same PCMBS treatment did not affect import of ¹⁴C-label into sink leaves during steady state labeling of a source leaf with ¹⁴CO₂. Lack of inhibition of import implies that sucrose did not pass through the free space during unloading. A passively transported xenobiotic sugar, l-[¹⁴C]glucose, imported by a sink leaf through the phloem, was evenly distributed throughout the leaf as seen by whole-leaf autoradiography. In contrast, l-[¹⁴C]glucose supplied to the apoplast through the cut petiole or into a vein of a sink leaf collected mainly in the vicinity of the major veins with little entering the mesophyll. These patterns are best explained by transport through the symplast from phloem to mesophyll.     

Sink Effect of Cytokinin in Detached Leaves Is Not Caused by Structural Rearrangements of Phloem

The sink effect of cytokinin is manifested as a decrease in source capacity and the induction of sink activity in the phytohormone-treated region of a mature excised leaf. In order to find out whether this effect was due to the direct action of cytokinin on the phloem structure, two types of phloem terminals were examined. In pumpkin (Cucurbita pepo L.) leaves, the phloem terminals are open; i.e., they are linked to mesophyll by numerous symplastic connections, which are located in narrow areas called plasmodesmal pit fields. In broad bean (Vicia faba L.) leaves, the phloem terminals belong to the closed type and have no symplastic links with mesophyll. The electron microscopic study of terminal phloem did not reveal any structural changes in the companion cells, which could account for the suppression of assimilate export. The treatment of leaves with cytokinin neither disturbed the structure of plasmodesmal pit fields in pumpkin leaves nor eliminated the wall protuberances (the ingrowths promoting phloem loading) in bean leaves. No evidence was obtained that the cytokinin-induced import of assimilates in mature leaves is caused by the recovery of meristematic activity, i.e., by either formation of new phloem terminals having immature sieve elements capable of unloading or by the development of new sieve elements within the existing veins. Cytokinin did not induce de novo formation of phloem elements. Structural characteristics of the leaf phloem, such as the number of branching orders in the venation pattern, the number of vein endings per areole, the number of areoles per leaf, the area of one areole, and the number of sieve elements per bundle remained unaltered. It is concluded that the sink effect of cytokinin in excised leaves cannot be determined by alteration of the phloem structure.     

Adhesion of Bacteroides succinogenes in pure culture and in the presence of Ruminococcus flavefaciens to cell walls in leaves of perennial ryegrass (Lolium perenne)

Bacteroides succinogenes and Ruminococcus flavefaciens are two of the most important cellulolytic bacteria in the rumen. Adhesion of B. succinogenes in pure culture, and in mixed culture with R. flavefaciens, to the various types of cell walls in sections of perennial ryegrass (Lolium perenne L. cultivar S24) leaves was examined by transmission and scanning electron microscopy. B. succinogenes adhered to the cut edges of most plant cell walls except those of the meta- and protoxylem. It also adhered, though in much smaller numbers, to the uncut surfaces of mesophyll, epidermal, and phloem cell walls. In mixed culture, both species adhered in significant numbers to the cut edges of most types of plant cell wall, but R. flavefaciens predominated on the epidermis, phloem, and sclerenchyma cell walls. B. succinogenes predominated on the cut edges and on the uncut surfaces of the mesophyll cell walls, and its ability to adhere to uncut surfaces of other cell walls was not affected by the presence of the ruminococcus. Both organisms rapidly digested the epidermal, mesophyll, and phloem cell walls. Zones of digestion were observed around bacteria of both species when attached to the lignified cell walls of the sclerenchyma, but not when attached to the lignified xylem vessels.     

VARIATIONS IN ANATOMY,ASSOCIATIONS, AND ORIGINS OF KRANZ TISSUE

Although the unique tissue required for C₄ photosynthesis in nonsucculent plants is often described as being modified leaf parenchyma sheath, which is positioned meaningfully between the mesophyll externally and the vascular tissues internally, the actual range of locations and known associations make that concept untenable. In origin the Kranz tissue develops from procambium as well as ground parenchyma. It is found in stems as well as leaves. In position Kranz tissue can lie in the parenchyma sheath, in the mestome sheath, isolated in the mesophyll, peripherally in some thick leaves, or within the veins. It can be associated with mesophyll only, mesophyll and colorless parenchyma, mesophyll and sclerenchyma, other Kranz tissue and vascular tissues, mesophyll and mestome sheath, mesophyll and phloem, mesophyll and xylem, epidermis, and, finally, mestome sheath and xylem and phloem. The use of the term Kranz is expounded.     

Adhesion of Bacteroides succinogenes in pure culture and in the presence of Ruminococcus flavefaciens to cell walls in leaves of perennial ryegrass (Lolium perenne).

Bacteroides succinogenes and Ruminococcus flavefaciens are two of the most important cellulolytic bacteria in the rumen. Adhesion of B. succinogenes in pure culture, and in mixed culture with R. flavefaciens, to the various types of cell walls in sections of perennial ryegrass (Lolium perenne L. cultivar S24) leaves was examined by transmission and scanning electron microscopy. B. succinogenes adhered to the cut edges of most plant cell walls except those of the meta- and protoxylem. It also adhered, though in much smaller numbers, to the uncut surfaces of mesophyll, epidermal, and phloem cell walls. In mixed culture, both species adhered in significant numbers to the cut edges of most types of plant cell wall, but R. flavefaciens predominated on the epidermis, phloem, and sclerenchyma cell walls. B. succinogenes predominated on the cut edges and on the uncut surfaces of the mesophyll cell walls, and its ability to adhere to uncut surfaces of other cell walls was not affected by the presence of the ruminococcus. Both organisms rapidly digested the epidermal, mesophyll, and phloem cell walls. Zones of digestion were observed around bacteria of both species when attached to the lignified cell walls of the sclerenchyma, but not when attached to the lignified xylem vessels.     

Rumen bacterial interrelationships with plant tissue during degradation revealed by transmission electron microscopy

The mode of rumen bacterial degradation of cell walls in coastal bermudagrass [Cynodon dactylon (L) Pers.] differed with the plant tissue type. Bacteria degraded thin, primary cell walls of mesophyll and phloem apparently by extracellular enzymes and without prior attachment; thick-walled bundle sheath and epidermal cells apparently were degraded after bacterial attachment, in some types by an extracellular substance, to the plant cell walls. Rumen bacteria split the nondegraded cuticle from the epidermis by preferentially attacking the cell just underneath the cuticle. The propensity for bacterial attachment to lignified cells of the vascular tissue was low, and bacterial degradation of these cells did not occur after 72 h of incubation.     

The effects of laurel (Laurus nobilis L.) on development of two mycorrhizal fungi

Hundreds of aromatic plant species are growing naturally around Mediterranean. Plant essential oils are incorporated in aromatic plant material and follow the litter fall. During litter degradation, the presence of essential oils can affect soil microorganisms. Mycorrhizal fungi have never been investigated so far under the presence of volatile oils. The aim of this study was to explore the effect of aromatic Laurus nobilis L. on development of two mycorrhizal species Glomus deserticola and Glomus intraradices. The response of fungi colonization and host growth were monitored under different concentrations of L. nobilis leaves and essential oil. The major compounds of L. nobilis essential oil were 1,8-cineole (49.6%), sabinene (7.8%), ??-pinene (6.0%), eugenole (5.6%), ??-terpinyl acetate (5.2%) and ??-pinene (5.1%). Both mycorrhizal fungi colonized successfully the host plants whose growth was positively influenced by mycorrhizal fungi. G. deserticola presented higher infection level than G. intraradices. The addition of L. nobilis leaves in the soil resulted in mycorrhiza inhibition. The level of inhibition was positively correlated with the added amount of aromatic leaves in the soil. The essential oil presented a little higher inhibition than the leaves. The presence of this aromatic plant in many different ecosystems could contribute in mycorrhiza inhibition and it is suggested, when it’s possible, reduction of laurel litter before reforestation programs.     

Phloem loading in squash

Squash (Cucurbita pepo L. var. melopepo torticalis, Bailey) leaves were supplied with ¹⁴C-sucrose, then specific radioactivities of the glucose and galactose moieties of translocated stachyose were determined. In every case, the specific radioactivity of the galactose moiety was greater than that of the glucose moiety. It is concluded that the stachyose was not synthesized at either the phloem-loading site or subsequent to phloem loading, but rather in cells that were not a part of the translocation system, possibly the mesophyll cells.     

Maize Brittle Stalk2-Like3, encoding a COBRA protein,functions in cell wall formation and carbohydrate partitioning

Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with ¹⁴C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (Bk2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, Bk2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning.

Mutations in Bk2L3 result in dwarfed plants with decreased anisotropic cell growth, cellulose deposition, phloem pressure, sucrose export, and carbohydrate hyperaccumulation in mature maize leaves.     

The leaf internal morphology and ultrastructure of Zostera muelleri irmisch ex aschers. (Zosteraceae): a comparative study of the intertidal and subtidal forms

The leaf anatomy, histochemistry and ultrastructure of the intertidal and subtidal seagrass Zostera muelleri Irmish ex Aschers. from Westernport Bay, Victoria were studied. Unusual anatomical and ultrastructural features are compared with other seagrasses and their functional significance is assessed. Subcuticular cavities are present in the young blade, but not observed in the older blade nor young and old leaf sheath. Wall ingrowths occur in the blade epidermal cells particularly on the inner tangential walls and the lower portions of the radial walls. Plasmodesmata are present between adjacent epidermal cells and between the epidermal and mesophyll cells, suggesting that solutes could transfer between these tissues both symplastically and apoplastically. Each leaf has three longitudinally aligned vascular bundles, each of which comprises a single xylem element isolated from the phloem tissue. The phloem consists of nacreous-walled sieve elements accompanied by phloem parenchyma cells which also process wall ingrowths. The xylem walls are completely hydrolysed and the middle lamella borders directly on the xylem lumen. Leaves have prominent air lacunae bisected transversely by septa at regular intervals along their length. Each septum consists of a file of small parenchyma cells with wall protuberances projecting into intercellular space. There are no major structural differences between the subtidal and intertidal plants, but the former have larger leaves and more leaves per shoot than the latter. In addition, a network of unusual reticulated fungal hyphae is present in the leaf intercellular spaces of the subtidal form and this network may facilitate solute transfer in these plants.     

Mode of Attack on Orchardgrass Leaf Blades by Rumen Protozoa

Leaf blade sections of orchardgrass were incubated with rumen fluid and examined by scanning and transmission electron microscopy for the mode of attack on tissues by rumen protozoa. Rumen protozoa resembling Epidinium ecaudatum from caudatum degraded forage tissue in diluted, whole rumen fluid suspensions of microbes containing 1.6 mg of streptomycin per ml, which inhibited bacterial fiber-digesting activity. Cell walls of mesophyll, parenchyma bundle sheath, and epidermis became swollen and frayed to reveal a microfibrillar network and loss of electron density, indicating partial degradation. Then the protozoa ingested whole cells and fragments of cell walls with the aid of their cilia. Plant cells with partially degraded walls as well as chloroplasts without walls were present within the protozoa. These entodiniomorphs digested orchardgrass leaves by partially degrading the plant cell walls apparently by extracellular enzymes and then ingestion of the plant cells and cell wall fragments.     

The role of sinigrin in host plant recognition by aphids during initial plant penetration

Plant penetration behaviour (probing) of the cabbage aphid, Brevicoryne brassicae, and the pea aphid, Acyrthosiphon pisum, was studied on excised leaves of broad beans, Vicia faba, kept in water or in a 1% aqueous solution of sinigrin. Using the DC EPG (Electrical Penetration Graph) technique it was shown that the cabbage aphid on sinigrin-untreated bean leaves showed numerous short probes into epidermis and mesophyll. None of these aphids showed either phloem salivation or ingestion waveforms on untreated leaves. In contrast, on sinigrin-treated bean leaves, 35% of the probing time was spent on phloem sap ingestion (E2) and almost all aphids reached phloem vessels and started feeding. The duration of phloem salivation before phloem ingestion and the mean duration of phloem ingestion periods were similar on a host and a sinigrin-treated non-host plant. However, the total probing time by B. brassicae was 10% longer, the total phloem sap ingestion time was twice as long, and the time to the first phloem phase within a probe was three times shorter on the host plant compared to sinigrin-treated broad beans. Acyrthosiphon pisum also responded to the addition of sinigrin to broad beans, but in this case sinigrin acted as a deterrent. On sinigrin-treated leaves, A. pisum terminated probes before ingestion from phloem vessels, and none of these aphids showed phloem salivation and ingestion on treated leaves. Glucosinolates were detected in the mesophyll cells of the brassicaceous plant, Sinapis alba. Based on this finding and in addition to the foregoing EPG analysis of aphid probing on these plants and broad beans, our hypothesis is that aphids may recognise their host plants as soon as they probe the mesophyll tissue and before they start ingestion from phloem vessels.     

Tropospheric ozone-induced structural changes in leaf mesophyll cell walls in grapevine plants

The structural changes in leaves of grapevine plants (Vitis vinifera L.) exposed to different ozone concentrations were investigated. Ozone fumigations were performed in open-top chambers at four different ozone levels (charcoal-filtered air (F), ambient air (N), ambient air + 25 mm³m⁻³ ozone (O-25) and ambient air + 50 mm³m⁻³ ozone (O-50)). The leaves of plants from chambers with increased ozone concentrations (O-25 and O-50) were significantly thicker than the controls (F), owing to increased thickness of the mesophyll layer. Observing O-50 leaves, it was found that the mesophyll cell wall displayed structural changes. In some places cell wall thickness increased up to 1 μm. We found callose deposits on the inner side of the cell walls of mesophyll cells. These data are in accord with the concept that the mesophyll cell wall acts as a barrier against the penetration of tropospheric ozone into the cells.     

Widespread cutaneous candidiasis and tinea infection masking mycosis fungoides

Seedlings of an inbred line of male-fertile corn possessing the gene rhm for resistance to Southern corn leaf blight were inoculated with conidia of Helminthosporium maydis race O. Histological observations at 1 day revealed that lesions were comprised of several dead mesophyll cells bordered by a pair of vascular bundles. By 4 days, lesions had only spread to a width of three vascular bundles. Ultrastructural observations revealed that although mesophyll cells degenerated at an early stage, bundle sheath and phloem cells remained intact even in 4-day-old lesions. It appears that the gene rhm imparts a resistance to bundle sheath and phloem cells against toxic substances released by the fungus. Addition key words: Zea mays L., ultrastructure, gene rhm, Southern corn leaf blight.     

The Effects of Fusicoccin,Abscisic Acid,and Benzyladenine on the Transport and Partitioning of Substances as Related to Cytokinin Sink Activity

We tested the possible cytokinin effect on the functioning of the active transport system involved in the assimilate loading into the phloem as a cause for the cytokinin sink and retention effect. This effect is manifested in the deceleration of substance export from and the stimulation of substance import to the sites of local phytohormone application to the mature detached leaf from untreated leaf areas. To affect the membrane mechanisms of the substance transport, we used leaf treatment with the phytotoxin fusicoccin, an enhancer of plasmalemmal H⁺-ATPase and a potential stimulator of assimilates export, and with the phytohormone ABA affecting transport, metabolism, and plant growth. However, fusicoccin did not enhance ¹⁴C-sucrose export from the leaf blade and did not interfere with the cytokinin-induced export deceleration. ABA reduced substantially ¹⁴C export from the leaf but eliminated the cytokinin effect on this process. Similar results were obtained for broad bean (Vicia faba L.) leaves with apoplastic phloem loading, involving H⁺-ATPase activity, and pumpkin (Cucurbita pepo L.) leaves with symplastic phloem loading, that is, occurring without sucrose transmembrane translocation and without H⁺-ATPase involvement. The conclusion is that the cytokinin-induced development of sink zones in source leaves is not related to the membrane mechanisms of the substance transport in the mesophyll–phloem system. The data obtained support the idea that the cause for the cytokinin sink and retention effect is the enhancement of elongation growth and total activation of metabolism in the mesophyll cells of the detached leaf.     

Symplastic Phloem Loading in Poplar

Sap is driven through phloem sieve tubes by an osmotically generated pressure gradient between source and sink tissues. In many plants, source pressure results from thermodynamically active loading in which energy is used to transfer sucrose (Suc) from mesophyll cells to the phloem of leaf minor veins against a concentration gradient. However, in some species, almost all trees, correlative evidence suggests that sugar migrates passively through plasmodesmata from mesophyll cells into the sieve elements. The possibility of alternate loading mechanisms has important ramifications for the regulation of phloem transport and source-sink interactions. Here, we provide experimental evidence that, in gray poplar (Populus tremula × Populus alba), Suc enters the phloem through plasmodesmata. Transgenic plants were generated with yeast invertase in the cell walls to prevent Suc loading by this route. The constructs were driven either by the constitutive 35S promoter or the minor vein-specific galactinol synthase promoter. Transgenic plants grew at the same rate as the wild type without symptoms of loading inhibition, such as accumulation of carbohydrates or leaf chlorosis. Rates of photosynthesis were normal. In contrast, alfalfa (Medicago sativa) plants, which have limited numbers of plasmodesmata between mesophyll and phloem, displayed typical symptoms of loading inhibition when transformed with the same DNA constructs. The results are consistent with passive loading of Suc through plasmodesmata in poplar. We also noted defense-related symptoms in leaves of transgenic poplar when the plants were abruptly exposed to excessively high temperatures, adding to evidence that hexose is involved in triggering the hypersensitive response.In the mid-1930s, several laboratories discovered that sugar concentrations are higher in the phloem than in mesophyll cells, where the sugar is synthesized (Crafts, 1961). These findings led to the concept of thermodynamically active phloem loading, in which Suc and other transport compounds are transferred into the sieve tubes against a concentration gradient. The idea was rapidly accepted, in part because it was consistent with the pressure flow hypothesis proposed earlier by Münch (1930). Münch (1930) had suggested that sap is propelled through the sieve tubes by a pressure gradient between the leaves (sources) and sinks (Patrick, 2012; De Schepper et al., 2013; Stroock et al., 2014), and because elevated solute levels increase hydrostatic pressure, it was reasonable to assume that the energy used to load the phloem generates the pressure at the source end of the transport stream needed to drive long-distance transport.However, it is important to note that the hypothesis by Münch (1930) predated the discovery of active phloem loading. Münch (1930) assumed that the upstream pressure is generated in the mesophyll cells and not the phloem and that carbohydrate is carried passively from the mesophyll into the sieve tubes (Münch, 1930). The two hypotheses, active and passive loading, lead to different perspectives on several important aspects of phloem physiology, including the regulated entry of ionic and molecular species into the transport system and the mechanisms of source-sink signaling.We provide evidence here that phloem loading of Suc in poplar (Populus tremula × Populus alba) is passive, as envisioned by Münch (1930). The reason for choosing poplar for study is that there is correlative evidence consistent with a passive loading mechanism in this species. First, the mesophyll cells and minor vein phloem of poplar are linked by plasmodesmata that are much more dense than those at the same interfaces in plants known to load through the apoplast (Russin and Evert, 1985). Second, the osmotic potential of the sieve element-companion cell complex in the minor veins, estimated by plasmolysis, is lower than commonly found in herbaceous plants and in the same range as that of the mesophyll cells (Russin and Evert, 1985). In species that load actively, the osmotic potential in the phloem is generally, but not always, well above that in the photosynthetic cells.Although these data are suggestive, they are only correlative and for several reasons, inconclusive (see “Discussion”). In the studies reported here, we experimentally tested the hypothesis of passive loading in poplar by introducing yeast invertase to the apoplast of transgenic plants. Invertase in the cell walls inhibits apoplastic loading by hydrolyzing Suc en route to the phloem (von Schaewen et al., 1990; Dickinson et al., 1991; Heineke et al., 1992). For comparison, we conducted the same experiments on alfalfa (Medicago sativa), which on the basis of low plasmodesmata numbers in the minor vein phloem (Gamalei, 1991), loads from the apoplast. Invertase-expressing alfalfa exhibited well-documented symptoms of loading inhibition: elevated foliar sugar and starch, leaf chlorosis, and slow growth. In contrast, transgenic poplar grew normally and accumulated little, if any, excess sugar and starch in the leaves, and it did so even under high light conditions, where sugar synthesis is most active and the loading mechanism is most challenged. The results are consistent with passive, symplastic (through plasmodesmata) phloem loading in poplar.     

Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies

Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and “malic enzyme” (EC 1.1.1.40) in bundle sheath cells. NADP-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations.