Multiple glycolytic enzymes are tightly bound to the fibrous sheath of mouse spermatozoa

The fibrous sheath is a cytoskeletal structure located in the principal piece of mammalian sperm flagella. Previous studies showed that glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS), a germ cell-specific glycolytic isozyme that is required for sperm motility, is tightly bound to the fibrous sheath. To determine if other glycolytic enzymes are also bound to this cytoskeletal structure, we isolated highly purified fibrous sheath preparations from mouse epididymal sperm using a sequential extraction procedure. The isolated fibrous sheaths retain typical ultrastructural features and exhibit little contamination by axonemal or outer dense fiber proteins in Western blot analyses. Proteomic analysis using peptide-mass fingerprinting and MS/MS peptide fragment ion matching identified GAPDHS and two additional glycolytic enzyme subunits, the A isoform of aldolase 1 (ALDOA) and lactate dehydrogenase A (LDHA), in isolated fibrous sheaths. The presence of glycolytic enzymes in the fibrous sheath was also examined by Western blotting. In addition to GAPDHS, ALDOA, and LDHA, this method determined that pyruvate kinase is also tightly bound to the fibrous sheath. These data support a role for the fibrous sheath as a scaffold for anchoring multiple glycolytic enzymes along the length of the flagellum to provide a localized source of ATP that is essential for sperm motility.     

Formation of hyaluronan- and versican-rich pericellular matrix by prostate cancer cells promotes cell motility

Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility.     

Structural and Biochemical Analysis of the Sheath of Phormidium uncinatum

The sheath of the filamentous, gliding cyanobacterium Phormidium uncinatum was studied by using light and electron microscopy. In thin sections and freeze fractures the sheath was found to be composed of helically arranged carbohydrate fibrils, 4 to 7 nm in diameter, which showed a substantial degree of crystallinity. As in all other examined motile cyanobacteria, the arrangement of the sheath fibrils correlates with the motion of the filaments during gliding motility; i.e., the fibrils formed a right-handed helix in clockwise-rotating species and a left-handed helix in counterclockwise-rotating species and were radially arranged in nonrotating cyanobacteria. Since sheaths could only be found in old immotile cultures, the arrangement seems to depend on the process of formation and attachment of sheath fibrils to the cell surface rather than on shear forces created by the locomotion of the filaments. As the sheath in P. uncinatum directly contacts the cell surface via the previously identified surface fibril forming glycoprotein oscillin (E. Hoiczyk and W. Baumeister, Mol. Microbiol. 26:699–708, 1997), it seems reasonable that similar surface glycoproteins act as platforms for the assembly and attachment of the sheaths in cyanobacteria. In P. uncinatum the sheath makes up approximately 21% of the total dry weight of old cultures and consists only of neutral sugars. Staining reactions and X-ray diffraction analysis suggested that the fibrillar component is a homoglucan that is very similar but not identical to cellulose which is cross-linked by the other detected monosaccharides. Both the chemical composition and the rigid highly ordered structure clearly distinguish the sheaths from the slime secreted by the filaments during gliding motility.     

Penetration into rice tissues by brown planthopper and fine structure of the salivary sheaths

The fine structure of the salivary sheaths in plant tissues can provide important information on homopteran probing and ingestion behaviors. Salivary sheaths secreted by the brown planthopper (BPH), Nilaparvata lugens (Stål) (Homoptera: Delphacidae), and their tissue pathway were investigated using light, scanning electron, and transmission electron microscopy. About half of the salivary flanges on the surface of the food substrate were connected with internal salivary sheaths. Only 43% of the salivary sheaths showed side branches. Many sculpture‐like protuberances and small cavities had been formed on the outer surface of the salivary sheath, but the sheath lumen circumferences were sealed. Brown planthoppers showed a preference for probing and leaving salivary sheaths in the susceptible rice variety TN1 rather than in the resistant variety B5 during the first 2 days of the experiments. The salivary sheaths in rice tissues reached the inner tissue layer of the leaf sheaths and stems, but were mostly observed to end in the first and second layer of the leaf sheaths. Brown planthoppers also preferred to probe into the thick segment of the outer leaf sheath. After ingestion by the insect, the cytoplasm in both phloem and companion cells degraded and the main organelles were lost. Numerous small vesicles were found in most of the phloem cells, but cell walls remained intact. Large numbers of symbiont‐like structures were observed inside the salivary sheath lumen. These results indicated that BPH has complicated feeding behaviors, which warrants further investigation.     

Formation of Gold Particles via Thiol Groups on Glycoconjugates Comprising the Sheath Skeleton of Leptothrix

Leptothrix, iron-oxidizing bacterium, produces microtubular sheaths that surround the catenulate cells. Organic nanofibrils excreted from the cell surfaces interweave and coalesce to form immature sheaths, which attract aqueous-phase inorganics to eventually form mature organic–inorganic sheaths. Such inorganic encrustation of the sheaths results from interactions between functional groups in the sheath skeleton and inorganics. Based on our previous findings that Leptothrix sheath skeleton sorbed 47 inorganics (Au was one of the most abundant adsorbates), we examined the sorption status of Au cations on cell-enclosing sheaths and their protein-free remnants and found that nano to sub-micron Au particles (AuNPs and AuSMPs, respectively) formed on the sheath-forming polymer consisting of a glycoconjugate (an amphoteric glycan modified with cysteine, glycine, and 3-hydroxypropionic acid). When the purified polymer was incubated in HAuCl₄ solution, AuNPs and AuSMPs formed on the polymer surfaces. Both particles formed also on cell-enclosing sheaths and protein-free sheath remnants incubated in HAuCl₄ solution. When SH groups in the cell-enclosing sheaths were masked with a fluorescent protein, Au particles did not form after incubation in HAuCl₄ solution. Results implicate that SH groups are at least partially involved in the reduction of Au cations to metallic Au and eventual formation of Au particles.     

In vivo structures of an intact type VI secretion system revealed by electron cryotomography

The type VI secretion system (T6SS) is a versatile molecular weapon used by many bacteria against eukaryotic hosts or prokaryotic competitors. It consists of a cytoplasmic bacteriophage tail‐like structure anchored in the bacterial cell envelope via a cytoplasmic baseplate and a periplasmic membrane complex. Rapid contraction of the sheath in the bacteriophage tail‐like structure propels an inner tube/spike complex through the target cell envelope to deliver effectors. While structures of purified contracted sheath and purified membrane complex have been solved, because sheaths contract upon cell lysis and purification, no structure is available for the extended sheath. Structural information about the baseplate is also lacking. Here, we use electron cryotomography to directly visualize intact T6SS structures inside Myxococcus xanthus cells. Using sub‐tomogram averaging, we resolve the structure of the extended sheath and membrane‐associated components including the baseplate. Moreover, we identify novel extracellular bacteriophage tail fiber‐like antennae. These results provide new structural insights into how the extended sheath prevents premature disassembly and how this sophisticated machine may recognize targets.     

Type VI secretion and bacteriophage tail tubes share a common assembly pathway

The Type VI secretion system (T6SS) is a widespread macromolecular structure that delivers protein effectors to both eukaryotic and prokaryotic recipient cells. The current model describes the T6SS as an inverted phage tail composed of a sheath‐like structure wrapped around a tube assembled by stacked Hcp hexamers. Although recent progress has been made to understand T6SS sheath assembly and dynamics, there is no evidence that Hcp forms tubes in vivo. Here we show that Hcp interacts with TssB, a component of the T6SS sheath. Using a cysteine substitution approach, we demonstrate that Hcp hexamers assemble tubes in an ordered manner with a head‐to‐tail stacking that are used as a scaffold for polymerization of the TssB/C sheath‐like structure. Finally, we show that VgrG but not TssB/C controls the proper assembly of the Hcp tubular structure. These results highlight the conservation in the assembly mechanisms between the T6SS and the bacteriophage tail tube/sheath.     

Observations on penetration of Barley leaves by the aphidRhopalosiphum maidis (Fitch)

Summary Penetration of leaves of barley,Hordeum vulgare L., by the corn leaf aphid,Rhopalosiphum maidis (Fitch), was studied with light, phase, and electron microscopes. Penetration of epidermis and mesophyll was largely intercellular, that of vascular bundles or veins largely intracellular. Like other aphids,R. maidis secretes a salivary sheath which surrounds the stylets. When mesophyll cells and parenchymatous elements of the veins were penetrated by stylets, their protoplasts were pushed to one side by intruding sheath material; hence, the protoplasts were not punctured by the stylets, although sometimes the plasmalemma of penetrated cells was ruptured by sheath material. The salivary sheaths ended more or less abruptly outside the walls of sieve elements being fed upon, the maxillary stylets projecting beyond the sheaths and into the sieve elements. Before penetrating a functional sieve element the aphid apparently flushes its stylets in order to clear them for ingestion of food. Salivary and food canals merge near the tips of the maxillary stylets to form a single canal, which ends short of the tips.This research was supported by the U.S. National Science Foundation (GB-8330).     

Genetic analysis of spirochete flagellin proteins and their involvement in motility, filament assembly, and flagellar morphology

The filaments of spirochete periplasmic flagella (PFs) have a unique structure and protein composition. In most spirochetes, the PFs consist of a core of at least three related proteins (FlaB1, FlaB2, and FlaB3) and a sheath of FlaA protein. The functions of these filament proteins remain unknown. In this study, we used a multidisciplinary approach to examine the role of these proteins in determining the composition, shape, and stiffness of the PFs and how these proteins impact motility by using the spirochete Brachyspira (formerly Treponema, Serpulina) hyodysenteriae as a genetic model. A series of double mutants lacking combinations of these PF proteins was constructed and analyzed. The results show the following. First, the diameters of PFs are primarily determined by the sheath protein FlaA, and that FlaA can form a sheath in the absence of an intact PF core. Although the sheath is important to the PF structure and motility, it is not essential. Second, the three core proteins play unequal roles in determining PF structure and swimming speed. The functions of the core proteins FlaB1 and FlaB2 overlap such that either one of these proteins is essential for the spirochete to maintain the intact PF structure and for cell motility. Finally, linear elasticity theory indicates that flagellar stiffness directly affects the spirochete's swimming speed.     

OBSERVATIONS ON PENETRATION OF LINDEN BRANCHES BY STYLETS OF THE APHID LONGISTIGMA CARYAE

Penetration of the bark of Tilia americana L., the linden tree, by Longistigma caryae (Harr.) is mainly intracellular. Like other aphids, L. caryae secretes a saliva sheath which encloses the path of the stylets, beginning with an external collar of sheath material on the surface of the periderm. Stylet sheaths within the bark gave positive reactions for callose, suggesting that, in reaction to wounding, punctured parenchyma cells secrete callose which diffuses throughout the stylet sheaths. Other, more conspicuous effects of wounding included: proliferation and enlargement of cells of the cortex and dilated rays bordering some stylet sheaths, formation of tylosoids in punctured sieve elements, deposition of massive amounts of callose in penetrated sieve elements and in sieve elements bordering penetrated cells, and stimulation of cambial activity and xylem differentiation. Stylet tips located in living sieve elements projected beyond their sheaths which terminated outside the sieve-element walls. It is suggested that such sieve elements can be considered to be functional. None of the living sieve elements containing stylet tips showed any signs of injury which could be attributed to the presence of the stylets. Stylet tips of feeding aphids were found in living sieve elements of both 1965 and 1966 phloem increments clearly indicating that L. caryae can feed on linden sieve elements more than 1 year of age.     

A cytological study of the ovary of Rhodnius prolixus. III. Cytoarchitecture and development of the trophic chamber

The tropharium of the telotrophic ovarioles of Rhodnius is syncytial with the nurse cell nuclei located in tortuous finger-like projections arborizing from a common cytoplasmic area, the trophic core. The nurse cell nuclei exhibit prominent nucleoli. Located adjacent to the nuclear envelope are masses of granular material both within the nucleus and adjoining cytoplasm. The cytoplasm consists primarily of ribosomes and mitochondria. The trophic core and the trophic cords that connect the core to individual oocytes characteristically possess parallel arrays of microtubules with ribosomes and mitochondria interspersed between. Surrounding the nurse tissue (germarium) is a thin layer of squamous cells comprising the inner sheath. The inner sheath is encompassed by the non-cellular tunica propria superficial to which are two external cellular sheaths. The syncytial nature of the tropharium appears to arise as a result of the fusion of many entangled nurse cell-oocyte complexes during the late fifth instar. The structural similarities, and possible homologies with the polytrophic type of ovariole is discussed.     

GLOEOCOCCUS TETIMSPORUS SP. NOV. (TETRASPORALES,PALM ELL ACEAE), FROM COLORADO MOUNTAIN LAKES1

A new species, Gloeococcus tetrasporus sp. nov., collected from mountain lakes, is described from unialgal culture. Vegetative cells are ellipsoid and Chlamydomo nas–like, occur in tetrad complexes within the general colonial matrix, and exhibit slow, limited motility within the confines of the individual gelatinous matrices. The colonial matrix is amorphous and structureless, without a definite bounding layer. Colonies may reach several centimeters in size. Vegetative cells have a parietal cup–shaped chloroplast with a central–basal pyrenoid and a small, linear stigma, two contractile vacuoles, and two short flagella. Cell division is by eleutheroschisis in nonflagellate cells. After two divisions, four daughter cells arc formed within the expanded parent wall that will become incorporated into the colonial matrix. Zoospores are formed either from transformed vegetative cells or after cytokinesis. Zoospore flagella are two to three times the length of vegetative cell flagella. Rapid flagellar movement ruptures the sheath and liberates the zoospores. When zoospores settle, they secrete new sheaths, and divide twice to initiate new colonies. Sexual reproduction and formation of resistant spores were not observed.     

THE FORMATION AND STRUCTURE OF MYELIN SHEATHS IN THE CENTRAL NERVOUS SYSTEM

The development and structure of myelin sheaths have been studied in the optic nerves of rats and of Xenopus laevis tadpoles. Both potassium permanganate- and osmium-fixed material was examined with the electron microscope. In the first stage of myelinogenesis the nerve fibre is surrounded by a cell process which envelops it and forms a mesaxon. The mesaxon then elongates into a loose spiral from which the cytoplasm is later excluded, so that compact myelin is formed. This process is similar to myelinogenesis in the peripheral nervous system, although in central fibres the cytoplasm on the outside of the myelin is confined in a tongue-like process to a fraction of the circumference, leaving the remainder of the sheath uncovered, so that contacts are possible between adjacent myelin sheaths. The structure of nodes in the central nervous system has been described and it is suggested that the oligodendrocytes may be the myelin-forming cells.     

Fine-structural and chemical analyses on inner and outer sheath of the cyanobacteriumGloeothece sp. PCC 6909

Cells of the unicellular cyanobacteriumGloeothece sp. PCC 6909 are surrounded by an inner (enclosing 1–2 cells) and an outer (enclosing cell groups) sheath. Using conventional Epon-embedding in combination with ruthenium-red staining, the inner and outer sheaths appeared similar and displayed multiple bands of electron-dense subunits. However, embedding in Nanoplast resin to avoid shrinkage led to the detection of two distinct zones (inner and outer zone) each with several distinct layers. The zone delimited by the electron-dense thick inner sheath layer, and the zone enclosed by the thin electron-dense outer sheath layer, are composed of a homogeneous material of little electron-contrast. Whereas the outer zone appears to be of even contrast, the inner zone is characterized by a distinct electron-transparent layer. Element distribution analysis revealed that the electron-transparent layer contained relatively large amounts of sulfur, carbon, and oxygen but only little nitrogen.Inner and outer sheath fractions were isolated by differential mechanical cell breakage and centrifugation. The outer sheath fraction was less hydrated than the inner one. The two fractions differed little in their contents of uronic acids, carbohydrate and protein, although the outer sheath fraction contained less sulfate. A soluble polysaccharide with a chemical composition similar to that of inner and outer sheath fractions was also obtained from the culture supernatant.     

Lipid polymers accumulate in the epidermis and mestome sheath cell walls during low temperature development of winter rye leaves

Summary Winter rye (Secale cereale L cv. Puma) was grown at 20 °C and at 5 °C and the development of epidermal and mestome sheath cells of leaves from plants grown at both temperatures was compared by electron microscopy. At 5 °C, the cells became densely packed with cytoplasm and small vacuoles after 41 days of growth. By day 56 at 5 °C, epidermal and mestome sheath cells were small in diameter and multivacuolate with asymmetrically thickened walls. By day 76 at 5 °C, a new developmental stage had been reached in epidermal and mestome sheath cells. The cells were larger in diameter although the thickened cell walls and multivacuolate cytoplasm were still present. As epidermal and mestome sheath cell walls thickened during low temperature growth of winter rye, an increase in cuticle thickness and the deposition of a lamellar layer could be observed in epidermal and mestome sheath cells, respectively. The lipid-derived polymers from the leaves of rye plants grown at 20 °C were shown by reductive depolymerization and GC-MS analysis to be comprised of 18-hydroxy-9, 10-epoxyoctadecanoic acid (47%) and dihydroxyhexa-decanoic acid (29%). The leaves of plants grown at 5 °C had two to four times as much lipid-derived polymeric material as those grown at 20 °C and the proportion of the major monomer, 18-hydroxy-9,10-epoxyoctadecanoic acid, increased to 73% of the polymeric material. Physical isolation of both epidermal tissue and vascular bundles followed by GC-MS analysis of the monomeric components released by reduction of the respective lipid polymers showed that 18-hydroxy-9,10 epoxyoctadecanoic acid was the major monomer in the polymer of both the epidermis and the mestome sheaths. The presence of this epoxide monomer in both the cuticles and mestome sheath cell walls of rye leaves was confirmed and visualized by using an epoxide-specific staining reaction.     

Ultrastructure of leaves in C4 Cyperus iria and C3 Carex siderosticta

The ultrastructural aspects ofCyperus iria leaves showing the C₄ syndrome and the typical C₃ species,Carex siderosticta, in the Cyperaceae family were examined.C. iria exhibited the chlorocyperoid type, showing an unusual Kranz structure with vascular bundles completely surrounded by two bundle sheaths. The cellular components of the inner Kranz bundle sheath cells were similar to those found in the NADP-ME C₄ subtype, having centrifugally arranged chloroplasts with greatly reduced grana and numerous starch grains. Their chloroplasts contained convoluted thyla-koids and a weakly-developed peripheral reticulum, although it was extensive mostly in mesophyll cell chloroplasts. The outer mestome bundle sheath layer was sclerenchymatous and generally devoid of organelles, but had unevenly thickened walls. Suberized lamellae were present on its cell walls, and they became polylamellate when traversed by plasmodesmata. Mesophyll cell chloroplasts showed well-stacked grana with small starch grains. InC. siderosticta, vascular bundles were surrounded by the inner mestome sheath and the outer parenchymatous bundle sheath with intercellular spaces. The mestome sheath cells degraded in their early development and remained in a collapsed state, although the suberized lamellae retained polylamellate features. Plastids with a crystalline structure, sometimes membrane-bounded, were found in the epidermal cells. The close interveinal distance was 35–50 μm inC. iria, whereas it was 157–218 μm inC. siderosticta. These ultrastructural characteristics were discussed in relation to their photosynthetic functions.     

COMPARATIVE BUNDLE SHEATH AND MESOPHYLL DIFFERENTIATION IN THE LEAVES OF THE C4 GRASSES PANICUM EFFUSUM AND P. BULBOSUM

The two C₄ Panicum species examined differ in C₄ acid decarboxylation type and in developmental origin of bundle sheaths in major veins of their leaf blades. In Panicum effusum R.Br. (NAD-malic enzyme type) both mesophyll (PCA) and chlorenchymatous bundle sheath (PCR) cells are derived from ground meristem. In contrast, in Panicum bulbosum H.B.K. (NADP-malic enzyme type), bundle sheath cells are derived from procambium, while mesophyll develops from ground meristem. To test the hypothesis that the developmental divergence of bundle sheath and mesophyll cells would occur earlier when these two tissues had different ontogenetic origins (in P. bulbosum) than when these tissues had the same origin (in P. effusum), the development of major veins in each species was investigated. We measured cell length and cross sectional area, plastid and mitochondrial number, plastid area, vacuole area fraction, wall thickness and fraction adjacent to intercellular space using direct and digitizer measurements of transmission electron micrographs of leaf cross sections of successive developmental stages. Many of the statistically significant changes in the structural parameters measured occurred late in development of both species. The magnitude of developmental change in P. effusum PCR cells was sometimes more dramatic, viz. changes in cell and PCR plastid area, and in mitochondrion number per cell. However, earlier divergence of PCR and PCA length and volume, and wall fraction adjacent to intercellular space in P. bulbosum than in P. effusum indicates that ontogenetic origin of PCR cells from procambium could determine the timing of at least some developmental events.     

Motility of the spirochete Leptospira

Spirochetes are a group of bacteria with a unique ultrastructure and a fascinating swimming behavior. This article reviews the hydrodynamics of spirochete motility, and examines the motility of the spirochete Leptospira in detail. Models of Leptospira motility are discussed, and future experiments are proposed. The outermost structure of Leptospira is a membrane sheath, and within this sheath are a helically shaped cell cylinder and two periplasmic flagella. One periplasmic flagellum is attached subterminally at either end of the cell cylinder and extends partway down the length of the cell. In swimming cells, each end of the cell may assume either a spiral or a hook shape. Translational cells have the anterior end spiral shaped, and the posterior end hook shaped. In the model of Berg et al., the periplasmic flagella are believed to rotate between the sheath and the cell cylinder. Rotation of the anterior periplasmic flagellum causes the generation of a gyrating spiral-shaped wave. This wave is believed sufficient to propel the cells forward in a low-viscosity medium. The cell cylinder concomitantly rolls around the periplasmic flagella in the opposite direction--which allows the cell to literally screw through a gel-like viscous medium without slippage. This model is presented, and it is contrasted to previous models of Leptospira motility.     

Rice Resistance to Sheath Blight Mediated by Potassium

This study investigated the effect of potassium (K) on sheath blight (Rhizoctonia solani) development on rice plants from cultivars BR‐IRGA 409 and Labelle grown in nutrient solution containing 0, 50 and 100 mm of K. Sheath blight progress on inoculated sheaths was evaluated by measuring the relative lesion length at 48, 72, 96 and 120 h after inoculation (hai). Data were used to calculate the area under relative lesion length progress curve (AURLLPC). The foliar K concentration on leaf sheaths tissue increased by 61.48 and 116.05% to cultivars BR‐IRGA 409 and Labelle, respectively, as the K rates increased from 0 to 100 mm . A linear model best described the relationship between the AURLLPC and the K rates. The AURLLPC decreased by 29.2 and 21.3% for cultivars BR‐IRGA 409 and Labelle, respectively, as the K rates in the nutrient solution increased. It can be concluded that high K concentration on leaf sheaths tissue was important to decrease sheath blight symptoms on rice leaf sheaths.     

Ultrastructural aspects of leaves inFestuca ovina andPoa sphondylodes (C-3 Poaceae)

Structural aspects of the leaves of two common festucoids,Festuca ovina andPoa sphondylodes, have been examined employing the electron microscopy. The nature of vascular bundles and of sheaths that surround vascular tissues was discussed in the study. The festucoids exhibited a non-Kranz C-3 anatomy with more than four mesophyll cells separating the bundle sheaths of a leaf blade. Vascular tissues in theseFestuca andPoa leaves were surrounded by a double sheath: an inner distinct mestome sheath (MST) and an outer indistinctive layer of parenchymatous bundle sheath (PBS) cells. The PBS cells were much larger than the MST and had thin walls. The MST cells were relatively small and rectangular inP. sphondylodes and more or less hexangular in transverse sections ofF. ovina. InP. sphondylodes, MST had conspicuously thickened inner tangential walls with asymmetrically uninterrupted suberized lamellae in radial and tangential walls. In most differentiated MST cells, all walls were highly suberized. During suberin deposition, MST cells were quite vacuolated and most of the cytoplasm was present as a thin peripheral layer. However, MST walls inF. ovina revealed very thin suberized lamellae with translucent striations. No chloroplasts were detected inP. sphondylodes, whereas the MST inF. ovina contained small chloroplasts. Plasmodesmata were well developed in the primary pit fields of walls between MST and vascular cells, and between adjacent MST cells. Plasmodesmata were less frequent in the walls between the inner and outer sheath cells. Suberized lamellae were totally absent from the PBS cell walls in all veins. External to the PBS, the mesophyll comprised thin walled cells with abundant intercellular spaces. Peripherally arranged chloroplasts in the mesophyll were numerous and often larger than those of PBS and MST cells. Characteristics associated with C-3 and other ultrastructural features were also discussed in the study.