GLANDULAR CUTICLE FORMATION IN CANNABIS (CANNABACEAE)

Formation of the cuticle from components of the secretory cavity and subcuticular wall was studied by transmission electron microscopy of glandular trichomes of Cannabis prepared by high pressure cryofixation-cryosubstitution. Secretory vesicles in the secretory cavity resembled those localized in the subcuticular wall as well as the vesicle-related material associated with the irregular inner surface of the cuticle and appeared to provide precursors for thickening of the cuticle. Some contiguous vesicles in the secretory cavity and subcuticular wall lacked a surface feature at their point of contact, supporting an interpretation of vesicle fusion. Fibrillar matrix from the secretory cavity contributed fibrillar matrix to the subcuticular wall, and persisted as residual fibrillar matrix associated with secretory materials coalesced to the thickened inner surface of the cuticle. Elongated fibrils arranged in uniformly spaced parallel pairs contributed to the organization of fibrillar matrix in the subcuticular wall. Striae were evident in the outer portion of the cuticle, and appeared to represent sites of degraded residual fibrillar matrix associated with secretory materials coalesced to the inner cuticular surface. This study supports an interpretation that contents of secretory vesicles from the secretory cavity contribute to formation of glandular cuticle.     

Cytochemical localization of cellulase in glandular trichomes ofcannabis (cannabaceae)

Cellulase reaction product was localized cytochemically at the ultrastructural level in the cell wall of disc cells, the secretory cavity and in the subcuticular wall of glands inCannabis. Cellulase reaction product was evident in the less dense region of the disc cell wall prior to secretory cavity formation. Reactivity in this region was associated with separation of an outer zone, forming the subcuticular wall, from the inner wall zone adjacent to the plasma membrane of the disc cells. Reaction product was associated with the disc cell wall and fibrillar matrix extending from it into the secretory cavity. Reactivity remained evident over the subcuticular wall throughout enlargement of the secretory cavity. Reaction product also was present over fibrillar matrix in the secretory cavity associated with both the inner wall and the subcuticular wall. The distribution of cellulase reaction product supports an interpretation that cellulase is involved in formation of the secretory cavity and subsequent redistribution of wall products to form the subcuticular wall during development of the secretory cavity.     

SECRETORY VESICLE FORMATION IN GLANDULAR TRICHOMES OF CANNABIS SATIVA (CANNABACEAE)

Formation of secretory vesicles in the noncellular secretory cavity of glandular trichomes of Cannabis saliva L. was examined by transmission electron microscopy. Two patterns of vesicle formation occurred during gland morphogenesis. 1) During initial phases of cavity formation small hyaline areas arose in the wall near the plasma membrane of the disc cell. Hyaline areas of elongated shape and different sizes were distributed throughout the wall and adjacent to the secretory cavity. Hyaline areas increased in size, some possibly fusing with others. These hyaline areas, possessing a membrane, moved into the cavity where they formed vesicles. As membraned vesicles they developed a more or less round shape and their contents became electron-dense. 2) During development of the secretory cavity and when abundant secretions were present in the disc cells, these secretions passed through the wall to accumulate as membraned vesicles of different sizes in the cavity. As secretions emerged from the wall, a membrane of wall origin delimited the secretory material from cavity contents. Vesicles released from the wall migrated in the secretory cavity and contacted the sheath where their contents permeated into the subcuticular wall as large or diffused quantities of secretions. In the subcuticular wall these secretions migrated to the wall–cuticle interface where they contributed to structural thickening of the cuticle. This study demonstrates that the secretory process in glands of Cannabis involves not only secretion of materials from the disc cell, but that the disc cell somehow packages these secretions into membraned vesicles outside the cell wall prior to deposition into the secretory cavity for subsequent structural development of the sheath.     

Immunochemical localization of tetrahydrocannabinol (THC) in cryofixed glandular trichomes of Cannabis (Cannabaceae)

Delta 9-tetrahydrocannabinol (THC) localization in glandular trichomes and bracteal tissues of Cannabis, prepared by high pressure cryofixation-cryosubstitution, was examined with a monoclonal antibody-colloidal gold probe by electron microscopy (EM). The antibody detected THC in the outer wall of disc cells during the presecretory cavity phase of gland development. Upon formation of the secretory cavity, the immunolabel detected THC in the disc cell wall facing the cavity as well as the subcuticular wall and cuticle throughout development of the secretory cavity. THC was detected in the fibrillar matrix associated with the disc cell and with this matrix in the secretory cavity. The antibody identified THC on the surface of secretory vesicles, but not in the secretory vesicles. Gold label also was localized in the anticlinal walls between adjacent disc cells and in the wall of dermal and mesophyll cells of the bract. Grains were absent or detected only occasionally in the cytoplasm of disc or other cells of the bract. No THC was detected in controls. These results indicate THC to be a natural product secreted particularly from disc cells and accumulated in the cell wall, the fibrillar matrix and surface feature of vesicles in the secretory cavity, the subcuticular wall, and the cuticle of glandular trichomes. THC, among other chemicals, accumulated in the cuticle may serve as a plant recognition signal to other organisms in the environment.     

CUTICLE DEVELOPMENT ON GLANDULAR TRICHOMES OF CANNABIS SATIVA (CANNABACEAE)

The dermal sheath of glandular trichomes of Cannabis sativa L., consisting of cuticle and a subcuticular wall, was examined by transmission electron microscopy. Cuticle thickened selectively on the outer wall of disc cells of each trichome prior to formation of the secretory cavity, whereas thickening was less evident on the dermal cells of the bract. Membraned secretory vesicles that differ in size and appearance in the secretory cavity were the source of precursors for synthesis of cuticle. Vesicle contents, released following the degradation of the vesicle membrane upon contact with the subcuticular wall, contributed to both structured and amorphous phases of cuticle development. The structured phase was represented by deposition and thickening of cuticle at the subcuticular wall-cuticle interface to form a thickened cuticle. In the amorphous phase precursors permeated the cuticle in a liquid state, as shown by fusion of cuticles and wax layers between contiguous glands, and may have contributed to growth in surface area of the expanding sheath. Disc cells are interpreted to control growth of secretory cavity by secretion of membraned vesicles into the cavity. The thickened cuticle, which increased eightfold in thickness during enlargement of the gland, provided structural strength for the extensive surface area of the dermal sheath. The gland of Cannabis in which vesicle contents contribute to the growth in thickness and surface area of the cuticle of the sheath is interpreted to represent a phylogenetically derived state as contrasted to secretory glands possessing only cuticle and lacking a complement of secretory vesicles.     

Developmental Ultrastructure of Glandular Trichomes of <Emphasis Type="Italic">Rosmarinus officinalis</Emphasis>: Secretory Cavity and Secretory Vesicle Formation

Glandular trichomes in the leaf lamina of Rosmarinus officinalis L. were examined by scanning and transmission electron microscopy. The leaves were characterized by an abundance of two types of glandular trichomes—small capitate and large peltate glandular trichomes. In addition to the glandular trichomes, numerous non-glandular trichomes were present on the abaxial surface of the leaf. These trichomes mainly predominated on the midrib, whereas glandular trichomes occurred on non-vein areas. At the initial phase of secretory cavity formation, hyaline areas were abundant in periclinal walls of head cells, while they were not observed in the anticlinal walls. The hyaline areas gradually increased in size, fusing with other areas throughout the wall. Loose wall material adjacent to hyaline areas was released from the head cell walls and migrated into the secretory cavities. As the secretory cavities continued to enlarge, the new vesicles emerging into the secretory cavities from the walls of head cells became surrounded with the surface of a typical membrane. They developed a round shape, but the contours of the vesicle surfaces appeared polygonal when tightly packed inside a cavity. These vesicles varied in size; small vesicles often possessed electron-dense contents, while large vesicles contained electron-light contents.     

Secretory vesicle formation in the secretory cavity of glandular trichomes of Cannabis sativa L. (Cannabaceae)

The disc cell wall facing the secretory cavity in lipophilic glands of Cannabis was studied for origin and distribution of hyaline areas, secretory vesicles, fibrillar matrix and particulate material. Secretions evident as light areas in the disc cell cytoplasm pass through modified regions in the plasma membrane and appear as hyaline areas in the cell wall. Hyaline areas, surrounded with a filamentous outline, accumulate near the wall surface facing the secretory cavity where they fuse to form enlarged hyaline areas. Fibrillar matrix is related to and may originate from the dense outer layer of the plasma membrane. This matrix becomes distributed throughout the wall material and contributes in part to the composition of the surface feature of secretory vesicles. Thickening of the cell wall is associated with secretions from the disc cells that facilitates movement of hyaline areas, fibrillar matrix and other possible secretions through the wall to form secretory vesicles and intervesicular materials in the secretory cavity. The outer wall of disc cells in aggregate forms the basilar wall surface of the secretory cavity which facilitates the organization of secretory vesicles that fill the secretory cavity.     

The fine structure of the tarsal glands of the honeybee Apis mellifera L. (Hymenoptera)

Summary Tarsal glands are located in the 6th tarsomere of adult honeybee queens, workers and drones. Their structural features are not cast or sex specific. The glandular epithelium is lined by a thin endocuticular layer. A cuticular pocket is formed from a postimaginal delamination of the cuticle secreted by the glandular epithelium. The apical plasma membrane of the glandular cells shows numerous cristae and microvilli lining large crypts that communicate with the subcuticular space. Pinocytotic vesicles, multivesicular bodies and residual dense bodies are present in the apical part of the glandular cells. The RER is well developed in perinuclear and basal parts of the glandular cells, but the Golgi apparatus is a discrete organelle without secretory granules. No exocytotic secretory structures were observed. To reach the glandular pocket, the non-proteinaceous secretory product must pass across the subcuticular space, the cuticular intima, the space between the intima and the cuticular wall, and the cuticular wall of the glandular pocket.     

Early Development of the Secretory Cavity of Peltate Glands in Humulus lupulus L. (Cannabaceae)

Early development of the secretory cavity of chemically fixed peltate glands in Humulus lupulus L. showed secretions with different densities, light, gray and dark, in the cytoplasm of disc cells and in the periplasmic space adjacent to the developing secretory cavity. Secretions were detected in the disc cell wall and subsequently in the developing secretory cavity under the subcuticular wall of the sheath. Light and gray secretions in the cavity possessed a membrane-like surface feature. Secretions were in contact with the irregular inner surface of the cuticle. Secretions contributed to the thickening of the cuticle, whereas the membrane-like surface feature contributed to a network of Cannabis striae distributed throughout the cuticle. This study supports an early development and organization of the secretory cavity in H. lupulus, parallel to those in Cannabis, and may represent common features for lipophilic glands in angiosperms.     

Foliar secretory trichomes of Ocimum obovatum (Lamiaceae): micromorphological structure and histochemistry

This study characterises the micromorphology, ultrastructure and main chemical constituents of the foliar glandular trichomes of Ocimum obovatum using light and electron microscopy and a variety of histochemical tests. Two types of glandular trichomes occur on the leaves: large peltate and small capitate. The head of each peltate trichome is made up of four broad head cells in one layer. The head of each capitate trichome is composed of two broad head cells in one layer (type I) or a single oval head cell (type II, rare). In peltate heads, secretory materials are gradually transported to the subcuticular space via fracture in the four sutures at the connecting walls of the head cells. Release to the head periphery occurs through opposite fracture in the four sutures in the head cuticle. In type I capitate trichomes, release of the secretions to the subcuticular space occurs via a pore between the two head cells, and release to the head periphery occurs through the opposite pore in the head cuticle. In type II capitate trichomes, the secreted material is released from the head cell through a ruptured particular squared area at the central part of the head cuticle. These secretion modes are reported for the first time in the family Lamiaceae. Histochemical tests showed that the secretory materials in the glandular trichomes are mainly essential oils, lipophilic substances and polysaccharides. Large peltate trichomes contain a large quantity of these substances than the small capitate trichomes. Ultrastructural evidence suggests that the plastids produce numerous lipid droplets, and the numerous polysaccharide small vesicles are derived from Golgi bodies.     

ULTRASTRUCTURAL DEVELOPMENT OF CAPITATE GLANDULAR HAIRS OF CANNABIS SATIVA L. (CANNABACEAE)

The capitate-sessile and capitate-stalked glands of the glandular secretory system in Cannabis, which are interpreted as lipophilic type glandular hairs, were studied from floral bracts of pistillate plants. These glands develop a flattened multicellular disc of secretory cells, which with the extruded secretory product forms the gland head and the auxiliary cells which support the gland head. The secretory product accumulates beneath a sheath derived from separation of the outer wall surface of the cellular disc. The ultrastructure of secretory cells in pre-secretory stages is characterized by a dense ground plasm, transitory lipid bodies and fibrillar material, and well developed endoplasmic reticulum. Dictyosomes and dictyosome-derived secretory vesicles are present, but never abundant. Secretory stages of gland development are characterized by abundant mitochondria and leucoplasts and by a large vacuolar system. Production of the secretory product is associated with plastids which increase in number and structural complexity. The plastids develop a paracrystalline body which nearly fills the mature plastid. Material interpreted as a secretion appears at the surface of plastids, migrates, and accumulates along the cell surface adjoining the secretory cavity. Extrusion of the material into the secretory cavity occurs directly through the plasma membrane-cell wall barrier.     

线纹香茶菜叶上腺毛发育的细胞学研究

为进行中药溪黄草基原植物的品种鉴定,采用光镜和电镜对线纹香茶菜(原变种)[Isodon lophanthoides var.lophanthoides]叶上腺毛的发育进行细胞学研究。结果表明,线纹香茶菜具有头状腺毛和盾状腺毛2种类型。头状腺毛无色透明,由1个基细胞、1个柄细胞和1或2个头部分泌细胞构成;盾状腺毛为红色,由1或2个基细胞、1个柄细胞和4~8个分泌细胞构成头部。2种腺毛均由原表皮细胞经两次平周分裂形成,后因柄细胞和头部细胞所处的分化状态不同而形成两类腺毛。2种腺毛超微结构表明,质体、高尔基体和粗面内质网为主要分泌物产生和运输的细胞器。当盾状腺毛成熟时,角质层下间隙充满了分泌物,其分泌物的性质很可能决定了线纹香茶菜腺毛的颜色。     

Morphological study of the development of the disseminate prostate in intact and castrated male pigs aged two to six months.

The light-microscopical and ultrastructural differentiation of the disseminate prostate was studied in 12 boars and 8 barrows, ranging in age from 8 to 28 and 12 to 27 weeks, respectively. The barrows had been castrated at the age of 8 weeks. In young boars the prostate is organized in an inner zone and an outer zone. The glandular tubules of the latter are more differentiated than those in the inner zone. Sexual maturation in the prostate is characterized by almost complete reduction of the inner zone and concomitant enlargement of the outer zone. This differentiation of the inner zone occurs progressively and is manifested by a higher density of the glandular tubules, by a decrease in the number of basal cells and by an increase in the secretory tubular cells containing more microvilli, organelles and secretory vesicles. A few weeks after castration, the characteristic morphology of the undifferentiated inner zone is found throughout the entire glandular layer. The number of glandular tubules in barrows has decreased, and their cells show minimal secretory activity, nuclear indentations and a decrease in their organelles. A further castration effect is vacuolization of the perinuclear cytoplasm in numerous glandular cells.     

Glandular Trichomes on Vegetative and Reproductive Organs of Leonotis leonurus (Lamiaceae)

The types of glandular trichomes, their ontogeny and patternof distribution on the vegetative and reproductive organs ofLeonotis leonurus at different stages of development, are studiedby light and scanning electron microscopy. Two morphologicallydistinct types of glandular trichomes (peltate and capitate)are described. Peltate trichomes, at the time of secretion,are characterized by a short stalk, which is connected witha large spherical head composed of eight cells in a single layer.Capitate trichomes can be divided into various types. Generally,they consist of a four-celled head supported by one or threestalk cells. The two kinds of trichomes differ in the secretionprocess. In the peltate trichomes, the secretory product seemsto remain accumulated in a subcuticular space, unless an externalfactor damages it. In the capitate trichomes, this product probablybecomes released through micropores. On the leaves peltate andcapitate trichomes are abundant, while on the flowers only thepeltate trichomes are numerous and the capitate are rare orabsent.Copyright 1995, 1999 Academic Press Leonotis leonurus R. Br., lion's ear, lion's tail, Lamiaceæ, glandular trichomes, morphology, ontogeny     

Glandular Trichomes on Alfalfa Impede Searching Behavior of the Potato Leafhopper Parasitoid

New cultivars of alfalfa, Medicago sativa L., have been released with glandular trichomes for resistance to potato leafhopper, Empoasca fabae (Harris). Yet, the impact of the glandular trichomes on the primary natural enemy of the leafhopper, Anagrus nigriventris Girault, is unknown. We compared the host searching behavior of the egg parasitoid on four alfalfa clones varying in trichome characters. Female wasps were videotaped on Ranger, a susceptible clone with relatively sparse trichomes, B14, a resistant clone with dense but nonglandular trichomes, and FG12 and FG18, two resistant clones with glandular trichomes. Although the number of leafhopper eggs per stem exposed to wasps did not significantly differ among the four clones, the frequency of foraging and total foraging time were less on the two clones with glandular trichomes than on the two clones with nonglandular trichomes. In addition, an analysis of covariance demonstrated that, although the number of ovipositional probes increased with egg density on a stem, the number of probes on stems with glandular trichomes was significantly less than that on stems without glandular trichomes. The allocation of time by wasps among drumming, probing, and grooming behaviors was similar among the clones. Wasps tended to fly off of clones with glandular trichomes more often than off of clones with nonglandular trichomes. This study suggests that cultivars with glandular trichomes may interfere with host searching by A. nigriventris.     

甘草腺毛的形态发生和组织化学研究

利用扫描电镜及薄切片技术对甘草的腺毛形态发生和发育过程进行了观察,并对腺毛发育过程中黄酮类成分积累进行了组织化学定位研究。结果表明：甘草腺毛为多细胞构成的盾状腺毛,有长柄和短柄2种类型;前者主要分布在花萼片上,而后者主要分布于叶片上。组化鉴定结果显示：腺毛中存在着黄酮类成分、其他亲脂类和非纤维素多糖类成分;在腺毛的发育过程中,黄酮类物质是随腺毛的发育成熟,在头部盘状结构的分泌细胞及角质层下腔中积累。研究结果对进一步探讨甘草叶中黄酮类成分的合成及其作用提供科学依据。     

The fine structure of the developing oocyst of Pterospora floridiensis (Apicomplexa,Urosporidae)

Developing oocysts of the gregarine Pterospora floridiensis Landers 2001 were examined by transmission electron microscopy. Each oocyst had an outer capsule and an inner capsule that contained 8 sporozoites. In early stages of development the inner capsular wall was separated from the developing sporozoites and residual mass, and was not appressed to the sporozoites. Early stage sporozoites were connected to a residual mass and were filled with endoplasmic reticulum, golgi and numerous developing secretory vesicles. In late stages of oocyst and sporozoite development, the inner capsular wall was closely appressed to the sporozoite surface. The inner capsular wall was ～60-100 nm thick and the outer capsular wall was ～160-320 nm thick. There were no extensions on the outer wall for which the genus was named. Late stage sporozoites had no residual mass connection, were more electron dense, and contained three distinct types of dense secretory structures: 1) small oval/spherical dense vesicles, 2) large (350-400 nm) vesicles near the anterior end, and 3) elongated dense tubular bodies that converged at the apex. Few ultrastructural reports exist of developing gregarine oocysts and sporozoites, and as more studies are completed these morphological characteristics may be important in interpreting molecular phylogenetic analyses.     

Development of peltate glandular trichomes of peppermint

Cryofixation and conventional chemical fixation methods were employed to examine the ultrastructure of developing peltate glandular trichomes of peppermint (Mentha x piperita). Our results are discussed in relation to monoterpene production and the mechanism of essential oil secretion. Peltate glands arise as epidermal protuberances (initials) that divide asymmetrically to produce a vacuolate basal cell, a stalk cell, and a cytoplasmically dense apical cell. Further divisions of the apical cell produce a peltate trichome with one basal cell, one stalk cell, and eight glandular (secretory) disc cells. Presecretory gland cells resemble meristematic cells because they contain proplastids, small vacuoles, and large nuclei. The secretory phase coincides with the separation and filling of the sub-cuticular oil storage space, the maturation of glandular disc cell leucoplasts in which monoterpene biosynthesis is known to be initiated, and the formation of extensive smooth endoplasmic reticulum at which hydroxylation steps of the monoterpene biosynthetic pathway occur. The smooth endoplasmic reticulum of the secretory cells appears to form associations with both the leucoplasts and the plasma membrane bordering the sub-cuticular oil storage cavity, often contains densely staining material, and may be involved with the transport of the monoterpene-rich secretion product. Associated changes in the ultrastructure of the secretory stage stalk cell are also described, as is the ultrastructure of the fragile post-secretory gland for which cryofixation methods are particularly well suited for the preservation of organizational integrity.     

Ultrastructure of the Accessory Glands in Female Genitalia of Pholcus phalangioides(Fuesslin, 1775) (Pholcidae; Araneae)

Pholcus phalangioidesdoes not possess receptacular seminis. The uterus externus (genital cavity) itself functions as a sperm storage structure. Two accessory glands are situated in the dorsal part of the uterus externus; they discharge their secretory product into the genital cavity. The secretion is considered to serve primarily as a matrix for sperm storage, i.e. to keep the spermatozoa in a fixed position. The accessory glands consist of numerous glandular units, each being composed of four cells: two secretory cells are always joined and surrounded twice by an inner and an outer envelope cell. Both envelope cells take part in forming a cuticular ductule that leads from the secretory cells to the pore plates of the uterus externus. The inner envelope cell produces the proximal part of the canal close to the microvilli of the secretory cells, whereas the outer envelope cell produces the distal part of the canal leading to the pore plate. Close to the pore the latter exhibits prominent microvilli that might indicate additional secretory activity.     

Cryo-electron microscopy reveals native polymeric cell wall structure in Bacillus subtilis 168 and the existence of a periplasmic space

Ultrarapid freezing of bacteria (i.e. vitrification) results in optimal preservation of native structure. In this study, cryo-transmission electron microscopy of frozen-hydrated sections was used to gain insight into the organization of the Bacillus subtilis 168 cell envelope. A bipartite structure was seen above the plasma membrane consisting of a low-density 22 nm region above which a higher-density 33 nm region or outer wall zone (OWZ) resided. The interface between these two regions appeared to possess the most mass. In intact and in teichoic acid-extracted wall fragments, only a single region was seen but the mass distribution varied from being dense on the inside to less dense on the outside (i.e. similar to the OWZ). In plasmolysed cells, the inner wall zone (IWZ)'s thickness expanded in size but the OWZ's thickness remained constant. As the IWZ expanded it became filled with plasma membrane vesicles indicating that the IWZ had little substance and was empty of the wall's polymeric network of peptidoglycan and teichoic acid. Together these results strongly suggest that the inner zone actually represents a periplasmic space confined between the plasma membrane and the wall matrix and that the OWZ is the peptidoglycan-teichoic acid polymeric network of the wall.