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.     

Essential Oil Gland Number and Ultrastructure During Mentha arvensis Leaf Ontogeny

Alterations in essential oil gland number, distribution and fine structure, and the oil content in the leaf of Mentha arvensis L. were examined during its growth and senescence. Accumulation of essential oil occurred predominantly during the rapid leaf expansion phase followed by a similar decline. The oil gland (trichome) number increased upto leaf maturation and declined thereafter. Initially, cuticle remains tightly apposed to the secretory head of oil glands but progressively a sub-cuticular space appears to be created for the oil. Considerable enlargement of vacuole with ageing is witnessed, whereas cytoplasm gradually decreases to a thin peripheral layer. Some secretory cells from senescing leaf were found almost empty, having only a few remnant oil droplets. This revised version was published online in June 2006 with corrections to the Cover Date.     

A New Histochemical Method for Localization of the Site of Monoterpene Phenol Accumulation in Plant Secretory Structures

A new method is reported for the histochemical localizationof monoterpene phenols in essential oil secretory structures.The method was adapted from a spot test originally devised forin vitro detection of phenolic compounds in organic analyses.Plant subjects were the Lamiaceae species Thymus vulgaris L.,Oreganum vulgare L. and Mentha x piperita L., which accumulateessential oil in glandular trichomes. A reagent consisting of4-nitrosophenol in conc. H₂SO₄was applied to sample leaves ofeach species. A positive test for phenol was indicated by theproduction of coloured indophenols. Using this method, monoterpenephenols were identified in the trichomes of T. vulgaris(thymol)and O. vulgare(carvacrol), indicated by colour changes to redand green respectively. No phenol was detected in trichomesof M. x piperita. Results were confirmed by GC-MS analysis ofleaf volatile extracts from each species, and in vitro testswith thymol and carvacrol. The method could be used in fieldsurveys for rapid identification of potential medicinal plantsand bioactive compounds. Copyright 2001 Annals of Botany Company Histochemistry, secretory structures, glandular trichomes, Lamiaceae, Thymus vulgaris, Oreganum vulgare, Mentha x piperita, essential oil, aromatic monoterpenes     

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.     

Floral elaiophores in Lockhartia Hook. (Orchidaceae: Oncidiinae): their distribution,diversity and anatomy

Background and Aims

A significant proportion of orchid species assigned to subtribe Oncidiinae produce floral oil as a food reward that attracts specialized bee pollinators. This oil is produced either by glabrous glands (epithelial elaiophores) or by tufts of secretory hairs (trichomal elaiophores). Although the structure of epithelial elaiophores in the Oncidiinae has been well documented, trichomal elaiophores are less common and have not received as much attention. Only trichomal elaiophores occur in the genus Lockhartia, and their distribution and structure are surveyed here for the first time.

Methods

Flowers of 16 species of Lockhartia were studied. The location of floral elaiophores was determined histochemically and their anatomical organization and mode of oil secretion was investigated by means of light microscopy, scanning electron microscopy and transmission electron microscopy.

Key Results and Conclusions –

All species of Lockhartia investigated have trichomal elaiophores on the adaxial surface of the labellum. Histochemical tests revealed the presence of lipoidal substances within the labellar trichomes. However, the degree of oil production and the distribution of trichomes differed between the three major groups of species found within the genus. All trichomes were unicellular and, in some species, of two distinct sizes, the larger being either capitate or apically branched. The trichomal cuticle was lamellate, and often appeared distended due to the subcuticular accumulation of oil. The labellar trichomes of the three species examined using transmission electron microscopy contained dense, intensely staining cytoplasm with apically located vacuoles. Oil-laden secretory vesicles fused with the plasmalemma and discharged their contents. Oil eventually accumulated between the cell wall and cuticle of the trichome and contained electron-transparent profiles or droplets. This condition is considered unique to Lockhartia among those species of elaiophore-bearing Oncidiinae studied to date.     

Isolation of secretory cells from plant glandular trichomes and their use in biosynthetic studies of monoterpenes and other gland products.

The natural products that accumulate in or exude from plant glandular trichomes are biosynthesized by secretory cells located at the apex of the trichome. To investigate the formation of glandular trichome constituents in several species of mints (Lamiaceae), a new procedure was developed for isolating large numbers of highly purified secretory cells. In this method, the leaf surface is gently abraded with glass beads in a way that fragments the glandular trichomes and yields clusters of intact secretory cells. The isolated, intact secretory cells and cell-free preparations derived from them are very active in monoterpene biosynthesis and provide useful starting materials for the purification of several key enzymes of monoterpene metabolism. The procedure described is adaptable to a broad range of plant species and should find wide application in the preparation of whole cell and cell-free systems for biosynthetic studies of plant natural products found in glandular trichomes.     

Morphology and histochemistry of the glandular trichomes of Lippia scaberrima (Verbenaceae)

BACKGROUND AND AIMS: Lippia scaberrima, an aromatic indigenous South African plant, with medicinal application, potentially has economic value. The production of essential oil from this plant has not been optimized, and this study of the chemico-morphological characteristics was aimed at determining the location of oil production within the plant. Furthermore, the locality of other secondary metabolites important in medicinal applications needed to be ascertained. This information would be useful in deciding the protocol required for isolation of such compounds. METHODS: The morphology of the glandular trichomes was investigated using a combination of scanning electron and light microscopy. Concurrently, the chemical content was studied by applying various chemical reagents and fluorescence microscopy. KEY RESULTS: Three types of trichomes were distinguished on the material investigated. Large, bulbous peltate glands containing compounds of terpenoid nature are probably the main site of essential oil accumulation. Small glands were found to be both peltate and capitate and fluorescent stain indicated the possible presence of phenolic compounds. The third type was a slender tapered seta with an ornamented surface and uniseriate base, and evidently secretory in nature. CONCLUSIONS: This study linking the chemical content and morphology of the glandular trichomes of L. scaberrima has contributed to the knowledge and understanding of secretory structures of Lippia spp. in general.     

Effects of light intensity on the anatomical structure,secretory structures,histochemistry and essential oil composition of Aeollanthus suaveolens Mart. ex Spreng. (Lamiaceae)

Aeollanthus suaveolens Mart. ex Spreng belongs to Lamiaceae family, and in the Amazon this species is cultivated by natives’ people, this medicinal plant is popularly known as Catinga-de-mulata, being used by the population for general pain treatment. The present study analyzed the effects of light intensity on the anatomy, secretory structures, histochemistry and composition of essential oil of leaf and stem of A. suaveolens. The anatomical structure were observed in response to two light intensities, namely 50% (half shade, HS) and 100% (full sun, FS) light. Histochemical analyses were performed to detect lipids, essential oils, alkaloids, phenolic compounds, sesquiterpene lactones, mucilage, and tannins. Secretory structures were observed under a scanning electron microscope. The results obtained in the present work indicate that the light intensity can affect the histochemistry and structures of A. suaveolens. Cross sections of the leaves and stem revealed glandular trichomes on both leaf surfaces as well as the stem surface. Essential oil was detected by histochemical analyses in all types of secretory trichomes. These anatomical and histochemical responses suggest modifications to protect the photosynthetic apparatus from excess light, in addition we note that in the chemical composition of the essential oil the class of hydrocarbons sesquiterpene prevailed.     

Glandular Trichomes on the Leaves and Flowers of Plectranthus ornatus: Morphology, Distribution and Histochemistry

The types of glandular trichomes and their distribution on leavesand flowers of Plectranthus ornatus were investigated at differentstages of their development. Five morphological types of glandulartrichomes are described. Peltate trichomes, confined to theleaf abaxial surface, have, in vivo, an uncommon but characteristicorange to brownish colour. Capitate trichomes, uniformly distributedon both leaf surfaces, are divided into two types accordingto their structure and secretory processes. In long-stalkedcapitate trichomes, a heterogeneous secretion (a gumresin) isstored temporarily in a large subcuticular space, being releasedby cuticle rupture, whereas, in the short-stalked capitate trichomes,the secretion, mainly polysaccharidic, is probably exuded viamicropores. On the leaves, digitiform trichomes, which do notshow a clear distinction between the apical glandular cell andthe subsidiary cells, occur with a similar distribution to thecapitate trichomes. They do not develop a subcuticular space,and secrete small amounts of essential oils in association withpolysaccharides. The reproductive organs, particularly the calyxand corolla, exhibit, in addition to the reported trichomes,unusual conoidal trichomes with long unicellular conical heads.A large apical pore, formed by tip disruption, releases thesecretion (a gumresin) stored in a rostrum-like projection.On the stamens and carpels, digitiform, capitate and conoidaltrichomes are absent, but peltate trichomes are numerous. Theyoccur between the two anther lobes, on the basal portion ofthe style, and between the four lobes of the ovary. The resultspresented are compared with those of other studies on Lamiaceaeglandular trichomes. Copyright 1999 Annals of Botany Company Plectranthus ornatus Codd, Lamiaceae, glandular trichomes, morphology, histochemistry, essential oils and mucilage secretion.     

Micromorphological and chemotaxonomical traits of Micromeria croatica (Pers.) Schott

A study on the types and distribution of trichomes, pollen morphology, chemical composition of essential oil (analyzed by GC-FID and GC/MS), and the content of macroelements (Na, K, Ca, and Mg) and trace elements (B, Fe, Cu, Mn, Zn, Al, Pb, Cr, Cd, Ni, Hg, and As) analyzed by ICP-AES (=inductively coupled plasma atomic emission spectroscopy) was conducted on Micromeria croatica (Pers.) Schott. Non-glandular trichomes, peltate trichomes (typical hairs of Lamiaceae), small capitate trichomes (composed of one basal epidermal cell and one head cell), and larger capitate trichomes (composed of one basal epidermal cell, two to three stalk cells, and one head cell with subcuticular space) were observed on leaves, stems, the calyx, and corolla. Pollen of M. croatica had six apertures (hexacolpate) which were set in the equatorial pollen belt (zonocolpate) and showed medium reticulate ornamentation (hetrobrochate type). The essential oil contained from 27 to 39?constituents, the most representative of which were caryophyllene oxide and E-caryophyllene. Among the investigated macroelements, the content of K was highest (8730-10080?mg/kg). The content of trace elements ranged from 0.12?mg/kg (Cr) to 78.00?mg/kg (Fe), while the content of Cd, Ni, Hg, and As were lower than the limit of quantification.     

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。     

Development and Essential Oil Content of Secretory Glands of Sage (Salvia officinalis)

Scanning electron microscopy of sage (Salvia officinalis L.) leaves confirmed the presence of two basic types of glandular trichomes consisting of a capitate stalked form containing a multicellular stalk and surmounted by a unicellular secretory head, and a capitate sessile form containing a unicellular stalk and unicellular, or multicellular, secretory head. In the latter type, secretory activity and filling of the subcuticular cavity may begin at virtually any stage of the division cycle affording fully developed glands containing from one to twelve cells in the secretory head. Gas liquid chromatographic analysis of the oil content of the most numerous gland species (capitate stalked, capitate sessile with one and with eight secretory cells) indicated only minor quantitative differences in essential oil composition. Thus, each gland type is capable of producing the four major monoterpene families (p-menthanes, pinanes, bornanes and thujanes) characteristic of sage.     

Analysis of monoterpenoids in glandular trichomes of the catmint Nepeta racemosa

Plants within the Lamiaceae are characterized by their production of essential oils, largely composed of monoterpenoids. They also possess on their aerial surfaces different types of trichomes, including relatively large peltate glandular trichomes. Observation of leaves of the catmint Nepeta racemosa , using cryogenic scanning electron microscopy, showed that around 3000 peltate trichomes are present on a single expanded leaf. An approach has been developed for sampling directly from the subcuticular cavity of such trichomes, in order to characterize qualitatively and quantitatively their contents. Gland samples from Nepeta spp. were analysed by combined gas chromatography-mass spectrometry. These analyses have confirmed that the monoterpenoids characteristic of these species (nepetalactones) accumulate within the subcuticular cavity of peltate glandular trichomes. Qualitative analysis showed that three different nepetalactone stereoisomers were accumulated in different proportions in peltate glands from individual N. racemosa plants. Quantitative analysis showed that individual peltate glands accumulate around 30 ng of nepetalactone by maturity, and that accumulation of nepetalactone probably occurs exclusively within this gland type.     

Glandular Trichomes in Satureja thymbra Leaves

The leaves of the aromatic plant Satureja thymbra have numerousglandular trichomes of two morphologically distinct types glandularhairs and glandular scales Investigations of the anatomy ofthese glandular trichomes with serial thick sections revealedthat the glandular hairs consist of three cells a foot, stalkand head cell Glandular scales also have a unicellular footand stalk Their heads, however, are composed of 12 cells Fourof these cells are small, occupying the central region of thehead, whereas the remainder are large and peripherally arrangedMorphometric analysis showed that, in leaf surface view, glandularscales are about 17-fold larger than glandular hairs In addition,glandular scales were found to occupy 5 7 % of the entire leafsurface area In each glandular scale the total amount of essentialoil, contained within both the subcuticular space and the interiorof the secretory cells, was calculated to be 2 51 x 10^–4mm³ The volume of the essential oil produced by all glandularscales on a single mature leaf was correspondingly determinedto be 0.059 mm³ Finally, the theoretical essential oil yieldof 100 g dry leaves of S thymbra was estimated to be 3 54 %(secretory activity of glandular scales only) Satureja thymbra, glandular trichomes, morphology, morphometry     

Glandular trichomes and essential oil composition of endemic Sideritis italica (Mill.) Greuter et Burdet from central Italy

Sideritis italica (Mill.) Greuter et Burdet belongs to the Lamiaceae family and is endemic to Italy. The glandular trichomes (morphology, distribution, histochemistry, and ultrastructure) of the plant were studied for the first time, along with the chemical composition of the essential oils. Abundant non-glandular hairs and peltate (type A) and capitate (types B, C(1), and C(x)) glandular trichomes were observed both on the vegetative and reproductive organs. The histochemical procedures and the ultrastructural investigation enabled specific location of the main site of essential oil production mainly in type-A peltate hairs. Particular emphasis is given to the release mechanism of the secreted material in all of the types of glands, and the potential taxonomic value of the indumentum in the Lamiaceae family is briefly discussed. Essential oils were hydrodistilled from flowering aerial parts of S. italica, and 136 compounds (112 in flowerheads, 79 in vegetative parts) were identified. The quantitative prevalence of diterpenoids (43.4% in flowerheads and 22.3% in vegetative parts) was the most significant characteristic of the essential oil of S. italica that could be classified as a diterpene-rich essential oil according to the classification of Kirimer.     

Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase

We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.     

The taxonomic value of leaf anatomy and trichome morphology of the genus Cyclotrichium (Lamiaceae) in Turkey

The genus Cyclotrichium (Boiss.) Manden. & Scheng. is represented by six species in Turkey: C. glabrescens, C. leucotrichum, C. longiflorum, C. niveum, C. origanifolium and C. stamineum. They are aromatic perennial subshrubs used as spices or herbal teas in traditional Turkish medicine. The leaf anatomy and tomentum morphology of leaves and calyces of Cyclotrichium species in Turkey was investigated by scanning electron microscopy (SEM) and light microscopy (LM). The investigated species have equifacial (C. niveum, C. origanifolium) or bifacial leaves (C. glabressens, C. leucotrichum, C. longiflorum, C. stamineum). All species have peltate and capitate glandular, and simple (all species) or branched (C. niveum) eglandular trichomes and diacytic stomata. Peltate trichomes consist of a basal cell embedded in the epidermis, a stalk cell, and a broad 12 (–13)‐celled secretory head arranged in two concentric circles. The capitate trichomes observed in Cyclotrichium can be grouped into five types, differing in structure and size. They consist of either a pear‐shaped or globose unicellular head and uni‐or bicellular stalk, or a bicellular head and bicellular stalk. The density of peltate, capitate and eglandular trichomes differs between species. Peltate trichomes are densely spaced only on the calyx and on the leaf surface of C. niveum and C. origanifolium and on the abaxial leaf surface of C. longiflorum and C. stamineum. The significance of trichome architecture for taxonomy in Cyclotrichium and Lamiaceae in general is discussed.     

Glandular hairs of Sigesbeckia jorullensis Kunth (Asteraceae): morphology,histochemistry and composition of essential oil

Long-stalked glandular hairs of outer and inner involucral bracts of Sigesbeckia jorullensis, which are important for epizoic fruit propagation, were investigated using light and scanning electron microscopy. The essential oil secreted by the hairs was analysed by chromatographic methods including gas chromatography/mass spectrometry and with a laser microprobe mass analyser. The glandular hairs consisted of a large multicellular stalk and a multicellular secreting head. The apical layer of glandular head cells was characterized by leucoplasts and calcium oxalate crystals. Below the apical cells there were up to six layers of cells containing many chloroplasts around the nucleus and surrounded by vacuoles filled with flavonoids and tannins. The essential oil originating in the head cells was secreted into the subcuticular space and may be liberated by rupture of the cuticle. It was mainly composed of sesqui- and diterpenes, with the sesquiterpene hydrocarbon germacrene-D as the main component. Monoterpenes, n-alkanes and their derivatives as well as flavonoid aglycones were also detected. The stickiness of the essential oil is probably associated with the high content of oxygenated sesqui- and diterpenes. In addition to long-stalked trichomes, small biseriate trichomes occurred, secreting small quantities of essential oil into a subcuticular space.     

Ultrastructure and cytochemistry of the pearl gland in Piper regnellii (Piperaceae)

Pearl glands are scattered throughout the lamina of developing leaves and rarely found on adult leaves of Piper regnellii (Piperaceae). The pearl gland is a bicellular secretory trichome composed of a short broad basal cell and a spatula-like, semiglobular apical cell. Four different stages of the pearl gland were determined during its ontogenesis: origin, pre-secretory, secretory and post-secretory. During the pre-secretory stage, mitochondria, ribosomes, dictyosomes, rough endoplasmic reticulum, and plastids with electron dense inclusions were present in the cytoplasm of the apical cell. During the secretory stage, the most remarkable characteristics of the apical cell are the proliferation of dictyosomes and their vesicles, rough endoplasmic reticulum, and modified plastids. At this stage, electron-dense oil drops occur in the plastids as well as scattered within the cytoplasm, proteins and polysaccharides are seen in the plastids, vesicles, and vacuoles. Only polysaccharides are present in the periplasmic space, wall cavities, and on the surface of the apical cell. The polysaccharides are one of the main components of the mucilagenous exudate that covers the developing leaf structures. The apical cell of the senescing trichomes undergoes a progressive degeneration of its cellular components, the plastids being the first organelles to undergo lysis.