Development, oil storage and dehiscence of peltate trichomes in Thymus vulgaris (Lamiaceae)

Alessandro Bruni and Paolo Modenesi

Bruni. A. & Modenesi. P. 1983. Develooment. oil storaee and dehiscence of Deltate trichomes in Thymus vulgarrs. - Nord. J. Bot. 3: 265-251. Copenhagen.’ ISSN 0107-055X.

The development, oil storage and dehiscence mechanism of the peltate oil-producing trichome in Thymas vulgaris L. was studied by conventional, fluorescence and elec- tron scanning microscopy. A single epidermal initial forms the glandular trichome whose primordium becomes distinguishable from non-glandular trichomes at its 3-celled stage. The further divisions determine the formation of three functional compartments, (a) a basal reservoir cell, (b) an “endodermal” cell, and (c) a group of secretory head cells. At first, the gland head shows a bowl-like shape with a folded cuticular covering that raises successively assuming a dome-like form. During the differentiation of the gland cells, histochemical tests reveal the occurrence of a pre- cise sequence of metabolic events: synthesis of RNA, proteins, glycoproteins, polysaccharides and, finally osmiophilic substances. At maturity, the cuticular sheath is formed by a non-cellulosic polysaccharide framework on which the cuticle is de- posed. Proceeding towards the senescence, the polysaccharide framework disappears and a large crescent shaped pore forms, whereby essential oils are released. Collected results are discussed in order to interpret gland function and essential oil production.

A. Bruni and P . Modmrsi, Inst. of Botany “Hanbury”, Univ. of Genova, Corm Dogali Ic, I-16136 C m o v a , Italy.

Introduction

Essential oils produced in the glandular trichomes of several species of Labiatae constitute an important group of economic plant products. They supply the raw material for a great variety of industries, e.g. for pre- paration of insecticides, antiseptics, pharmaceutical products, perfumes and spices. Although many studies have been carried out on the chemical composition of essential oils in Labiatae (see Guenther 1964, Erickson 1976), a comparatively restricted amount of experi- mental research was devoted to define the sequence of the development and secretive mechanism of oil-pro- ducing trichomes (see Fahn 1979).

Lipophilic glandular trichomes show a great mor- phological variety that presumes a different develop- mental sequence and, likely, different secretive mechanisms (Luttge 1071). Recent studies (Hammon & Mahlberg 1977, 1078, Vermeer & Peterson 1979a,b)

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emphasize that the lipophilic secretion involves many cellular compartments, and conclude that the possible sites of synthesis, dynamic secretion mechanism, and oil storage are not yet established. In addition, the reports mentioned state that the major problem in interpreting the gland function in a complex system, such as in trichomes, is the localization of a specific substance at a specific cellular compartment. A tentative manner to overcome this histochemical problem is offered by using high sensitive histofluorescent procedures.

This study, carried out by conventional, fluorescence and electron scanning microscopy, concerns some as- pects of the development and oil storage in peltate trichomes of Thymus vulgaris L., a mediterranean plant commonly grown in Europe. There are several reasons for studying the glandular trichomes of thyme; firstly, the importance of this plant as drug for pharmaceutical purposes; secondly, the structure, oil storage and dehis- cence of its glandular trichomes are scantly known (Up-

245

hof 1962); thirdly, the morphological microcharacters could have taxonomic relevance in the evaluation of the thyme chemical races (Metcalfe & Chalk 1950, Granger & Passet 1973).

Materials and methods

Plant material

The observations were made on leaves at various stages of development from plants of Thymus vulgaris L. col- lected in their natural habitat in the eastern coast of Liguria and in the Botanical Garden of the Genova University (Italy).

Conventional and fluorescence microscopy

Small pieces of leaves were fixed either in 10% neutral formalin for 12 h or in FAA for 24 h (Sass 1958) and then embedded in a methyl-butyl methacrylate mixture. Transverse sections in sets, 1-2 ym thick, which were cut using a Reichert Om-U2 ultramicrotome, were hydrated by means of a decreasing alcoholic series, and immediately stained with toluidine blue 0 (TBO) as general stain (Feder & O’Brien 1968).

For histochemical investigations, embedded sections or sections obtained by a cryostat microtome were pro- cessed with the following methods: a) acridine orange (AO) for nucleic acids (Bush & Smetana 1970); b) Hg-fluorescamine reaction for proteins (Bruni et al. 1976, 1977); c) aniline blue (Smith & McCully 1978) for (1 + 3) polysaccharides; d) periodic acid-Schiff reagent (PAS) (Jensen 1962) and periodic acid-fluores- cent Schiff reagent (F-PAS) for polysaccharides (Pearse 1972, Bruni & Vannini 1973); e) fluorescent test for plant glycoproteins (Bruni 1979); f) ruthenium red (Jensen 1962) for pectin-like substances; g) osmium tetroxide (Jensen 1962) and Sudan Black B (Bronner 1975) for lipophilic substances. Distribution of chloro- plasts in living glandular trichomes was determined by the use of a Zeiss fluorescence microscope (Fasulo et al. 1976). By the same instrument, the autofluorescence of suberin-like substances was explored (Jensen 1962).

For all the above-cited histochemical methods, con- trol reactions were made following the suggestions of the respective authors. Additional data on the nature of the material which forms PAS positive inclusions were obtained by the control procedures proposed by Swift & Saxton (1967) and by treating sections with 5% a-amylase, so as to hydrolize specifically the glycosidic a-bond (Ling-Lee et al. 1977).

Some clarifications are added about the fluores- cent-PAS reaction. The F-PAS is an histofluorescent test for polysaccharides employed in plant histochemis- try because of its high sensitivity and selectivity in com- parison to the conventional PAS (Bruni & Vannini 1973). By this procedure in standardized procedural conditions, the cellulosic cell wall fluoresces green, while the starch and other non-cellulosic polysac- charides emit an orange fluorescence. However, when the cellulosichon cellulosic polysaccharide ratio in the cell wall is modified, the fluorescence changes from green to orange (Bruni et al. 1982). The specificity of the reaction has been studied in detail (see Pearse 1972); the colour of fluorescence is due to the specific accumulation of fluorochrome that is related to the number and distribution of fluorochrome-polyanion bond sites in a complex macromolecular structure (Pearse 1972).

After staining, sections were mounted in a rapid non- fluorescent medium (UV inert Einschlussmittel, Serva, Heidelberg, Rm) and observed with a bright field using a standard equipment. A Zeiss microscope fitted with an epi-illuminator, UV exciting illumination and Neo- fluar objectives was used for examination of fluores- cence.

Scanning electron microscopy

Whole leaves or pieces of leaves approximately 2 mm long were placed in a solution containing 2% glutaral- dehyde in 0.07 M phosphate buffer, pH 6.8, and fixed for 2 h. They were subsequently dehydrated with acetone, and critical-point dried from acetone with liquid-CO,. Once dried, the samples were coated with a I:1 gold-palladium alloy and viewed with a Siemens Autoscan microscope.

Fig. 1. Longitudinal section of the initial development stage of a trichome in a young leaf of Thyrnur vulgaris. The protruding part of the initial cell (arrow) increases in height and width producing a pear-shaped cell. TBO. ~ 3 0 0 . -Fig. 2. Longitudinal section of a glandular trichome at the stage of 3-cells. Observe the large uppermost cell. F-PAS. ~ 4 5 0 . - Fig. 3. Longitudinal section of a young lipophilic glandular trichome just after the division of the uppermost cell. F-PAS. X450. - Fig. 4. Transverse section of a young peltate trichome at the level of the 4-celled head. TBO. X850. - Fig. 5. Transverse section of developing gland head of a lipophilic trichome when cell divisions are finished. The fluorescence reveals an active protein content of the gland cells. Hg-fluorescamine. ~ 6 0 0 . - Fig. 6. Transverse section of the developing gland head of a peltate trichome. The starch (arrows) emits a strong orange fluorescence. F-PAS. ~ 6 0 0 . - Fig. 7. Longitudinal section of developing trichomes. The secretory head becomes bowl-like in structure and the cuticular sheath raises in the edges of the gland head. F-PAS. X 580. - Fig. 8. SEM micrograph showing a gland head whose concave surface exhibits a folded cuticular cover. ~ 7 0 0 . -Fig. 9. Longitudinal section of maturing peltate trichome. During the raising of the cuticular sheath, a separation of the secretory cell walls occurs. The outermost layer (double arrows) fluoresces orange and follows the cuticle during its raising, while the innermost one (single arrows) adheres to the head cells. F-PAS. x 1350. - Fig. 10. Longitudinal section of the trichome in which the raising of the cuticular sheath is occurring. Arrows indicate the presence of an amorphous flocculent substance that emits an orange fluorescence. F-PAS. ~ 6 5 0 .

246 Nord. J . Bot. 3 (2) 1983

Observations

Three types of glandular trichomes are present on the abaxial and adaxial surface of young leaves of Thymus vulgaris: a clubbed-bulbous type producing mucilage, a clubbed type having an hydatode function, and a big peltate type producing essential oil. At an early stage of the development, it is not possible to distinguish the primordium of each type of gland. All trichomes initiate with the outgrowth of a single epidermal cell which in- creases in heigth and width becoming a pear-shaped cell (Fig. 1). After two periclinal divisions, it is possible to distinguish the 3-celled primordium (Fig. 2 ) , which will form an oil-producing trichome, from the ones which will produce the other types of glandular trichomes. The glandular primordium shows a conspicuous difference in size between the two upper cells: (1) the sub-terminal cell is small and coin-like. It will differentiate into the endodermal cell; ( 2 ) the terminal cell is large and globe-shaped. By a series of up to seven anticlinal divi- sions (Figs 3, 4) the uppermost cell will form the 10-14-celled secretory head of the oil-producing trichome. When cell divisions end, the glandular head assumes a bowl-like form (Fig. 7) whose concave sur- face exhibits a folded cuticular cover which firmly adheres to the cell walls (Fig. 8). The secretory cells have a dense cytoplasm with amoeboid chloroplasts that fluoresce red under UV-light. At this stage, the cytop- lasm of these cells is sequentially positive to the histof- luorescent tests for RNA, proteins and glycoproteins (Fig. 5). Later on, a conspicuous amount of starch is synthetized, as visualized by PAS and F-PAS (Fig. 6). With further maturity, the general histochemical reac- tivity decreases, starch disappears, plastidial degenera- tion occurs, and various osmiophilic droplets can be observed. Slowly a Sudan Black B-positive secretory product fills up the sub-cuticular space, at first in the edges (Fig. 7), then in the centre of the bowl-like struc- ture of the glandular head (Fig. 10). During this phase, the cuticle swells and rises to form a tight dome owing to the buildup of secreted material between the cell wall and cuticular sheath (Figs 10, 12).

Once collected beneath the cuticular sheath, the es- sential oils become autofluorescent (Fig. 13), thus per-

mitting an evaluation of the distribution and differen- tiation of trichomes in living leaves: at their apex, the trichomes are precociously differentiated and emit a strong orange fluorescence; in their middle part, they are not completely differentiated and fluoresce yellow; at their base, the trichomes are immature and not au- tofluorescent.

At maturity, the oil-producing trichomes possess a turgid reservoir cell, and a flat “endodermal” cell whose wall appears positive to the histofluorescent test for suberin-like substances. Most likely, a Casparian-like strip envelops the lateral and partially, the basal part of the endodermal cell. The glandular head cells have large nuclei located in the lower part of the cells, radial walls labyrinthine in appearance, and osmiophilic droplets in the ground cytoplasm.

When the essential oil begins to fill the sub-cuticular space, a separation between the outermost and inner- most layers of the secretory cell walls occurs (Fig. 9). At the same time, an amorphous flocculent substance which fluoresces in orange with F-PAS and reacts posi- tively with ruthenium red, becomes evident in the inter- vening space. During its raising, the cuticle is accom- panied by the outermost layer of the cell wall, which fluoresces in orange by F-PAS. The innermost wall layer adheres to the head cells and emits a strong green fluorescence (Fig. 9). It shows metachromatic staining with TBO at pH 5.6 (Fig. l l ) , and is negative to ruthenium red. These reactions and the control proce- dures employed indicate that the cellulosic component is prevalent in the innermost wall layer, while the out- ermost one does not appear cellulose-like, and reacts negatively to the histofluorescent test for fl (1 + 3) polysaccharides and a-amylase treatment. In favourable sections, i.e. when the cuticular sheath is cut and form a spherical segment, many points, strongly fluorescent in orange by F-PAS, are observed (Fig. 14). When a dome-like portion of the cuticular sheath is cut and observed in front, a suggestive polysaccharide lattice is evident (Fig. 15). These observations indicate that the cuticular sheath of the gland is provided with a framework on which the cuticle is deposed. This framework originated from the outermost wall layer of the head cells, it exhibits orange fluorescence with

Fig. 11. Longitudinal section of a mature peltate trichome in Thymus vulgaris. To note: the cuticular sheath (c) entirely raised; the flocculent polysaccharide material (0 in the intervening space (s); the endodermal coin-like cell (e); and the reservoir cell (r). TBO. ~ 5 6 0 . -Fig. 12. SEM micrograph showing a mature peltate trichome on the abaxial surface of a young leaf. ~ 7 1 0 . - Fig. 13. Autofluorescence emitted from essential oils built up in the secretive head of peltate trichomes on the abaxial surface of a young leaf. The epi-illuminator equipment permits a localization of the mature trichomes. X 100. - Fig. 14. Transverse section of the cuticular sheath of a mature trichome. Note the points (1) strongly fluorescent (orange) on the cuticle (pale green). F-PAS. X750. - Fig. 15. Transverse section of a mature trichome. A dome-like portion of the cuticular sheath is observed in front. A polysaccharide lattice, strongly fluorescent ( l ) , is evident. F-PAS. ~800. - Fig. 16. Longitudinal section of a peltate trichome becoming senescent. The amount of polysaccharide material (1) that forms the cuticular sheath, gradually decreases. F-PAS. ~ 4 3 0 . - Fig. 17. Transverse section of a senescent peltate trichome at the level of the cuticular sheath. Note the absence of the polysaccharide lattice. F-PAS. ~ 7 0 0 . - Fig. 18. Longitudinal section of a senescent peltate trichome before the essence release. F-PAS. X430. -Fig. 19. SEM micrograph showing the dehiscence of the oil-producing trichomes. Note the formation of a regular large pore whereby essential oils are released. ~ 4 9 0 .

248 Nord. I . Bot. 3 (2) 1983

F-PAS, and it is positive to ruthenium red and conven- tional PAS. These reactions and the negativity to the test for p (1 -+ 3) polysaccharides indicate that the cuticular framework is not cellulosc-like or callose-like, but it shows some similarity to the middle lamella. With increasing senescence, the cuticular sheath shows a de- creasing reactivity to the polysaccharide tests and, in old glands, it is limited to the cuticle only (Figs 16, 17, 18).

The mature peltate trichomes possess a dehiscence mechanism whereby stored essential oils are released. This mechanism appears to be formed by the develop- ment of a line of frailty in the diametrical region of the dome-like structure covering the secretory head. Fig. 19 shows the crescent shaped pore by which the sub-cuticular secreted material is released. The large pore is not an artifact due to fixation, since it was observed that it also forms in living field-collected leaves after a sunny day or under the action of warm light from a tungsten lamp.

Discussion

In contrast to most Labiatae plants which have more than one type of lipophilic trichomes (Amelunxen 1964, 1965, Schnepf 1972, Heinrich 1973), Thymus vulgaris has only got one kind of oil-producing trichome. At maturity, the lipophilic peltate trichome is composed of a reservoir cell that supplies nutrients to the developing head, an “endodermal” cell that prevents backflow of secreted substances through the apoplast, and a gland head formed by 10-14 cells whose prominent cytologi- cal characteristics are a relatively large nucleus if com- pared to the cell volume, and a great amount of small osmiophilic vacuoles. These aspects, and the ones con- cerning the structural changes accompanying the stages of development of the gland complex, are similar to those described in Salvia and Mentha for peltate trichomes (Amelunxen 1964, 1965, Schnepf 1972, 1974). In thyme, however, during the final maturation of the gland head, there are aspects that provide new comments.

As shown by the histochemical tests employed, a pre- cise and concatenate sequence of metabolic events occurs during the differentiation: enhancement of RNA; synthesis of proteins; assembling of glycopro- teins; increase of the polysaccharide content; plastid degeneration; and, finally, appearance of osmiophilic droplets. Many of these events were already shown during the differentiation of lipophilic glands in some plants (Schnepf 1974, Fahn 1979, Kristen 1980), but an ordered sequence comparable to that cited above was described only in Cannabis sativa (Hammond & Mahlberg 1978). In thyme, where the trichome dif- ferentiation is a slow process, the single phases of the developmental sequence are easily followed. The sequ- ence observed is consistent with the current view that during the trichome differentiation an increase of

polysaccharides occurs and that plastids are involved in the biosynthesis and secretion of essential oils (Green- wood et al. 1963, Amelunxen & Gronau 1969, Akers et al. 1978).

A raising process of the cuticular sheath comparable to that observed in thyme was described in Cannabis (Hammond & Mahlberg 1973, De Pasquale 1974), while in Monarda it was observed that the head cells shrink and no cuticular distension occurs (Heinrich 1973). In thyme the formation of a sub-cuticular space causes distinctive cell wall changes. The relatively thick cuticular sheath consists not only of cuticle, but of a cuticle and a polysaccharide framework originated by the separation of the gland cell wall in two layers. The wall separation presumably begins with the enzymatic break down of a middle wall layer rich in pectin and with a process similar to that described in Chrysan- themum (Vermeer & Peterson 1979).

A cuticular sheath derived from cell wall separation was observed in capitate glands of Cannabis (Hammond & Mahlberg 1978), Silene (Fridvalsky et al. 1970), and Mentha (Amelunxen 1965), but no precise indication was provided concerning the chemical nature of the wall portion associated with the cuticle. In thyme the cuticular sheath is formed by a non-cellulose-like framework with a high pectic component. On the con- trary, the cell wall layer that adheres to gland cells maintains a chemical composition prevalently cellulose- like.

The secretion occurs in many oil-producing trichomes through small pores in the cuticle (Hulsbruch 1961, Szentpetery et al. 1969, Schnepf 1972, Dell & McComb 1975). In Cannubis, the cuticular sheath is broken down by external factors that provoke the ablation of the cuticular envelope (De Pasquale 1974, Hammond & Mahlberg 1978). In Thymus vulgaris, the large crescent shaped pore is comparable in appearance to that de- scribed for the trichomes of Chrysanthemum (Vermeer & Peterson 1979). It is not clear how the gland dehis- cence takes places. However, since, in thyme, the dis- charge pore is very regular in shape, the presence of a line of frangibility in the diametrical region of the cuticular sheath is tenable. This is in accordance with the observation that, during the raising of the cuticular sheath, the polysaccharide framework decreases in thickness up to its total disappearance in old glands. In this way, the resistance of the cuticular sheath di- minishes, so that the increase of internal pressure or external factors provoke the pore formation and, con- sequently, the discharge of essential oils.

In conclusion, in thyme only one type of oil-produc- ing trichome is present. The dehiscence mechanism of the gland head occurs by a large pore, thus the essential oil is rapidly released. This fact could be considered in the evaluation of the favourable time for collecting this plant, since external factors, such as climatic conditions, can provoke the trichome dehiscence and, therefore, the decay of oil yield in this aromatic plant.

250 Nord. J . Bot. 3 (2) 1983

Acknowlrdgcwienrs -This study was supported by a grant from the Italian Research Council (CNR). We should like to thank our colleagues of the Institute of Botany and Electron Micros- copy Centre of the University of Ferrara for allowing us to perform some of the experiments in their laboratories and for stimulating discussions.

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