Ovary Peltate Trichomes of Zeyheria montana (Bignoniaceae): Developmental Ultrastructure and Secretion in Relation to Function

Abstract

• Background and Aims Nectar production in the Bignoniaceae species lacking a nectariferous functional disc is ascribed to trichomatic glands around the ovary base and/or on the inner corolla wall. Nevertheless, knowledge about the secretion and function of these glands is very incomplete. The purpose of this paper is to study, from a developmental viewpoint, the ultrastructure, histochemistry and secretory process of the peltate trichomes on the ovary of Zeyheria montana, a species in the Bignoniaceae which has a rudimentary disc.

• Methods Samples of the gynoecium at various developmental stages were fixed and processed for light and electron microscopy. Histochemistry and cytochemistry tests were performed to examine the chemical composition of exudates. Thin layer chromatography was used to determine the presence of alkaloids and terpenes in gynoecium and fruit extracts, and in fresh nectar stored in the nectar chamber.

• Key Results Peltate trichomes at different developmental stages appear side by side from floral budding up to pre-dispersal fruit. Large plastids with an extensive internal membrane system consisting of tubules filled with lipophilic material, abundant smooth endoplasmic reticulum, few Golgi bodies, lipophilic deposits in the smooth endoplasmic reticulum and mitochondria, and scattered cytoplasmic oil droplets are the main characteristics of mature head cells. The secretion which accumulates in the subcuticular space stains positively for hydrophilic and lipophilic substances, with lipids prevailing for fully peltate trichomes. Histochemistry and thin layer chromatography detected terpenes and alkaloids. Fehling's test to detect of sugars in the secretion was negative.

• Conclusions The continuous presence and activity of peltate trichomes on the ovary of Z. montana from early budding through to flowering and fruiting set, and its main chemical components, alkaloids and terpenes, suggest that they serve a protective function and are not related to the floral nectar source or to improving nectar quality.

INTRODUCTION

The Bignoniaceae family, represented by more than 100 genera and about 800 species, is mainly neotropical, with most of its members found in tropical America (Gentry, 1980, 1982). Most Bignoniaceae species have showy flowers with a well-developed nectariferous annular disc surrounding the base of the ovary (Gentry, 1982; Thomas and Dave, 1992; Galetto, 1995; Rivera, 2000). In a few genera, such as Clytostoma, Cydista, Phryganocydia, Lundia and Zeyheria, a functional disc is absent and the source of floral nectar has been ascribed to trichomatic glands around the ovary and/or on the inner corolla wall (Bureau and Schumann, 1897; Gentry, 1980; Machado et al., 1995; Rivera, 1996, 2000; Lopes et al., 2002; Bittencourt and Semir, 2004). In spite of the taxonomic and ecological value of the glandular trichomes on the reproductive organs of the Bignoniaceae, little is known about the development and structural details of these glands and the chemical composition of their exudates. Detailed ultrastructural studies and chemical analysis may aid in the interpretation of the function of these glands (Ascensão et al., 1999).

Zeyheria montana (syn. Z. digitalis) is an important component of cerrado vegetation, a savanna-like tropical ecosystem in central and south-east Brazil. It possesses yellow, nectariferous, odourless tubular flowers, which are pollinated by hummingbirds (Yanagizawa et al., 1982; Bittencourt and Semir, 2004). Copious amounts of nectar are stored in a nectar chamber, and its production was ascribed to peltate glands around the ovary base (Machado et al., 1995). A previous ultrastructural analysis of peltate trichomes on the ovary from functional flowers of Z. montana showed features associated primarily with lipophilic secretion (Castro and Machado, 2003). Since then, Bittencourt and Semir (2004) have described the pollination ecology and floral anatomy of Z. montana and found differences in distribution, number and staining properties between glandular trichomes of the gynoecium and those of the nectar chamber. According to these authors, nectar secretion in Z. montana is performed by corolla-borne glandular trichomes that are more abundant on the bulging filament bases than ovary glandular trichomes. However, the lack of more detailed structural and chemical studies added doubts as to the true function of the ovary glands. Therefore, this study describes the ultrastructure and chemical composition of the secretion of Z. montana ovary glandular trichomes from a developmental viewpoint.

MATERIALS AND METHODS

Plant material

Flowers and fruits at various stages of development were collected from Zeyheria montana Mart. [syn. Z. digitalis (Vell.) Hoehne] plants growing in cerrado vegetation around Botucatu, São Paulo State, Brazil (22°55′S, 48°30′W). Voucher specimens were deposited in the Herbarium at the Department of Botany (BOTU), Botucatu Campus, São Paulo State University, Brazil. Nine stages were considered, six of the developing flower, and three of the fruit: stage 1, very young flower buds (5–15 mm long, approx. 20 d before anthesis); stage 2, flower buds (20–25 mm long, approx. 4–5 d before anthesis); stage 3, flower buds (35–45 mm long, 1 d before anthesis); stage 4, fresh, open flowers; stage 5, flowers 2 d after anthesis; stage 6, flowers 4 d after anthesis; stage 7, immature fruits (green pericarp, about 5 mm long); stage 8, immature fruits (pericarp turning brown, about 40 mm long); and stage 9, mature fruits (brown pericarp, fully ripened, about 80 mm long). Ten samples from each stage were collected for anatomical, chemical and ultrastructural studies.

Light microscopy

Gynoecium samples were fixed with 2·5 % glutaraldehyde in 0·1 m phosphate buffer, pH 7·3, for 6–12 h at 4 °C; post-fixed with 1 % osmium tetroxide, in the same buffer, for 2 h at room temperature; dehydrated in a graded series of ethanol solutions and embedded in Araldite resin. Semi-thin sections (1 μm) were stained for general histology using 1 % toluidine blue in 1 % aqueous sodium tetraborate solution (O'Brien et al., 1965). The slides were sealed with Entellan and examined with a Zeiss light microscope.

Histochemistry

Fresh hand-sections of all materials were subjected to the following histochemical tests.

Periodic acid–Schiff (PAS) reaction to water-insoluble polysaccharides (Jensen, 1962)

The fresh sections were incubated in a blocking saturated solution of dimedone in 5 % acetic acid at 60 °C for 18 h, followed by oxidation with 1 % periodic acid (10 min), reduction by rinsing with potassium iodide–sodium thiosulphate, and finally a reaction with Schiff reagent (0·5 g of basic fuchsin, 0·5 g of sodium metabisulfite in 100 mL of 0·15 n HCl; the mixture was shaken, 300 mg of fresh decolorizing charcoal added, and then it was filtered). This reaction depends on 1,2-glycol linkage oxidation within the sugar molecule with periodic acid, resulting in the formation of free aldehydes that stain with Schiff reagent producing a red or magenta coloration. Omission of the periodic acid treatment was used as a negative control.

Ruthenium red to detect mucilage/pectin (Johansen, 1940)

Fresh sections were placed in an aqueous ruthenium red solution (1 : 5000) for 10 min, with a pink coloration reflecting the presence of acidic polysaccharides.

Sudan black B (Lison, 1960) for total lipids

Fresh sections were placed in 50 % ethanol for 5 min, stained in a saturated and filtered solution of Sudan black B in 70 % ethanol for 15 min at room temperature, washed with 70 % ethanol followed by rinses with distilled water. All cell lipids, including fats, oils, waxes, free fatty acids and phospholipids, stained blue to black.

Nile blue (Cain, 1947) for total lipids

Fresh sections were stained in 1 % Nile blue at 37 °C for 1 min, differentiated in 1 % acetic acid at 37 °C for 1 min, and rapidly rinsed with distilled water. Neutral lipids (fats, oils and waxes) stained red; acidic lipids (free fatty acids and phospholipids) stained blue.

Osmium tetroxide for unsaturated lipids (Jensen, 1962)

Sections were treated with 1 % osmium tetroxide for 60 min and then washed with distilled water. A black compound of osmium is formed which indicates the location of lipids with a relatively large number of double bonds.

NADI reagent (David and Carde, 1964) for terpenes

Sections were incubated in the dark for 60 min at room temperature in NADI (naphthol + dimethyl-paraphenylenediamine) reagent, prepared immediately prior to staining. After incubation, the sections were rinsed for 2 min in a sodium phosphate buffer (0·1 m, pH 7·2). By oxidation this reagent forms indophenol blue that changes colour with variation in pH and makes it possible to distinguish between essential oils (blue) and resin acids (intense red).

For all histochemical lipid tests, sections were previously treated with a methanol, chloroform, water and chloride acid mixture (66 : 33 : 4 : 1) for 3 h at room temperature to remove lipids, providing negative controls (Gahan, 1984).

Ferric trichloride for phenolic compounds (Johansen, 1940)

Fresh sections were placed in 10 % aqueous ferric trichloride plus a dash of sodium carbonate, for 15 min at room temperature. In this test, orto-dihydroxiphenols react with ferric ions producing deep green or black deposits.

Dragendorff reagent for alkaloids (Svendsen and Verpoorte, 1983)

Fresh sections were placed in 5 % diluted solution of Dragendorff reagent (stock solution: 25 mL 12·5 % Bismuth nitrate in 25 % acetic acid and 10 mL 40 % potassium iodide) for 5 min, washed with 5 % sodium nitrite and then distilled water. This test produces reddish brown deposits of alkaloids (tertiary or quaternary amines). Fresh sections previously treated with 5 % tartaric acid in 95 % ethanol for 48 h at room temperature were used to provide negative controls.

Mercuric bromophenol blue for total proteins (Mazia et al., 1953)

Fresh sections were immersed for 15 min in bromophenol blue solution (95 % ethyl alcohol using 10 g of HgCl₂ and 100 mg of bromophenol blue per 100 mL), rinsed for 20 min in 0·5 % acetic acid, and treated with a sodium phosphate buffer (0·1 m, pH 7·0) for 3 min to yield a blue coloration. Fresh sections previously treated with a solution of acetic anhydrous in 10 % pyridine were used as negative controls.

Fehling's solution test to detect reducing sugars (Purvis et al., 1964)

Fresh sections were immersed in a mixture of equal volumes of solution A (1 L water and 79·28 g copper sulfate) and solution B (1 L water, 346 g sodium potassium tartrate and 100 g sodium hydroxide) and then heated to boiling. A brick-red precipitate indicates reducing sugars.

Standard control procedures were performed simultaneously. For all histochemical tests, sections were mounted in glycerine under a coverslip and then viewed on a Zeiss light microscope.

Thin layer chromatography (TLC)

Two extraction procedures were performed for detecting alkaloids and terpenes: (1) 80 % hydromethanol extracts were made from fresh gynoecium of flowers at stage 4 and fruit at stages 8 and 9—these extracts were concentrated in a rotary evaporator apparatus (Buchi) and an acid-base extraction (Ferri, 1996) was performed to obtain three concentrated fractions rich in terpenoid, alkaloid and quaternary alkaloid compounds; (2) freshly harvested gynoecium samples, at the same developmental stages as procedure 1, were immersed in chloroform for 30 min at room temperature because it causes the trichome cuticule to rupture and subsequently liberate the secretion (Siebert, 2004). Samples of each extract were spotted onto TLC plates with a glass capillary tube. Fresh nectar collected with a glass capillary tube from the nectar chamber was spotted directly onto TLC plates to determine the presence of alkaloids and terpenes. TLC was performed using Silica gel 60 F₂₅₄ plates (Merck). The plates were developed in toluene/ethyl acetate (93 : 7) for terpenoids and in toluene/ethyl acetate/isopropanol (70 : 28 : 2) for alkaloids. Terpenoids were visualized by spraying the plates with AS (anisaldehyde–sulphuric acid) reagent, heated at 100 °C for 10 min, and then evaluated in visible light. Reference samples of monoterpene alkene (myrcene), hydroxylated monoterpene (geraniol), sesquiterpene alkene (trans-caryophyllene) and diterpenes (crotonin and trans-dehydrocrotonin) were run simultaneously with Z. montana samples. Alkaloids were visualized by spraying the plates with Dragendorff reagent, sprayed with 10 % ethanol sulphuric acid, and then visualized in visible light (Wagner and Bladt, 1996).

Analysis for triterpenoids

The Lieberman–Burchard test [based on Matos (1988) and Aryantha et al. (2002)] was used for detecting triterpenoids in Z. montana because the solvent system used on TLC was inappropriate for triterpene standards. The filtered extracts were added to the Liberman–Buchard reagent (1 mL conc. H₂SO₄, 20 mL acetic anhydride and 50 mL CHCl) for 15 min at room temperature. They were then observed under visible light. This test allows identification of free steroids (the solution turns to blue-green) and free triterpenoids (the solution turns to brownish-red).

Scanning (SEM) and transmission (TEM) electron microscopy

For scanning electron microscopy, intact gynoecia at various developmental stages were fixed in glutaraldehyde (2·5 % with 0·1 m phosphate buffer, at pH 7·3, overnight at 4 °C), dehydrated in a graded acetone series, critical point dried, mounted on aluminum stubs, gold-coated and examined with a Philips 515 scanning electron microscope.

For routine observations with the transmission electron microscope, ultra-thin sections from material fixed in glutaraldehyde/osmium tetroxide were stained with uranyl acetate and lead citrate (Reynolds, 1963). Four cytochemical tests were used: 1 % ruthenium red for pectins (Luft, 1971); imidazole-buffered osmium tetroxide to enhance the preservation and contrast of lipids (Angermüller and Fahimi, 1982); ammoniacal silver for basic proteins (Souza, 1989); and the PATAg (periodic acid–thiocarbohydrazide–silver proteinate) test to detect polysaccharides containing 1,2-glycol groups. For the last method, sections were either treated with periodic acid or, to provide controls, solutions not containing periodic acid and thiocarbohydrazide (Thiéry, 1967). Sections were observed on a Philips CM 100 transmission electron microscopy at 80 kV.

RESULTS

Morphology and distribution of the peltate trichomes

The mature gynoecium of Zeyheria montana is covered by a dense indumentum formed by non-glandular stellate trichomes and peltate glandular trichomes (Fig. 1A and C). Peltate trichomes occur all over the gynoecium surface, while non-glandular trichomes are found only around the ovary (Fig. 1A). Non-glandular trichomes are multicellular and consist of a basal cell, a long uniseriate stalk with entangled terminal branches, forming a dense layer that covers the peltate glandular trichomes (Fig. 1C). They are scanty or even absent on very young ovaries (Fig. 1B), but increase in quantity and size as the gynoecium matures; on the mature ovary (stages 3–5) they are extremely numerous (Fig. 1A). Peltate trichomes are already numerous on very young floral buds (stage 1, Fig. 1B). The very young peltate trichome head has a pyramidal shape and the smooth cuticle follows the contour of the cell wall (Fig. 1D). Mature peltate trichomes present a round, broad, spherical head with a smooth surface (Figs 1E and F and 5A). Wrinkled cuticle is seen in the peltate trichomes at the earliest stages of secretion (Figs 1E and 4A). Peltate trichomes consist of a basal cell, a short stalk cell with a thick lateral wall, and a head with eight secretory cells arranged in a disc (Fig. 1F). Examination by scanning electron and light microscopy did not reveal cuticle rupture, pores or crater-like formation, and showed that the cuticle remains intact and distended even in fully secretory trichomes. Eventually, cuticle disruption was observed during the manipulation or piercing of fresh, unfixed trichomes.

Fig. 1.

Morphology and distribution of peltate trichomes on the gynoecium of Zeyheria montana. (A–E) (SEM): (A) stellate non-glandular trichomes on the ovary, and glandular trichomes on the upper part of the mature gynoecium; (B) immature gynoecium with abundant peltate glandular trichomes; (C) mature ovary in cross-section showing peltate trichomes (arrows) covered by stellate non-glandular trichomes; (D) peltate trichome (^*) with a pyramidal multicellular head; (E) portion of an immature gynoecium covered with abundant peltate trichomes showing variable degrees of cuticle distension. (F) Light microscopy: section through a mature peltate trichome showing one basal cell, one stalk cell, a large head formed by secretory cells disposed in a single layer, and a large subcuticular space (Ss) filled with lipid droplets. Note the intact elevated cuticle (arrow).

Histochemistry

The secretion stored in the subcuticular space of mature peltate trichomes contains hydrophilic and lipophilic components (Table 1). The presence of these compounds was independent of the flower development stage, but it is dependent on the trichome development stage. In young peltate trichomes, with an undeveloped subcuticular space, only polysaccharides and proteins were detected. In mature peltate trichomes, positive reactions to alkaloids and lipids were observed in addition to proteins and polysaccharides. A reaction positive to NADI reagent indicates the presence of terpenes; the strength of this positive reaction reached a maximum in the full peltate trichomes. The ruthenium red test detected a thin pectin layer under the distended cuticle. Treatments with ferric trichloride for phenolic compounds and with Fehling's solution for reducing sugars proved negative in all stages.

Table 1.

Histochemistry of the mature peltate trichome on the ovary of Z. montana

			Reactivity*
Staining procedure	Target compounds	Colour observed	Head cells	Secretion
Sudan black B	Total lipids	Dark blue to black	++	++
Nile blue A	Neutral and acidic lipids	Blue	+	++
Osmium tetroxide	Unsaturated lipids	Brownish to black	++	++
NADI	Terpenes	Violet-blue	++	++
Ruthenium red	Mucilage/pectin	Red to pink	+	+
Schiff (PAS)	Neutral polysaccharides	Red	+	+
Dragendorff	Alkaloids	Brownish to red	+	++
Mercuric bromphenol blue	Proteins	Blue	++	+
Ferric trichoride	Phenolic compounds		–	–
Fehling's solution	Sugars	Pink to read	+	–

^*−, negative; +, slightly positive; ++, strongly positive.

Thin layer chromatography

TLC revealed alkaloids and monoterpenes in the gynoecium and fruit extracts. Although both 80 % hydromethanol (concentrated in a rotary evaporator) and chloroform extracts revealed these two classes of compounds, with the same spots and the same lipid and alkaloid profile, the chloroform extract produced a better resolution. With AS reagent, terpenes stained pinkish-purple with an R_f of 0·76, the same as the monoterpene alkene, myrcene, which was mainly in gynoecium extracts (Fig. 2). Using Dragendorff reagent, alkaloids spontaneously gave orange-brown zones in the 0·20–0·25 R_f range (Fig. 2). Plates with quaternary alkaloid extracts showed no reaction with Dragendorff reagent. Also, the plate with fresh nectar did not react with Dragendorff or AS reagent.

Fig. 2.

Thin layer chromatograms of chloroform extracts. (A) Detection of terpenoids (pinksh-purple zones): 1, gynoecium from flowers at stage 4; 2, fruit at stage 8; 3, fruit at stage 9; 4, myrcene; 5, geraniol; 6, trans-caryophyllene; 7, crotonin; 8, trans-dehydrocrotonin. (B) Alkaloid detection (orange-brown zones): 1, gynoecium from flowers at stage 4; 2, fruit at stage 8; 3, fruit at stage 9.

The Lieberman–Burchard test gave a positive reaction in all stages analysed, indicating the presence of triterpenoids.

Transmission electron microscopy and cytochemistry

Peltate trichomes at various development stages were seen together from the floral bud stages up to early fruit setting. However, the proportion of mature/immature trichomes increased with ovary maturity; immature trichomes were absent on mature fruits. Actively secreting trichomes had already been observed on 5- to 8-mm-long floral buds. Thus, the developmental stages described here are primarily related to head cells during the trichome secretion period, and are independent of the flower and fruit development stages considered in this study.

Stage 1

Head cells of the young peltate trichomes, with no visible appearance of secretory activity, show abundant cytoplasm with many polyribosomes, mitochondria with well-developed membranes, rough endoplasmic reticulum (RER) and multi-shaped plastids with small starch grains (Fig. 3A). A thick cuticle attached to the outer cell wall is a feature of these cells (Fig. 3B). As differentiation progresses the plastids show a decrease in number and size of starch grains, and are characterized by the appearance of vesicles or tubules; some plastids are encircled by endoplasmic reticulum element (Fig. 3B). In these cells the Golgi bodies are scarce and poorly developed (Fig. 3C). Portions of RER elements and electron-translucent vesicles close to the plasma membrane are visible (Fig. 3D and E). The presence of vesicles close to the plasma membrane (Fig. 3D and E), formation of a periplasmic space containing various lamellar bodies (Fig. 3C and E) and appearance of the subcuticular space (Fig. 3F) denote the start of secretory activity. Formation of this space commences with the onset of secretory activity, and it develops from the cuticle detachment and outer cell wall loosening in certain regions of the head cells (Fig. 3C). After full separation, the cuticle is bordered on the lower surface by a thin, dense layer that represents a layer of pectin (Fig. 3F). The secretion stored in the small initial subcuticular space is finely granular and homogeneous in aspect, and consists of hydrophilic substances only.

Fig. 3.

Aspects of the young peltate trichomes on the ovary of Z. montana (TEM). (A) Portion of two head cells showing electron-dense cytoplasm with polyribosomes, mitochondria (M), multishaped plastids (Pl), and rough endoplasmic reticulum (Rer). (B) Upper region of a head cell showing thick cuticle (C) closely attached to the dense cell wall, and cytoplasm with mitochondria (M), round plastids (Pl) with a few vesicles and degrading starch grains, and periplastidial endoplasmic reticulum element (arrow). (C) Upper part of a head cell showing initial detachment of the thick cuticle (C), electron-dense cell wall (Cw), periplasmic space with lamellar bodies (arrow), and cytoplasm with Golgi body (Gb), vesicles and voluminous mitochondria (M). (D) Portion of a head cell showing rough endoplasmic reticulum cisternae (arrowhead) and vesicles (arrows) close to the plasma membrane. (E) Cluster of vesicles (arrows) located close to the plasma membrane, and lamellar bodies in the periplasmic space. (F) Portion of a head cell showing finely granular material inside the undeveloped subcuticular space (^*). The arrow indicates an electron-dense layer (pectin) adhered to the thick cuticle (C).

Stage 2

This is characterized by the appearance of large oil drops throughout the cytoplasm (Fig. 4A and C) and inside the subcuticular space (Fig. 4B). The subcuticular space is undeveloped and the thick cuticle shows folds. Head cells are usually tetrahedral or wedge-shaped and thin-walled, and have a spherical central nucleus and abundant cytoplasm (Fig. 4A). Polyribosomes are markedly increased, and RER and Golgi bodies are less visible than before (Fig. 4C). The plastids become larger, round, filled with tubular/vesicle structures and osmiophilic inclusions, and are encircled by smooth endoplasmic reticulum (SER) profiles (Fig. 4D).

Fig. 4.

Aspects of the Z. montana peltate trichomes on the ovary at the onset of secretion (TEM). (A) Peltate trichome with a small subcuticular space and head cells with central spherical nucleus (N), abundant cytoplasm and scattered oil droplets (^*). The arrows indicate cuticle folds. (B) Detail of the peltate trichome showing thick cuticle (C) subtended by the subcuticular space (Ss) filled with finely granular hydrophilic matrix and electron-light zones containing lipids (^*). (C) Detail of the cytoplasm with many polyribosomes and large oil droplets (^*). (D) Plastid encircled by endoplasmic reticulum element (arrow). (E) Plastid showing a central constriction (arrow), and a network of tubules and electron-dense stroma. (F) Osmiophilic smooth endoplasmic reticulum (Ser) and Golgi body (Gb). (G) Portion of a peltate trichome showing subcuticular space filled with finely granular hydrophilic matrix and electron-light lipid deposits (^*). Note lipid globules scattered in the cytoplasm of the head cells and vacuoles (V).

As secretion progresses, the plastids show densification of the stroma and an increase in inner tubular membranes, some containing an osmiophilic core and/or black granulations and a central constriction and/or thin isthmus (Fig. 4E) suggesting proliferation. Tubules and SER vesicles filled with osmiophilic deposits are abundant in the cytoplasm (Fig. 4F). The subcuticular space is larger than at the previous stage and contains hydrophilic and lipophilic components (Fig. 4G). Lipophilic secretion is visible as electron-translucent large inclusions of irregular outline; they gradually increase in size by coalescence and are surrounded by fine granular hydrophilic substances.

Stage 3

At this stage, the subcuticular space reaches maximum development and gives the trichome head a spherical shape. The head cells show a spherical nucleus, abundant electron-dense cytoplasm and small vacuoles (Fig. 5A). Plasmodesmata are seen in the anticlinal walls, indicating simplastic connections. The cytoplasm contains polyribosomes, mitochondria with well-developed inner membranes, plastids and SER (Fig. 5B). Oil drops are seen close to the plasma membrane and inside the periplasmic space (Fig. 5C). Plastids are large and show a peripheral stack of membrane cisternae filled with dense deposits, a profusion of tubules and vesicles, and strongly electron-dense globules. Small black granulations can also be seen within the mitochondrial matrix (Fig. 5D). Golgi bodies and RER are not evident at this stage. At maximum secretion, head cells presented SER proliferation and much larger, rounded plastids (Fig. 5E). Plastids show a reduction in electron density and are filled with tubular membranes, some of them devoid of osmiophilic inclusions.

Fig. 5.

Aspects of the Z. montana peltate trichomes on the ovary at maximum secreting stage (TEM). (A) Peltate trichome showing intact cuticle (arrow), the large subcuticular space, and head cells with prominent nucleus (N) and abundant cytoplasm. (B) Portion of the anticlinal wall showing plasmodesmata (arrowheads), and plastids filled with tubules/vesicles and osmiophilic inclusions. (C) Lipid droplets inside the plastid and in the periplasmic space (arrowheads). (D) Plastid with a peripheral stack of membrane cisternae (arrow). Note small osmiophilic granules inside the mitochondrion (M) and abundant SER tubules/vesicles in the cytoplasm. (E) Portion of a head cell at the maximum secreting stage with abundant larger round plastids (Pl) and SER proliferation, and developed vacuole (V) with membranes debris. (F) Lipid deposits (^*) in the upper part of the subcuticular space. The arrow indicates the thick cuticle. (G) Portion of the bottom of the subcuticular space showing vesicle-like structures embedded in a finely granular matrix. The arrow indicates osmiophilic core.

At this stage, the secretion stored inside the subcuticular space consists of finely granular hydrophilic material mixed with large electron-opaque oil droplets and vesicle-like structures. Oil droplets vary in size and make up most of the accumulated secretion in the upper region of the subcuticular space (Fig. 5F). Vesicle-like structures of different sizes and having an irregular outline are either translucent or contain an osmiophilic core, and are located typically at the bottom of the subcuticular space (Fig. 5G).

Cytochemical staining of the polysaccharides revealed positive reactions in the cytosol, vacuoles (Fig. 6A) and plastids (Fig. 6B). Protein is seen in the subcuticular space as small black granules over the outer cell wall and underneath the cuticle, and as a loose fibrillar mesh connecting the cell wall to the cuticle (Fig. 6C), in the cytoplasm, periplasmic space, cell walls (Fig. 6D), vacuoles and plastids (Fig. 6E). Lipids were observed in the subcuticular space (Fig. 6F), plastids (Fig. 6G), cytosol (Fig. 6H), SER (Fig. 6I), in the peripheral cytoplasm underlying the plasma membrane and in the periplasmic space (Fig. 6J).

Fig. 6.

Cytochemistry of fully secreting Z. montana peltate trichomes on the ovary. (A and B) Positive reactions for polysaccharides: (A) electron-dense droplets (arrows) in the cytoplasm and vacuole (V); (B) polysaccharides in plastids (Pl). (C–E) Positive reactions for basic proteins: (C) protein (arrowheads) in the subcuticular space; (D) protein in the cytoplasm and cell walls; (E) protein in the vacuole (V) and plastid (Pl). (F–J) Positive reactions for lipids: (F) electron-dense bodies in the subcuticular space; (G) electron-dense inclusions in the plastids; (H) electron-dense inclusions in the cytosol; (I) electron-dense inclusions in the SER and plastid peripheral membrane stacks (arrow); (J) lipid inclusions in the peripheral cytoplasm and periplasmic space (arrows).

DISCUSSION

The developmental and chemical study of the peltate trichomes on the ovary of Zeyheria montana contributes to a better understanding of the functional and ecological significance of floral glands in the Bignoniaceae. It was observed that the peltate trichomes on the ovary differentiate and start secreting at a very early stage of flower development, and accumulation of the secreted material continues during the growth of the organ. Similar findings were described for the peltate glandular trichomes in the Lamiaceae (Werker, 1993). At different flower stages, both juvenile and full peltate trichomes occur side by side from floral budding up to pre-dispersal fruit, with immature trichomes being absent on mature fruit. The production of glands on vegetative organs of other plants cease early during leaf growth (Hammond and Mahlberg, 1977), while peltate trichomes on the ovary of Z. montana are continually produced during all floral stages.

The secretion which accumulates in the subcuticular space of the peltate trichomes on the ovary of Z. montana stains positively for hydrophilic and lipophilic substances, with lipids (terpenes) prevailing in fully secreting trichomes. In spite of the low specificity of the histochemical tests, NADI reagent has been widely used to locate terpenes in plant secretions (Ascensão et al., 1997; Corsi and Botega, 1999; Sacchetti et al., 1999; Kolb and Müller, 2004). In this study, NADI proved to be a good test and TLC and the Lieberman–Burchard test confirmed its positive results. The osmium tetroxide test has some problems, including the simultaneous staining of unsaturated lipids, proteins, alkaloids and phenolic compounds (Jensen, 1962); however, the limitations of this staining technique were overcome by careful observation and control tests, as well as to lipid-, protein- and phenol-specific tests.

TLC analysis confirmed the presence of alkaloids and indicated monoterpenes in the secretion from the peltate trichomes on the ovary of Z. montana. Chloroform immersion proved to be a remarkably simple, fast and efficient method for releasing glandular secretions from fresh samples in Z. montana, as reported by Siebert (2004).

TLC analysis for terpenoids showed spots with the same retention value (R_f) as the myrcene standard, indicating the presence of similar substance in Z. montana reproductive organs. Additional spots in the flower and fruit samples on TLC plates indicate the presence of additional compounds, providing preliminary information. Further steps using the HPLC technique or even GCMS for quantitative analysis and for chemical structure elucidation of these substances are required.

The analysis of the development of peltate trichomes on the ovary of Z. montana showed several morphological changes which have been described for lipophilic glands of different species. These modifications include the formation of a large subcuticular storage space by the progressive elevation of the thick cuticle, together with a thin residual cell wall layer. A similar pattern of subcuticular space formation was described in oil glands of species in the Lamiaceae (Werker, 1993; Ascensão et al., 1997; Turner et al., 2000) and Cannabis sativa (Kim and Mahlberg, 1995). This wall reinforcement along the cuticle may provide resistance and stability to the subcuticular space when a large amount of secretion is stored (Ascensão et al., 1997). Basic proteins, detected in the subcuticular space by histochemistry and cytochemistry, may contribute to subcuticular space stability. The wrinkled surface observed in the juvenile peltate trichomes with an undeveloped subcuticular space may be related to progressive subcuticular space formation (Hammond and Malberg, 1977; Venkatachalam et al., 1984). It is unlikely that this feature is due to the escape of highly volatile secretory products, such as monoterpenes, by cuticular exudation as has been suggested for other plants (Ascensão et al., 1997; Sacchetti et al., 1999), since these substances were detected in all ovary stages, including mature fruit that possesses only fully secreting peltate trichomes.

In the peltate trichome of the ovary, the secretion remains trapped in the subcuticular space and probably, under natural conditions, mechanical contact with herbivores causes cuticle rupture with exsudate liberation, as described for other surface glands (Werker, 1993; Werker et al., 1993; Ascensão et al., 1995, 1997; Serrato-Valenti et al., 1997; Sacchetti et al., 1999; Pichersky and Gershenzon, 2002).

In young peltate trichomes on the ovary of Z. montana, the abundance of polyribosomes and RER elements could be related to an intense cytoplasmic enzyme synthesis needed for cell metabolism, monoterpene biosynthesis (Turner and Croteau, 2004) and alkaloid production (Roshchina and Roshchina, 1993; Vassilyev, 1994). Alkaloids, nitrogen-containing metabolites, are all ultimately derived from the protein amino acids, so that there is always competition for precursors which are required for more important processes, such as protein synthesis (Harborne, 1997). RER elements adjoining the plasma membrane, as well as an abundance of proteins in both cytosol and cell walls, can be related to enzymatic synthesis needed for wall loosening observed during the beginning of subcuticular space development (Ascensão et al., 1997). Golgi bodies and translucent vesicles seen at the earliest stages of secretion could be related to the production of the hydrophilic component of secretion (Findlay, 1988; Meyberg, 1988; Figueiredo and Pais, 1992; Jian et al., 1997) as well as being involved in the glucosylation of secretion compounds, especially some toxic essential oil components (Venkaiah, 1992; Figueiredo and Pais, 1994).

As reported for lipophilic glands, plastids were the organelles that showed the most striking changes during the development of the peltate trichomes on the ovary of Z. montana. Depletion of starch accumulations in the plastids observed during the early stages of gland development is usually associated with a source of energy for highly metabolic processes or as a precursor of the hydrophilic components of secretion (Monteiro et al., 1999). The extensive development of the plastid compartment with profuse tubular osmiophilic structures, together with the high proliferation of SER at the maximum stage of secretion, have been described in many secretory structures, particularly in those secreting monoterpenes (Gleizes et al., 1982, 1983; Cheniclet and Carde, 1985; Kleining, 1989; Figueiredo and Pais, 1994; Ascensão et al., 1997; Monteiro et al., 1999; Turner et al., 1999). Turner and Croteau (2004) using immuno-cytochemical methods demonstrated a high degree of compartmentalization within the secretory cells of peltate trichomes in Mentha, and the involvement of leucoplasts, SER membranes, mitochondrial matrix and the cytosol in monoterpene biosynthesis.

In the ovary glands of Z. montana, lipophilic material (both translucent and electron-dense droplets) was first observed to occur inside plastids which are surrounded by periplastidial ER cisternae. Later, lipophilic material occurs in the SER, scattered in the peripheral portion of the cytoplasm, close to the plasma membrane, and inside the periplasmic space; it then moves through the walls to the subcuticular space. The transmission electron microscopy images and cytochemistry results of the present study are consistent with the findings of Turner and Croteau (2004) concerning the subcellular compartmentalization of lipid (terpenoids) biosynthesis, and fit in with their assumptions about the directional movement of these substances in the gland cells.

The main ultrastructural characteristics of the fully secreting peltate trichomes on the ovary of Z. montana, such as numerous plastids with a well-developed tubular membrane system filled with osmiophilic inclusions, mitochondria and abundant SER with lipophilic deposits, periplastidial SER, poorly developed Golgi bodies and abundant cytoplasmic oil droplets, together with chemical analysis, supply evidence that they are typical lipid secreting glands and not nectariferous trichomes, as previously suggested by Machado et al. (1995). Although nectaries could show variable ultrastructural features according to the diverse nectar composition described by Baker and Baker (1975), they have basic features contrasting with the lipid gland cells, such as an abundance of active Golgi bodies and plastids with prominent starch grains (Fahn, 1988; Fahn and Shimony, 1998), which were not observed in mature peltate glands on the ovary of Z. montana. In addition, the lack of sugars in the exudate, associated with the absence of terpenes and alkaloids in the nectar confirm that the peltate trichomes on the ovary of Z. montana are not involved with either nectar production or nectar quality improvement, respectively. This finding substantiates the hypothesis of Bittencourt and Semir (2004) about the involvement of ovary trichomes with secretory functions other than of nectar secretion.

Bittencourt and Semir (2004), based on the distribution, number, and stain properties of nectar chamber wall and gynoecium glandular trichomes, suggested that nectar secretion in Z. montana is performed mainly by corolla-borne glandular trichomes. However, these authors report that their study does not present any histochemical and/or biochemical evidence on the site of nectar secretion. Ultrastructural studies of corolla-borne glandular trichomes, rudimentary disc and ovary lobes, together with chemical analysis of secretions, are needed to elucidate the true source of Zeyheria nectar.

The present data showed that the peltate trichomes on the ovary of Z. montana produce terpenes (mono- and triterpenes) and also appear to be the site of synthesis and/or storage of alkaloids. This constitutes a new finding for glands on reproductive organs in the Bignoniaceae. These substances may be involved in chemical defence. There is good evidence that alkaloids are efficiently used as defensive agents and they may be moved around the plant to those parts needing greater protection during growth, development and flowering (Harborne, 1993, 1997). Terpenoids (monoterpenes, sesquiterpenes, diterpenes, triterpenes) are toxicants, antibiotics, feeding deterrents and oviposition deterrents (Nishida, 2002). Triterpenes are a major group of terpenoids in plants and their various properties include toxicity to mammals, molluscicidal activity, bitter taste repellent, antifeedant activity and hormonal interference in insects (Harborne, 1997). Plant volatiles, as myrcene, have the potential to influence not only animals but also other plants in the vicinity, providing a detailed language for communication. Recent advances in biochemistry and molecular biology of plant volatile biosynthesis have provided new tools for evaluating the natural roles of these substances (Pichersky and Gershenzon, 2002).

Fruit protection in some Bignoniaceae species has been described as the secondary function of the extrafloral nectaries (Elias and Prance, 1978). In other plant families, secondary compounds, such as terpenoids, are involved in fruit and seed protection (Harborne, 1989, 1993).

On the ovary of Z. montana, non-glandular stellate trichomes are abundant and larger at anthesis, creating a thick mechanical layer and, presumably, collaborating with peltate trichomes to protect against herbivory or desiccation (Corsi and Bottega, 1999; Mello and Silva-Filho, 2002; Kolb and Müller, 2004). In addition, they would maximize any protective advantage afforded to the developing fruit and seed against biotic and abiotic agents.

The presence of peltate glandular trichomes, filled with terpenoids and alkaloids, through flower development from early budding up to pre-dispersal fruit, appears to be a mechanism to protect organs, increasing the reproductive potential of Z. montana.