In this work, the localization, density, morphology and ultrastructure of secretory structures in aerial organs of Flourensia campestris (FC) and F. oolepis (FO) (Asteraceae) by means of a combination of light, fluorescence, transmission (TEM) and scanning electron microscopy (SEM) were examined. The possible role of secretory structures in the production and secretion of the phytotoxic sesquiterpene (-)-hamanasic acid A ((-)HAA) in both species was also assessed. Capitate glandular trichomes were found in all reproductive organs of FC and FO, and were being reported for the first time. These glandular trichomes, typically associated to edges and veins, were of the same type as those already described for vegetative organs, and were abundant in involucral bracts and corolla of tubulose and ligulate flowers. Their density in reproductive organs of both species was similar (ca. 30/mm 2) and lower than that found in leaves (ca. 100/mm 2) and stems (ca. 160/mm 2 in FC, and up to 650/mm 2 in FO). Glandular trichomes in vegetative organs followed a species-specific pattern of distribution. TEM and SEM observations suggest that each species differs in the way in which secretory materials are released to the outside: through cracks or pores in FC, or through a loose cuticle in FO. Similar inspections of the secretory ducts revealed lipophilic vacuoles localized in subepithelial and epithelial cells, in which secretions accumulated before being transferred to the duct. The presence of wall ingrowths in subepithelial cells suggests that granulocrine secretion operates in these species. Secretory ducts varied in density and diameter among the organs in both species, with the combination being maximal in woody stems. (-)HAA was only detected in surface secreted resins of both species, and its concentration (2D-TLC, GC-FID) was intimately associated with the distribution and density of glandular trichomes in each organ (capitula, leaves, and stems with primary or secondary growth). In addition, no (-)HAA was detected internally in the resins collected from secretory ducts. The composition of these resins showed distinctive profiles for FC and FO, and only four from ca. 30 compounds detected (GC/MS) were shared by both species. In addition to the elucidation of ultrastructural traits, distribution and density of secretory structures in aerial organs of FC and FO, present findings suggest a functional role for glandular trichomes in the secretion of the putative phytotoxic allelochemical (-)HAA.
The Asteraceae are among the largest families of flowering plants with ca. 1700 genera and 23,000 - 26,000 species in all continents except for Antartica [
Flourensia campestris (FC) and Flourensia oolepis (FO) are easily distinguished by the characteristic glossy aspect of their leaves due to resinous exudates of unknown composition, which represent ca. 40% and 20% of the biomass in young and adult leaves, respectively (Silva and Scopel, Personal communication, 2005). Plant resins, typically constituted by mixtures of volatile and non-volatile phenolic and/or terpenoids secondary compounds, are usually secreted in specialized structures located either internally or on the surface of the plant [
FC has been shown to produce a phytotoxic compound identified as (-)-hamanasic acid A {(-)HAA; 7-car- boxy-8-hydroxy-1(2), 12(13)-dien-bisabolene} [
In Asteraceae, two secreting systems occur: glandular trichomes at the surface and secretory ducts inside the organs [
In this work, we examined the localization, morphology, density and ultrastructure of secretory structures―in vegetative and reproductive organs―of FC and FO by means of a combination of light, fluorescence, transmission and scanning electron microscopy. We also assessed the possible role of secretory structures in the production and secretion of (-)HAA in FC and FO. The presence and distribution of (-)HAA in different organs were studied by means of 2D-TLC, GC-FID and GC-MS.
Plant materials were collected in natural areas corresponding to the Punilla Valley, Córdoba province, Argentina. The study areas were located in shrub communities (total plant cover 70% - 90%), dominated by the evergreen shrubs Flourensia campestris Griseb. (FC) and F. oolepis S.F. Blake (FO). Specimens of FC were collected in “El Dragón” (La Falda), and FO plants were collected in “Dique El Cajón” (Capilla del Monte), in summer (February) of 2009 and 2010. The voucher specimens (BAA 26.498-FC- and BAA 26.499-FO-) are deposited at the Herbario “Gaspar Xuárez” of the Facultad de Agronomía, Universidad de Buenos Aires, Argentina.
For anatomical and morphological studies, cross and longitudinal section samples of fully developed capitula (common receptacle, phyllaries, paleae, and ligulate and tubular flowers), young and mature leaves, and stems from each species were obtained. Three developmental stages were considered in stems: primary growth (PG)- corresponding to the early growth of stems during the growing season; secondary growth (with early phellogen activity (SGph)-corresponding to stems between 3 months to one year old, and woody stems (WS)-correspond- ing to stems of 2 + years. The three categories were processed separately. The materials for light microscopy (LM) were fixed in FAA and embedded in paraffin [
or with aqueous toluidine blue (Figures 1B and Figures 1C) [
Quantitative Analysis
Density of glandular trichomes (number of secretory structures per tissue surface unit area) in aerial organs was measured in fresh material previously submerged in EtOAc for 20 s in order to remove surface-secreted compounds. Epidermal layers were manually removed, mounted in 50% glycerine, and trichomes were counted under LM, using a digital videomicroscopic camera with the corresponding software (MotiCam 1.3 MP). Density and diameter of secretory ducts were measured in cross sections of common receptacle and stems (PG, WS) while ducts length was measured in longitudinal sections of tissues.
Quantitative density analysis of described parameters was performed in 5 different individuals, in 5 different samples of 1 mm2 from each one. Diameter and length of the ducts were measured in 5 different individuals, in 5 different secretory ducts from each one.
For TEM studies, plant materials were pre-fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.2) overnight and then post-fixed in OsO4 at 2˚C in the same buffer for 3 h. Following dehydration in a graded ethanol series,
the material was embedded in Spurr’s resin. Thin sections (1 µm) were made with a Reichert-Jung ultramicrotome and stained with toluidine blue for observation with light microscope. Ultrathin sections (750 - 900 nm) were made with the same ultramicrotome and then stained with uranyl acetate and lead citrate [
Plant materials were fixed with FAA (2.2.1), dehydrated in a graded ethanol series and coated with a thin layer of gold. Observations were carried out on a Zeiss scanning electron microscope at 15 kV.
Based on the results of type, distribution and density of secretory structures in the plant organs studied (capitula, leaves and stems), different extraction methods were performed in order to investigate the possible role of the two different secretory structures in (-)HAA storage and secretion.
The presence and content of (-)HAA in FC and FO was assessed in whole tissues, on the tissues surface (surface secreted resins), and in the resins collected directly from secretory ducts in pooled materials harvested from 5 different individuals.
Whole Tissue: For whole organs, tissue extracts were obtained from grinded fresh material (50 mg), extracted twice using EtOAc (1.5 mL and 0.5 mL), vortexed for 2 min and centrifuged (10 min, 3000 g). The EtOAc phases were pooled, dried at 30˚C under N2 flow and weighed [
Surface Secreted Resins: Fresh organs were washed with gentle agitation in cold EtOAc during 20 s (short dips) [
Duct Resins: Resins produced in secretory ducts were collected from fresh stems of FC and FO, after peeling the external tissues and allowing the products to exude for ca. 10 min at ambient temperature. Resins were picked up using a micro syringe, weighed, and stored at −20˚C until analysis.
(-)HAA was identified and measured by means of GC-FID and 2D-TLC [
(-)HAA was also investigated in duct resins by GC-MS on a Perkin Elmer Clarus 600, and TurboMass 5.4.2 for data acquisition. The GC column was DB5 (60 m, 0.25 mm ID, 0.25 µm particle size) (Perkin Elmer) and the carrier gas used was He (49.6 psi). The initial temperature of 60˚C was gradually increased after 2 min to 300˚C by a ramp of 5˚C∙min−1 and held for 10 min at 300˚C. The injector was used in split mode (20 mL∙min−1) with the inlet temperature set to 250˚C. Samples were diluted in acetone or in EtOAc as follows: in 100 µL of solvent and 1/50 µL of solvent, the injection volume was 1 µL. Some peaks were identified by comparison of mass spectra using NIST MS Search 2.0., and using previous own data from volatiles in the same species [
One way ANOVA or Mann-Whitney nonparametric test for the significance of the difference between the distributions of independent samples for ordinal data were applied.
Secretory structures in the capitula of both species were composed by glandular trichomes and secretory ducts (
Species | Bracteae | Flowers | Leaves | Stems | |||||
---|---|---|---|---|---|---|---|---|---|
Phyllaries | Paleae | Corolla of ligulate flowers | Corolla of tubular flowers | Adaxial surface | Abaxial surface | PG | SGph | WS | |
FC | 25 ± 2e | 40 ± 4d | 14 ± 1f | 26 ± 2e | 123 ± 8b | 93 ± 5c | 174 ± 12a | 162 ±12a | ND |
FO | 31 ± 4d | 45 ± 4d | 19 ± 1e | 20 ± 2e | 95 ± 3c* | 85 ± 4c | 656 ± 22a* | 344 ± 16b* | ND |
Data (media ± SE, n = 25) represent the number of glandular trichomes per mm2. PG: stems with primary growth; SGph: stems with secondary growth and incipient phellogen activity; WS: woody stems; Letters (a - f) indicate significant differences among tissues; asterisks (*) show differences between species (p < 0.05); ND: not detected.
In leaves, glandular trichome density on the adaxial surfaces of FC was higher than in FO (
Secretory ducts were observed in all the floral pieces of both species and were similar in shape to those observed in the cross section of the common receptacle (data not shown). Density and diameter of secretory ducts present in the common receptacle were significantly different in FC as compared to FO (
There were no ultrastructural differences between the glandular trichomes of FC and FO, with the exception of a broken cuticle commonly observed in FC. The secretory cells of the trichome head have a dense cytoplasm and small vacuoles (
Capitula | Stems | |||||
---|---|---|---|---|---|---|
Common receptacle | PG | WS | ||||
Cortex | Pith | Cortex | Secondary phloem | |||
FC | Density (N˚/mm2) | 8.8 ± 1.3a* | 13.2 ± 1.2a | 5.9 ± 0.9b | 13.8 ± 0.8a | 57.1 ± 4.6c |
Diameter (μm) | 131.4 ± 12.1a* | 39.5 ± 2.9b | 25.6 ± 1.3c | 165.5 ± 18.6a* | 58.9 ± 9.5b | |
FO | Density (N˚/mm2) | 34.7 ± 2.2a | 15.5 ± 0.4b | 5.5 ± 0.3c | 12.2 ± 1.0b | 45.2 ± 2a |
Diameter (μm) | 78.6 ± 1.6a | 31.3 ± 2.8b | 17.5 ± 1.8c | 89.1 ± 7.2a | 53.9 ± 3.1d |
Data are expressed as mean ± SE, n = 25. PG: stems with primary growth; WS: woody stems. Letters (a - d) indicate significant differences among tissues; asterisks (*) show differences between species (p < 0.05).
and ultimately breaks releasing the products (
In secretory ducts, the lumen is surrounded by a layer of specialized cells, the epithelium (
Whole tissue concentrations of (-)HAA in all aerial organs of FO were significantly lower than those originally reported in FC (
(-)HAA | ||||||
---|---|---|---|---|---|---|
FC | FO | |||||
Whole tissue mg∙g−1 DW | Surf. secreted resins mg % | Duct resins mg % | Whole tissue mg∙g−1 DW | Surf. secreted resins mg % | Duct resins mg % | |
Capitula | 17.0 ± 0.6a* | 21.0 ± 0.6b* | NA | 1.2 ± 0.1c | 1.02 ± 0.03b | NA |
Leaves | 17.9 ± 0.1a* | 26.6 ± 0.7a* | NA | 0.90 ± 0.01c | 0.76 ± 0.02c | NA |
Stems PG SGph WS | 10.5 ± 0.5b* 4.9 ± 0.2c* 0.60± .03d* | 21.4 ± 0.6b* 17.3 ± 0.3c* 2.0 ± 0.1d* | ND ND ND | 5.3 ± 0.2a 1.8 ± 0.1b 0.30 ± 0.02d | 6.45 ± 0.19a 1.20 ± 0.03b 0.65 ± 0.02d | ND ND ND |
(-)HAA levels in whole tissues of FC and FO (mean ± SE; N = 3). Values in FC (Silva et al. 2012) are shown for comparison purposes. NA: not applicable; ND: not detected; PG: primary growth; SGph: secondary growth and incipient phellogen activity (last season); WS: woody stems (2 + yrs). Letters (a - d) indicate significant differences among tissues; asterisks (*) show differences between species (p < 0.05).
Surface secreted resins obtained from short dip extractions with EtOAc revealed the presence of (-)HAA in all aerial organs; concentrations being within the range of those found in whole tissue (
In duct resins collected from fresh stems of both species, (-)HAA was not detected by means of 2D-TLC or GC-MS (FC:
The 2D-TLC chromatographic profiles of whole tissue of PG stems also revealed the presence of a fast chromatographic fraction represented by less polar compounds relative to (-)HAA, which was also detected in duct produced resins (cf.
showed distinctive profiles. From a total of 30 compounds found in each species only four (13%) were present in both; the volatiles santolinatriene and spathulenol could be identified by this analysis. Remarkably only 7 of these metabolites accounted for 75% (FC) and 90% (FO) of the total duct resins composition.
Results showed that both species present two types of secretory structures: glandular trichomes-capitate wit3 h a biseriate-pluricelular head, and secretory ducts, confirming the observations of Delbón et al. [
Present observations demonstrate, for the first time in the genus Flourensia, the existence of glandular trichomes in reproductive organs. These were morphologically identical to those found in the vegetative organs of both species studied, and were also similar to those described in the vegetative organs of other species of the genus, like F. hirta, F. tortuosa, F. niederleinii and F. leptopoda [
Despite the density of glandular trichomes found in capitula was lower than that found in leaves, and much lower than that found in stems, these trichomes may still play relevant ecophysiological functions in the reproductive organs. In this sense, secretions of glandular trichomes in floral structures have been shown to act as pollinator attractants and/or to be involved in the chemical defense against herbivore insects and microbes [
The densities of glandular trichomes in the leaves of both species were very similar to those previously reported by Delbón et al. [
Detailed observations of SEM and TEM images suggest that each species would differ in the way they release the secretions to the plant surface. In FC, cuticle rupture was commonly observed. The products would accumulate temporarily in the interfibrillar spaces of the cell wall, and would be transferred and stored in the subcuticular chamber until the cuticle fully distends and ultimately breaks releasing the products. In FO, instead, cuticle rupture or pore formation was not observed by SEM in any of the materials inspected. In this species, the secreted products seem to be released to the plant surface through a loose cuticle.
Storage of secretions in the subcuticular space and cuticle rupture has been reported for other Asteraceae species [
A growing body of experimental evidence shows that terpene biosynthesis takes place within glandular trichomes [
Differences in the chemical composition of glandular trichomes, even at within-plant level, have also been reported. In this sense, Appezzato-da-Glória et al. [
The mild dipping extraction method of intact organs, which did not damage the epidermis or external tissues, proved that (-)HAA is only present in the surface secretions found in glandular trichomes bearing organs. The presence of small amounts of (-)HAA on the surface of WS may derive from the product being lixiviated by rain from the younger portions of the branch. The fact that (-)HAA is easily leached out by water [
In order to further investigate the possible role of secretory structures specifically related to the storage and secretion of (-)HAA, we compared the concentrations of (-)HAA found internally (secretory ducts) with those found on the surface of the plant (glandular trichomes). In organs such as leaves and capitula, in which resins from internal tissue were very difficult to obtain without surface contamination, whole tissue extractions were compared against surface resins. Results from 2D-TLC and GC-MS (as shown in
Based on the above, we propose that glandular trichomes in FC and FO are the secretory structures involved in the production of this putative phytotoxic allelochemical. These results are also consistent with those originally described by Hashidoko et al. [
In respect to secretory ducts, the importance of resin production in Asteraceae has been long recognized by Carlquist [
Albeit the importance and widespread existence of secretory ducts in woody shrubs of arid environments, morphometric data is rarely presented. In both Flourensia species, the highest density of secretory ducts was observed in stems with SG, specifically in the secondary phloem, while the larger diameters corresponded to ducts in the cortex of SG stems. Estimations based on the diameters and densities of ducts indicate that ducts in SG stems may represent as much as 2.6% per unit of volume (1 µm3), emphasizing the importance of this secretory system in FC and FO. In two varieties of Senecio filaginoides (Asteraceae), Feijóo et al. [
In FC and FO, the localization of the ducts in the secondary phloem, close to the cork, would facilitate the secretion of products to the surface of stems through the lenticels or cracks (by the new phellogen activity near the surface). In fact, in plants growing in their natural environment, stems usually present sticky depositions (bulk resins) that have been secreted to the surface as resin drops of different colors (yellowish to reddish) and viscosity, probably due to different degrees of oxidation.
The ultrastructural analysis of secretory ducts of FC and FO revealed the presence of chloroplasts with starch accumulation in the epithelial and subepithelial cells. The presence of abundant lipid globules in the dense cytoplasm strongly suggests their involvement in the synthesis of resins. Wall ingrowths, typically associated with transfer processes [
Preliminary results from GC-MS analysis of resins collected directly from stem ducts of FC and FO showed very distinct profiles for each species, and no (-)HAA was detected as discussed above. Only some monoterpenic and sesquiterpenic volatile compounds already reported (FC: [
Reproductive organs of FC and FO presented the same type of secretory structures (glandular trichomes and secretory ducts) described in vegetative organs. Our observations demonstrate, for the first time in the genus Flourensia, the presence of glandular trichomes in reproductive organs. These capitate glands were morphologically identical to those found in vegetative organs of all Flourensia species studied so far, and might constitute an important trait for the taxonomy of the genus.
Glandular trichome’s density was very similar in reproductive organs of both species. In contrast, densities of glandular trichomes in vegetative organs were significantly different between the two species; the highest density corresponded to FO stems.
Secretory ducts varied in density and diameter among the organs of both species, with the combination being maximal in stems with SG. TEM and SEM observations of glandular trichomes suggested that each species differed in the way in which secretory materials were released to the outside: through cracks or pores in FC, or through a loose cuticle in FO. In secretory ducts, the localization of lipophilic vacuoles and the presence of wall ingrowths in subepithelial cells suggest the existence of a granulocrine mechanism of secretion. Similar inspections performed since early stages of ducts development revealed a schizogenous origin.
Our studies are the first to report the distribution of the sesquiterpenoid (-)HAA in different organs of FO. Concentration of (-)HAA in all organs was significantly lower than in FC, as measured in whole tissues, with the highest contents being present in stems with primary growth. In both species, (-)HAA was only detected in surface secreted resins from those organs where glandular trichomes were also present (capitula, leaves and stems). The concentration of (-)HAA was closely and positively related to the density of the glandular trichomes. In addition, no (-)HAA was detected in the resins collected from secretory ducts. These findings strongly suggest that glandular trichomes are the sites of accumulation and secretion of (-)HAA. Biochemical investigations on the biosynthetic capacity of isolated glandular trichomes to produce (-)HAA are under way. Preliminary data on resins composition―from resins extracted directly from secretory ducts in stems― show distinctive profiles for FC and FO; only 13% of the compounds detected are shared by both species.
In addition to the elucidation of ultrastructural traits, distribution and density of secretory structures in reproductive and vegetative organs of FC and FO, our findings suggest a functional role for glandular trichomes in the secretion of the putative phytotoxic allelochemical (-)HAA.
This research was supported financially by grants from the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) Grant PICT 0411, the Universidad de Buenos Aires, UBACyT 0566, and the Ministerio de Ciencia y Tecnología, Córdoba, Grant PID 2010. We are most grateful to Augusto Maillet for his dedication and technical assistance in the laboratory.