Polyphenol Deposition in Leaf Hairs of Olea europaea (Oleaceae) and Quercus ilex (Fagaceae)

by George Karabourniotis, George Kofidis, Costas Fasseas, Vally Liakoura, Ioannis Drossopoulos
Polyphenol Deposition in Leaf Hairs of Olea europaea (Oleaceae) and Quercus ilex (Fagaceae)
George Karabourniotis, George Kofidis, Costas Fasseas, Vally Liakoura, Ioannis Drossopoulos
American Journal of Botany
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ZLaboratory of Plant Physiology, Department of Agricultural Biotechnology; and
'Laboratory of Electron Microscopy, Department of Agricultural Biotechnology, Agricultural University of Athens,
Iera Odos 75, 11855, Botanikos, Athens, Greece

The subcellular localization (cytoplasm, vacuoles, cell walls) of polyphenol compounds during the development of the multicellular nonglandular leaf hairs of Olea europaea (scales) and Quercus ilex (stellates), was investigated. Hairs of all developmental stages were treated with specific inducers of polyphenol fluorescence, and the bright yellow-green fluores- cence of individual hairs was monitored with epifluorescence microscopy. During the early ontogenetic stages, bright flu- orescence was emitted from the cytoplasm of the cells composing the multicellular shield of the scales of 0. europaea. Transmission electron micrographs of the same stages showed that these cells possessed poor vacuolation and thin cell walls. The nucleus of these cells may be protected against ultraviolet-B radiation damage. The progressive vacuolation that occurred during maturation was followed by a shifting of the bright green-yellow fluorescence from the perinuclear region and the cytoplasm to the cell walls. The same trends were observed during the development of the nonglandular stellate hairs of Quercus ilex, in which maturation was also accompanied by a considerable secondary thickening of the cell walls. Despite the differences in morphology, high concentrations of polyphenol compounds are initially located mainly in the cytoplasm of the developing nonglandular hairs, and their deposition on the cell walls takes place during the secondary cell wall thickening. These structural changes during the development of the leaf hairs make them a very effective barrier against abiotic (uv-B radiation) and probably biotic (pathogenic) stresses.

Key words: cell walls; development; leaf hairs; Olea europaea; phenolics; Quercus ilex; ultraviolet-B radiation damage.

Leaf pubescence is a defensive barrier against biotic andlor abiotic stresses. A dense indumentum provides protection against insects and pathogens (Levin, 1973; Johnson, 1975; Smith, 1989; Allen et al., 1991). It may also reflect radiant energy received by the leaf or help reduce transpiratory water losses (Ehleringer, 1984; No- bel, 1991; Schuepp, 1993). Hair layers can also absorb in the ultraviolet-B (uv-B) region of the spectrum, pro- tecting the leaves against uv-B radiation damage (Kara- bourniotis et al., 1992; Karabourniotis, Kyparissis, and Manetas, 1993; Skaltsa et al., 1994). As other protective tissues that first intercept radiation, including the epi- dermis and cuticle (Wollenweber and Dietz, 1980; Cald- well, Robberrecht, and Flint, 1983; Tevini, 1994), leaf hairs contain uv-B absorbing compounds, primarily phe- nolics such as flavonoids (Karabourniotis et al., 1992; Skaltsa et al., 1994). Thus, they can screen radiation pen- etrating into more sensitive tissues (Karabourniotis al., 1992; Skaltsa et al., 1994). Leaves without this protective filter (artificially dehaired) become more sensitive to the uv-B damage than intact ones (Karabourniotis, Kyparis- sis, and Manetas, 1993; Gramrnatikopoulos et al., 1994; Skaltsa et al., 1994; Karabourniotis, Kotsabassidis, and Manetas, 1995). In many cases the protective function of the trichome may be transient and limited only to the more sensitive, developing young leaves. In these leaves, both the density and the uv-B protective capacity of the trichome are considerably higher than in the mature

I ~ ~~ ~ ~ ~ ~

received 6 M~~ 1997; revision accepted 21 ~~~~~b~~ 1997. Author for correspondence.

leaves, when the epidermis seems to be inadequately de- veloped to assume its protective role (Karabourniotis, Kotsabassidis, and Manetas, 1995; Karabourniotis and Fasseas, 1996).

The polyphenol compounds in the mature leaf hairs of Olea europaea L. (Oleaceae), as well as of Quercus ilex

L. (Fagaceae), are diffusely located in the hair cell walls (Karabourniotis and Fasseas, 1996). Since these compounds are synthesized in the cytoplasm it is of interest to determine the means by which these compounds are localized in the cell wall. The green-yellow fluorescence emitted by polyphenol compounds after treatment with specific fluorescence inducers was used to investigate the relative subcellular distribution of these compounds dur- ing the development of the nonglandular leaf hairs.


Plant material-All experiments were performed during spring 1995. Twigs of the two trees were collected from Athens Agricultural Uni- versity experimental olive grove (0. europaea), or from Mount Parnis, near Athens (Q. ilex). The twigs were placed in plastic bags and taken immediately to the laboratory in a portable coolbox. During spring new, develpping leaves co-exist with mature leaves of the previous growth season. The young leaves were pubescent on both surfaces, while the mature ones were mainly on the abaxial surface. For hair isolation, developing leaves of the first, second and third node from the apex were used. Preliminary observations showed that these leaves possessed hairs of all ontogenetic stages.

Microscopy-For fluorescence microscopy, hairs of all ontogenetic

i~t stages were isolated from fresh leaves by gently scraping the leaf sur- face with a razor blade. The isolated hairs were immediately immersed in a buffer solution, containing HEPES (N-[2-Hydroxyethyllpiperazine

N'-[2-ethane-sulfonic acid])-KOH 20 mmol/L (pH 7.2), CaC1, 0.2 mmolk, and PVP (polyvinylpyrrolidone) 0.6 % (wlv). Diphenylboric acid-ethanolamine (Naturstoff Reagent [NR] Sigma Chemical Co., St. Louis, MO, USA) and 1% aqueous solutions of AlCl, were used as inducers of polyphenol fluorescence. Both compounds are staining re- agents for the detection of flavonoids in paper and thin layer chroma- tography (Markham, 1982; Harborne, 1989). NR was first dissolved in a small volume of methanol, the buffer solution was added, and the solution was filtered through a Whatrnan Number 3 paper. For plas- molysis induction, mannitol (final concentration 1 molk) was added to the buffer solution when needed. Epifluorescence micrographs were taken with a Zeiss Axiolab fluorescent microscope equipped with a BP 450-490 exciter filter and a FT 510 chromatic beam splitter. Similar observations, but with weaker fluorescence emission, were taken with G-365 as exciter filter and FT-395 as a chromatic beam splitter. Samples of isolated hairs with the corresponding fluorescence inducer were viewed immediately, without prior fixation. Samples without inducers were used as controls. Preliminary experiments showed that aqueous solutions of fluorescence inducers, did not produce the characteristic green-yellow fluorescence (Fig. 9) probably due to the occurrence of a cuticle (see Fahn, 1986), which is a physical barrier against the pene- tration of aqueous solutions into the hair cells. On the other hand treat- ment with inducers in organic solvents or ammonia fumes (Karabour- niotis and Fasseas, 1996) was not advisable because of extensive mem- brane damage and subsequent cell lysis. The cuticle was removed by immersing intact leaves of both trees in chloroform (Wollenweber,

1985) for 30 sec (Fig. 10). Then we used aqueous solutions of fluores- cence inducers to observe fluorescence.

For TEM (transmission electron microscope) and LM (light micro- scope) examination fresh leaf samples were cut and fixed in 3% glu- taraldehyde in 0.1 mom sodium cacodylate buffer at pH 7.3 for 3 h at 4"C, postfixed in 1% OsO, for 2 h, dehydrated in an ethanol dilution series followed by propylenoxide, embedded in Spurr epoxy resin and polymerized at 70°C for 36 h Ultrathin sections were cut with a Reich- ert OM-U ultramicrotome, placed in pyroxylin-coated copper grids, and stained with uranyl acetate and lead citrate. Micrographs were taken with a Zeiss 9-S TEM.

For LM, semithin sections (1.5-2 pm) were stained with toluidine blue. Specimens for microscopy were processed from at least ten dif- ferent leaves, and at least three specimens were cut from each leaf.


Embedded and sectioned samples of leaf hairs of both trees did not emit the characteristic green-yellow fluores- cence in the presence of fluorescence inducers (data not shown). The reason for this may be the extraction of the polyphenol compounds during fixation, dehydration, and embedding processes. These compounds -are noncovalently bound to the cell walls of the mature hairs and thus solvent extractable (Karabourniotis et al., 1992). Extra- protoplasmic low molecular mass phenolic compounds contained in the cell walls are widespread in plants (Wal- lace and Fry, 1994).

Isolated hairs from fresh material of all developmental stages were immersed in a hypertonic solution containing the appropriate fluorescence inducer. Plasmolysis induc- tion was important for the discrimination of the particular cell component emitting the observed fluorescence. The stages of development of the multicellular shield of the leaf hairs of Olea (Figs. 1-6) are similar to those de- scribed by Galati (1982). During the stages of the mul- ticellular shield formation (Figs. 1-6) fluorescence was emitted mainly by the cytoplasm of the cells; cell walls did not fluoresce. The concentration of the polyphenol compounds within young, developing hairs might be con- siderably higher compared to those of the mature leaves (Karabourniotis. Kotsabassidis. and Manetas. 1995). Dur- hgthe early de;elopmental (correspdnding to the stages in Figs. 1-3) there were many small vacuoles, oc- cupying a small portion of the total volume of the cells (Figs. 16-18). During hair development one large vacu- ole progressively expanded, finally restricting the cyto- plasm to a thin band pressed against the cell walls (Figs. 19-21). The same characteristics were observed during the development of the apical cell of the nonglandular leaf hairs of Helichrysum aureonitens (Afolayan and Meyer, 1995).

As the multicellular shield reached its final form, the fluorescence seemed to be emitted diffusely from the cell walls and/or cuticle (Fig. 8). If the fluorescence had de- rived from the cytoplasm and/or the vacuole, it might have been more restricted to distinct regions, because of the existing plasmolytic conditions (Fig. 8). On the other hand, the cuticle of the leaf hairs of Olea did not contain considerable amounts of uv-B absorbing compounds (Karabourniotis et al., 1992) and, therefore, it is possible that the emitted fluorescence came mainly from the cell walls. The transfer of the polyphenol compounds to the cell walls took place during the short period of final hair development, which corresponded to secondary wall

Figs. 1-15. Fluorescence micrographs of leaf hairs of 0. europaea and Q. ilex of different stages of development. 1-8. Successive stages of development of the multicellular shield of the nonglandular hairs of olive leaves. Hairs were immersed in a plasmolytic medium containing either NR (Figs. 1-6 and 8), or AlCl, (Fig. 7) as fluorescence inducers (see Materials and Methods). In the stages of the formation of the multicellular shield (Figs. 1-6), the emitted fluorescence appears to derive mainly from the cytoplasm (since vacuoles are limited, see Figs. 16-18), whereas in the final stage (Fig. 8), where the shield possesses its final form, the emitted fluorescence is diffuse and derives mainly from the cell walls. In Figs. 6 and 7 the young developing hairs are at a similar ontogenetic stage (the 32-cell shield). In Fig. 7, note that the region of the nuclei emitted brighter fluorescence than the surrounding cytoplasm. Figs. 9-10. Stellate hairs of Q. ilex at similar developmental stages. NR was used as the fluorescence inducer. 9. Hairs isolated from intact leaves, after 30 min in NR. 10. Hairs isolated from leaves, immersed for 30 sec in chloroform, and followed by 2 min in NR. Extensive parts (arrows) of the hairs of the intact leaves do not emit the characteristic bright green-yellow fluorescence, in spite of a prolonged (30 min) period of NR treatment. 11-15. Stellate leaf hairs of Q. ilex of different developmental stages. Hairs were plasmolyzed. NR was used as the fluorescence inducer. In Fig. 11, only the cytoplasm emits fluorescence, since vacuoles are limited (as in stage 1, Fig. 22). In Fig. 14 the arms of the hairs have reached their final length and the secondary thickening has started, indicated by the fluorescence of the cell walls, which is more evident in Fig. 15. The lumen of the mature hairs (arrowheads in Fig. 15) does not fluoresce. Scale bar: Figs. 1- 7, 11-14 = 25 p.m; Figs. 8, 15 = 50 pm; Figs. 9-10 = 100 p.m.

Figure Abbreviations: BC, basal cell; C, cytoplasm; CU, cuticle; CW, cell walls; E, epidermal cells; H, hair; SC, stalk cell; SH, shield cells; SI, shield initials; V, vacuole.

thickening. The absorption spectra of the methanolic ex- tracts of olive hairs differed during the different stages of leaf development (Karabourniotis, Kotsabassidis, and Manetas, 1995). It is probable, therefore, that these dif- ferences may be related to biochemical changes during the polyphenol deposition on the cell walls. Fahn (1986) observed an extensive suberin deposition on the cell walls of the trichome bases in the hairs of the mature leaves of 0. europaea. This suberization may be respon- sible for the isolation of each hair from the rest of the leaf tissues and the subsequent death of the cells com- posing the multicellular shield. Heide-Jorgensen (1980) described a similar pattern for the nonglandular hairs of

Hakea suaveolens.

The same trends of polyphenol localization were ob- served during the development of the nonglandular stel- late hairs of Q. ilex (Figs. 11-15). However, maturation was accompanied by an extensive secondary thickening of the cell walls of their arms (Figs. 22-25). As a result, subcellular organelles were restricted within the thin lu- men.

Reagents induced different fluorescence emission pro- files of individual ,developing hairs (Figs. 6-7). Brighter fluorescence was observed from the area occupied by the nucleus of each cell after treatment with AlC1, (Fig. 7), whereas the emission was more uniform after treatment with NR (Fig. 6). This organelle may be protected by specific polyphenol compounds. The nuclei of pollen and superficial cells of protective tissues such as epidermis may require special protection (Caldwell, Robberrecht, and Flint, 1983). The pollen walls contain uv-B absorb- ing compounds that apparently serve a protective func- tion during pollination (Tevini, 1993). The different flu- orescence profile, after treatment with AlC1, or NR in our experiments, probably results from the distribution of the various polyphenol compounds within the cells.

The diffuse polyphenol deposition on the cell walls of the nonglandular hairs makes the trichome layer(s) a very effective filter against the uv-B radiation. Hairs of older leaves contain lower concentrations of polyphenol com- pounds per milligram of dry mass than the younger, fully expanded ones, thus having a reduced uv-B absorption capacity (unpublished results). Phenolic constituents can be leached from damaged hairs (unpublished results). Therefore the thin films of surface rainwater can contain these compounds. Flavonoids and other polyphenol con- stituents may be toxic to bacteria (Waage and Hedin, 1984; Wang et al., 1989; Ruiz-Barba et al., 1990), fungi (Aguinagalbe, Perez-Garcia, and Gonzalez, 1990; Wei- denboerner et al., 1990; Perez-Garcia et al., 1992), insects (Elliger, Chan, and Waiss, 1980) andlor have allelopathic properties (Rice, 1979). Therefore it would be of interest to investigate the possible effectiveness of hair constitu- ents against some biotic stresses.

In conclusion, we show that structural changes during the development of the nonglandular hairs are associated with the diffuse deposition of polyphenol compounds on their cell walls. This phenomenon may be part of an ef- fective mechanism protecting against abiotic and possibly biotic stresses.



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