Localization of Partially Methylated Flavonol Glucosides in Chrysosplenium americanum: Immunogold Labeling

Localization of Partially Methylated Flavonol Glucosides in Chrysosplenium americanum: Immunogold Labeling L. MARCHAND'), P. M. CHAREST'), and RAGAI K. IBRAHIM') I) Departement de Phytologie, Universite Laval, Quebec GIK7P4 2) Plant Biochemistry Laboratory, Department of Biology, Concordia University, Montreal, Quebec H3G 1M8 (Canada) Received May 11, 1987 . Accepted May 15, 1987

Abstract Leaves of Cbrysosplenium ammcanum acccumulate a variety of partially O-methylated flavonol glucosides which are hydrophobic in nature. The intracellular localization of these metabolites was studied by immunocytochemical labeling of flavonoids, using the protein A-gold

post-embedding technique on thin sections with transmission electron microscopy. Antibodyspecific labeling was observed mainly on the walls of both epidermal and mesophyll cells. There was significant amount of labeling associated with the plasmalemma, but none with other organelles such as the endoplasmic reticulum, Goigi or chloroplasts. Control sections treated with preimmune serum or with flavonoid preabsorbed antibody were devoid of label. These results provide strong evidence for the localization of partially methylated flavonol glucosides in the cell walls of this tissue. The physiological significance of this finding is discussed in relation to the hydrophobic nature of these metabolites and their possible role in the survival of this semiaquatIc spectes.

Key words: Chrysosplenium americanum, cell wails, epidermis immunogo/d labeling, localization, O-methylated flavonol glucosides.

Introduction C.nrysosplenium americanum Schwcin ex Hooker (Saxifragaceae) is a semi-aquatic plant which accumulates a number of tri- to penta-O-methylated flavonol glucosides as the major phenolic constituents (Collins et aI., 1981). These are derived from 2'hydroxyquercetin and its 6-hydroxy analog, 2'-hydroxyquercet agetin (Fig.!). The enzymes involved in their O-methylation (De Luca and Ibrahim, 1982; !985 a, b; Khouri et al., 1986) and O-glucosylation (Bajaj et aI., 1983; Khouri and Ibrahim, 1984; Latchinian et aI., 1987) have recently been characterized. Despite the fact that these flavonoids arc monoglucosides, they are sparingly soluble in water or alcohol since they contain three to five methoxyl groups on the flavonoid ring system (Fig. 1). Previous work seems to indicate that plant secondary metabolites usually accumulate in the cellular vacuole (for review see Wagner, 1982), in specialized cells (Bal and Savory, 1980; Chariere-Ladreix, 1979; Mersey and Cutler, 1984; Zobel, 1986), or may be secreted on the plant surface as farina (Wollenweber, 1984) or gummy exudate (Chariere-Ladreix, 1977). Because of the hydrophobic nature of Chrysosplenium ~ To whom reprint requests should be addressed.

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OMe OR OH

OMe Oglu

OH

R:H.A R:Me.B

c

Fig. 1: Three of the major flavonoid constituents (A - C) of Chrysosplenium americanum.

flavonoids, it was considered important to study their intracellular localization within the leaves of this species. Precise localization of plant secondary metabolites in general, and flavonoid com~ pounds in particular, poses numerous problems to the cell biologist (for review see Luckner et al., 1980; Ibrahim et al., 1986) because of the lack of specific histochemical reagents for their visualization, and the possible contamination of tissues or organ~ elles with these metabolites during preparation. Recent ultrastructural studies of Chrysosplenium leaves, treated with caffeine as pre. fix and visualizing agent, suggested the presence of electron-dense deposits in the walls of epidermal and mesophyll cells, as well as various membrane profiles and associated vesicles (Charest et al., 1986). Furthermore, we have raised an antibody in rabbits (Lamoureux et aI., 1986) which exhibited high specificity for the 2'-O-glucosides of partially methylated flavonols (compounds A and B, Fig. I), although it showed some cross reactivity against the 5'-O-glucoside C (Fig. 1); both of which are major flavonoid constituents of Chrysosplenium. This antibody was used for the immunofluorescence localization of flavonoids, at the tissue level, which suggested that the latter compounds were associated with the peripheral areas of epidermal and mes~ ophyll cells; i.e. the cell wall or the periplasm of these cells (Brisson et al., 1986). Since these methods have not provided unequivocal evidence as to the precise loca~ lization of Chrysosplenium flavonoids, we had to resort to the use of an immunocytochemical technique which is known for its specificity and high resolution. Antibodies labelled with electron-dense markers, such as colloidal gold or ferritin, have recently been used in the localization of enzymes (Amann et al., 1985; Bergman et al., 1985; Doman and Trelease, 1985; Smart and Amrhein, 1987), seed proteins (Craig and Goodchild, 1984; Herman and Shannon, 1984), phytoagglutenins (Greenwood et aI., 1984; Greenwood and Chrispeels, 1985; Raikhel et aI., 1984) and of low molecular weight compounds, such as plant growth substances (Zavala and Brandon, 1983; Sossountzov et al., 1986) and cell wall polysaccharides (Moore et al., 1986). This report describes the intracellular localization of flavonoids in Chrysosplenium leaves using a post-embedding immunogold labeling technique, coupled with transmission electron microscopy.

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Materials and Methods Plane material Chrysosplenium americanum Schwein ex Hooker (Saxifragaceae) was collected from Huntington, Quebec, and was maintained in the greenhouse under conditions simulating its natural habitat.

Tissue preparation Small leaf segments (ca. 1-2mm in length) were fixed in 3% glutaraldehyde (in 0.1M cacodylate buffer, pH 7.2) overnight, followed by post-fixation in 1 % OS04 (in the same buffer) for one h. After rinsing in cacodylate buffer and dehydrating in a series of EtOH, the tissue was embedded in Epon. Ultrathin sections (ca. 90nm) were mounted on 200-mesh nickel grids coated with Formvar. Immunocytochemical methods

For immunocytochemical labeling of flavonoids, the protein A-gold post-embedding technique {Roth et ai., 1978} was used with thin sections. The colloidal gold suspension (16nm) was prepared according to Ferns (1973) and was complexed with protein A as described by Roth et al. (1978). Ultrathin sections were floated on fetal calf serum for 5 min at room temperamre. They were then incubated with 20ILI of antiserum, which was diluted 1 :60 (v/v) with 0.05M phosphate-buffered saline, pH 7.4 for 60 min at room temperature. The grids were thoroughly washed with phosphate-buffered saline, in order to remove the unbound antibody molecules, then incubated with the protein A-gold complex for 30 min at room temperature. Finally, the grids were successively washed with phosphate-buffered saline and double distilled water, then stained with uranyl acetate followed by lead citrate. The sections were examined with a Siemens Elmiskop 102 electron microscope. Controls for immunolabeling

Tn order to assess the specificity of antibody labeling, the following immunocytochemical controls were carried out: (a) inactivation of the antiserum with a solution (2mg/ml in 5% dimethyl sulfoxide) of the corresponding antigen (5,5'-dihydroxy-3,7,4'-trimethoxyflavone-2'-Oglucoside, Fig. 1 A) for 30 min at room temperature, before its application to the sections, (b) incubation of the sections with preimmune serum for one h at room temperature, followed by protein A-gold as described above, and (c) incubation of the sections with the protein A-gold complex, or with colloidal gold stabilized with polyethylene glycol (MW 20,000) for 30 min at room temperature.

Results Preservation of ultrastructure Chrysosp/enium leaves exhibit simple cellular organization, which consists of three to four layers of highly vacuolated mesophyll cells, bordered by two epidermal layers. The ultrastructure of both cell types, which had previously been described in detail (Charest et aI., 1986), appears to be well preserved as indicated by the integrity of the various organelles and membrane profiles (Figs. 2-4). These cells are characterized by extensive undulation of the plasmalemma (Figs. 2 A, B; 3 C; 4 A, B), as well as the: presence of membrane-surrounded vesicles of various size (Figs. 3 C; 4A-C), which are suggestive of their secretory nature.

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Abbreviations in Figs. 2-4: Ch, chloroplast; CW, cell wall; Ee, epidermal cell; G, Golgi apparatus; IS, intercellular space; M, mitochondrion; Me, mesophyU cell; Pi, plasmalemma; RER, rough endoplasmic reticulum; V, vacuole; Ve, vesicle. Fig. 2: Epidermal cells. A, Control from sections where the antiserum added was inactivated by the corresponding antigen (as described in the Methods section). B. Treated sections. The gold labeling occurs mostly along the cell wall and, to a much lesser extent, in the vacuole. x 25,000.

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distinctly labeled whereas fig. }, Epidermal and mesophyll cells. A, The mesophyll cell wall is s and chloropl asts, the labeling observed in other cellular organel1es, such as the Golgi apparatu label in the intergold of absence the Note level. und backgro the to is negative or corresponds l cells and one mescellular space. x 41,000. B, An intercellular space between two epiderma x 19,400. C, Epidermal ophyll cell. Control from sections treated with preimmune serum. w vesicles between cells exhibiting undulation of the plasmalemma and the presence of numero gold panicles, ~ with labeled highly is lanef The wall cell the and the retracted plasmale mma compared with the vacuo k x 45,000.

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Fig. 4: A, Epidermal cells. A, The cell wall and the periplasmic space are positively labeled, while few gold particles can be observed in the cytoplasm. The plasmalemma is largely undulated and numerous vesicles are present in between it and the cell wall, x 25,700. B, The plasmalemma of the lower epidermal cell appears positively labeled as well as the cell wall of both adjacent cells, x 34,500. C, Many gold particles can be observed in the periplasmic area of an epidermal cell, but none in the vesicles, x 48,700. D, The RER and Golgi of both epidermal and mesophyll cells are consistently devoid of specific labeling, x 55,300.

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specificity of labeling The immunocytochemical method used in this study, when coupled with the appropriate controls, appears to be quite reliable as indicated by the density, specificity

and uniformity of labeling observed on the walls of both mesophyll (Fig. 3 A) and epidermal (Figs. 2 B; 4 A) cells. However, external cell wall junctions between epidermal cells showed significantly lower labeling intensity (not illustrated). There was some labeling observed in the vacuoles of mesophyll cells (Fig. 2 B; 3 C), as well as over the periplamic area (Fig. 4 A, 4 C) and the plasmalemma (Fig. 4 B, 4 C) of epidermal cells. On the other hand, the two latter areas of mesophyll cells appeared labeled with lower intensity (Fig. 3 A, 3 C). No labeling was associated with other cellular organelles, such as chloroplasts, Golgi, rough endoplasmic reticulum (Figs. 3 A, 4 D) or the numerous vesicles included between the plasmalemma and the cell

wall (Figs. 3 C; 4 D). The near absence of gold particles from the different controls used (Figs. 2 A; 3 B) attests to the specificity of the labeling observed in the treated sections. Discussion The problems usually encountered with the use of conventional methods for intracellular localization of secondary metabolites can be overcome by using the in situ

immunogold labeling, which is sensitive and highly specific. The results presented here clearly demonstrate that the hydrophobic flavonol glucosides of Chrysosplenium leaves are mainly localized in the walls of epidermal and mesophyll cells. Although the chemical nature of flavonoid association with cell wall material has yet to be investigated, however, these results support earlier postulations which were based on

ultrastructural (Charest et a1., 1986) and immunofluorescence (Brisson et a1., 1986) studies. The low density of labeling observed in the mesophyll vacuole (Fig. 2 B) suggests that the latter is a minor compartment for flavonoid accumulation. This is in agreement with earlier observations where leaf protoplasts exhibited very weak autofluorescence and immunofluorescence as compared to that of the cell walls (Brisson et

aI., 1986). Since the antibody used in this study was highly specific for the 2'-glucosides of partially methylated flavonols, though less so for the 5 1-glucosides (Lamoureux et aI., 1986), it is not unexpected, therefore, that other cellular organelles such as

the Golgi apparatus (Figs. 3 A, 4 D) or membrane vesicles (Figs. 2 B, 3 C, and 4 A, B) are not labelled. The latter structures may be involved in the early steps of flavonoid biosynthesis, where some metabolic intermediates may be compartmented. However, the presence of gold labeling in the plasmalemma as well as the periplasmic area (Fig. 4 A - C) is consistent with the secretory nature of this tissue (Charest et al.,

1986) and suggests the channeling of these metabolites towards the cell walls. These observations lend further support to the recently proposed model for compartmentation of the enzymes involved in the methylation-glucosylation sequence of these fla-

vonoids (Ibrahim et a1., 1987) which was based on biosynthetic, enzymic, kinetic and ultrastructural considerations. However, this view must await further immunocytochemical evidence using antibodies raised against the key enzymes of this pathway.

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In contrast with the vacuolar accumulation of flavonoid compounds reponed in most plants (Wagner, 1982 and refs. therein) or their secretion as farinose (Wol· lenweber, 1984) or gummy exudates (Chariere-Ladreix, 1977) in others, the physiological significance of cell wall localization of these metabolites is difficult to explain. The diversity in the chemical nature of flavonoid compounds (glycosides, hydroxyor methoxy aglycones) and the various roles they assume in different plants may explain the lack of one common accumulation site. However, in view of the semiaquatic nature of this plant and its lack of lignified tissues, cell wall localization of flavonoids may seem to be consistent with their possible role as antiviral agents (e.g. Van Hoof, et al., 1984), as defence mechanism against predators andlor protection against ultraviolet radiation {Harhorne, 1977}. In conclusion, the use of immunogold labeling provides unequivocal evidence for the localization of flavonoids in the cell walls of Chrysosplenium leaves, and can be easily applied to other secondary metabolites. To our knowledge, this is the first repon of immunocytochemical localization of flavonoids in plants. Acknowledgements This work was supported in part by grants from the Natural Sciences and Engineering Research Council of Canada and the Fonds FeAR, Government of Quebec, for which we are grateful. We wish to thank S. Lamoureux for the preparation of the antibody used in this study, and A. Goulet for technical assistance.

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