In situ localization of chalcone synthase in tannin-containing plants

Phytochemistry, Vol . 32,

No. 3, pp . 585-590, 1993

0031-9422/93 $6.00+0.00 © 1993 Pergamon Press Ltd

Printed in Great Britain.

IN SITU LOCALIZATION OF CHALCONE SYNTHASE IN TANNIN-CONTAINING PLANTS B. KARWATZKI,* A. HERGET, L. BEERHUESt and R . WIERMANN$ Institut fur Botanik, Universitat Munster, Schlol3garten 3, D-4400 Munster, F.R.G.; *Institut far Entwicklungs- and Molekularbiologie der Pflanzen, Universitat Diisseldorf, D-4000 Dusseldorf, F.R.G.; f lnstitut fur Pharmazeutische Biologie, Universitat Bonn, NuBallee 6, D-5300 Bonn, F .R.G. (Received in revised form

16 September 1992)

IN HONOUR OF PROFESSOR MEINHART ZENK'S SIXTIETH BIRTHDAY Key Word Index-Kalanchoe tubiflora; K. daigremontiana; Crassulaceae; Acorus calamus ; Araceae; tannin cells ; chalcone synthase; immunofluorescence localization .

Abstract-Chalcone synthase in leaves of Kalanchoe daigremontiana and Acorus calamus, and in phyllodes of Kalanchoe tub flora was detected both enzymatically and immunochemically. In situ localization by indirect immunofluorescence revealed that, in all three systems, chalcone synthase is highly expressed in tannin-containing idioblasts . In A . calamus the enzyme is additionally present in oilcells . The spatial distribution of chalcone synthase did not change during organ development .

INTRODUCTION

Chalcones are the central C 15 intermediates in flavonoid and isoflavonoid biosynthesis [1, 2] . Their formation is catalysed by chalcone synthase (CHS) . Thus, this enzyme resides at an important regulatory position. In situ localization of CHS gives valuable information about histological and cellular compartmentation of flavonoid metabolism. In leaves of various dicotyledonous plants, CHS is present in epidermal and to a lesser extent in subepidermal tissue [3, 4] . Needles of Larix decidua contain varying amounts of CHS in epidermis and mesophyll dependent on the developmental stage [5] . In roots of pea and bean the enzyme is present in rhizodermis, cortex and the lateral regions of the calyptra [6] . Occurrence of CHS in anthers of Tulipa is confined to the tapetum [7]. To date nothing is known about expression of CHS in tannin-containing plants . Plant tannins are phenolic metabolites that are well known for their ability to form complexes with proteins . Tannins of higher plants are subdivided into hydrolysable and condensed tannins [8, 9] . In brown algae a third tannin class, the phlorotannins, occurs [10] . Condensed tannins or proanthocyanidins are derived from flavonoid metabolism [8, 9] . They are polyflavanoids consisting of polyhydroxy-flavan-3-ol units. The precursors of these, the flavan-3,4-diols or leucoanthocyanidins, are highly reactive and condense with themselves or the flavan-3-ols to yield oligomers and polymers . tAuthor to whom correspondence should be addressed.

We have used antibodies directed against CHS to localize this enzyme in tannin-containing plants . Accompanying histochemical studies showed where tannins accumulate in plant tissue . The investigations were carried out on leaves of Kalanchoe daigremontiana, phyllodes of Kalanchoe tubiflora and leaves of Acorus calamus . Occurrence of tannin cells in these plants has been reported [11-13] . RESULTS

CHS activity and immunoblotting

CHS activity in crude extracts from 0 .5-1 .5-cm-long phyllodes of K. tubifiora and leaves of K. daigremontiana was 55 nkat kg - t and 107 nkat kg -1, respectively. After concentration by 80% ammonium sulphate precipitation, the protein extracts were used for immunoblotting and enzyme assays with subsequent product analysis . The major product formed in the enzyme tests under the selected experimental conditions was naringenin, indicating the presence of CHS in the two Kalanchoe species. Two by-products also formed were not further analysed . They accounted for about 10 and 35% of the radioactivity associated with the products of the CHS reaction . CHS activity in crude extracts from basal parts of A. calamus leaves was 9 nkat kg - ' . The protein fraction obtained by ammonium sulphate precipitation between 55 and 80% saturation was used for immunoblotting and enzyme assays . The only product formed in the enzyme assays was naringenin. 585



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Immunoblotting following SDS-PAGE demonstrated that the antibodies used recognized only one polypeptide species in protein extracts from all three plants (Fig, 1). The M, of the visualized polypeptides was about 42 000 which corresponds to the subunit molecular mass of chalcone synthases purified to homogeneity [14-19] . Incubation of protein blots with preimmune-IgG did not result in protein band detection . Thus, the antibodies used cross-reacted monospecifically with the CHS proteins from the three systems examined and were suitable for use in in situ localization experiments . Localization of CHS and tannin cells in K. tubiflora and K . daigremontiana

In phyllodes of K. tubiflora and leaves of K. daigremontiana numerous cells in the second subepidermal cell layer were selectively stained with ferric chloride, ferrous sulphate and potassium dichromate (Fig. 2a). When the nitroso reagent was applied these cells turned brown, suggesting accumulation of prodelphinidins [20]. When cross-sections of phyllodes and leaves were incubated with antibodies, the idioblasts exhibited intense immunofluorescence (Fig . 2b, d) . The other mesophyll cells and the epidermal cells did not immunofluoresce. Inside the idioblasts immunofluorescence appeared to be confined to the cytoplasm (Fig . 2t) . Incubations with preimmune-IgG only mediated a low background level, indicating the specificity of the immunofluorescence observed after antibody treatment (Fig . 2c, e, g) . Analysis of phyllodes and leaves of varying age showed that immunofluorescence distribution did not change during development of the organs . Immunofluorescence was always associated with the idioblasts in the second subepidermal

cell layer. Its intensity was low in young organs (stage I ; see Experimental), highest in about 1-cm-long phyllodes and leaves (stage II) and decreased with increasing age (stages III and IV) . Localization of CHS and idioblasts in A . calamus

In leaves of A . calamus, tannin-containing idioblasts are scattered in mesophyll (Fig. 2h). They are absent from epidermal tissue. Some idioblasts were observed in xylem and phloem of vascular bundles (Fig . 2k). With the nitroso reagent the tannin cells were stained red, indicating the presence of procyanidins [20] . Incubation of leaf cross-sections with antibodies mediated strong immunofluorescence in the tannin cells in mesophyll and vascular bundles (Fig. 2i,1) . Cross-sections through all three sectioning levels (see Experimental) contained immunofluorescent tannin cells. However, fluorescence intensity varied greatly between these cells . Inside the tannin cells immunofluorescence appeared to be restricted to the cytoplasm . Besides tannin cells A. calamus leaves contain oil cells, as reported previously [11, 12, 21] . These cells are present as big bubbly cells at the branch points of the aerenchymatic tissue and as big rhomb-shaped cells in the outward-oriented epidermis (Fig . 2m). They are absent in the upper epidermis. Histochemical staining showed that oil cells do not contain tannins . When leaf cross-sections through the sectioning levels A and B were incubated with antibodies, both the epidermal and aerenchymatic oil cells immunofluoresced (Fig . 2n). Oil cells at sectioning level C, however, were devoid of immunofluorescence . Fluorescence in mesophyll and epidermal oil cells again appeared to be confined to the cytoplasm . Incubation of leaf cross-sections with preimmune-IgG estab-

Acorus calamus

Kalanchoe daigremontiana (K .d.) Kalanchoe tubiflora (K.t.)

anti-CHS IgG preimmune IgG

anti-CHS I9G preimmune IgG

97 .4 670 430-

MID

K .d .

0-55 55-80 0-55 55-80

K .t .

K .d .

K .t .

0-80 0-80 0-80 0-80

% (NH4)2SO4 saturation

Fig. 1. Immunoblotting of protein fractions obtained by ammonium sulphate precipitation of crude protein extracts. The SDS-PAGE was performed in a 9-16 .5% gradient gel . MP, marker proteins .



Immunofluorescence localization of chalcone synthase

Fig. 2. Immunofluorescence localization of CHS in phyllodes of Kalanchoe tubiflora (a-c), leaves of K. daigremontiana (d-g), and leaves of Acorus calamus (h-n). (a) Cross-section under visible light . Tannin cells are stained brown . x 90, bar: 50 µm. (b, c) Cross-sections incubated with anti-CHS IgG (b) and preimmune IgG (c) . x 90. (d, e) Cross-sections after treatment with anti-CHS IgG (d) and preimmune IgG (e). x 70, bar. 50 µm. (f, g) Details from d and e, respectively, showing cells in the second subepidermal layer at higher magnification . x 195, bar : 15 µm. (h) Cross-section under visible light . The lumen of tannin cells is stained brown . x 280, bar : 15 µm. (i, j) Cross-sections incubated with anti-CHS IgG (i) and preimmune IgG (j). (k, l) Cross-sections of vascular bundles with tannin cells under visible light (k ; x 280, bar: 15 µm) and after incubation with anti-CHS IgG (1; x 720, bar : 5 µm) . (m, n) Cross-sections with aerenchymatic oil cells under visible light (m) and after treatment with anti-CHS IgG (n) . x 280, bar : 15 µm.

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Immunofluorescence localization of chalcone synthase lished that immunofluorescence in both kinds of idioblasts was specific (Fig . 2j). Following this treatment, the specialized cells only exhibited a faint background fluorescence. DISCUSSION

Leaves of K. daigremontiana and A . calamus and phyllodes of K. tubiflora contain tannin-accumulating cells that highly express CHS . This indicates that the deposited tannins most likely represent proanthocyanidins, as suggested by the histochemical studies and previous data [11, 22, 23], and that these compounds accumulate in the cells where they are synthesized. A similar observation has been made for flavonoids in leaves of spinach, bean, pea and parsley, where the site of accumulation also coincided with the site of synthesis, i.e. epidermal tissue [3,24] . In contrast, CHS activity in oat leaves was mainly detected in mesophyll, whereas flavonoids accumulated predominantly in epidermal cells [25] . As a consequence the authors postulated an intercellular transport of the flavonoids . Condensed tannins usually accumulate in the central vacuole of the cell [20]. Their additional occurrence in cell walls, as reported for K . daigremontiana idioblasts [13], may be a secondary site of deposition which may be due to tonoplast rupture upon cell death [20] . In all plant species studied here, CHS appears to be located in the cytoplasm. In A. calamus, cytoplasm is obviously protected from the deposited tannins by suberization of the tonoplast membrane [12] . Proanthocyanidin synthesis appears to be associated with vesicles probably derived from endoplasmic reticulum. These vesicles coalesce and fuse with the central vacuole [12, 26-28] . Localization experiments at the subcellular level would be necessary to elucidate the intracellular compartmentation of the proanthocyanidin pathway . Using immunogold labelling the two chalcone synthase forms . in spinach leaves were shown to be cytoplasmic enzymes ; however, these proteins were not significantly associated with endoplasmic reticulum [3] . In contrast, an association of CHS with endoplasmic reticulum membranes was concluded from localization experiments with buckwheat hypocotyls [29]. Occurrence of CHS in the tannin cells is associated with organ development . The enzyme is present in higher concentration at an early developmental stage . Studies on K. blossfeldiana have shown that the concentration of total phenolics is highest in young leaves and then decreases with leaf age [30]. Compared to other plants, the phenolic content in this species was found to be particularly high, and tannins, most likely proanthocyanidins, constituted the main phenolic fraction . The developmental control of CHS in leaves and phyllodes studied is similar to the temporal regulation of the enzyme in leaves of spinach, parsley and oat and roots of pea and bean [3, 4, 6, 25] . These plants also contain CHS only at an early developmental stage. In the course of organ development the spatial distribution of CHS in the investigated leaves and phyllodes

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did not change . The enzyme was always present in the idioblasts . Similarly, no change in tissue distribution of CHS during development has been observed in leaves of spinach, parsley and oat [3, 4, 25], whereas in needles of Larix decidua CHS is stage-specifically expressed in epidermis and mesophyll [5] . In leaves of A . calamus CHS-containing tannin cells additionally occur in phloem and xylem of vascular bundles. Their occurrence in phloem has previously been reported [11] . Recently, expression of CHS in tannin cells of vascular bundles has also been observed in needles of Larix decidua [5]. Tannins serve as feeding deterrents and defence compounds against pathogens [8, 31] . To fulfil these functions they are preferentially accumulated in peripheral tissue [32] . Interestingly, the tannin idioblasts in leaves and phyllodes of the Kalanchoe species are present in the organ periphery . In addition, these idioblasts are more abundant at the abaxial, i .e. the outside oriented surface of the young leaf organs. Acorus calamus leaves contain epidermal and aerenchymatic oil cells in addition to tannin cells . This type of idioblast does not contain tannins although it expresses CHS, suggesting that monomeric flavonoids are deposited together with the essential oil . Lipophilic flavonoid aglycones are frequently encountered in plants producing essential oils [33] . Expression of CHS in oil cells is also developmentally regulated . CHS is only present in oil cells of young tissue, i .e. tissue near the leaf base . The walls of these oil cells are reinforced during differentiation by suberization, finally leading to death of these idioblasts [12] . In this work we have demonstrated the occurrence of CHS in tannin cells of the two Kalanchoe species and tannin and oil cells of A . calamus by immunohistochemical methods . At present we do not know whether other tissues or cell types of these plants, e .g. epidermal tissue, may synthesize and accumulate flavonoids under certain conditions . However, we did not detect any significant immunofluorescence outside the idioblasts in our experiments . EXPERIMENTAL

Plant material . Kalanchoe tubifiora and K. daigremontiana were grown in a greenhouse . Phyllodes and leaves with a length of 0.3 cm (stage I), 1 .0 cm (stage II), 2.5 cm (stage III) and 4 .0 cm (stage IV) were used for the enzymatical and immunochemical studies . Acorus calamus was collected near Munster and then maintained in a growth chamber . Young leaves of this plant were crosssectioned at the base (sectioning level A), 0 .5 cm from the base (sectioning level B), and 1 .0 cm from the base (sectioning level C) . Chemicals . [2- 14C]Malonyl-CoA (1 .71 GBgmmol -1) was purchased from N.E.N ., Boston, MA, U .S.A . 4Coumaroyl-CoA was synthesized by Dr P .A. Baumker, Munster as described previously [34]. Enzyme assay and product analysis . CHS activity was determined according to ref. [18] . Product analysis was

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carried out either by TLC [5] or HPLC. When HPLC was employed, non-radioactively labelled malonyl-CoA was used and the volume of the enzyme assay increased 3fold . After incubation the complete reaction mixture (375 p1) was applied to the HPLC-column (LiChrosorbRP-18, 5 µm, 8 x 120 mm) . Separation was performed with a linear gradient from 10 to 50% acetonitrile in 1 HOAc (25 min, flow 2 ml min - '). Detection was at 285 nm. The R, value of naringenin was 22 min. Histochemical analyses . Histochemical stainings were performed on fresh tissue . Freehand cross-sections of leaves and phyllodes were treated with the following reagents : (A) 3% ferric chloride, (B) 1 or 10% ferrous sulphate, (C) 10% potassium dichromate, (D) the nitroso reagent [26, 35, 36] . Immunoblotting and immunolocalization. Immunochemical procedures were carried out according to ref . [3]. The antibodies used had been raised against CHS from spinach leaves (anti-CHS IgG=anti-AII IgG ; [18]). Acknowledgements-The financial support by the 'Deu-

tsche Forschungsgemeinschaft' and the 'Fonds der Chemischen Industrie' is gratefully acknowledged . We thank Mrs S . Schmidt, Bonn, for revising the English . REFERENCES

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