Localization of Sesquiterpene Lactones Biosynthesis in Flowers of Arnica Taxa

Abstract

Arnica montana is a valuable plant with high demand on the pharmaceutical and cosmetic market due to the presence of helenalin (H) and 11α, 13-dihydrohelenalin (DH) sesquiterpene lactones (SLs), with many applications and anti-inflammatory, anti-tumor, analgesic and other properties. Despite the great importance of these compounds for the protection of the plant and their medicinal value, the content of these lactones and the profile of the compounds present within individual elements of florets and flower heads have not been studied so far, and attempts to localize these compounds in flower tissues have also not been conducted. The three studied Arnica taxa synthesize SLs only in the aerial parts of plants, and the highest content of these substances was found in A. montana cv. Arbo; it was lower in wild species, and a very small amount of H was produced by A. chamissonis. Analysis of dissected fragments of whole inflorescences revealed a specific distribution pattern of these compounds. The lactones content in single florets increased from the top of the corolla to the ovary, with the pappus calyx being a significant source of their production. Histochemical tests for terpenes and methylene ketones indicated the colocalization of lactones with inulin vacuoles.

Keywords: A. chamissonis; Arnica montana; biosynthesis localization; helenalin and dihydrohelenalin esters; histochemical study; sesquiterpene lactones.

Figure 1

Reactive helenalin groups α,β-unsaturated carbonyl groups, from the left: cyclopentenone and α- methylene-γ-butyrolactones.

Figure 2

Examined derivatives of helenanolides (H) and dihydrohelenanolides (DH).

Figure 3

Concentration of helenanolides and dihydrohelenanolides ± SD (mg/g dw) in different parts of ray flowers (a–d), disc flowers (f–i), and green parts of flowers (e–j).

Figure 4

The image of single disc floret from A. montana cv. Arbo flower heads. (a–e) Polarization of glandular and non-glandular trichomes results from fatty aldehydes, inulin, secondary metabolites, or cellulose. (a) the upper part of floret; (b1,b2) the middle part of floret; (c1,c2) pappus calyx with single bristle; (d) the middle part of the floret with bristle whorl; and (e) the lower part of the floret–ovary with twin covering and glandular trichomes.

Figure 5

The histochemical analysis of A. montana cv. Arbo single florets from the flower head. (a) Zimmermann’s reaction for methyl ketones and aldehydes—positive reaction visible in the head of glandular trichomes (arrowheads); (b,c) Zimmermann’s positive reaction is visible in the bristles of the pappus. (b) Ray floret, (c) disc floret; (d–f) the Legal test for methylene and methyl ketones, (d) phyllary bracts,(e) pappus bristle, (f) middle part of the floret; (g) test for ketones/aldehydes with 2,4-dinitrophenylhydrazine on the lower part of the ovary; (h) the bristle of pappus stained with Schiff reagent in UV light excitation at 395 nm, using a Zeiss barrier filter (FT 396, LP 420 barrier filter) showing the presence of aldehydes in the lumen of inulin vacuoles (arrowheads); (i,j) inulin spherocrystals in dried bristles of pappus (arrowheads), in white transmitted (i) and polarized light (j).

Figure 6

The histochemical analysis of A. montana cv. Arbo single florets from the flower head, peduncle, and leaf. (a–d) Schiff’s reagent for aldehydes, (a,b) ovary with stained glandular and twin covering trichomes. (c) Bristle of pappus, (d) glandular, and non-glandular trichomes on the middle part of the floret; (e) fluorescence in blue light excitation at 450–490 nm, using a Zeiss barrier filter LP (520) of pappus whorl of bristles treated with AlCl₃ for lactones; (f) green autofluorescence of pappus, glandular and non-glandular trichomes of dry achene in blue light excitation at 450–590 nm, using a Zeiss barrier filter LP (520); (g–k) reaction with NADI for cytochrome oxidase, (g) glandular and twin-celled trichomes on the ovary, (h) glandular trichomes on the peduncle, (i) glandular and non-glandular trichomes on phyllary bracts, (j) pappus bristle, (k) bristle whorl of pappus, non-glandular and glandular trichomes (arrowheads) on the ovary; (l) succinate dehydrogenase activity in covering trichomes of leaf, and oil body surrounded with leucoplast (arrowheads); (m–o) immunolocalization of farnesyl antibody (yellow-green fluorescence), (m) covering trichome on petal epidermis, (n) glandular trichome on adaxial leaf epidermis, (g) pappus bristle.