Evolutionary Aspects of Hypericin Productivity and Endogenous Phytohormone Pools Evidenced in Hypericum Species In Vitro Culture Model

Abstract

Shoot cultures of hypericin non-producing H. calycinum L. (primitive Ascyreia section), hypericin-producing H. perforatum L., H. tetrapterum Fries (section Hypericum) and H. richeri Vill. (the evolutionarily most advanced section Drosocarpium in our study) were developed and investigated for their growth, development, hypericin content and endogenous phytohormone levels. Hypericins in wild-growing H. richeri significantly exceeded those in H. perforatum and H. tetrapterum. H. richeri also had the highest hypericin productivity in vitro in medium supplemented with 0.2 mg/L N⁶-benzyladenine and 0.1 mg/L indole-3-butyric acid and H. tetrapterum-the lowest one in all media modifications. In shoot culture conditions, the evolutionarily oldest H. calycinum had the highest content of salicylic acid and total jasmonates in some of its treatments, as well as dominance of the storage form of abscisic acid (ABA-glucose ester) and lowest cytokinin ribosides and cytokinin O-glucosides as compared with the other three species. In addition, the evolutionarily youngest H. richeri was characterized by the highest total amount of cytokinin ribosides. Thus, both evolutionary development and the hypericin production capacity seemed to interact closely with the physiological parameters of the plant organism, such as endogenous phytohormones, leading to the possible hypothesis that hypericin productivity may have arisen in the evolution of Hypericum as a means to adapt to environmental changes.

Keywords: Hypericum evolution; endogenous phytohormones; hypericin; in vitro culture; wild habitats.

Figure 1

In situ growing H. calycinum L. (A); H. tetrapterum Fries. whole plant habitus (B) and blossoms with visible dark glands (D.gl.) on petal and sepal ribs (C); H. perforatum L. blossoms with dark glans (D); H. richeri Vill. leaves with scarce dark glands visible on leaves margins of non-blossoming aerial parts (E); and H. richeri Vill. blossoms with abundant dark glands visible on petals and sepals, as well as leaf ribs in proximity of blossoms (F).

Figure 2

In vitro cultivated shoot cultures of H. calycinum L. in M_0 culture medium (A) with well visible translucent glands (Tr. gl.) on the leaves (B); stimulation of axillary shoot formation (C) and rooting (D) in M_1 medium; stimulation of the height of auxillary shoots formed in M_2 medium (E); further stimulation of the height of axillary shoots and inhibition of rooting in M_3 medium (F); intensive biomass formation (G) and dark glands on leaves margins (H) in in PGR-free M_0 medium H. tetrapterum Fries.; biomass formation (I) and dark and translucent glands on leaves (J) in H. perforatum L. in M_0 medium; growth inhibition and necrosis in PGR-free M_0 medium in H. richeri Vill. (K); biomass formation stimulation H. richeri Vill. (L) with well visible translucent and dark glands on the leaves (M,N) in M_2 medium. Space bar = 1 cm.

Figure 3

Effect of plant growth regulators on tissue hydricity in Hypericum ssp. studied in vitro. M_0—PGR-free (A), M_1—0.2 mg/L BA (B), M_2—0.2 mg/L BA + 0.1 mg/L IBA (C) and M_3—0.1 mg/L BA + 0.2 mg/L IBA (D) supplemented media, HR—H. richeri Vill., HP—H. perforatum L., HT—H. tetrapterum Fries. and HC—H. calycinum L. Same letters denote statistically non-significant differences and different letters denote statistically significant differences in mean values when comparing the parameter between all samples.

Figure 4

Total hypericin content of wild growing Hypericum ssp. samples. HT—H. tetrapterum Fries., HP—H. perforatum L., HR Vitosha—H. richeri Vill. from Vitosha Mountain accession, HR Rila—H. richeri Vill. from Rila Mountain accession. All measurements were performed in triplicate and the values are given as mean ± SD. Different letters denote statistically significant differences of the means.

Figure 5

Total hypericin content in the three Hypericum ssp. in M_0—PGR-free, M_1—0.2 mg/L BA, M_2—0.2 mg/L BA + 0.1 mg/L IBA and M_3—0.1 mg/L BA + 0.2 mg/L IBA supplemented media. HP—H. perforatum L., HR—H. richeri Vill. and HT—H. tetrapterum Fries. All measurements were performed in triplicate and the values are given as mean ± SD. Same letters denote non-significant differences and different letters denote statistically significant differences of the means.

Figure 6

Representation of the total sum of the two abscisic acid (ABA) forms: ABA and its storage conjugate ABA–glucose ester (ABA–GE) in the four Hypericum species in M_0—PGR-free, M_1—0.2 mg/L BA, M_2—0.2 mg/L BA + 0.1 mg/L IBA and M_3—0.1 mg/L BA + 0.2 mg/L IBA supplemented media, HR—H. richeri Vill., HP—H. perforatum L., HT—H. tetrapterum Fries. and HC—H. calycinum. Same letters indicate statistically non-significant differences in mean values when comparing the parameter between all samples.

Figure 7

Total sum of salycilic acid (SA) and benzoic acid (BA) in the four Hypericum species in M_0—PGR-free, M_1—0.2 mg/L BA, M_2—0.2 mg/L BA + 0.1 mg/L IBA and M_3—0.1 mg/L BA + 0.2 mg/L IBA supplemented media. HR–H. richeri Vill., HP–H. perforatum L., HT–H. tetrapterum Fries. and HC–H. calycinum. Same letters indicate statistically non-significant and different letters indicate statistically significant differences in mean values when comparing the parameter between all samples.

Figure 8

The sum of jasmonic acid (JA) and its bioactive conjugate JA-isoleucine in the four Hypericum species in M_0—PGR-free, M_1—0.2 mg/L BA, M_2—0.2 mg/L BA + 0.1 mg/L IBA and M_3—0.1 mg/L BA + 0.2 mg/L IBA supplemented media. HR–H. richeri Vill., HP–H. perforatum L., HT–H. tetrapterum Fries. and HC–H. calycinum. Same letters indicate statistically non-significant and different letters indicate statistically significant differences in mean values when comparing the parameter between all samples.

Figure 9

Total sum of cytokinin ribosides in the four Hypericum species in M_0—PGR-free, M_1—0.2 mg/L BA, M_2—0.2 mg/L BA + 0.1 mg/L IBA and M_3—0.1 mg/L BA + 0.2 mg/L IBA supplemented media, HR–H. richeri Vill., HP–H. perforatum L., HT–H. tetrapterum Fries. and HC–H. calycinum. Same letters indicate statistically non-significant and different letters indicate statistically significant differences of the mean values when comparing the parameter between all samples.

Figure 10

Total sum of cytokinin N- and O-glucosides (upper and lower figure, respectively) in the four Hypericum species in M_0—PGR-free, M_1—0.2 mg/L BA, M_2—0.2 mg/L BA + 0.1 mg/L IBA and M_3—0.1 mg/L BA + 0.2 mg/L IBA supplemented media, HR–H. richeri Vill., HP–H. perforatum L., HT–H. tetrapterum Fries. and HC–H. calycinum. Same letters indicate statistically non-significant and different letters indicate statistically significant differences in mean values when comparing samples separately for each of the two parameters (comparisons of O-glucoside CKs—distinguished by an asterisk from N-glucosides comparisons).

Figure 11

Phenylacetic acid (PAA) levels in the four Hypericum species in M_0—PGR-free, M_1—0.2 mg/L BA, M_2—0.2 mg/L BA + 0.1 mg/L IBA and M_3—0.1 mg/L BA + 0.2 mg/L IBA supplemented media. HR–H. richeri Vill., HP–H. perforatum L., HT–H. tetrapterum Fries. and HC–H. calycinum. Same letters indicate statistically non-significant and different letters indicate significantly significant differences in mean values when comparing the parameter between all samples.