Spatial and developmental synthesis of endogenous sesquiterpene lactones supports function in growth regulation of sunflower

Abstract

Tissue-specific occurrence and formation of endogenous sesquiterpene lactones has been assessed and suggests physiological function as antagonists of auxin-induced plant growth in sunflower. Sunflower, Helianthus annuus, accumulate high concentrations of bioactive sesquiterpene lactones (STL) in glandular trichomes, but in addition, structurally different STL occur in only trace amounts in the inner tissues. The spatial and temporal production of these endogenous STL during early phases of plant development is widely unknown and their physiological function as putative natural growth regulators is yet speculative. By means of HPLC and MS analysis it was shown that costunolide, dehydrocostuslactone, 8-epixanthatin and tomentosin are already present in dry seeds and can be extracted in low amounts from cotyledons, hypocotyls and roots of seedlings during the first days after germination. Semi-quantitative and RT-qPCR experiments with genes of the key enzymes of two independent routes of the endogenous STL biosynthesis confirmed the early and individual expression in these organs and revealed a gradual down regulation during the first 72-96 h after germination. Light irradiation of the plants led to a fast, but transient increase of STL in parts of the hypocotyl which correlated with growth retardation of the stem. One-sided external application of costunolide on hypocotyls conferred reduced growth of the treated side, thus resulting in the curving of the stem towards the side of the application. This indicates the inhibiting effects of STL on plant growth. The putative function of endogenous STL in sunflower as antagonists of auxin in growth processes is discussed.

Keywords: Gene expression; Growth inhibition; Helianthus; Light induction; Sesquiterpene lactone biosynthesis.

Fig. 1

Examples of trichome-based (above line) and endogenous (below line) sunflower sesquiterpene lactones

Fig. 2

Proposed biosynthetic pathway of costunolide, dehydrocostuslactone, xanthanolides and 8β-substituted germacrolides and heliangolides in sunflower. Solid arrows mark enzymatically proven steps in sunflower. FPP, farnesylpyrophosphate synthase; HaGAS, Helianthus annuus germacrene A synthase; HaGAO, Helianthus annuus germacrene A oxidase; HaG8H, Helianthus annuus 8β-hydroxylase, HaCOS, Helianthus annuus costunolide synthase

Fig. 3

Temporal and spatial distribution of sesquiterpene lactones in cotyledons, hypocotyls and roots of sunflower seedlings between 48 and 192 h after imbibition. Quantification was performed by HPLC–UV using standard curves of reference compounds and the relative concentration of STL was corrected applying tissue- and compound-specific extraction efficiency. Mean values ± SE (n = 3)

Fig. 4

Semi-quantitative gene expression in organs of sunflower seedlings between two (2d), three (3d) and five days (5d) after imbibition. Ubiquitin expression was used as house-keeping gene. The data (generated by capillary electrophoresis) represent the relative expression of FPS (farnesyl-pyrophosphate-synthase), HaGAO (Helianthus annuus germacrene A oxidase) and HaG8H (Helianthus annuus germacrene A acid 8β-hydroxylase) of three independent experiments. Capitate glandular trichomes of sunflower were used as a positive control for biosynthetic activity of genes involved in trichome-based sesquiterpene lactone biosynthesis. co cotyledon, hy hypocotyl, rh root hair zone, rt root tip

Fig. 5

Quantitative expression of genes involved in sesquiterpene lactone biosynthesis of sunflower in a seeds and b seedlings at different times after imbibition. Expression values are normalized to three house-keeping genes (α-tubulin, elongation factor 1α, and ubiquitin) and are given relative the youngest stage of seeds (ungerminated seeds at 0 h) or the youngest cotyledon stage (48 h), respectively. For every biological replicate, three technical replicates were analyzed. Mean values ± SE (n = 3). HaGAO, Helianthus annuus germacrene A oxidase; HaG8H, Helianthus annuus germacrene A acid 8β-hydroxylase; HaCOS, Helianthus annuus costunolide synthase

Fig. 6

Relative concentration of sesquiterpene lactones in the hypocotyl of sunflower seedlings after 2 h (L2h) and 4 h (L4h) of illumination in comparison to dark controls (D2h, D4h; defined as 100%; blue horizontal line). Mean values ± SE (n = 3) are displayed. COS costunolide, DCL dehydrocostuslactone, 8-EPI 8-epixanthatin, TOM tomentosin

Fig. 7

Time course of sunflower hypocotyl bending (deviation in circle from starting position as mean values of n = 14 plants per sample and time) after unilateral application of 500 µM costunolide in vaseline (blue lines). Positive values represent left sided, negative values right-sided application. DMSO (orange lines) enforced the effect of costunolide but did not affect the reaction of controls (green lines for samples with and without DMSO, respectively)