Biotransformations of putative phytoecdysteroid biosynthetic precursors in tissue cultures of Polypodium vulgare

Abstract

Incubation of calli and prothalli of Polypodium vulgare with different tritium-labelled ecdysteroids has led to modification of some previous assumptions about the biosynthesis of ecdysteroids in plants. Thus,-hydroxyecdysone was transformed efficiently in both tissues into 20-hydroxyecdysone (20E), but no 25-deoxyecdysteroids such as pterosterone and inokosterone were formed. Likewise, incubation of 2-deoxyecdysone (2dE) produced exclusively ecdysone (E) and 20E, indicating a high 2-hydroxylase activity in both tissues, despite calli not producing phytoecdysteroids. This 2-hydroxylation was also evident in the transformation of 2,22-dideoxyecdysone (2,22dE) into 22-deoxyecdysone (22dE). Different ecdysteroids that do not occur in P. vulgare were formed in the incubation of 3-dehydro-2,22,25-trideoxyecdysone (3D2,22,25dE) by 3a-reduction and 3b-reduction and 25-hydroxylation processes. The fact that 22,25-dideoxyecdysone and 22dE were the only 2-hydroxylated products formed in this case suggests that only compounds bearing a 3b-hydroxyl group are substrates for the 2-hydroxylase. Surprisingly, 22-hydroxylation was never observed with either 2,22dE or 3D2,22,25dE, raising the possibility that it could occur at an early step in the biosynthetic pathway. In this respect, labelled 22R-hydroxycholesterol was efficiently converted into E and 20E, whereas 22S-hydroxycholesterol was not transformed into ecdysteroids, because of its unsuitable configuration at C22. Finally, the conversion of 25-hydroxycholesterol into E and 20E was greatly enhanced after thermal treatment of prothalli which induces the release of previously stored ecdysteroids. Thus, P. vulgare prothalli and calli appear to be particularly suitable models for the study of ecdysteroid biosynthesis and its regulation in plants.Keywords: 20-hydroxyecdysone; fern; hydroxycholesterol derivatives; in vitro cultures; induction.Many plant species produce C 27 ±C 29 ecdysteroids of a wide structural diversity [1±5]. These analogues of insect molting hormone (20-hydroxyecdysone, 20E) are secondary metabolites which are expected to provide some protection to plants against non-adapted phytophagous insect species [6]. Although the presence of such compounds in plants was demonstrated more than 30 years ago, little has been learnt since about their biosynthetic pathway(s), and whether it operates in a sequence that is similar to or different from that in Arthropods is not known.In vivo and in vitro labelling experiments have established that these compounds are biosynthesized from mevalonate via C 27 , C 28 and C 29 phytosterols. Cholesterol has been proved to be the intermediate in the biosynthesis of C 27 phytoecdysteroids in Taxus baccata [7]. In biosynthetic studies with Ajuga reptans roots transformed with Agrobacterium rhizhogenes, 24-methylcholesterol and 24-ethylcholesterol have been proposed as intermediates for the biosynthesis of C 28 and C 29 ecdysteroids, respectively [8]. Adler & Grebenok [9] proposed lathosterol (5a-cholest-7-en-3b-ol) as the logical ...

Phytoecdysteroid C2‐hydroxylase is microsomal in spinach, Spinacia oleracea L.

“…Taking together, the current results with those of previous experiments on ring-B functionalization (Davies et al, 1980;Fujimoto et al, 2000) and on radioactive putative precursor metabolism (Reixach et al, 1999;Bakrim et al, 2008), we conclude that plant and insect biosynthetic pathways differ markedly, which suggests that they are the result of a convergent evolution rather than having a common origin.…”

“…Our knowledge of the ecdysteroid biosynthetic pathway in plants is rather poorly documented. There is evidence that the first steps would differ from those of insects and even possibly differ between plant species (Reixach et al, 1999;Fujimoto et al, 2000;Dinan et al, 2009), but the types of enzymes may be similar. Thus, the different hydroxylations are expected to be catalyzed by CYP enzymes, as is the case for the biosynthesis of steroid plant growth hormones sharing some structural features with PEs, the brassinosteroids (Fujioka et al, 2002;Nomura and Bishop, 2006).…”

The first cytochrome P450 in ferns

“…This result suggests that these calli lines lack the biosynthetic machinery required for the first steps of PE biosynthesis. However, some of the enzymes involved in E oxidation, such as E 2‐, 20‐ and 25‐hydroxylases, can also be expressed [9].…”

“…Polypodium vulgare is a fern with a worldwide distribution that usually grows in dark and humid areas, and produces PE, the highest content being in rhizomes (up to 0.4% dry weight). Different culture lines of prothalli and calli from this plant have been obtained and their PE content has been described by our group [5–10]. Among those models, a calli line with undetectable levels of PE, but that is able to transform E into 20E, was selected for this study.…”

Distribution and Biosynthesis of 20-Hydroxyecdysone in Plants ofAchyranthes japonicaNakai

“…atropurpurea. [8][9][10][11][12][13] Plants, but not animals, synthesize phytoecdysteroids from mevalonic acid in the mevalonate pathway of the plant cell using acetyl-CoA as a precursor.…”