An assay for secologanin in plant tissues based on enzymatic conversion into strictosidine

Abstract

The secoiridoid glucoside secologanin is the terpenoid building block in the biosynthesis of terpenoid indole alkaloids. A method for its determination in plant tissues and in cell suspension cultures has been developed. This assay is based on the condensation of secologanin with tryptamine, yielding strictosidine, in a reaction catalysed by the enzyme strictosidine synthase (STR; E.C. 4.3.3.2). Subsequently, the formation of strictosidine is quantified by high performance liquid chromatography (HPLC). STR was isolated from transgenic Nicotiana tabacum cells expressing a cDNA-derived gene coding for STR from Catharanthus roseus. The high specificity of STR for secologanin, in combination with a sensitive and selective HPLC system, allows a simple extraction of secologanin from plant tissue. The detection limit of this method is 15 ng secologanin. Using this assay, secologanin contents were determined in tissues of various plant species; Lonicera xylosteum hairy roots were found to contain 1% of secologanin on a dry weight basis.

Regeneration of Lonicera tatarica plants via adventitious organogenesis from cultured stem explants

“…This result was unexpected as it has been postulated that secologanin is accumulated in young leaves to provide protection against pathogen attack. Indeed, Hallard et al (1998) reported relatively high levels of secologanin in leaves of L. xylosteum whereas in the present study the highest levels of secologanin were detected in stems of L. tatarica plants (Fig. 3b).…”

“…3b). However, loganin, as well as secologanin, can act as a feeding deterrent against herbivores (Hallard et al 1998;Rimpler 1991).…”

“…Members of the Caprifoliaceae family, including Lonicera spp., are naturally rich in iridoids including secologanin (Hallard et al 1998;Tanahashi et al 1984). Previous studies using plants such as Catharanthus roseus have shown that secologanin is a limiting precursor in the synthesis of alkaloids .…”

“…Previous studies using plants such as Catharanthus roseus have shown that secologanin is a limiting precursor in the synthesis of alkaloids . Therefore, if a transformation system was available for L. tatarica together with the appropriate cloned genes encoding enzymes of secologanin biosynthesis, transgenic approaches could be applied not only to the study of metabolic flux control in secologanin biosynthesis, but also in studies to expand the biosynthetic capability of the plant by introducing genes encoding secologaninmetabolising enzymes involved in, for example, terpenoid indole alkaloid biosynthesis Hallard 2000). However, before metabolic engineering in L. tatarica becomes a reality, a reliable system for transformation and regeneration of whole plants must be developed.…”

“…Cloned cDNAs encoding enzymes of secologanin biosynthesis will provide useful tools for understanding regulation of the pathway flux using transgenic approaches applied to a suitable target plant species, such as L. tatarica, the plant chosen in our laboratory. Furthermore, it might be possible to extend secondary metabolite biosynthesis in L. tatarica plants by introducing genes encoding enzymes acting beyond secologanin, which are involved in alkaloid biosynthesis in other plant species, together with the action of endogenous, non-specific enzymes on unnatural substrates produced by transgenic enzymes (Hallard 2000). An important step in the development of a transformation system for plant species is to establish a facile regeneration procedure.…”