A Catharanthus roseus mutant with trace levels of secologanin and monoterpenoid indole alkaloids does not express BIS1/BIS2 transcription factors and fails to activate iridoid biosynthesis.

Abstract

The Madagascar periwinkle (Catharanthus roseus) is the sole source of the monoterpenoid indole alkaloids (MIAs) that result in several essential anti-cancer chemotherapies as well as being an important source for other MIA derived pharmaceutical agents. Most of the alkaloid and pre-alkaloid iridoid pathway has been elucidated, but some critical areas remain uncharacterized. The early iridoid pathway is localized to internal phloem associated parenchyma (IPAP) cells, with the latter part of the pathway localized to the epidermal cells indicating intercellular transport within the leaf tissue does occur. However, possible transport or translocation of MIAs or pre-MIAs between organs within Catharanthus roseus has not been studied. Previously, 3600 EMS (ethyl methane sulfonate) mutagenized C. roseus plants had been screened with a simple TLC (thin layer chromatography) to identify mutants with altered MIA profiles yielded one plant with trace MIA production. This trace MIA status was confirmed using UPLC-MS (Ultra Performance Liquid Chromatography and Mass Spectrometry) and the plant had a thick, stocky, short root system, small leaves, a rigid stem, premature senescence and was susceptible to infection resulting in its rescue to in-vitro. Quantitative real-time PCR analysis of iridoid gene expression showed significant downregulation of several iridoid pathway genes as well as the downregulation of transcription factors BIS1 and BIS2. Feeding the trace MIA mutant roots the iridoid secologanin resulted in alkaloid production in the leaves. While grafting trace MIA mutant shoots onto MIA producing WT roots also resulted in alkaloid production in the mutant leaves. This study establishes that MIAs or pre-MIA iridoids, such as secologanin, may be translocated between the plant organs and this may be useful for the identification of novel transporters, enzymes, and transcription factors.