Insights into carotenoid accumulation using VIGS to block different steps of carotenoid biosynthesis in petals of California poppy.

KEY MESSAGE: Viral-induced gene silencing of selected biosynthetic genes decreased overall carotenoid accumulation in California poppy. Regulation of carotenogenesis was linked with pigment sequestration, not changes in biosynthetic gene expression. Genes of carotenogenesis are well described, but understanding how they affect carotenoid accumulation has proven difficult because of plant lethality when the pigments are lacking. Here, we used a Tobacco Rattle Virus-based virus-induced-gene-silencing (VIGS) approach in California poppy (Eschscholzia californica) to investigate how silencing of the carotenoid biosynthetic pathway genes affects carotenoid metabolite accumulation and RNA transcript abundance of the carotenoid biosynthetic pathway genes. VIGS of upstream (PDS and ZDS) and downstream (ßOH and ZEP) genes reduced transcript abundance of the targeted genes in the poppy petals while having no effect on abundance of the other carotenogenesis genes. Silencing of PDS, ZDS, ßOH and ZEP genes reduced total pigment concentration by 75-90% and altered petal colour. HPLC and LC-MS measurements suggested that petal colour changes were caused by substantially altered pigment profiles and quantity. Carotenoid metabolites were different to those normally detected in wild-type petals accumulated but overall carotenoid concentration was less, suggesting the chemical form of carotenoid was important for whether it could be stored at high amounts. In poppy petals, eschscholtzxanthin and retro-carotene-triol were the predominant carotenoids, present mainly as esters. Specific esterification enzymes for specific carotenoids and/or fatty acids appear key for enabling petal carotenoids to accumulate to high amounts. Our findings argue against a direct role for carotenoid metabolites regulating carotenogenesis genes in the petals of California poppy as transcript abundance of carotenogenesis genes studied was unchanged, while the petal carotenoid metabolite profile changed substantially.

Virus-induced gene silencing of the alkaloid-producing basal eudicot model plant Eschscholzia californica (California Poppy).

Eschscholzia californica (California poppy), a member of the basal eudicot family of the Papaveraceae, is an important species to study alkaloid biosynthesis and the effect of alkaloids on plant metabolism. More recently, it has also been developed as a model system to study the evolution of plant morphogenesis. While progress has been made towards establishing methods for generating genetically modified cell culture lines, transcriptome data and gene expression analysis, the stable transformation and subsequent regeneration of transgenic plants has proven extremely time consuming and difficult. Here, we describe in detail a method to transiently down regulate expression of a target gene by virus-induced gene silencing (VIGS) and the subsequent analysis of the VIGS treated plants. VIGS in E. californica allows for the study of gene function within 2 to 3 weeks after inoculation, and the method proves very efficient, enabling the rapid analysis of gene functions.