Title: Transgene-based anthocyanin hyper-pigmentation as a visual reporter of gene silencing in plants Author
Submitted to: Meeting Abstract
Publication Type: Proceedings
Publication Acceptance Date: March 25, 2008
Publication Date: March 30, 2008
Citation: Velten, J.P. 2008. Transgene-based anthocyanin hyper-pigmentation as a visual reporter of gene silencing in plants[abstract]. Keystone Symposium: RNAi, MicroRNA, and Non-Coding RNA. Whistler, Canada. March 25-30, 2008. Technical Abstract: “Co-suppression” associated loss of flower pigmentation in transgenic petunia plants was one of the first clear indicators of the natural process of RNA-associated gene silencing in plants. We have been exploring the use of genetically engineered anthocyanin over-production in vegetative tissues as a visual indicator of both gene induction and silencing. Constitutive over-expression of the Arabidopsis PAP1 or PAP2 genes, encoding transcription factors from the myb family (AtMYB75 and AtMYB90, respectively), can result in ectopic over-production of anthocyanin pigments in Arabidopsis and other plant species. The resulting pigment accumulation produces a clearly visible red-to-purple color in the leaves, stems, flowers and roots of select transgenic lines. In order to examine the utility of MYB-induced pigmentation as a visible reporter we have generated transgenic (Agrobacterium transformation) purple tobacco lines that over-express the Arabidopsis PAP2 gene. Agro-infiltration of purple hemizygous PAP2 transgenic plants with a T-DNA construct that produces dsRNA against PAP2 results in a distal loss of anthocyanin pigmentation (green phenotype) that begins near the veins of the 2-5 most infusion-proximal newer leaves. The initiating signal for this 'systemic' silencing moves transiently upward through the plant, with younger leaves eventually returning to the PAP2 purple phenotype. However, the dsRNA-induced silenced areas of these plants are stable, remaining green throughout their continued development and senescence. A plant-wide silencing of the purple phenotype is also associated with a doubleing of PAP2 gene dosage, with homozygotes at the PAP2 T-DNA locus showing a green phenotype similar to that seen in the dsRNA-induced silenced leaves. In all cases examined, the silenced green phenotype is correlated with a significant reduction in steady-state PAP2 mRNA levels. The gene-dosage associated silencing of the purple phenotype is further influenced by developmental and environmental factors, providing valuable insight into physiological mechanisms that impact/regulate post transcriptional gene silencing in plants.