Molecular analysis of flower senescence using virus-induced gene silencing

Flower senescence is a genetically controlled program, and genes whose transcript abundance is associated with this process have been identified from a range of flowers. Previously, the need for time consuming transformation and regeneration has limited our understanding of the function of these genes. I have employed virus-induced gene silencing (VIGS) as a rapid alternative technique for analyzing gene function during floral senescence. Tobacco rattle virus (TRV) was chosen for my studies because TRV infects floral tissues, has only mild symptoms, and has a broad host range. A range of host plants was tested using TRV containing a fragment of the phytoene desaturase (PDS) cDNA to provide a visible reporter of gene silencing. In most cases, infection resulted in no apparent phenotype. In petunia, infection resulted in systemic infection and silencing of the host PDS as indicated by photobleaching of the leaves. The silencing phenotype was non-uniform and cultivar-dependent. Infection of purple-flowered cultivars with TRV containing fragments of PDS and chalcone synthase (CHS) resulted in leaf photobleaching and white patterns on the flowers. The abundance of transcripts of CHS and PDS was reduced, confirming simultaneous silencing of both genes by the construct. I further confirmed the efficacy of tandem constructs by testing the effects of infection with TRV CHS/ACO4. Abundance of transcripts encoding ACC oxidase (ACO) was reduced in white sectors of infected flowers, and these sectors lasted longer than purple uninfected sectors. These results indicate the value of VIGS with tandem constructs containing CHS as reporter and a target gene as a tool for examining the function of floral-associated genes.;Prohibitin was identified using our VIGS system to be a gene that impacts flower longevity. Prohibitins have been shown to play important roles in cell cycling and senescence in animals and yeast. However, their function has not yet been demonstrated in plants. I identified two prohibitin genes, PhPHB1 and PhPHB2, in petunia. Their deduced amino acid sequences show high similarity, respectively, to prohibitin-1 and prohibitin-2 from yeast and other systems. Down-regulating prohibitin-1 from petunia using VIGS, I observed plants with smaller and distorted leaves and flowers. Cells in the silenced flowers were larger, indicating a substantial reduction in cell number. The life of petunia flowers was shortened by prohibitin-1 silencing. The respiration of the silenced flowers was higher than that of the controls, and I observed a marked increase in transcripts of a catalase and a small heat-shock protein in the silenced flowers. The data indicate that prohibitins play a key role in plant development and senescence.