In plants that rely on animals for the transfer of pollen, the cells of the epidermis of flower petals are specialized to attract pollinators. The differentiation program of these cells includes the synthesis of anthocyanin pigments, their accumulation in the central vacuole of these cells, the display of the color via modulation of the pH in the lumen of the vacuole, the building of a “papillary” cell shape that provides a handy landing surface to the pollinator and contributes to the final color of the petal by affecting the refraction angle of the light. All these mechanisms are controlled by a group of transcription factors that activate different sets of target genes (1). A set of genes has been shown to encode for the biosynthetic enzymes of the anthocyanin pathway, while two other target genes encode for two different P-ATPases (PH1 and PH5) that together acidify the lumen of the central vacuole and some 10 other target genes still have not been assigned a function (2); (3). Localization studies for the tonoplast proteins PH1 and PH5 has brought to the discovery that in petal epidermal cells, the sorting of proteins to the vacuole includes an intermediate organelle on which proteins reside before reaching the central vacuole. As this organelle looks like a small vacuole and is marked by vacuolar proteins (including vacuolar SNAREs), we called it “vacuolino”. In transient expression assays, the membrane of vacuolinos is marked by the PH5-GFP fusion 24 hours after cell transformation, while the tonoplast of the central vacuole shows fluorescence only ~48 hrs after transformation. In different cell types (like leaf cells or unpigmented petal mesophyll cells) vacuolinos are absent and PH5-GFP appears on the tonoplast already 24 hrs after transformation. All other vacuolar proteins we have observed in their sorting pathway to the vacuole in petal epidermal cells, reach the final destination after a short permanence on the vacuolinos. Mutants for any of the above mentioned transcription factors do not show vacuolinos implying that a set of their target genes is involved in the genesis of these compartments. We have used different methods to compare the transcriptomics of petals mutant for each of the transcription factors involved in the presence of vacuolinos (AN1, PH3 and PH4) and we have isolated a number of target genes of these regulators to isolate candidate genes involved in vacuolinos biogenesis. We are now isolating mutants for each of these genes by the screening of petunia BLASTABLE collections of transposon insertions and by RNAi technology. The first three genes for which we could see disappearance of vacuolinos in the mutants are involved in different steps of the vacuolino pathway to the vacuole: - in mutants for one of this gene vacuolar proteins directly go to the vacuole without passing through the vacuolino (like in mesophyll cells), - in another they get stuck in small vesicle-like structures, - in the third the markers remain on the vacuolinos as vacuolinos seem not to be able to fuse to the central vacuole. We are at the moment also studying when the vacuolinos appear during bud development as this could give some clue of their function. Analysis of the localization of vacuolar GFPs in epidermal petal cells of buds at different developmental stages shows that: - in young buds, the petal epidermal cells are rather small (compared to open flowers) and have small vacuoles with huge folded tonoplast - during development the cells enlarge and the tonoplast unfold - -vacuolinos only show up after flower opening, when petals are approaching senescence All three these genes encoded unexpected players in the genesis, physiology and fusion of membrane compartments. 1. Koes R, Verweij CW, & Quattrocchio F (2005) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci. 5:236-242. 2. Faraco M, et al. (2014) Hyperacidification of Vacuoles by the Combined Action of Two Different P-ATPases in the Tonoplast Determines Flower Color. Cell reports 6(1):32-43. 3. Verweij W, et al. (2008) An H+ P-ATPase on the tonoplast determines vacuolar pH and flower colour. Nature cell biology 10(12):1456-1462.
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