Functie van de eiwitten die de synthese van anthocyanine en de pH van de vacuole regelen
04 / 2001 - onbekend
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)
The last years significant new insights have been obtained into the mechanisms by which developmental patterns are formed in groups of apparently similar cells in plant meristems. However, we know little or nothing about the mechanisms by which cells begin to differentiate once patterns have been established. Anthocyanins are pigments that accumulate in specific floral tissues of higher plants, which makes them an ideal marker for differentiation. Our goal is to understand how the (patterning) genes that establish the identity of a flower petal, (ultimately) activate tissue-specific genes such as those involved in pigmentation. By genetic methods we identified and isolated five regulatory genes (aril, an2, an4, an11 andjafl3) that control the transcription of (i) structural genes encoding enzymes of the anthocyanin pathway and of (ii) yet unknown target genes determining the pH of the vacuole and cell shape. In addition we isolated two genes (ph2 and ph4) that specifically affect intracellular pH but not anthocyanin synthesis. Genetic experiments revealed a complex pattern of interactions between these an and ph genes and their products. The bHLH proteins AN1 and JAF13 can directly interact with each other and form complexes with one or more of the MYB proteins AN2, AN4, PH4 and MYB-X. This results in the formation of at least two distinct transcription complexes that activate a pH pathway (a complex with PH4) and anthocyanin synthesis (a complex without PH4). The MYB proteins AN2 and AN4 have an additional function in activating transcription of the anl gene. AN11 is a cytosolic WD40 protein that appear to regulate the activity of these transcription complexes post-translationally. We propose to study by a combination of biochemical and genetic approach how different complexes of these regulators can activate seemingly unrelated pathways. Specific issues that will be studied are (i) the composition of the protein complex(es) in which the different proteins are present, (ii) the effect of removal of one regulatory protein (by mutation) on the stability, complexation and subcellular localisation of the other regulators, (iii) complex formation of isolated proteins in vitro by BIACORE and by pull-down assays, (iv) the subcellular location where complex formation occurs.