The structure and function of PETAL LOSS, a trihelix transcription factor that regulates sepal and petal development in Arabidopsis

In Arabidopsis thaliana the trihelix transcription factor family is composed of 29 members. This plant specific protein family is believed to have evolved from an ancestor of the Myb transcription factors because of similarities within their DNA binding domain. Members of the trihelix family encode proteins with either one or two DNA binding domains. The PETAL LOSS gene encodes a transcription factor with two trihelix domains. When mutated, yvtl plants show a decrease in petal numbers as the inflorescence meristem grows older. The flowers also have defects in their first whorl, showing boat shaped and sometimes fused sepals. In flowers the expression pattern of PTL mostly coincides with the sites of disruption in ptl mutant plants. Expression in early flower primordia is located at the inter-sepal zone, and PTL is also expressed in margins of newly arising organs, such as leaves, sepals, petals and stamens. Gene transcript levels are relatively low in wild type plants, and over-expression of PTL causes a halt to growth where-ever it is expressed, supporting a role for PTL in growth suppression. In this study I have analyzed the protein sequence of PTL and of other members of the trihelix family in Arabidopsis, as well as of orthologues in other flowering plants. This revealed the presence of a conserved central domain between the two DNA binding domains. In addition putative nuclear localization signals occur in each DNA binding domain, and an additional monopartite NLS is present in the conserved central domain. Alanine scanning mutagenesis of each NLS in a PTLGFP fusion context revealed that the NLS in the C-terminal trihelix suffices for its nuclear transport. Interestingly the remaining two NLSs can confer nuclear transport only when both are present. Once in the nucleus, I found that the PTL transcription factor can function as a transcriptional activator in yeast as well as in planta. Protein deletions revealed that the trans-activation domain of PTL is located at its C-terminal sequence. Protein interactions were analysed by the yeast two hybrid system revealing a direct interaction between PTL and members ofthe transcription pre-initiation complex. Furthermore these analyses, although preliminary, disclosed a connection between PTL function and the auxin vi signalling pathway, and a direct interaction between PTL and the meristem maintenance transcription factor SHOOT MERISTEMLESS. By putting all these findings together a model for PTL mode of action as a localized growth suppressor is postulated.