Functional domains of the PETAL LOSS protein, a trihelix transcription factor that represses regional growth in Arabidopsis thaliana

PETAL LOSS (PTL) is a trihelix transcription factor that represses growth, especially between sepal primordia. As one of 30 trihelix proteins in Arabidopsis, it falls in the GT2 clade with duplicated trihelix DNA-binding domains and a long α-helical central domain. PTL orthologs occur in all angiosperm genomes examined except grasses, and sequence comparisons reveal that there are two further short conserved domains at each end. GT2 itself carries two nuclear localization sequences, but PTL has an additional nuclear localization sequence (NLS). We show that PTL can act as a transcriptional activator in yeast and in planta, with the latter tested by two different functional assays. Specific deletions revealed that the activation region is C-terminal. Site-directed mutagenesis of the DNA-binding domains has shown that a conserved tryptophan and two downstream acidic amino acids in the second trihelix, predicted to promote folding, are each required for PTL function. Also, three basic residues in the third helix, near the DNA interaction sites, support its function. PTL was found to dimerize in yeast. This was confirmed and extended by jointly expressing differentially tagged forms of PTL in a transient expression system in Nicotiana benthamiana leaves. Cytoplasmic PTL (with mutant NLS sequences) was carried into the nucleus upon binding with nuclear-localized PTL, providing each partner carried intact central domains. As this 90-amino acid domain is conserved in most trihelix family members, it seems likely that they all function in dimeric form.