Mutant analysis, protein–protein interactions and subcellular localization of the Arabidopsis Bsister (ABS) protein

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Abstract
Recently, close relatives of class B floral homeotic genes, termed Bsister genes, have been identified in both angiosperms and gymnosperms. In contrast to the B genes themselves, Bsister genes are exclusively expressed in female reproductive organs, especially in the envelopes or integuments surrounding the ovules. This suggests an important ancient function in ovule or seed development for Bsister genes, which has been conserved for about 300 million years. However, investigation of the first loss-of-function mutant for a Bsister gene (ABS/TT16 from Arabidopsis) revealed only a weak phenotype affecting endothelium formation. Here, we present an analysis of two additional mutant alleles, which corroborates this weak phenotype. Transgenic plants that ectopically express ABS show changes in the growth and identity of floral organs, suggesting that ABS can interact with floral homeotic proteins. Yeast-two-hybrid and three-hybrid analyses indicated that ABS can form dimers with SEPALLATA (SEP) floral homeotic proteins and multimeric complexes that also include the AGAMOUS-like proteins SEEDSTICK (STK) or SHATTERPROOF1/2 (SHP1, SHP2). These data suggest that the formation of multimeric transcription factor complexes might be a general phenomenon among MIKC-type MADS-domain proteins in angiosperms. Heterodimerization of ABS with SEP3 was confirmed by gel retardation assays. Fusion proteins tagged with CFP (Cyan Fluorescent Protein) and YFP (Yellow Fluorescent Protein) in Arabidopsis protoplasts showed that ABS is localized in the nucleus. Phylogenetic analysis revealed the presence of a structurally deviant, but closely related, paralogue of ABS in the Arabidopsis genome. Thus the evolutionary developmental genetics of Bsister genes can probably only be understood as part of a complex and redundant gene network that may govern ovule formation in a conserved manner, which has yet to be fully explored.