Abstract
The FILAMENTOUS FLOWER gene from Arabidopsis thaliana is a member of a gene family whose role is to specify abaxial cell fate in lateral organs. Analysis of the amino-terminal region of the FILAMENTOUS FLOWER protein suggests that seven cysteine residues at positions 14, 26, 30, 33, 54, 56, and 57, and two histidine residues at positions 18 and 24 contribute to a putative zinc finger motif, Cys-X(3)-His-X(5)-His-X-Cys-X(3)-Cys-X(2)-Cys-X(20)-Cys-X-Cys-Cys. Zinc determination experiments revealed that the FILAMENTOUS FLOWER protein binds two zinc ions per molecule. Chemical modification was required to release one zinc ion, whereas the other was released spontaneously or more rapidly in the presence of metallochromic indicator. The loss of a zinc ion and the subsequent structural change of the zinc finger domain were correlated with the multimerization of the FILAMENTOUS FLOWER protein. A cysteine residue at position 56 in the FILAMENTOUS FLOWER protein potentially interferes with zinc ligation within the zinc finger and causes this zinc release. In support of this, substitution of the Cys(56) by alanine suppressed both the zinc release and the multimerization of the FILAMENTOUS FLOWER protein. Deletion analysis showed that the region between positions 45 and 107 functions in the intermolecular contacts between FILAMENTOUS FLOWER proteins. This region corresponds to the carboxyl-terminal half of the zinc finger domain and the following hydrophobic region containing two putative alpha-helices. Our results suggest that the FILAMENTOUS FLOWER protein forms a range of different conformers. This attribute may lead to a greater degree of functional flexibility that is central to its role as an abaxial cell fate regulator.
Highlights
In higher plants, lateral organs produced from the flanks of the apical meristem exhibit defined adaxial-abaxial polarity
In this study we have investigated the role of zinc ions in the FIL zinc finger domain and the structural organization of the protein to understand the functional properties of the FIL protein
It is proposed that FIL is a member of the gene family including CRABS CLAW (CRC) [7], INNER NO OUTER (INO) [8], YAB2, YAB3, and YAB5 [3]
Summary
Cells and Plasmids—Plasmid pET28a and Escherichia coli BL21(DE3) were from Novagen, Inc. The gene was amplified by PCR using the synthetic DNA oligomers P1 (5Ј-ATGTCGTCCATGGCCTCCCCTTCCTCAGCTG-3Ј) and P2 (5Ј-TTTATCCTCGAGATAAGGAGTCACACCAACG-3Ј) as primers. Truncation of the FIL gene was performed via PCR with the synthetic DNA oligomers carrying BamHI and EcoRI sites as primers, and using KOD DNA polymerase, according the procedures recommended by the supplier. After the column was washed with buffer A (20 mM Tris-HCl, pH 8.0, 0.1 M NaSCN, 5 mM -mercaptoethanol, 0.6% sodium deoxycholate (DOC), and 10% glycerol), the FIL protein was eluted with buffer A containing 100 mM imidazole. The protein solution was dialyzed against a buffer containing 10 mM Tris-HCl, pH 8.0, 250 M TCEP, 0.6% DOC, and 10% glycerol to remove -mercaptoethanol. Zinc release in the presence of PAR only was analyzed by measuring the absorbance change at 500 nm.
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