Function and regulation of INNER NO OUTER, a gene required for the polar development of ovules in Arabidopsis thaliana

The ovule, the precursor to a seed, is a useful model for the elucidation of the molecular mechanisms of plant development. In studies on ovules of Arabidopsis thaliana, one gene, INNER NO OUTER ( INO), was found to be necessary for the correct growth of the outer integument, a structure that encases the entire ovule and becomes the seed coat. INO is a member of a small group of genes, the YABBY family, that are hypothesized to function as transcription factors to specify abaxial identity in plants. Using reporter gene fusions of β- GLUCURONIDASE and the GREEN FLUORESCENT PROTEIN to the INO promoter (P-INO), we show that INO expression is limited to the outermost cell layer of the outer integument. We demonstrate that previously described regulators of ovule development, AINTEGUMENTA, BELLI, SUPERMAN (SUP) and INO, can influence expression of the INO gene. Transgenic analysis using INO, SUP and CRABS CLAW ( CRC, a YABBY family member) indicate that INO is involved in a positive autoregulatory circuit that is attenuated by SUP. Expression of each YABBY family member with P-INO demonstrated that although several could promote outer integument growth, only INO is sensitive to the effects of SUP. Analysis of INO-CRC chimeric proteins showed that regions of INO that are both conserved and divergent relative to the other YABBY family members could influence the sensitivity to SUP. Ectopic expression of INO could produce both similar and distinct phenotypes relative to constitutive expression of other YABBY members. Using promoter deletion analysis, three positive regulatory regions were identified within P-INO. One such element, POS9, was sufficient to reproduce the endogenous pattern of INO expression and appeared to consist of three distinct sub-elements. A heterologous assay identified two distinct protein families that could interact with POS9; members of one family contained a canonical C2H2 zinc finger motif whereas members of the other contained a novel DNA-binding region. This second family appears to be plant specific but its in vivo function is unclear. In summary, we have expanded the current models of both ovule development and the transcriptional regulation of polar determinants by a combination of genetic and molecular techniques.