ETTIN regulates genes involved in cell wall modification

ETTIN (ETT) encodes a member of the Auxin Response Factor (ARF) family of transcription factors. The gynoecia of strong ett mutants show a partial breakdown in abaxial-adaxial (internal -external) polarity, and a shift in tissue boundaries along the apico-basal axis of the gynoecium. This latter effect increases the size of the stigma, style and stipe in ett mutants at the expense of ovary tissue. Among the putative direct targets of ETT, we found several genes encoding either pectin methylesterases (PMEs) or their inhibitors. PMEs may function to increase the extensibility of the cell wall and thus contribute directly to cellular growth. The identification of genes encoding PMEs and related molecules as direct targets of ETT therefore suggests this factor to be situated near the end of a hierarchy of genetic regulation acting on growth through cell wall modification. Other putative direct targets of ETT encode two members of the Aux/IAA protein family, which have been found to negatively regulate ARFs as an inverse function of auxin concentration. ETT lacks the protein domains, present in most ARFs, that are necessary for negative regulation by Aux/IAA proteins and its own activity should not therefore be subject to regulation by auxin concentration. However, the regulation of two Aux/IAA proteins by ETT may represent an input of this protein into an auxin-regulated network involving other ARFs. To identify the direct target genes of ETT, we generated transgenic plants in which the translocation to the nucleus of a constitutively produced ETT fusion protein could be induced by exogenous application of the hormone analogue dexamethasone (DEX). Inflorescence tissues of transformed plants were treated either with DEX and cycloheximide (CYC) to modify the transcription of ETT-target genes while blocking protein synthesis, or with CYC alone to provide a reference sample. RNA was harvested 2 h after treatments and processed for expression analyses on CATMA microarrays, representing most of the genes in the A. thaliana genome. Microarray analyses were performed from three completely independent experiments (one pair of dye-swaps for each experiment) and the results of these were compared statistically to derive a list of putative ETT-targets.