CRABS CLAW positively regulates genes involved in the establishment of both abaxial and adaxial identity in Arabidopsis

CRABS CLAW (CRC) encodes a transcription factor of the plant-specific YABBY class in the model plant Arabidopsis thaliana. This gene is highly expressed in the abaxial (external) domain of the gynoecium wall and contributes to the establishment of abaxial-adaxial (external-internal) polarity in that tissue. Here we derive a list of putative target genes of CRC, which include AUXIN RESPONSE FACTOR4 (ARF4) and ASSYMETRIC LEAVES1 (AS1), both of which are known to be involved in the establishment of abaxial-adaxial polarity in lateral organs. ETTIN (ETT), which is partially redundant with ARF4, was not identified as a direct target of CRC, and this observation led us to predict that crc/ett double mutants might resemble ett/arf4 double mutants, which show a very strong breakdown of abaxial-adaxial polarity in the gynoecium wall. We have confirmed this prediction by constructing double mutants using several available mutant alleles of crc, ett and arf4. Interestingly, AS1 plays a role in the establishment of adaxial tissue identity in lateral organs, though its expression is not restricted to the adaxial domain. The observed positive regulation of AS1 by CRC may thus occur very early in gynoecium development, before CRC expression becomes polarised, or at later stages of development, specifically in the abaxial domain. To identify the direct target genes of CRC, we generated transgenic plants in which the translocation to the nucleus of a constitutively produced CRC fusion protein could be induced by exogenous application of the hormone analogue dexamethasone (DEX). The transgene construction used in this procedure included a VP16 viral transactivation domain to amplify the effects of CRC fusion protein on transcription, thereby increasing the sensitivity of the analysis. Inflorescence tissues of transformed plants were treated either with DEX and cycloheximide (CYC) to induce the transcription of CRC-target genes while blocking protein synthesis, or with CYC alone to provide a reference sample. RNA was harvested 2 or 4h after treatments and processed for expression analyses on CATMA microarrays, representing most of the genes in the A. thaliana genome. Further experiments were performed without CYC treatment (mock/DEX), permitting the identification of genes regulated indirectly be CRC.