ABSTRACT: One of the key innovations of the flowering plants is their female reproductive organ, the carpel. Here we show that a mechanism controlling carpel margin development in the model flowering plant Arabidopsis thaliana was recruited from light-regulated processes. This recruitment followed the loss from the basic Helix-Loop-Helix transcription factor SPATULA (SPT) of a domain previously responsible for its negative regulation by phytochrome. We propose that the loss of this domain was a prerequisite for the light-independent expression in female reproductive tissues of a genetic module that also promotes shade avoidance responses in vegetative organs. Striking evidence for this proposition is provided by the restoration of wild type carpel development to spt mutants by low red/far-red light ratios, simulating vegetation shade, which we show to occur via PHYB, PIF4 and PIF5. Our data illustrate the potential of modular evolutionary events to generate rapid morphological change, and thereby provide a molecular basis for neo-Darwinian theories that describe this non-gradualist phenomenon. Furthermore, the effects shown here of light quality perception on carpel development lead us to speculate on the potential role of light-regulated mechanisms in plant organs that, like the carpel, form within the shade of surrounding tissues. The SPATULA (SPT) coding sequence was fused to the 5M-bM-^@M-^Y-extremity of a sequence encoding the 75 C-terminal residues of the viral VP16 transcriptional activator and inserted into the pG0229-35S:GR plant transformation vector between the CaMV-35S promoter and sequences encoding the rat glucocorticoid receptor (GR), so as to conserve the entire reading frame. The resulting plasmid was transferred to Agrobacterium tumefaciens C58/pMP90 cells and used to transform A. thaliana Col-0 plants by standard methods. A homozygous, single-copy transformant was identified, from which two populations of ten T3 descendents were grown and treated by dipping of inflorescences for 2 min in cycloheximide (CHX; 10 M-BM-5g/ml) containing Silwet L-77 surfactant (0.01% v/v). This treatment reduced translation to approximately 5% of its native level in inflorescence tissues, as measured by the in vivo incorporation of [35S]-methionine into proteins. One hour later, inflorescence tissues of the two plant populations were dipped for 2 min in CHX solution, as described above, with and without dexamethasone (DEX; 10 M-BM-5M). After a further 2h, treated inflorescences, excluding open flowers, were harvested and pooled prior to RNA extraction for global gene expression analyses. Microarray analyses were performed using CATMA microarrays each containing 31776 gene-specific tags corresponding to 22089 A. thaliana genes. Three biological replicates were performed, based on three entirely separate induction experiments involving T3 plants from the same T2 parent. A transformed line containing a construction in which SPT had been replaced by an initiation codon and nuclear localization signal (NLS) was used to verify that the targets identified did not respond to the nuclear translocation of VP16-GR protein alone (35S:NLS-VP16-GR plants). In this NLS control experiment, two biological replicates were performed. One technical replicate with fluorochrome reversal was performed for each biological replicate. The labeling of cRNAs with Cy3-dUTP or Cy5-dUTP (Perkin-Elmer-NEN Life Science Products), the hybridization of these cRNAs to microarrays, and the subsequent scanning of microarrays, was performed as described by Lurin et al. (2004) Plant Cell 16, 2089-2103.
