Down-regulation of DR12, an auxin-response-factor homolog, in the tomato results in a pleiotropic phenotype including dark green and blotchy ripening fruit

Following differential screening of gene expression during tomato fruit development, we isolated developmentally regulated (DR) clones, including several putative transcription factors. Based on sequence homology, DR1, DR3, DR4 and DR8 are members of the Aux/IAA family, and DR12 belongs to the auxin response factor (ARF) family of transcription factors. Importantly, mRNA accumulation for the Aux/IAA-like genes was regulated by ethylene in tomato fruit but not in the leaves, indicating that these putative auxin response components also participate to the ethylene-dependent regulation of gene expression in a tissue-specific manner. The functional significance of DR12, the ARF-like gene, was investigated by cellular biology and reverse genetics approaches. Heterologous protein targeting studies, carried out using a DR12-GFP gene fusion construct, revealed specific nuclear localization of the DR12-encoded protein, in accordance with its putative function as a transcriptional regulator. Transgenic plants over- and under-expressing DR12 were generated in order to explore the physiological role of the gene. Both antisense and sense co-suppressed DR12-inhibited lines displayed a pleiotropic phenotype that included dark-green immature fruit, unusual cell division in the fruit pericarp, blotchy ripening, enhanced fruit firmness, upward curling leaves and increased hypocotyl and cotyledon growth. While a perturbation of the response to auxin may explain some of the phenotypes, surprisingly, the expression of members of four classes of early auxin-regulated genes was unaffected in the DR12-inhibited plants. The involvement of this ARF-like encoded protein in mediating the auxin response is discussed along with the possibility that it might affect responsiveness to other phytohormones in the tomato.