Identification of Auxin Response Factor-Encoding Genes Expressed in Distinct Phases of Leaf Vein Development and with Overlapping Functions in Leaf Formation

Based on mutant phenotypes the MONOPTEROS (MP)/Auxin Response Factor 5 (ARF5) gene acts in several developmental processes including leaf vein development. Since overlapping functions among ARF genes are common, we assessed the related ARF 3-8 and 19 genes for potential overlap in expression during vein development using in-situ hybridization. Like MP/ARF5, ARF3 was expressed in preprocambial and procambial cells. ARF7 was also expressed in procambial cells, close to and during vein differentiation. ARF19 was expressed in differentiating vessel elements. To assess if genes with vein expression have overlapping functions, double mutants were generated. While arf3, 5 and 7 mutants formed leaves normally, double mutant combinations of mp/arf5 with arf3 or arf7 resulted in a breakdown of leaf formation. Instead, novel structures not present in any of the single mutants formed. The results implicate ARF3 and ARF7 in rosette leaf formation and suggest that their functions overlap and act in parallel with MP/ARF5 in this process. The observed vascular expression patterns suggest unique functions (ARF7 and 19) and potentially overlapping functions (ARF3 and 5) in vein development. Since arf3 arf5 double mutants do not form leaves, assessment of their potential combined action in vein development will require the use of conditional mutants.