DEFECTIVELY ORGANIZED TRIBUTARIES 5 is not required for leaf venation patterning in Arabidopsis thaliana

ABSTRACT The search for genetic regulators of leaf venation patterning started over thirty years ago, primarily focussed on mutant screens in the eudicotyledon Arabidopsis thaliana . Developmental perturbations in either cotyledons or true leaves led to the identification of transcription factors required to elaborate the characteristic reticulated vein network. An ortholog of one of these, the C2H2 Zn finger protein DEFECTIVELY ORGANIZED TRIBUTARIES 5 (AtDOT5), was recently identified through transcriptomics as a candidate regulator of parallel venation in maize leaves. To elucidate how AtDOT5 orthologs regulate vein patterning, we generated three independent loss of function mutations by gene editing in Arabidopsis. Surprisingly, none of them exhibited any obvious phenotypic perturbations. To reconcile our findings with earlier reports, we re-evaluated the original Atdot5-1 and Atdot5-2 alleles. By genome sequencing, we show that reported mutations at the Atdot5-1 locus are actually polymorphisms between Landsberg erecta and Columbia ecotypes, and that other mutations present in the background most likely cause the pleiotropic mutant phenotype observed. We further show that a T-DNA insertion in the Atdot5-2 locus has no impact on leaf venation patterns when segregated from other T-DNA insertions present in the original line. We thus conclude that AtDOT5 plays no role in leaf venation patterning in Arabidopsis. Significance statement An understanding of gene function is often derived on the basis of loss of function mutant phenotypes and thus correct identification of mutated loci is crucial. Through gene editing we reveal previous mis-identification of a causative mutation that led to inappropriate functional assignation for the DEFECTIVELY ORGANIZED TRIBUTARIES 5 gene in Arabidopsis thaliana .