Identification and characterization of several putative interacting genes in AXR4 related pathways

The axr4 mutation causes defects in gravitropism and lateral root formation as well as resistance to actively-imported auxins, and has been shown to result in mislocalization of the auxin import carrier AUX1 in certain cell types. However, the biochemical function of the AXR4 protein is unclear. To address this problem, we identified second-site mutations that enhance the auxin resistant root elongation phenotype of axr4 mutants in Arabidopsis thaliana. During this study, two such axr4-Enhancers, namely 17.4.1 and 37.5.1 were characterized. When these Enhancers are combined with axr4, they confer enhanced resistance to the imported auxin, 2,4-D but no change in resistance to the diffusible auxin, NAA or in aerial morphology. The two Enhancer mutations are allelic, auxin-sensitive in the absence of the axr4 mutation. However, these double mutants do not significantly alter the delayed gravitropic feature, the defects in lateral root formation, Sulfur sensing, responses to osmotic stress and auxin transport. The 37.5.1 Enhancer showed a stronger resistance to 2,4-D when compared to 17.4.1. This axr4-Enhancer was roughly mapped to the long arm of chromosome 5. However, lack of formation of recombinants which is speculated to be caused by chromosomal rearrangements in this region hindered fine mapping and cloning of the gene. But this putative Enhancer gene appears to define a new locus which is important for auxin influx in plants. Cloning and characterization of the putative axr4-Enhancer gene and characterization of the putative AXR4-interactors identified through the split-ubiquitin screen will shed light in identifying auxin influx pathway in more detail and expand the current knowledge on membrane trafficking in plants.