Pleiotropic and Non-redundant Effects of an Auxin Importer in Setaria and Maize1

ABSTRACT Directional transport of auxin is critical for inflorescence and floral development in flowering plants, but the role of auxin influx carriers (AUX1 proteins) has been largely overlooked. Taking advantage of available AUX1 mutants in Setaria viridis and maize, we uncover previously unreported aspects of plant development that are affected by auxin influx, including higher order branches in the inflorescence, stigma branch number, and glume (floral bract) development, and plant fertility. However, disruption of auxin flux does not affect all parts of the plant, with little obvious effect on inflorescence meristem size, time to flowering, and anther morphology. In double mutant studies in maize, disruptions of ZmAUX1 also affect vegetative development. A GFP-tagged construct of SvAUX1 under its native promoter showed that the AUX1 protein localizes to the plasma membrane of outer tissue layers in both roots and inflorescences, and accumulates specifically in inflorescence branch meristems, consistent with the mutant phenotype and expected auxin maxima. RNA-seq analysis finds that most gene expression modules are conserved between mutant and wildtype plants, with only a few hundred genes differentially expressed in spp1 inflorescences. Using CRISPR-Cas9 technology, we disrupted SPP1 and the other four AUX1 homologs in S. viridis. SvAUX1/SPP1 has a larger effect on inflorescence development than the others, although all contribute to plant height, tiller formation, leaf, and root development. The AUX1 importers are thus not fully redundant in S. viridis. Our detailed phenotypic characterization plus a stable GFP-tagged line offer tools for future dissection of the function of auxin influx proteins. One sentence summary Mutations in a single auxin importer gene Spp1/SvAUX1 uncover broad and unexpected effects in nearly all aspects of the development of shoots, inflorescences, and flowers. Footnotes 1 C.Z., M.S.B. and D.T. were supported by National Science Foundation (NSF) grant IOS-1413824 to E.A.K. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Elizabeth A. Kellogg, ekellogg{at}danforthcenter.org. E.A.K., C.Z. and M.S.B. designed the project. Experiments and data analysis were carried out by C.Z., M.S.B., D.T., H.H., A.N.D., and Y.Y. C.Z. drafted the paper with subsequent comments and review by E.A.K. and A.N.D. All authors read and approved the final manuscript.