Pleiotropic and nonredundant effects of an auxin importer in Setaria and maize

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 green millet (Setaria viridis) and maize (Zea mays), we uncover previously unreported aspects of plant development that are affected by auxin influx, including higher order branches in the inflorescence, stigma branch number, 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 green fluorescent protein (GFP)-tagged construct of the Setaria AUX1 protein Sparse Panicle1 (SPP1) under its native promoter showed that SPP1 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 indicated that most gene expression modules are conserved between mutant and wild-type plants, with only a few hundred genes differentially expressed in spp1 inflorescences. Using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology, we disrupted SPP1 and the other four AUX1 homologs in S. viridis. SPP1 has a larger effect on inflorescence development than the others, although all contribute to plant height, tiller formation, and 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.