DOI: 10.5061/dryad.0zpc86701
title: Phenotype data for: Pleiotropic and non-redundant effects of an auxin importer in Setaria and maize

description: 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 Spp1 under its native
 promoter showed that the SPP1 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.  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.
description: <b>Plant growth, phenotyping, and statistical
 comparisons</b> <i>Setaria
 viridis</i> accessions A10.1 and ME034V were grown in growth chamber
 and greenhouse conditions, respectively, following Acharya et al. (2017) and Zhu et
 al. (2018). The original <i>spp1</i> mutation was isolated
 from an A10.1 background; ME034V was chosen for CRISPR confirmation of the
 mutant phenotype because of its high transformation efficiency. Plant
 height, leaf number, panicle length, and branch number were measured as
 described in Huang et al. (2017) and Zhu et al. (2018). Fertility was
 measured as the ratio of spikelets with a fully developed
 upper floret to total spikelets; bristles were ignored for fertility
 measurements. Tillers were
 counted at 37 days after sowing (DAS) and plant height measured at 40 DAS.
 Stigma and style number
 were assessed by dissecting one floret from each of ten spikelets per
 plant, five plants for <i>spp1</i> and three for A10, for a total of 50 mutant florets and 30 wildtype.  We recorded number of styles and number of stigmas for each floret.  Numbers varied as much within as between plants so the numbers were pooled among all plants to estimate the frequency of each number. Histology and SEM followed Zhu et al. (2018). Inflorescence length, meristem width and height were measured using ImageJ (Schneider et al., 2012) from SEM photos. For root phenotyping, sterilized seeds were grown either in Murashige and Skoog (MS) medium or germination pouches as described in Huang et al. (2017) and Acharya et al. (2017), respectively. Auxin rescue experiments followed Marchant (1999) and Yu et al. (2015). 2, 4-D (from Plant Media, in 1mM stock with pure ethanol) and NAA (from Sigma-Aldrich, in 10mM stock with pure ethanol) were added to the medium to a final concentration of 0.1 mM.  MS medium containing 0.1% ethanol was used as a mock control. Seeds were grown on MS medium for three days and then transferred to media containing appropriate concentrations of auxin or mock for three more days. Root hairs were imaged at 4x magnification on a Leica DM750 microscope. Root hair number was counted in the focal plane on the side of the root facing the observer and normalized to root length. Experiments were repeated three times. In maize, <i>zmaux1</i> mutant plants were crossed to <i>vt2</i>, <i>bif2</i> and <i>Bif4</i> mutants, and F2 segregating populations were grown in the field in Columbia, Missouri in 2017. Plants were genotyped to identify single and double mutants using primers listed in Table S10 and were phenotyped at the eighth week. For the dominant mutant <i>Bif4</i>, both heterozygotes and homozygotes were included for mutant phenotyping analysis. For each mutant and mutant combination we assessed traits of the tassel (length from flag leaf to tassel tip, number of branches, spikelets on main spike, spikelet number per cm) and ear (kernel number, ear row number), and three vegetative traits (height of flag leaf, number of leaves above the lowest elongated internode, and number of tillers). All pairwise comparisons used Welch’s t-test as implemented in R (R Core Team, 2020).  Single, double and higher order mutants were compared to each other and to wildtype by one-way or two-way Type I or Type II ANOVA as appropriate, followed by Tukey’s HSD test using standard programs in R (R Core Team, 2020).  Comparisons with p&gt;0.05 were considered non-significant.
description: A ReadMe file has been included with the uploaded data. The files
 are organized according to the figure numbers in the manuscript.  Each
 figure number has its own directory, within which are one or more excel
 files (.xls) presenting the raw measurements.  These are unprocessed
 although they have been transferred to the excel files in a format for
 easy conversion to a dataframe compatible with R. Also within each
 directory are multiple .txt files that present the output of the
 statistical analyses, whether t-tests, ANOVA, or linear.  Mean and s.d.
 and p values from the t-test or ANOVA are reported in the appropriate
 supplemental tables in the manuscript.  Most are also presented as box
 plots in the figures in the paper.