AUXIN AND BRASSINOSTEROID REGULATION OF PRIMARY ROOT DEVELOPMENT IN MAIZE

The growth of the maize root has been previously shown to be controlled by hormones, specifically auxin and brassinosteroids. However, there is a gap in understanding the downstream interdependent regulatory mechanisms that these hormones utilize to signal and control maize root growth. Plant hormones have shown to have tremendous effects on plant development and modulation of their biosynthesis, transport, signaling, or perception have previously been utilized to improve plant growth and improve yield. However, investigation into their role on the below ground growth, specifically roots, has not been well studied. Identifying regulators of root growth will have tremendous effects for future maize yields because the root system is necessary to acquire water and nutrients from the soil. The goal of this proposal is to identify the mechanisms that two plant hormones utilize to control primary root growth by using a combination of developmental genetics, genome wide association studies, and mutant screens. Previously identified mutants that are deficient in the synthesis of auxin or brassinosteroids will identify the primary developmental mechanisms, cell division and/or elongation, that these hormones influence to control root growth. The genome wide association study will test the response of 282 diverse maize lines to treatment with auxin or brassinosteroids to identify downstream regulators that directly affect root growth. Lastly, a mutant screen for insensitive mutants to auxin and brassinosteroids will be conducted. This will identify specific genes that control auxin and brassinosteroid response to control root growth. The results from this work could be targets for future breeding programs to improve root development, an under studied aspect of plant growth.

Aim1a- Root kinematics of auxin and brassinosteroid (BR) mutants coupled with chemical treatments: A biochemical and genetic approach will be undertaken to determine how auxin and BRs regulate primary root growth in maize. The auxin mutant (vt2) has long roots and the BR mutant (brd1) has short roots. F1 progeny of a cross between vt2 and brd1 are currently being selfed to create double mutants. WT, vt2, brd1, and double mutants will be treated with a control treatment (0.05% dimethyl sulfoxide), 10 µM IAA (applied 3 d after germination), 10 µM IBA, or 10 nm BL for 5 d in the dark to determine the interdependency of these two hormones on root growth. The seedlings will be grown in paper towels using this cigar method. This approach includes 4 genetic samples with 4 treatments, totaling 16 different observations. The seedlings will be photographed and the primary root length will be measured using ImageJ.The 2 cm distal end of the root will be collected from 10 seedlings of each category and fixed in FAA to determine root kinematics. Cell division primarily occurs in the 2.5 mm distal region of the root, whereas cell elongation primarily occurs in the 2.5-8 mm region. Fixed roots will be infiltrated with resin, longitudinally sectioned, and mounted. The distal 1 cm end of the root will be split into 1 mm segments and 15 cortex cells within each segment will be measured to obtain an average cell length. The average local cell length will be used to calculate the displacement velocity (mm·h-1) plotted against distance from the tip of the root over the length of the distal end using the Origin graphing software. Combining genetic and biochemical treatments will dissect the auxin and BR interdependent regulation of primary root development in maize and acquiring this knowledge will measure the success of this portion of the project.Aim1b- Quantification of auxin and BRs in root division and elongation zone: Quantification of auxin and BRs in root division and elongation zone: Additional WT, vt2, brd1, and double mutants will be grown to collect the distal 2.5 mm and 2.5-8 mm regions of the root for hormone measurements. BRs will be extracted and measured using a targeted LC/MS/MS method. Free IAA and IBA will be measured using a method being developed by the MU Metabolomics Center. A total of 3 biological replicates will be extracted using a 2:1:0.002 Isopropanol:H2O:HCl buffer. A spike-in of deuterated IAA and IBA standards will be added to the samples. Samples will be purified using an Oasis HLB column, re-dissolved in 1:1 methanol:1% acetic acid, and injected into a LC/MS/MS machine for quantification. Results will be evaluated by determining the differences in hormone accumulation in the mutants to identify if there is a compensation effect of either hormone.Aim1c- Auxin and BR fluorescent reporters: To investigate the genetic interactions of auxin and BRs on root development in vivo, auxin and BR fluorescent protein reporters are currently being introgressed into the brd1 and vt2 mutant backgrounds. Using confocal microscopy, subcellular hormonal effects can be observed by fluorescent reporters. The auxin accumulation DR5::RFP and auxin transporter ZmPIN1:YFP are able to identify in vivo changes of auxin amount and transport, respectively. The BR transcription factor ZmBES1::YFP localizes to the nucleus when BRs are present and will act as an in vivo BR reporter in the vt2 and double mutants. The reporters will be assayed using confocal microscopy in the University of Missouri Microscopy Core. Combining these constructs with genetically deficient mutants for BR and auxin will test how perturbing one hormone affects the protein localization or expression of signaling components of the other hormone. Testing these hypotheses will act as a measurable of the success of this portion.Aim2a- Genomic wide association studies: To identify natural variants affecting BR and auxin root development a genome wide association (GWA) study is proposed. The 282 maize inbred lines will be obtained from collaborating mentor Ruthie Angelovici's lab at MU. The panel will be grown in paper towels using the cigar method and will be treated with or without 0.05% DMSO, 15 µM IBA, 3 µM brassinolide (BL), or 1 µM IBA and 0.1 µM BL for 5 d in the dark. The association panel will be grown in replicates of three with fifteen seeds each at 30°C in an incubator purchased as part of this fellowship. The purchased incubator can accommodate half of the association panel with four treatments. Each replicate requires two separate growing periods resulting in a total of six growing periods for the entire project. The seeds will be surface sterilized using a chlorine gas treatment. All seedlings will be photographed after 5 d of growth and root lengths will be measured using ImageJ.The average percent decrease and length difference between treatments and control will be used as input phenotypic data to perform GWA. Genotypic variant data for the 282 association panel is available from genotyping by sequencing (GBS) at 7X coverage from the Panzea consortium at CyVerse.org. To calculate variants associated with response to the three independent treatments the Fixed and random model Circulating Probability Unification (FarmCPU), will be run on the biocluster2 server at MU. Identifying candidate loci and genes will indicate a success of this portion; however, testing candidate genes is beyond the scope of this proposal but will act as preliminary data for the PD in his future lab.Aim2b- Forward genetic mutant screen to identify novel auxin and BR insensitive loci: A total of 1,800 M1 seeds were created by treating B73 pollen with ethyl methanesulfonate (EMS) to create M2 families this summer to allow for identification of recessive mutations. A total of 20 seeds from each M2 family will be grown in paper towels as described in Aim 1a. Families will be treated with 50 µM IBA or 5 µM BL. The M2 families will have mutants segregating in a 3:1 fashion, therefore any families that have 3-7 long root seedlings with the treatment will be selected for further analysis. All families that are selected in the screen will have 30 seeds replanted and tested to confirm insensitivity and segregation of the mutant phenotype. I hypothesize that I will identify mutants that are insensitive to only auxin or BR and families that are insensitive to both hormones. One mutant from each of the three categories that exhibits the highest insensitivity to hormone treatment will be carried forward for molecular characterization as part of this project.The three mutant lines that are selected will be molecularly characterized by bulk segregant analysis and next-generation sequencing approach (NGS). DNA will be extracted from a pool of 5 mutants in each family, RNase treated, and sequenced on the HiSeqX by Macrogen, Corp. The 50 Gb of sequenced reads per mutant will be aligned to the B73 reference genome (v4) using BWA in coding sequence only. SNPs between mutant samples and the B73 reference will be called using the mpileup function within the SAMtools software. Identified SNPs will be filtered for being 100% non-reference, strand bias, and G to A or C to T transitions. Remaining SNPs will be characterized for their effect on protein coding sequence using the snpEff software. Identified highly deleterious mutations will be annotated by using the maize functional annotation file at gramene.org and BLAST will be utilized to identify the closest ortholog in Arabidopsis and rice. Candidate genes will be tested for co-segregation with the mutant phenotype to confirm linkage of the SNP. To identify additional alleles, candidate loci will be sequenced in the other insensitive M2 families identified. If additional alleles are not identified then a targeted mutagenesis approach will be used.