Auxin is a key regulator of root morphogenesis across angiosperms. To better understand auxin-regulated networks underlying maize root development, we have characterized auxin-responsive transcription across two time points (30 and 120 min) and four regions of the primary root: the meristematic zone, elongation zone, cortex and stele. Hundreds of auxin-regulated genes involved in diverse biological processes were quantified in these different root regions. In general, most auxin-regulated genes are region unique and are predominantly observed in differentiated tissues compared with the root meristem. Auxin gene regulatory networks were reconstructed with these data to identify key transcription factors that may underlie auxin responses in maize roots. Additionally, Auxin-Response Factor subnetworks were generated to identify target genes that exhibit tissue or temporal specificity in response to auxin. These networks describe novel molecular connections underlying maize root development and provide a foundation for functional genomic studies in a key crop.

Root system architecture is a critical factor in maize health and stress resilience. Determining the genetic and environmental factors that shape maize root system architecture is an active research area. However, the ability to phenotype juvenile root systems is hindered by the use of field‐grown and soil‐based systems. An alternative to soil‐ and field‐based growing conditions for maize seedlings is a controlled environment with a soil‐free medium, which can facilitate root system phenotyping. Here, we describe how to grow maize under soil‐free conditions for up to 12 days to facilitate root phenotyping. Maize seeds are sterilized and planted on specialized seed germination paper to minimize fungal contamination and ensure synchronized seedling growth, followed by imaging at the desired time point. The root images are then analyzed to quantify traits of interest, such as primary root length, lateral root density, seminal root length, and seminal root number. In addition, juvenile shoot traits can be quantified using manual annotation methods. We also outline the steps for performing rigorous hormone response assays for four classical phytohormones: auxin, brassinosteroid, cytokinin, and jasmonic acid. This protocol can be rapidly scaled up and is compatible with genetic screens and sample collection for downstream molecular analyses such as transcriptomics and proteomics. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Maize seedling rolled towel assay and phenotyping Basic Protocol 2: Maize seedling hormone response assays using the rolled towel assay

Auxin is a key regulator of root morphogenesis across angiosperms. To better understand auxin regulated networks underlying maize root development we have characterized auxin responsive transcription across two time points (30 and 120 minutes) and four regions of the primary root: the meristematic zone, elongation zone, cortex, and stele. Hundreds of auxin-regulated genes involved in diverse biological processes were quantified in these different root regions. In general, most auxin regulated genes are region unique and are predominantly observed in differentiated tissues compared to the root meristem. Auxin gene regulatory networks (GRNs) were reconstructed with these data to identify key transcription factors that may underlie auxin responses in maize roots. Additionally, Auxin Response Factor (ARF) subnetworks were generated to identify target genes which exhibit tissue or temporal specificity in response to auxin. These networks describe novel molecular connections underlying maize root development and provide a foundation for functional genomic studies in a key crop.