Validation of a high-confidence regulatory network for gene-to-NUE phenotype in field-grown rice

Nitrogen (N) and Water (W) - two resources critical for crop productivity – are becoming increasingly limited in soils globally. To address this issue, we aim to uncover the gene regulatory networks (GRNs) that regulate nitrogen use efficiency (NUE) - as a function of water availability - in Oryza sativa, a staple for 3.5 billion people. In this study, we infer and validate GRNs that correlate with rice NUE phenotypes affected by N-by-W availability in the field. We did this by exploiting RNA-seq and crop phenotype data from 19 rice varieties grown in a 2x2 N-by-W matrix in the field. First, to identify gene-to-NUE field phenotypes, we analyzed these datasets using weighted gene co-expression network analysis (WGCNA). This identified two network modules ("skyblue" & "grey60") highly correlated with NUE grain yield (NUEg). Next, we focused on 90 TFs contained in these two NUEg modules and predicted their genome-wide targets using the N-and/or-W response datasets using a random forest network inference approach (GENIE3). Next, to validate the GENIE3 TF→target gene predictions, we performed Precision/Recall Analysis (AUPR) using nine datasets for three TFs validated in planta. This analysis sets a precision threshold of 0.31, used to "prune" the GENIE3 network for high-confidence TF→target gene edges, comprising 88 TFs and 5,716 N-and/or-W response genes. Next, we ranked these 88 TFs based on their significant influence on NUEg target genes responsive to N and/or W signaling. This resulted in a list of 18 prioritized TFs that regulate 551 NUEg target genes responsive to N and/or W signals. We validated the direct regulated targets of two of these candidate NUEg TFs in a plant cell-based TF assay called TARGET, for which we also had in planta data for comparison. Gene ontology analysis revealed that 6/18 NUEg TFs - OsbZIP23 (LOC_Os02g52780), Oshox22 (LOC_Os04g45810), LOB39 (LOC_Os03g41330), Oshox13 (LOC_Os03g08960), LOC_Os11g38870, and LOC_Os06g14670 - regulate genes annotated for N and/or W signaling. Our results show that OsbZIP23 and Oshox22, known regulators of drought tolerance, also coordinate W-responses with NUEg. This validated network can aid in developing/breeding rice with improved yield on marginal, low N-input, drought-prone soils

Local Changes in Chromatin Accessibility and Transcriptional Networks Underlying the Nitrate Response in Arabidopsis Roots

Transcriptional regulation, determined by the chromatin structure and regulatory elements interacting at promoter regions, is a key step in plant responses to environmental cues. Nitrate (NO3-) is a nutrient signal that regulates the expression of hundreds of genes in Arabidopsis thaliana. Here, we integrate mRNA sequencing, genome-wide RNA polymerase II (RNPII), chromatin immunoprecipitation sequencing, and DNase sequencing datasets to establish the relationship between RNPII occupancy and chromatin accessibility in response to NO3- treatments in Arabidopsis roots. Genomic footprinting allowed us to identify in vivo regulatory elements controlling gene expression in response to NO3- treatments. NO3--modulated transcription factor (TF) footprints are important for a rapid increase in RNPII occupancy and transcript accumulation over time. We mapped key TF regulatory interactions and functionally validated the role of NAP, an NAC-domain containing TF, as a new regulatory factor in NO3- transport. Taken together, our study provides a comprehensive view of transcriptional networks in response to a nutrient signal in Arabidopsis roots.This work is funded by Instituto Milenio iBio - Iniciativa Científica Milenio MINECON, Chile; by grants from the Fondo de Desarrollo de Areas Prioritarias (FONDAP) Center for Genome Regulation (15090007), Chile; and Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) (1180759), Chile; to R.A.G. J.M.A. is supported by postdoctoral grant FONDECYT (3140336), Chile.J.J. is funded by grant MCB-1412948 from the National Science Foundation, United States; and J.M. by funding from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Spain (RTA2015-00014-c02-01). We also want to acknowledge the “Severo Ochoa Program for Centers of Excellence in R&D” from the Agencia Estatal de Investigación of Spain (SEV-2016-0672 (2017–2021)) for supporting the scientific services used in this work.Peer reviewe

Local changes in chromatin accessibility and transcriptional networks underlying the nitrate response in Arabidopsis roots

Transcriptional regulation, determined by chromatin structure and regulatory elements interacting at promoter regions, is a key step in plant responses to environmental cues. Nitrate (NO3-) is a nutrient signal that regulates the expression of hundreds of genes in Arabidopsis thaliana. Here we integrate mRNA-seq, genome-wide RNA polymerase II (RNPII), ChIP-Seq and DNase-seq data sets to establish the relationship between RNPII occupancy and chromatin accessibility in response to NO3- treatments in Arabidopsis root organs. Genomic footprinting allowed us to identify in vivo regulatory elements controlling gene expression in response to NO3- treatments. NO3--modulated TF footprints are important for a rapid increase in RNPII occupancy and transcript accumulation over time. We mapped key TF regulatory interactions and functionally validated the role of NAP, a NAC-domain containing TF, as a new regulatory factor in NO3- transport. Our strategy provides a comprehensive view of transcriptional networks in response to a nutrient signal in Arabidopsis roots.Fil: Alvarez, José M.. Pontificia Universidad Católica de Chile; ChileFil: Moyano, Tomás C.. Pontificia Universidad Católica de Chile; ChileFil: Zhang, Tao. Yangzhou University; ChinaFil: Gras, Diana Ester. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Herrera, Francisco J.. University of California at Berkeley; Estados UnidosFil: Araus, Viviana. Pontificia Universidad Católica de Chile; ChileFil: OBrien, José A.. Pontificia Universidad Católica de Chile; ChileFil: Carrillo, Laura. Universidad Politécnica de Madrid; EspañaFil: Medina, Joaquín. Universidad Politécnica de Madrid; EspañaFil: Vicente-Carbajosa, Jesús. Universidad Politécnica de Madrid; EspañaFil: Jiang, Jiming. University Of Wisconsin-madison; Estados UnidosFil: Gutiérrez, Rodrigo A.. Pontificia Universidad Católica de Chile; Chil