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

Abstract

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.

Nitrogen sensing and regulatory networks: it's about time and space

“…These conclusions were determined using linear models that analyzed RNA-seq and phenotypic data from rice exposed to a factorial matrix of N-by-W conditions of different rice varieties in both laboratory and field conditions ( Swift et al 2019 ). Using this N-by-W expression and phenotype dataset, Shanks et al 2022 identified the TFs, OsbZIP23, and Oshox22 as regulators of NUE grain yield (NUEg) by developing gene regulatory networks that linked TFs to target genes to field NUEg phenotypes ( Swift et al 2019 ; Shanks et al 2022 ).…”

“…To facilitate these analyses, the open-source ConnecTF web platform includes the validated TF-target gene data generated using the plant cell-based TARGET system, along with published in planta TF perturbation data, ChIP-seq, and DAP-seq data. Examples of how ConnecTF can be used to develop high-confidence networks using AUPR analysis have been published for Arabidopsis ( Brooks et al 2021 ) and rice ( Shanks et al 2022 ).…”

Insights into molecular links and transcription networks integrating drought stress and nitrogen signaling

“…Nonetheless, recent research has revealed regulatory links between N, ABA, and drought in rice. Specifically, the rice ABF1 (OsABF1) TF plays a role in governing the expression of genes influenced by both drought and N (Shanks et al ., 2022). OsABF1 functions within a network module that bridges gene expression to phenotypic responses under conditions of N and drought (Shanks et al ., 2022).…”

Overexpression ofPpGL2fromPrunus persicaenhanced soybean drought tolerance

“…Studies on the expression of four genes in yellow cauliflower reveal that CpHB4 and CpHB5 are downregulated by drought but unresponsive to ABA, whereas CpHB6 and CpPH7 are induced by both drought and ABA stress [20]. In rice, the HD-ZIP transcription factor OsHOX22 affects ABA biosynthesis and regulates drought and salt stress via ABA-mediated signaling pathways [21,22]. Ariel et al [23] found that salt stress induces the expression of HD-ZIP protein MtHB1 in the protoplast and lateral root meristem of Medicago truncata.…”