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Summary: Plant root system is significantly influenced by high soil levels of ammonium nitrogen, leading to reduced root elongation and enhanced lateral root branching. In Arabidopsis, these processes have been reported to be mediated by phytohormones and their downstream signaling pathways, while the controlling mechanisms remain elusive in crops.Through a transcriptome analysis of roots subjected to high/low ammonium treatments, we identified a cytokinin oxidase/dehydrogenase encoding gene, CKX3, whose expression is induced by high ammonium. Knocking out CKX3 and its homologue CKX8 results in shorter seminal roots, fewer lateral roots, and reduced sensitivity to high ammonium.Endogenous cytokinin levels are elevated by high ammonium or in ckx3 mutants. Cytokinin application results in shorter seminal roots and fewer lateral roots in wild‐type, mimicking the root responses of ckx3 mutants to high ammonium. Furthermore, CKX3 is transcriptionally activated by type‐B RR25 and RR26, and ckx3 mutants have reduced auxin content and signaling in roots under low ammonium.This study identified RR25/26‐CKX3‐cytokinin as a signal module that mediates root responses to external ammonium by modulating of auxin signaling in the root meristem and lateral root primordium. This highlights the critical role of cytokinin metabolism in regulating rice root development in response to ammonium. [ABSTRACT FROM AUTHOR]

SUMMARY: Lateral roots are crucial for plant growth and development, making them an important target for research aiming to improve crop yields and food security. However, their endogenous ontogeny and, as it were, stochastic appearance challenge their study. Lateral Root Inducible Systems (LRIS) can be used to overcome these challenges by inducing lateral roots massively and synchronously. The combination of LRISs with transcriptomic approaches significantly advanced our insights in the molecular control of lateral root formation, in particular for Arabidopsis. Despite this success, LRISs have been underutilized for other plant species or for lateral root developmental stages later than the initiation. In this study, we developed and/or adapted LRISs in rice, Medicago, and Arabidopsis to perform RNA‐sequencing during time courses that cover different developmental stages of lateral root formation and primordium development. As such, our study provides three extensive datasets of gene expression profiles during lateral root development in three different plant species. The three LRISs are highly effective but timing and spatial distribution of lateral root induction vary among the species. Detailed characterization of the stages in time and space in the respective species enabled an interspecies co‐expression analysis to identify conserved players involved in lateral root development, as illustrated for the AUX/IAA and LBD gene families. Overall, our results provide a valuable resource to identify potentially conserved regulatory mechanisms in lateral root development, and as such will contribute to a better understanding of the complex regulatory network underlying lateral root development. Significance Statement: Transcriptome and gene co‐expression analysis during lateral root development in three different species provided datasets that can be used to identify key regulatory genes and pathways involved in lateral root development in rice and Medicago, and could assist cereal and legume translational research. [ABSTRACT FROM AUTHOR]

SUMMARY: High levels of ammonium nutrition reduce plant growth and different plant species have developed distinct strategies to maximize ammonium acquisition while alleviating ammonium toxicity through modulating root growth. To date, the mechanisms underlying plant tolerance or sensitivity towards ammonium remain unclear. Rice (Oryza sativa) uses ammonium as its main N source. Here we show that ammonium supply restricts rice root elongation and induces a helical growth pattern, which is attributed to root acidification resulting from ammonium uptake. Ammonium‐induced low pH triggers the asymmetric distribution of auxin in rice root tips through changes in auxin signaling, thereby inducing a helical growth response. Blocking auxin signaling completely inhibited this root response. In contrast, this root response is not activated in ammonium‐treated Arabidopsis. Acidification of Arabidopsis roots leads to the protonation of indole‐3‐acetic acid and dampening of the intracellular auxin signaling levels that are required for maintaining root growth. Our study suggests a different mode of action by ammonium on the root pattern and auxin response machinery in rice versus Arabidopsis, and the rice‐specific helical root response towards ammonium is an expression of the ability of rice to moderate auxin signaling and root growth to utilize ammonium while confronting acidic stress. Significance Statement: In this study, we discovered a helical response of the rice root in response to the acidic stress resulting from root ammonium uptake. Ammonium‐induced acidic stress triggers the asymmetric distribution of auxin at rice root tips through modulating auxin signaling pathways, thereby inducing a helical root response. [ABSTRACT FROM AUTHOR]