Summary Ammonium (NH4+) is toxic to root growth in most plants, even at moderate concentrations. Transcriptional regulation is one of the most important mechanisms in the response of plants to NH4+ toxicity, but the nature of the involvement of transcription factors (TFs) in this regulation remains unclear. Here, RNA‐seq analysis was performed on Arabidopsis roots to screen for ammonium‐responsive TFs. WRKY46, the member of the WRKY transcription factor family most responsive to NH4+, was selected. We defined the role of WRKY46 using mutation and overexpression assays, and characterized the regulation of NUDX9 and indole‐3‐acetic acid (IAA)‐conjugating genes by WRKY46 via yeast one‐hybrid and electrophoretic mobility shift assays and chromatin immunoprecipitation‐quantitative real‐time polymerase chain reaction (ChIP‐qPCR). Knockout of WRKY46 increased, while overexpression of WRKY46 decreased, NH4+‐suppression of the primary root. WRKY46 is shown to directly bind to the promoters of the NUDX9 and IAA‐conjugating genes (GH3.1, GH3.6, UGT75D1, UGT84B2) and to inhibit their transcription, thus positively regulating free IAA content and stabilizing protein N‐glycosylation, leading to an inhibition of NH4+ efflux in the root elongation zone (EZ). We identify TF involvement in the regulation of NH4+ efflux in the EZ, and show that WRKY46 inhibits NH4+ efflux by negative regulation of NUDX9 and IAA‐conjugating genes.

Rice is known for its superior adaptation to ammonium (NH 4 + ) as a nitrogen source. Compared to many other cereals, it displays lower NH 4 + efflux in roots and higher nitrogen-use efficiency on NH 4 + . A critical role for GDP-mannose pyrophosphorylase (VTC1) in controlling root NH 4 + fluxes was previously documented in Arabidopsis, but the molecular pathways involved in regulating VTC1-dependent NH 4 + efflux remain unclear. Here, we report that ETHYLENE-INSENSITIVE3-LIKE1 (OsEIL1) acts as a key transcription factor regulating OsVTC1-3-dependent NH 4 + efflux and protein N-glycosylation in rice grown under NH 4 + nutrition. We show that OsEIL1 in rice plays a contrasting role to Arabidopsis-homologous ETHYLENE-INSENSITIVE3 (AtEIN3) and maintains rice growth under NH 4 + by stabilizing protein N-glycosylation and reducing root NH 4 + efflux. OsEIL1 constrains NH 4 + efflux by activation of OsVTC1-3, but not OsVTC1-1 or OsVTC1-8. OsEIL1 binds directly to the promoter EIN3-binding site (EBS) of OsVTC1-3 in vitro and in vivo and acts to increase the transcription of OsVTC1-3. Our work demonstrates an important link between excessive root NH 4 + efflux and OsVTC1-3-mediated protein N-glycosylation in rice grown under NH 4 + nutrition and identifies OsEIL1 as a direct genetic regulator of OsVTC1-3 expression.

Plant growth and development depends on sufficient nutrient availability in soils. Agricultural soils are generally nitrogen (N) deficient and thus, soils need to be supplemented with fertilizers. Ammonium (NH4  +) is a major inorganic N source. However, at high concentrations ammonium nutrition becomes a stress situation that inhibits plant growth. The cause of ammonium stress or toxicity is multi-factorial but the interaction with other nutrients is among the main determinants of plants sensitivity towards high NH4  + supply. In addition, NH4  + uptake and assimilation provokes the acidification of the cell external medium (apoplast/rhizosphere), which has a clear impact on nutrients availability. This review summarizes current knowledge, at both physiological and molecular level, of ammonium nutrition interaction with essential mineral elements absorbed as cations: macronutrients (K +, Ca 2+, Mg 2+) and micronutrients (Fe 2+/3+, Mn 2+, Cu +/2+, Zn 2+, Ni 2+). We hypothesize that considering these nutritional interactions, and soil pH, when formulating fertilizers may be key in order to boost the use of NH4  +-based fertilizers, which have less environmental impact compared to the nitrate-based ones. In addition, we are convinced that better understanding these interactions will help to identify novel targets with potential to improve crop productivity.