Nitrate alleviates ammonium toxicity in Brassica napus by coordinating rhizosphere and cell pH and ammonium assimilation

Abstract

In natural and agricultural situations, ammonium ( ${\mathrm{NH}}_4^{+}$ ) is a preferred nitrogen (N) source for plants, but excessive amounts can be hazardous to them, known as ${\mathrm{NH}}_4^{+}$ toxicity. Nitrate ( ${\mathrm{NO}}_3^{-}$ ) has long been recognized to reduce ${\mathrm{NH}}_4^{+}$ toxicity. However, little is known about Brassica napus, a major oil crop that is sensitive to high ${\mathrm{NH}}_4^{+}$ . Here, we found that ${\mathrm{NO}}_3^{-}$ can mitigate ${\mathrm{NH}}_4^{+}$ toxicity by balancing rhizosphere and intracellular pH and accelerating ammonium assimilation in B. napus. ${\mathrm{NO}}_3^{-}$ increased the uptake of ${\mathrm{NO}}_3^{-}$ and ${\mathrm{NH}}_4^{+}$ under high ${\mathrm{NH}}_4^{+}$ circumstances by triggering the expression of ${\mathrm{NO}}_3^{-}$ and ${\mathrm{NH}}_4^{+}$ transporters, while ${\mathrm{NO}}_3^{-}$ and H⁺ efflux from the cytoplasm to the apoplast was enhanced by promoting the expression of ${\mathrm{NO}}_3^{-}$ efflux transporters and genes encoding plasma membrane H⁺ -ATPase. In addition, ${\mathrm{NO}}_3^{-}$ increased pH in the cytosol, vacuole, and rhizosphere, and down-regulated genes induced by acid stress. Root glutamine synthetase (GS) activity was elevated by ${\mathrm{NO}}_3^{-}$ under high ${\mathrm{NH}}_4^{+}$ conditions to enhance the assimilation of ${\mathrm{NH}}_4^{+}$ into amino acids, thereby reducing ${\mathrm{NH}}_4^{+}$ accumulation and translocation to shoot in rapeseed. In addition, root GS activity was highly dependent on the environmental pH. ${\mathrm{NO}}_3^{-}$ might induce metabolites involved in amino acid biosynthesis and malate metabolism in the tricarboxylic acid cycle, and inhibit phenylpropanoid metabolism to mitigate ${\mathrm{NH}}_4^{+}$ toxicity. Collectively, our results indicate that ${\mathrm{NO}}_3^{-}$ balances both rhizosphere and intracellular pH via effective ${\mathrm{NO}}_3^{-}$ transmembrane cycling, accelerates ${\mathrm{NH}}_4^{+}$ assimilation, and up-regulates malate metabolism to mitigate ${\mathrm{NH}}_4^{+}$ toxicity in oilseed rape.

Keywords: Brassica napus; ammonium assimilation; ammonium toxicity; nitrate transmembrane cycling; pH balance; proton flux.