Friend or foe: how nitrate antagonizes ammonium toxicity

Nitrogen acquisition underpins plant growth. Plants take up a significant amount of inorganic nitrogen, notably ammonium (${\text{NH}}_4^{+}$) and nitrate (${\text{NO}}_3^{-}$), from the soil. Intensive application of nitrogen fertilizers considerably improves crop yield but causes serious environmental problems, such as acidification of soil and aquifer contamination (Wang et al., 2012). Therefore, it is essential to resolve the molecular mechanisms underlying the nitrogen nutrition of plants to improve nitrogen use in agriculture.

Plants can directly assimilate ammonium after absorption, whereas nitrate needs to be reduced to ammonium before assimilation. However, culturing plants on medium with ${\text{NH}}_4^{+}$ as the exclusive nitrogen source can lead to pleiotropic deleterious effects on growth, commonly referred to as ammonium toxicity syndrome (Britto and Kronzucker, 2002). Little is known about the primary events leading to ammonium toxicity at the cellular level. As uptake of ${\text{NH}}_4^{+}$ is usually coupled with proton efflux, ammonium toxicity may be induced by acidification of the rhizosphere (Esteban et al., 2016).

Considerable attention has been given to developing strategies that enable plants to tolerate ammonium. Scientists have long recognized the presence of ${\text{NO}}_3^{-}$, even at low concentration, can effectively lessen ammonium toxicity; however, the molecular basis of nitrate-mediated ammonium tolerance is unclear (Britto and Kronzucker, 2002; Esteban et al., 2016). In Arabidopsis (Arabidopsis thaliana), the nitrate efflux channel, so-called Slow Anion Channel Homologue 3 (SLAH3), plays a key role in ammonium tolerance, as demonstrated by hypersensitivity of slah3 knockout mutants to high-ammonium/low-nitrate/acidic stress (Zheng et al., 2015).

In this issue of Plant Physiology, Sun et al. (2021) describe post-translational regulation of SLAH3 during ammonium detoxification. In a search for SLAH3-interacting partners, they identified SnRK1.1, a member of the sucrose nonfermenting-related protein kinases, by a yeast two-hybrid screen. Detailed protein interaction analyses, including bimolecular fluorescence complementation assays, revealed SnRK1.1 interacts with the C-terminus of SLAH3 at the plasma membrane (Sun et al., 2021). Although a wealth of functions have already been assigned to SnRK1 in energy homeostasis, growth, and various stress responses (Crepin and Rolland, 2019), direct involvement of SnRK1.1 in nitrate-directed ammonium detoxification had not been previously identified.

In vitro kinase assays in conjunction with LC-MS/MS analyses showed SnRK1.1 phosphorylates serine 601 (S601) localized at the C-terminal tail of SLAH3. Nitrate efflux capacity was abolished in phospho-mimicking SLAH3-S601D-expressing lines that exhibited a severe toxic phenotype under high-ammonium/low-nitrate/acidic condition. By contrast, phospho-dead SLAH3-S601A-expressing lines displayed a wild-type-like phenotype. These data show C-terminal phosphorylation by SnRK1.1 negatively regulates SnRK1.1. By contrast, N-terminal phosphorylation of SLAH3 by protein kinases, such as calcium protein kinases, SnRK2s, calcineurin B-like-interacting protein kinases, and PBL27, is essential for activation of SLAH3 (Liu et al., 2019; Geiger et al., 2011; Kudla et al., 2018).

Subcellular localization of SnRK1.1 is tightly regulated by cellular energy status. Low energy stress caused by darkness, arrested photosynthesis, or hypoxia can trigger the migration of SnRK1 from the cytoplasm to the nucleus (Ramon et al., 2019). Sun et al. showed nuclear translocation of SnRK1.1 also occurs under high-ammonium/low-nitrate/acidic condition.

Taken together, these results support a model for SLAH3-directed ammonium detoxification (Figure 1). In the absence of high-ammonium stress, negative regulation of SLAH3 by SnRK1.1 prevents nitrate loss in the root. In response to high-ammonium/low-nitrate/acidic stress, nuclear translocation of SnRK1.1 prevents its inhibitory effect on SLAH3, activating SLAH3 channel activity to pump nitrate out of the root cell. The authors propose this regulated nitrate efflux is a strategy to alleviate the inhibitory acidification of the rhizosphere, because most nitrate influx transporters are ${\text{NO}}_3^{-}$/H⁺ symporters, mediating ${\text{NO}}_3^{-}$ uptake accompanied by H⁺ influx (Wang et al., 2012). They hypothesize the accumulation of nitrate in the rhizosphere accompanied by proton uptake is sufficient to alleviate ammonium toxicity.

Figure 1.

Model for SnRK1.1-mediated post-translational regulation of SLAH3. A, In the absence of high-ammonium stress, SnRK1.1 phosphorylates S601 at the C-terminus of SLAH3 at the plasma membrane to inhibit SLAH3 activity and in turn prevent nitrate loss. Currently, it is unclear when SnRK1.1 is activated, but SnRK1.1 could be active under low sugar conditions. B, In response to high-ammonium/low-nitrate/acidic stress, nuclear translocation of SnRK1.1 releases its inhibition on SLAH3. Meanwhile, an unknown protein kinase phosphorylates the N-terminus of SLAH3 to activate SLAH3 activity, leading to nitrate efflux. High-ammonium stress may be coupled with severe energy deficiency, which further triggers the nuclear translocation of SnRK1.1 and activates SnRK1.1 to promote the expression of stress-responsive genes through the phosphorylation of an SnRK1.1-targeted transcriptional factor (TF). Figure based on figure 11 from Sun et al. (2021).

In addition to improving our understanding of SLAH3-mediated ammonium detoxification, this study also raises several important questions. First, although several protein kinases phosphorylate different sites of SLAH3, the phosphatase responsible for dephosphorylation of SLAH3, particularly in response to high-ammonium stress, remains to be elucidated.

Furthermore, given activation of SnRK1.1 is regulated by cellular energy status, which conditions activate SnRK1.1 to phosphorylate SLAH3 remain unknown. Notably, Sun et al. observed supplementation of lower concentration (<1%) of metabolizable sugars, such as sucrose and glucose, in the medium antagonized the toxic phenotype of slah3 mutants and the SnRK1.1 overexpression line under the same high-ammonium/low-nitrate/acidic condition. These results imply the SnRK1-derived low energy response may participate in nitrate-dependent alleviation of ammonium toxicity. Therefore, it is worthwhile to investigate whether nuclear-localized SnRK1.1-derived transcriptional regulation contributes to ammonium detoxification, with the potential also to decipher the coordination between energy homeostasis and nutrient usage in plants.