New Molecular Mechanisms of Plant Response to Ammonium Nutrition

Ammonium (NH₄⁺) and nitrate (NO₃⁻) are two major inorganic nitrogen (N) forms for plants. In contrast to NH₄⁺, NO₃⁻ is highly mobile in soils and is easily leached into groundwater and open water bodies, contributing to eutrophication [1]. Furthermore, NO₃⁻ must be reduced to NH₄⁺ in plant cells before it can be utilized by plants in metabolism. This process can consume 12–26% of photosynthetically generated energy [2]. Recent studies have shown that elevated carbon dioxide (CO₂) in the atmosphere can inhibit the assimilation of NO₃⁻ in crop plants and algae, potentially further decreasing the utilization efficiency for NO₃⁻ in future climates. However, it is puzzling that the theoretically preferred source of N is instead toxic to plants [3]. Therefore, it is important to study the molecular mechanisms of plant responses to NH₄⁺ nutrient stress, and to coordinate NH₄⁺ uptake and mitigate NH₄⁺ toxicity. In addition, the effective mitigation of NH₄⁺ toxicity and improvements in crop NH₄⁺ tolerance are also crucial for the application of NH₄⁺-N fertilizers in agriculture. In recent years, many advances have been made in the study of plant responses to NH₄⁺ nutrient stress.

(i)

Which compound, NH₄⁺ or glutamine (Gln), accumulates and leads to NH₄⁺ toxicity?

High intracellular concentrations of free NH₄⁺ can lead to NH₄⁺ toxicity, which may also be exacerbated by metabolic disturbances from excessive NH₄⁺ assimilation. A recent study indicates that the knockout of AtGLN2 enhances tolerance to elevated NH₄⁺ stress, despite the associated increase in free NH₄⁺ accumulation [4]. Under high NH₄⁺ conditions, the reaction of NH₄⁺ to Gln catalyzed by shoot AtGLN2 produced an abundance of H⁺ within the cytosol, thereby inducing acidic stress in shoots. This observation suggests that acidic stress may serve as a principal contributor to NH₄⁺ toxicity [4]. In rice, mutations in argininosuccinate lyase (ASL) render the roots more susceptible to NH₄⁺ stress. Subsequent investigations reveal that ASL mitigates NH₄⁺ toxicity by promoting the conversion of excess Gln to arginine in the presence of high NH₄⁺ stress [5]. In summary, the data imply that the metabolic consequences of excess NH₄⁺, specifically Gln and H⁺ accumulation, may pose a greater risk to plants under high NH₄⁺ stress than the accumulation of free NH₄⁺ alone.

(ii)

How do plants modulate NH₄⁺ uptake in response to elevated NH₄⁺ concentrations?

Optimal regulation of NH₄⁺ uptake from the environment plays a pivotal role in minimizing intracellular NH₄⁺ concentrations, thereby alleviating potential NH₄⁺ toxicity. A previous study has demonstrated that that the protein kinase AtCIPK23 can inhibit NH₄⁺ uptake through the phosphorylation of a conserved threonine residue located in the C-terminus of AtAMT1;1 and AtAMT1;2 [6]. Subsequent studies have elucidated that the transcription factor AtSTOP1 is capable of directly binding to the AtCIPK23 promoter, leading to its transcriptional activation. This, in turn, suppresses the transcription of both AtAMT1;1 and AtAMT1;2 [7]. Furthermore, another protein kinase, AtCIPK15, has been identified to phosphorylate the C-termini of AtAMT1;1, AtAMT1;2, and AtAMT1;3, consequently inhibiting their NH₄⁺ transport functions [8].

(iii)

How do phytohormones function in response to NH₄+ toxicity?

Previous research has demonstrated that elevated NH₄⁺ levels lead to a reduction in endogenous free IAA content, while the application of low concentrations of exogenous IAA can mitigate NH₄⁺ toxicity [9]. Our recent studies indicate that elevated NH₄⁺ diminishes free IAA content by enhancing IAA conjugation rather than by inhibiting IAA biosynthesis. Additionally, the transcription factor AtWRKY46 plays a pivotal role, as it directly suppresses the transcription of IAA-conjugating genes [10]. In addition, exogenous BR treatment significantly suppresses AtPIN2 expression and the nuclear auxin signal under high NH₄⁺ stress, indicating that auxin operates downstream of the BR signaling pathway in response to elevated NH₄⁺ stress [9].

Beyond IAA and BR, ABA is required for protecting chloroplast and root growth under high NH₄⁺ stress [11]. Our recent study shows that high NH₄⁺ induces ABA biosynthesis and activates the ‘OsSAPK9-OsbZIP20’ module, which in turn augments reactive oxygen species (ROS) scavengers and bolsters NH₄⁺ assimilation, thereby minimizing ROS and free NH₄⁺ accumulation in rice roots [11]. Intriguingly, the ABA signaling elicited by high NH₄⁺ levels can also disrupt the interaction between AtABI1 and AtCIPK23 by inactivating AtABI1. As a consequence, the liberated AtCIPK23 is then able to further phosphorylate and inhibit AtAMTs in Arabidopsis [12].

(iv)

How does NH₄⁺ stress induce a burst of ROS?

High-NH₄⁺-triggered ROS accumulation has been reported in a previous study; however, the mechanisms underlying the NH₄⁺-induced ROS burst remain unclear [13]. Interestingly, two recent studies have proposed that iron (Fe) accumulation might be the primary driver of the NH₄⁺-induced ROS burst. When compared to NO₃⁻, NH₄⁺ induces greater Fe accumulation in the apoplast of the phloem, resulting in a ROS burst. This process appears to involve a cell wall-localized ferroxidase known as LPR2. Both the knockout of AtLPR2 or reduction in Fe supplementation can significantly enhance root growth tolerance to high NH₄⁺ stress [14]. Moreover, the acidification of the apoplast, caused by NH₄⁺ uptake, results in Fe precipitation in the elongation and differentiation zones of the root tip, which, in turn, induces hydrogen peroxide (H₂O₂) accumulation and NH₄⁺ toxicity. The knockout of AtPDX1.1, an enzyme involved in vitamin B6 biosynthesis, or application of exogenous vitamin B6 could quench ROS and partially rescue root growth under NH₄⁺ stress [15].

(v)

How do roots regulate NH₄⁺/H⁺ efflux in response to NH₄⁺ uptake?

The capacity of NO₃⁻ to mitigate NH₄⁺ toxicity has been documented in many species; however, the underlying molecular mechanism remains elusive. Earlier investigations have demonstrated that NH₄⁺ uptake results in rhizosphere acidification, whereas NO₃⁻ uptake leads to its alkalinization [16,17]. Thus, it is worth exploring whether NO₃⁻ counteracts the rhizosphere acidification instigated by NH₄⁺ absorption and subsequently mitigates NH₄⁺ toxicity. Recently, a team from China found that AtSLAH3 (a NO₃⁻ efflux channel) interacts with AtNRT1.1 (a NO₃⁻/H⁺ symporter) to form a ‘transporter-channel’ complex to manipulate NH₄⁺ influx, NO₃⁻ influx/efflux, and H⁺ influx through the membrane [18,19]. Consequently, the AtSLAH3/AtNRT1.1 complex enhances tolerance against NH₄⁺ uptake-induced rhizosphere acidification.

Beyond the rhizosphere, futile NH₄⁺ efflux, triggered by NH₄⁺ absorption, is also a predominant factor contributing to NH₄⁺ toxicity [10]. Our team recently identified a transcription factor, AtWRKY46, that curtails this futile NH₄⁺ efflux in two distinct ways: Firstly, AtWRKY46 directly downregulates AtNUDX9, preserving the levels of N-glycosylated proteins and subsequently impeding futile NH₄⁺ efflux. Secondly, AtWRKY46 directly suppresses the IAA-conjugating genes AtGH3.1/GH3.6/UGT75D1/ UGT84B2, ensuring the maintenance of free IAA levels and inhibiting the futile NH₄⁺ efflux from the roots [10].

As previously established, NH₄⁺ uptake leads to an increase in H⁺ efflux, and IAA also promotes H⁺ efflux by phosphorylating the proton pump. Therefore, it is of interest to further identify the relationships between free IAA, H⁺ and NH₄⁺ efflux. Our recent results further show that H⁺ secreted in the extracellular compartment can promote the NH₄⁺ efflux, while the PM H⁺-ATPase knockout mutants Ataha1-7 and Ataha2-5 exhibit lower NH₄⁺ efflux compared with wild-type. Intriguingly, a mutation in AtPIN5, an IAA transporter responsible for IAA translocation from the cytosol to the ER lumen, leads to enhanced cytosolic IAA accumulation, consequentially increasing H⁺ efflux and decreasing NH₄⁺ flux. The application of the PM H⁺-ATPase inhibitor, vanadate, in the medium further attenuates both H⁺ and NH₄⁺ efflux in Atpin5. This suggests that AtPIN5 may play a pivotal role in synchronously modulating H⁺ and NH₄⁺ efflux by regulating free IAA levels in the cytoplasm [20].

Additionally, our study identified a transcription factor, OsEIL1, in rice, which has been demonstrated to directly associate with the promoter of OsVTC1.3 (a homolog of AtVTC1). This binding promotes transcription, maintaining the levels of N-glycosylated proteins in roots, which in turn mitigates unnecessary NH₄⁺ efflux. Notably, this regulatory paradigm is absent in Arabidopsis, potentially offering an explanation as to why rice exhibits greater NH₄⁺ tolerance compared to Arabidopsis [21].

In summation, significant advancements have been made in discerning the molecular underpinnings of NH₄⁺ toxicity in recent times. This not only provides a theoretical groundwork for future explorations into plant NH₄⁺ response mechanisms but also holds promise for enhancing plant NH₄⁺ resilience in agriculture.