Nitrogen is both the basis of metabolism and the primary determinant of growth and yield (Lawlor, 2001). Nitrogen is a major component of nucleic acids, proteins, chlorophyll, and other substances, and is involved in many physiological and biological processes in plant growth and metabolism, including photosynthesis, carbohydrate allocation, and root formation (Ohyama, 2010, Viktor and Cramer, 2005). Plant nitrogen metabolism can also regulate the antioxidant system (Zhao, 2009). Thus, nitrogen is clearly an essential nutrient for plant growth.
Soils include inorganic nitrogen in the form of ammonium and nitrate, and organic nitrogen as amino acids, peptides, and proteins, with inorganic nitrogen more readily absorbed by plants (Jackson et al., 2008, Patterson et al., 2010). Ammonium is a major inorganic nitrogen source in most soils, its assimilation by plants requires less energy than nitrate, and plants prefer to take up ammonium when the external nitrogen concentration is low (Bloom, 1997, Noctor et al., 1998).
Inorganic nitrogen can be used for the metabolism of organic compounds in the form of ammonium, and non-ammonium nitrogen sources are generally converted to ammonium before amino synthesis (Xu et al., 2012). Oxidative deamination of glutamate is catalyzed by glutamate dehydrogenase (GDH), and is generally involved in the oxidative decomposition of amino acids rather than their synthesis (Lopes et al., 2015). Asparaginase (ASN) catalyzes the hydrolysis of asparagine to produce aspartate and ammonia, and participates in nitrogen fixation (Lopes et al., 2015, Atkins et al., 1975). Glutaminase (GLS) catalyzes the formation of glutamate from glutamine as part of amino acid catabolism (Yang et al., 2017). These enzymes play important roles in the process of amino acid cycling in plants, indirectly facilitating the uptake and fixation of ammonium.
Ammonium transporters (AMTs) belong to the Ammonium transporter/Methylammonium permease/Rhesus (AMT/MEP/Rh) gene family, members of which have been identified in plants, microorganisms, and animals, indicating that ammonium transporter proteins are widely distributed in living organisms (Marini et al., 1997). Two major groups of ammonium transporter proteins have been identified in plants: the AMT1 and AMT2 subfamilies (Couturier et al., 2007). The plant AMT2 subfamily is more distantly related to the plant AMT1 subfamily (Guether et al., 2009), and in Arabidopsis, AtAMT2 is likely to play a significant role in moving ammonium (Sohlenkamp et al., 2002). Additional members of these families have also been characterized in Arabidopsis. AtAMT1;3, AtAMT1;4 and AtAMT1;5 exhibit high affinity for ammonium (Lopez-Pedrosa et al., 2006, Yuan et al., 2007), and AtAMT1;2 exhibits a relatively low affinity for ammonium (Neuhauser et al., 2007). A plasma membrane NH4+ channel Ammonium Facilitator 1 (AMF1) has also been found to regulate plasma membrane permeability to NH4+ and NH4+ uptake indirectly through AMT/MEP/Rh (Mazurkiewicz, 2013).
The transcriptional regulation of ammonium uptake and utilization is driven by a series of transcription factors. In rice, transcription factor Indeterminate domain 10 (OsIDD10) binds to a cis-element motif present in the promoter region of OsAMT1;2 to specifically activate expression. In Arabidipsis, transcription factor Long Hypocotyles 5 (HY5) negatively regulates the expression of AtAMT1;2, an orthologous gene of OsAMT1;2 (Huang et al., 2015). Another group of plant-specific transcription factors, DNA binding with one finger (OsDOF) transcription factors, positively regulate ammonium uptake, assimilation, and significantly increase amino acid content by regulating the transcript abundance of OsAMTs (Yanagisawa et al., 2004, Santos et al., 2012, Wu et al., 2017, Yanagisawa, 2000). OsMYB55, a member of the R2R3-MYB gene family, plays a positive role in amino acid metabolism by promoting the expression of OsGS1;2 and related genes (El-Kereamy et al., 2012).
A membrane-localized basic helix-loop-helix (bHLH) transcriptional factor, Glycine max Symbiotic Ammonium Transporter 1 (GmSAT1), encodes a novel regulatory gene involved in ammonium uptake during soybean root tumor development (Chiasson et al., 2014). GmSAT1 is involved in the regulation of nitrogen signaling regulatory networks related to nitrogen transport and metabolism (Dehcheshmeh, 2013). GmSAT1 activates the transcription of plasma membrane NH4+ channel ScAMF1, which indirectly enhances NH4+ permeability and finally promotes ammonium uptake (Chiasson et al., 2014, Mazurkiewicz, 2013).
The growth and yield of plants are highly dependent on environmental nutrient factors, including nitrogen. However, in pursuit of unilateral high yield, excessive input of nitrogen fertilizer has led to reduced nitrogen efficiency and decreased fruit quality, leading to lower agricultural production efficiency (Miao et al., 2011). The over application of ammonium fertilizer presents a significant burden to both soil and plants (Dawar et al., 2021), therefore, investigating the mechanism of ammonium utilization is an important goal in plant production (Rubio-Asensio and Bloom, 2017). Additionally, study of the tight regulation of transcription factors on nitrogen uptake can enable genetic engineering strategies to improve nutrient uptake regulation in plants (Wei et al., 2019). In this study, we identified an ammonium-responsive MdSAT1 gene in apple and found that MdSAT1 regulates the expression of genes related to ammonium uptake and the enzymatic activities of ammonium assimilation-related proteins. MdSAT1 also can affect root conformation and root hair development, to ultimately promote nitrogen uptake. Overall, these findings provide insight into the mechanisms by which MdSAT1 controls ammonium uptake as well as plant growth and development in apple.