Functional identification of bHLH transcription factor MdSAT1 in the ammonium response in apple

Functional identification of bHLH transcription factor MdSAT1 in the ammonium response in apple | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Functional identification of bHLH transcription factor MdSAT1 in the ammonium response in apple Tong Li, Zi-Quan Feng, Bai-Hui Zhu, Ming-Li Li, Guo-Dong Li, Wen-Sheng Gao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1520208/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Plants mainly uptake inorganic nitrogen from soil as ammonium and nitrate. Less energy is required to assimilate ammonium compared to nitrates, and plants prefer to take up ammonium when the external nitrogen concentration is low. Investigating the patterns and mechanisms of ammonium absorption can help improve crop nitrogen utilization. In this study of apple, we isolated MdSAT1 , a gene encoding an ammonium-responsive bHLH transcription factor. MdSAT1 promoted the growth and development of lateral roots and root hairs. Overexpression of MdSAT1 increased the transcript levels of genes related to ammonium uptake and assimilation and promoted the activities of ammonium assimilation-related enzymes, indicating that MdSAT1 can enhance ammonium uptake and utilization. MdSAT1 also can modulate ROS accumulation to ultimately regulate plant growth. Taken together, these findings provide insight into the mechanisms by which MdSAT1 controls ammoniun utilization as well as plant growth and development in apple. Apple Ammonium MdSAT1 Functional identification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key message The bHLH transcription factor MdSAT1 can enhance ammonium uptake and utilization in apple. 1. Introduction 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 NH 4 + channel Ammonium Facilitator 1 (AMF1) has also been found to regulate plasma membrane permeability to NH 4 + and NH 4 + 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 NH 4 + channel ScAMF1 , which indirectly enhances NH 4 + 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. 2. Materials And Methods 2.1 Plant materials and growth conditions Apple seedlings ( Malus domestica ) were cultured in a plant growth chamber under 25°C/22°C, 14 h/8 h temperature, and photoperiod. Apple group culture seedlings were grown in Murashige & Skoog (MS) medium (pH = 6.0) containing 6-Benzylamino Purine (6-BA, 0.5 mg/L), Naphthaleneacetic Acid (NAA, 0.1 mg/L), and Gibberellin (GA, 0.5 mg/L) for succession every 30 days. For the nitrogen treatment experiment, 1 month-old apple seedlings were selected for rooting in ½MS rooting medium containing 1 mg/L 3-Indoleacetic acid (IAA). When rooting was completed, the seedlings were transferred to a nutrient bowl and cultured for about 30 days. Seedlings of uniform growth were selected and pre-treated in hydroponic conditions with ddH 2 O for 1 week. The seedlings were then treated with 2 mM KCl (represents 0 N), KNO 3 (represents nitrate), or NH 4 Cl (represents ammonium), and sampled after 0, 3, 6, 9, 12, and 24 h of treatment. Arabidopsis seeds were disinfected with 75% alcohol and 3% sodium hypochlorite, and then sown on ½ MS medium solid culture plates (15 g L − 1 sucrose and 8.0 g L − 1 agar powder, pH adjusted to 5.9 with 1.0 M sodium hydroxide). The plates were incubated at 4°C with dark vernalization for 4 d. Seeds were germinated and grown at 22°C with a 16 h/8 h light/dark cycle. Different types of Arabidopsis ( MdSAT1-OE , Col) seedlings used for gene expression analysis by RT-PCR were germinated on ½ MS medium solid culture plates for 7 days before being transplanted to vermiculite, irrigated with tap water, and then watered weekly with a modified Hoagland’s nutrient solution with either Low NH 4 + (0.5 mM NH 4 Cl) or High NH 4 + (5 mM NH 4 Cl). The basic nutrient solution contained 1.0 mM CaCl 2 , 1.0 mM NaH 2 PO 4 , 1.0 mM MgSO 4 , 0.1 mM FeNa 2 EDTA, 50 µM MnSO 4 ·H 2 O, 50 µM H 3 BO 3 , 0.05 µM CuSO 4 ·5H 2 O, 0.5 µM Na 2 MoO 4 ·2H 2 O, 15 µM ZnSO 4 ·7H 2 O, 2.5 µM KI, and 0.05 µM CoCl·6H 2 O with low or high concentration of NH 4 Cl, and the pH was adjusted to 5.9 with 1.0 M sodium hydroxide. The final K + concentration was adjusted to be the same in both solutions by addition of K 2 SO 4 . After growing for four weeks, the Arabidopsis ( MdSAT1-OE , Col) seedlings were subjected to phenotype observation and physiological and biochemical analysis. One or two days after germination, different types of Arabidopsis ( MdSAT1-OE , Col) seedlings were transplanted and grown on Low NH 4 + (0.5 mM NH 4 Cl) or High NH 4 + (1.5 mM NH 4 Cl) modified solid medium containing the above modified nutrient solution, plus organic matter (2 µM C 6 H 12 O 6 ·2H 2 O, 0.02 µM NC 5 H 4 COOH, 0.001 µM C 12 H 17 ClN 4 OS·HCl, 0.01 µM C 8 H 11 O 3 N·HCl, 0.1 µM NH 2 CN 2 ·COOH), 30 g L − 1 sucrose, and 8.0 g L − 1 agar powder, with the pH adjusted to 5.9 with addition of 1.0 M sodium hydroxide. After three days, the root hairs of the seedlings grown on the above treatment medium were observed, and after seven days, the primary and lateral roots were observed. 2.2 Transgenic materials MdSAT1-OE and ProMdSAT1::GUS Arabidopsis seeds were obtained as described (Fig. S1) (Yang et al. , 2021). 2.3 Bioinformatics analysis 2.3.1 Multiple sequence alignment and phylogenetic tree construction The protein sequences of SAT1 from different species were obtained using blastp at the NCBI website ( https://blast.ncbi.nlm.nih.gov/ ). The obtained sequences were used to construct a neighbor-joining phylogenetic tree with 1000 bootstrap replicates in MEGA-X (Kumar et al. , 2018) using the built-in ClustalW algorithm, Poisson model, and parameter settings for partial deletion (95%). 2.3.2 Prediction of conserved domains The conserved domains of the SAT1 protein were predicted using Phyre 2 ( http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi? id = index) (Kelley et al. , 2015). 2.4 Extraction of plant genomic DNA and RNA Genomic DNA Reagent Kit and Omni Plant RNA Kit (tDNase I) were used to extract plant DNA and RNA (Tiangen, Beijing, China), respectively. 2.5 Real-time quantitative RT-PCR analysis of gene expression From the extracted RNA, cDNA required for quantitative PCR was synthesized using the PrimeScript First Chain cDNA Synthesis Kit (Takara, Dalian, China). These synthesized products were used as templates for real-time quantitative RT-PCR to detect the expression levels of selected genes. Apple 18S rRNA and Arabidopsis actin rRNA genes were used as controls. PCR analysis was performed using specific primer sequences designed using Primer3Plus ( http://primer3plus.com/cgi-bin/dev/primer3plus.cgi ) (Untergasser et al. , 2007) and listed in Supplemental Table S1. The qRT-PCR analysis performed in triplicate, and relative gene expression was calculated using the 2 −ΔΔCt method. 2.6 Physiological measurements 2.6.1 Determination of substance content Ammoninum, Oxygen-derived free radicals (OFR), and Malondialdehyde (MDA) levels were measured by UV spectrophotometry as described below. Ammonium interacts with hypochlorite and phenol in a strong alkaline medium to produce the water-soluble dye indophenol blue. Indophenol blue has a characteristic absorption peak at 625 nm and the absorbance value is proportional to the ammonium nitrogen content. OFR react with hydroxylamine hydrochloride to form NO 2 − , which in the presence of p-aminobenzenesulfonic acid and α-naphthylamine produces a red azo compound with a characteristic absorption peak at 530 nm. The content of OFR in the sample can be calculated by measuring the change in absorbance at 530 nm. MDA condenses with thiobarbituric acid (TBA) to produce a red product with a maximum absorption peak at 532 nm that can be used to estimate the amount of lipid peroxide in the sample. The absorbance at 600 nm was also measured, and the difference between the absorbance at 532 nm and 600 nm was used to calculate the amount of MDA. 2.6.2 Determination of enzymatic activities Weigh 0.1g of plant material, GDH, ASN, GLS, Peroxidase (POD), Catalase (CAT), and Superoxide dismutase (SOD) activities were measured using activity assay kits (Comin, Suzhou, China) based on the below principles. GDH catalyzes the formation of glutamate and NAD + from NH 4 + , α-ketoglutarate and NADH. This causes a decrease in absorbance at 340 nm, so GDH activity can be calculated by measuring the rate of decrease in absorbance at 340 nm. ASN catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia, and its enzymatic activity can be calculated by detecting the rate of ammonia increase using Nessler's reagent. GLS catalyzes the hydrolysis of glutamine to L-glutamate and ammonia, and the rate of increase of ammonia was measured by using Nessler's reagent to calculate the enzymatic activity. POD catalyzes the oxidation of specific substrates by H 2 O 2 and exhibits characteristic light absorption at 470 nm. CAT catalyzes the decomposition of H 2 O 2 by CAT with characteristic light absorption at 405 nm. SOD can scavenge OFR, and OFR can reduce azotetrazolium to produce blue methanamine, which exhibits absorption at 560 nm. 2.6.3 Nitroblue tetrazolium staining Nitroblue tetrazolium (NBT) staining was performed according to existing methods (CORPAS, 2004). 2.7 GUS staining and enzyme activity assay Transgenic Arabidopsis ( ProMdSAT1::GUS ) seedlings were immersed in GUS staining buffer consisting of 1 mM 5-bromo-4-chloro-3-indolyl-β-glutamic acid, 100 mM sodium phosphate (pH 7.0), 0.1 mM EDTA, 0.5 mM ferricyanide, and 0.1% (v/v) Triton X-100 37°C for 1 h in the dark. To quantify GUS activity, proteins were extracted from the seedlings with 1 mL of extraction buffer (50 mM Na 2 HPO 4 /NaH 2 PO 4 [pH 7.0], 10 mM β-mercaptoethanol, 10 mM Na 2 -EDTA, 0.1% (v/v) Triton X-100), and 1 mL of RIPA lysis buffer. A protein assay kit (Bio-Rad) was used to determine the total protein concentration. To measure GUS, 100 µL of the protein extract was added to 900 µL of GUS reaction buffer containing 1 mm 4-methylumbelliferone glucuronide and the mixture was incubated at 37°C for 0, 5, 10, 15, 30, and 60 min. Then, 100 µL of the reaction mixture was added to 900 µL of the termination solution (1 M sodium carbonate). Fluorescence values were measured using a VersaFluor Spectrofluorometer (Bio-Rad) at an excitation wavelength of 365 nm and an emission wavelength of 455 nm. Arabidopsis ( ProMdSAT1::GUS ) seedlings (seven days-old) were pre-treated with ddH 2 O for two days, treated with 1 mM KCl, KNO 3 , or NH 4 Cl for different time periods, immersed in the GUS staining solution, and photographed. 2.8 Root system analysis Arabidopsis taproots were observed and photographed under a body view microscope. Digimizer software was used to measure and calculate the number and length of root hairs in a 4-mm area starting 2 mm from the root tip. 2.9 Data analysis All experiments were repeated independently three times, unless otherwise indicated. The data are expressed as mean and standard deviation. Data were analyzed by one-way analysis of variance, and means were compared using Duncan's multiple range test. Different letters indicate significant differences at the p < 0.05 level. 3. Results 3.1 Phylogenetic relationships, multiple sequence alignment, and protein structure analysis of MdSAT1 The MdSAT1 (MD10G1115500) gene was identified from the NCBI website according to the GmSAT1 sequence of soybean ( Glycine max ). A phylogenetic tree was constructed, and apple MdSAT1 was most closest related to pear PbSAT1 (Rosaceae) (Fig. 1A), indicating that these genes diverged recently in evolution. We compared the SAT1 protein sequences of apple with those of other plant species, and the results showed that all 13 proteins had high sequence similarity and belonged to the plant bHLH transcription factor superfamily, members of which contain a bHLH domain and an H-E-R DNA binding region (Fig. 1B-C). The high-level structure of the MdSAT1 protein was predicted by homology model, and the results indicated that the secondary and tertiary structures of MdSAT1 match those of the core conserved domain (Fig. 1C-D). 3.2 MdSAT1 is an ammonium-responsive gene MdSAT1 is homologous to GmSAT1, which is involved in ammonium uptake (Chiasson et al., 2014), and qRT-PCR was next used to detect the expression of MdSAT1 in response to different nitrogen forms (KCl, KNO 3 , and NH 4 Cl). The expression of MdSAT1 was significantly induced by NH 4 Cl both in shoots and roots, however, the transcript level of MdSAT1 showed little change in response to nitrate (Fig. 2A-B), suggesting that MdSAT1 was specifically responsive to ammonium. ProMdSAT1::GUS transgenic Arabidopsis seedlings were treated with different forms of nitrogen, and GUS staining results suggested that the highest GUS activity was observed under NH 4 Cl treatment (Fig. 2C-D). With increasing time of different treatments, the expression activity of ProMdSAT1::GUS was specifically induced by NH 4 Cl (Fig. S2). Taken together, these results suggest that MdSAT1 is specifically responsive to ammonium. 3.3 Overexpression of MdSAT1 regulates ammonium uptake Given that MdSAT1 is an ammonium-responsive gene, we next treated MdSAT1-OE and wild type (Col) in MS medium containing 0.5 mM NH 4 Cl (Low NH 4 + ) or 5 mM NH 4 Cl (High NH 4 + ) for four weeks and then assessed the effects on plant growth and ammonium content. Under low NH 4 + conditions, ectopic expression of MdSAT1 promoted seedling growth compared with Col, and MdSAT1-OE showed greater fresh weight and increased ammonium content (Fig. 3A-C). In contrast, under high NH 4 + conditions, ectopic expression of MdSAT1 reduced fresh weight and accumulated higher ammonium (Fig. 3A-C). These results indicate that MdSAT1 promotes ammonium uptake to regulate plant growth. To further evaluate the role of MdSAT1 in ammonium uptake, the effects of MdSAT1 on the expression of genes related to ammonium uptake were analyzed. The result showed that transcript levels of AtAMTs were not increased in MdSAT1-OE lines, however, expression of AtAMF1;3 was significantly induced in the MdSAT1-OE lines (Fig. 3D). AMF proteins promote NH 4 + permeable transport (Chiasson et al., 2014), so these results indicated that MdSAT1 promoted ammonium uptake by increasing the expression levels of genes related to ammonium uptake. 3.4 Overexpression of MdSAT1 affects the enzymatic activities of ammonium assimilation-related proteins After taken up by plant roots, NH 4 + is then assimilated to amino acids or amides through the action of GDH, ASN, and GLS (Yang et al., 2017, Lea, 2006, Lopes et al., 2015, Atkins et al., 1975). Therefore, we measured the activities of these ammonium assimilation-related enzymes in MdSAT1 transgenic Arabidopsis . The results showed that overexpression of MdSAT1 promoted GDH, ASN, and GLS activities in vivo , independent of ammonium treatment concentration (Fig. 4). The expression levels of ammonium assimilation-related genes showed the same trend (Fig. S3). Therefore, the results demonstrate that MdSAT1 increases the activities of ammonium assimilation-related enzymes to influence ammonium assimilation. 3.5 Overexpression of MdSAT1 promotes lateral root development Ammonium in the soil is actively taken up by the roots mainly by ammonium ion transporters (von Wittgenstein et al. , 2014). The tissue-specific localization of MdSAT1 was detected using ProMdSAT1::GUS transgenic Arabidopsis . GUS staining results showed that MdSAT1 was differentially expressed during lateral root growth, with the highest expression observed at the time of lateral root primordium genesis (Fig. S4). There was no significant difference of primary root length, but the lateral root numbers were significantly increased in the MdSAT1-OE lines compared with those of Col (Fig. 5A-D). These results suggest that MdSAT1 overexpression promotes lateral root growth and development. 3.6 Overexpression of MdSAT1 regulates root hair growth and development Root hairs play an important role in nutrient uptake (Moon et al. , 2019), and we next observed the phenotypes of root hairs in MdSAT1-OE and Col. MdSAT1 significantly increased the number and length of root hairs under low NH 4 + treatment (Fig. 6A, C-D), and the number and length of root hairs were inhibited under high NH 4 + treatment (Fig. 6B-D). The expression levels of genes related to root hair development were also detected. The results showed significantly down-regulated transcript levels of AtEGL3 , AtGL3 , and AtTTG1 genes that inhibit root hair development under low NH 4 + treatment (Fig. 7A-C) (Bernhardt et al. , 2003, Schiefelbein et al. , 2014) and significantly up-regulated levels of AtSCM , a positive regulator in root hair development (Fig. 7D) (Kwak and Schiefelbein, 2014); the expression patterns were reversed under high NH 4 + treatment (Fig. 7A-D). These results suggest that MdSAT1 promotes the growth and development of root hairs by regulating the transcript levels of root hair development-related genes. 3.7 MdSAT1 promotes the ROS accumulation ROS plays a role in root hair development (Monshausen et al. , 2007), so we next measured ROS content by NBT staining. The results showed that overexpression of MdSAT1 increased ROS accumulation of leaves compared with the level in Col (Fig. 8A). OFR content was promoted in the MdSAT1-OE lines, both in high and low NH 4 + treatment (Fig. 8C), while the MDA content was only promoted in high NH 4 + treatment (Fig. 8B). CAT, SOD, and POD are important enzymes for ROS deconstruction (Waszczak et al. , 2018, Miller et al. , 2008) so the activities of these enzymes were measured. Under low NH 4 + treatment, overexpression of MdSAT1 resulted in higher CAT and SOD activities but significantly lower POD activity compared to Col (Fig. 8D-F). Overall, these results suggest that overexpression of MdSAT1 promotes OFR production by affecting the activity of ROS-deconstruction-related enzymes. 4. Discussion When the soil nitrogen concentration is low, plants are more likely to take up ammonium (Bloom et al. , 1992). However, the absorption and utilization efficiency of nitrogen by wild-type plants (Col) is not ideal when only ammonium is applied as nitrogen fertilizer. Moreover, the growth of plants is significantly inhibited at low concentration of single ammonium fertilizer, and the use of a higher concentration of single ammonium fertilizer will cause ammonium toxicity to plants. These trends will guide the application of single ammonium as nitrogen fertilizer in agricultural production. Determination of the appropriate intermediate concentration of single ammonium for use as nitrogen fertilizer is essential to ensure the effective utilization of plants and also avoid the waste of resources and environmental pollution caused by excessive fertilization. Alternatively, combined application with nitrate nitrogen may be more appropriate. Therefore, it is important to study the law and mechanism of ammonium absorption by crops to optimize nitrogen utilization. GmSAT1 was functionally identified in soybean root nodule development (Dehcheshmeh, 2013). In this study, phylogenetic and conserved domains analysis indicated that the MdSAT1 protein may be similar in function to GmSAT1 (Fig. 1). The function of MdSAT1 was characterized and the results showed that it is highly expressed, mainly in nutrient organs (Fig. S5), and plays a key role in ammonium uptake and assimilation (Fig. 2; Fig. 3D; Fig. 4). MdSAT1 is phenotypically similar to GmSAT1, further confirming the genetic relationship of MdSAT1 with GmSAT1. MdSAT1 also regulates the accumulation of ROS and ultimately plant growth (Fig. 3A-C; Fig. 5–8). Several studies have shown that GmSAT1 is important for the symbiosis of soybean rhizobia and acts in NH 4 + uptake during soybean rhizome development (Chiasson et al., 2014, Dehcheshmeh, 2013). The Gmsat1 mutant negatively regulates nitrogen deficiency-induced genes to reduce nitrogen uptake (Dehcheshmeh, 2013). Given that MdSAT1 is an ammonium-responsive gene that is induced by NH 4 + expression (Fig. 2; Fig. S2), we evaluated the role of MdSAT1 in ammonium uptake and found that ectopic expression of MdSAT1 promoted seedling growth under low NH 4 + conditions compared with the wild type (Fig. 3A-C). Previous study showed that SAT1 can regulate the nitrogen starvation response and coordinates related signaling regulatory networks (Dehcheshmeh, 2013). SAT1 transcriptionally activates a unique plasma membrane NH 4 + channel AMF1, indirectly enhancing NH 4 + permeability, which also affects the regulation of MEP by SAT1 (Mazurkiewicz, 2013). Interestingly, SAT1 was unable to enhance the expression of MEP3 in the absence of AMF1, and SAT1 affects NH 4 + uptake by indirect regulation of the AMT/MEP/Rh family through activation of AMF1 (Mazurkiewicz, 2013). Given this, we analyzed the expression of genes related to ammonium uptake and found that overexpression of MdSAT1 significantly promoted the expression of AtAMF1;3 (Fig. 3D), suggesting that MdSAT1 may promote ammonium uptake by affecting the expression levels of AMF1;3 . Most of the NH 4 + absorbed by plant roots is enzymatically assimilated to glutamate in the root system, and glutamate is then converted to other amino acids and subsequently transported to various parts of the plant organs and tissues via the xylem (Lea PJ, 2006, Comadira et al. , 2015). ASN is a amide hydrolase that participates in amino acid metabolism and GDH and GLS catabolize ammonia by deamination (Yang et al., 2017, Lopes et al., 2015, Atkins et al., 1975). Our experiments showed that independent of the external NH 4 + concentration, overexpression of MdSAT1 promoted the activities of GDH, ASN, and GLS, consistent with the observed changes in transcript levels of the corresponding genes (Fig. 4; Fig. S3). In this way, MdSAT1 can help regulate the free ammonia level in vivo and promote the plant's own ammonia cycle. NH 4 + in soil is actively taken up by the root system mainly through ammonium ion transporters (Wang et al. , 2012). NH 4 + can affect root system conformation, including primary roots, lateral roots, and root hairs. Previous work found that the primary root length, lateral root length, and root surface area gradually decreased with increasing NH 4 + concentration (Li and Sun, 2007) and addition of 0.1–10 mM of NH 4 + could promote the number and elongation of lateral roots (Yang, 2010). In this study, we found that the primary root lengths of Arabidopsis seedlings overexpressing MdSAT1 were not significantly different from those of Col (Fig. 5C), but the number of lateral roots increased significantly compared with Col (Fig. 5D). We also found that GUS activity of ProMdSAT1::GUS transgenic Arabidopsis seedlings was expressed at the highest level at the beginning of lateral root primordia (Fig. S4). The above results suggest that MdSAT1 is involved in the process of lateral root genesis and growth. A treatment of 0.1 mM NH 4 + promotes the increase of root hair density and root hair number, but at NH 4 + concentrations higher than 1 mM, the root hair density and root hair number gradually decrease with increasing NH 4 + concentration (Yang, 2010). Our results showed that under low NH 4 + treatment, MdSAT1 significantly promoted root hair development (Fig. 6A, C-D), but under high NH 4 + treatment, it showed the opposite inhibitory effect (Fig. 6B-D). AtEGL3 , AtGL3 , and AtTTG1 , which inhibit root hair development, were significantly down-regulated in MdSAT1-OE Arabidopsis under low NH 4 + treatment (Fig. 7A-C), but the AtSCM genes were significantly up-regulated (Fig. 7D). These results suggest that MdSAT1 regulates root development by regulating the transcript levels of root hair-related genes in an ammonium dosage-dependent manner. A role for ROS in root hair growth and formation mechanisms was previously reported (Monshausen et al., 2007). To ask if MdSAT1 regulates root hair development through the ROS pathway, we performed NBT staining and measured MDA and OFR content. The results suggested that overexpression of MdSAT1 accumulated more OFR (Fig. 8A-C). Therefore, we speculate that OFR may regulate root hair development by ROS, a process that requires the involvement of the ammonium-responsive gene MdSAT1 . In conclusion, the results of this study showed that overexpression of MdSAT1 promotes plant growth and biomass accumulation. These findings provide theoretical guidance to resolve the mechanisms by which MdSAT1 regulates ammonium uptake and plant growth and provide a reference for future selection of superior germplasm with more efficient nitrogen uptake. Declarations Author Contributions: Xiao-Fei Wang and Wen-Sheng Gao designed the experiments. Tong Li, Zi-Quan Feng, Bai-Hui Zhu, Ming-Li Li and Guo-Dong Li performed the research. Tong Li, Zi-Quan Feng, Bai-Hui Zhu and Chun-Xiang You analyzed the data. Tong Li, Xiao-Fei Wang and Wen-Sheng Gao wrote the paper. Funding: This work was supported by the National Natural Science Foundation of China (31972378), Agricultural Variety Improvement Project of Shandong Province (2019LZGC007), China Agriculture Research System of MOF and MARA (CARS-27), Shandong Province Key R&D Program (2021CXGC010802) and Taishan scholar project (LJNY2020266). Acknowledgments: We sincerely thank our team leader Dr. Yu-Jin Hao, who will be remembered for his great achievement and for the support and help in our work. Competing Interest: The authors declare no conflict of interest. References ATKINS, C. A., PATE, J. S. & SHARKEY, P. J. (1975), "Asparagine metabolism-key to the nitrogen nutrition of developing legume seeds.", Plant Physiology, Vol. 56 No. 6, pp. 807-812. BAOHAI, L. & WEIMING, S. (2007), "EFFECTS OF ELEVATED NH 4 + ON ARABIDOPSIS SEEDLINGS DIFFERENT IN ACCESSIONS", Journal of Soil, Vol. 44 No. 3, pp. 508-515. BERNHARDT, C., LEE, M. M., GONZALEZ, A., ZHANG, F., LLOYD, A. & SCHIEFELBEIN, J. (2003), "The bHLH genes GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the Arabidopsis root", Development, Vol. 130 No. 26, pp. 6431-9. BLOOM, A. J. (1997), "Nitrogen as a Limiting Factor: Crop Acquisition of Ammonium and Nitrate", Ecology in Agriculture, 145-172. BLOOM, A. J., SUKRAPANNA, S. S. & WARNER, R. L. (1992), "Root respiration associated with ammonium and nitrate absorption and assimilation by barley", Plant Physiol, Vol. 99 No. 4, pp. 1294-301. CHIASSON, D. M., LOUGHLIN, P. C., MAZURKIEWICZ, D., MOHAMMADIDEHCHESHMEH, M., FEDOROVA, E. E., OKAMOTO, M., MCLEAN, E., GLASS, A. D., SMITH, S. E., BISSELING, T., TYERMAN, S. D., DAY, D. A. & KAISER, B. N. (2014), "Soybean SAT1 (Symbiotic Ammonium Transporter 1) encodes a bHLH transcription factor involved in nodule growth and NH 4 + transport", Proc Natl Acad Sci U S A, Vol. 111 No. 13, pp. 4814-9. COMADIRA, G., RASOOL, B., KARPINSKA, B., MORRIS, J., VERRALL, S. R., HEDLEY, P. E., FOYER, C. H. & HANCOCK, R. D. (2015), "Nitrogen deficiency in barley (Hordeum vulgare) seedlings induces molecular and metabolic adjustments that trigger aphid resistance.", Journal of experimental botany, Vol. 66 No. 12, pp. CORPAS, F. J. S. L. (2004), "Cadmium-induced subcellular accumulation of O 2 − and H 2 O 2 in pea leaves-ROMERO-PUERTAS ", Plant, Cell & Environment, Vol. 271122-1134. COUTURIER, J., MONTANINI, B., MARTIN, F., BRUN, A., BLAUDEZ, D. & CHALOT, M. (2007), "The expanded family of ammonium transporters in the perennial poplar plant", New Phytol, Vol. 174 No. 1, pp. 137-150. DAWAR, K., SAIF-UR-RAHMAN, FAHAD, S., ALAM, S. S., KHAN, S. A., DAWAR, A., YOUNIS, U., DANISH, S., DATTA, R. & DICK, R. P. (2021), "Influence of variable biochar concentration on yield-scaled nitrous oxide emissions, Wheat yield and nitrogen use efficiency", Sci Rep, Vol. 11 No. 1, pp. 16774. DEHCHESHMEH, M. M. (2013) Regulatory control of the symbiotic enhanced soybean bHLH transcription factor, GmSAT1. Adelaide; 2013. EL-KEREAMY, A., BI, Y. M., RANATHUNGE, K., BEATTY, P. H., GOOD, A. G. & ROTHSTEIN, S. J. (2012), "The rice R2R3-MYB transcription factor OsMYB55 is involved in the tolerance to high temperature and modulates amino acid metabolism", PLoS One, Vol. 7 No. 12, pp. e52030. GUETHER, M., NEUHAUSER, B., BALESTRINI, R., DYNOWSKI, M., LUDEWIG, U. & BONFANTE, P. (2009), "A mycorrhizal-specific ammonium transporter from Lotus japonicus acquires nitrogen released by arbuscular mycorrhizal fungi", Plant Physiol, Vol. 150 No. 1, pp. 73-83. HUANG, L., ZHANG, H., ZHANG, H., DENG, X. W. & WEI, N. (2015), "HY5 regulates nitrite reductase 1 (NIR1) and ammonium transporter1;2 (AMT1;2) in Arabidopsis seedlings", Plant Sci, Vol. 238330-9. JACKSON, L. E., BURGER, M. & CAVAGNARO, T. R. (2008), "Roots, nitrogen transformations, and ecosystem services", Annu Rev Plant Biol, Vol. 59341-63. KELLEY, L. A., MEZULIS, S., YATES, C. M., WASS, M. N. & STERNBERG, M. J. (2015), "The Phyre2 web portal for protein modeling, prediction and analysis", Nat Protoc, Vol. 10 No. 6, pp. 845-58. KUMAR, S., STECHER, G., LI, M., KNYAZ, C. & TAMURA, K. (2018), "MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms", Mol Biol Evol, Vol. 35 No. 6, pp. 1547-1549. KWAK, S. H. & SCHIEFELBEIN, J. (2014), "TRIPTYCHON, not CAPRICE, participates in feedback regulation of SCM expression in the Arabidopsis root epidermis", Plant Signal Behav, Vol. 9 No. 11, pp. e973815. LAWLOR D. W., L. G. G. F. (2001), Plant Nitrogen. Springe, Berlin/Heidelberg, Germany. LEA PJ, A. R. (2006), "Nitrogen use efficiency. 1. Uptake of nitrogen from the soil", Annals of Applied Biology, Vol. 149243-347. LOPES, A. M., OLIVEIRA-NASCIMENTO, L. D., RIBEIRO, A., TAIRUM, C. A., BREYER, C. A., OLIVEIRA, M. A. D., MONTEIRO, G., SOUZA-MOTTA, C. M. D., MAGALHES, P. D. O. & AVENDAO, J. G. F. (2015), "Therapeutic l-asparaginase: upstream, downstream and beyond", Critical Reviews in Biotechnology, 82-99. LOPEZ-PEDROSA, A., GONZALEZ-GUERRERO, M., VALDERAS, A., AZCON-AGUILAR, C. & FERROL, N. (2006), "GintAMT1 encodes a functional high-affinity ammonium transporter that is expressed in the extraradical mycelium of Glomus intraradices", Fungal Genet Biol, Vol. 43 No. 2, pp. 102-10. MARINI, A. M., SOUSSI-BOUDEKOU, S., VISSERS, S. & ANDRE, B. (1997), "A family of ammonium transporters in Saccharomyces cerevisiae", Mol Cell Biol, Vol. 17 No. 8, pp. 4282-93. MAZURKIEWICZ, D. (2013) Characterisation of a Novel Family of Eukaryotic Ammonium Transport Proteins. Adelaide; 2013. MIAO, Y., STEWART, B. A. & ZHANG, F. (2011), "Long-term experiments for sustainable nutrient management in China. A review", Agronomy for Sustainable Development, Vol. 31 No. 2, pp. 397-414. MILLER, G., SHULAEV, V. & MITTLER, R. (2008), "Reactive oxygen signaling and abiotic stress", Physiol Plant, Vol. 133 No. 3, pp. 481-9. MONSHAUSEN, G. B., BIBIKOVA, T. N., MESSERLI, M. A., SHI, C. & GILROY, S. (2007), "Oscillations in extracellular pH and reactive oxygen species modulate tip growth of Arabidopsis root hairs", Proc Natl Acad Sci U S A, Vol. 104 No. 52, pp. 20996-1001. MOON, S., CHO, L. H., KIM, Y. J., GHO, Y. S., JEONG, H. Y., HONG, W. J., LEE, C., PARK, H., JWA, N. S., DANGOL, S., CHEN, Y., PARK, H., CHO, H. S., AN, G. & JUNG, K. H. (2019), "RSL Class II Transcription Factors Guide the Nuclear Localization of RHL1 to Regulate Root Hair Development", Plant Physiol, Vol. 179 No. 2, pp. 558-568. NA, Y. (2010) Interactions between nitrogen (NH 4 + ,NO 3 - ) and phytohormones in the regulation of root conformation in Arabidopsis thaliana ., Nanjing Agricultural University. NEUHAUSER, B., DYNOWSKI, M., MAYER, M. & LUDEWIG, U. (2007), "Regulation of NH 4 + transport by essential cross talk between AMT monomers through the carboxyl tails", Plant Physiol, Vol. 143 No. 4, pp. 1651-9. NOCTOR, G, FOYER & CH (1998), "Review article. A re-evaluation of the ATP:NADPH budget during Cphotosynthesis: a contribution from nitrate assimilation and its associated respiratory activity?", Journal of Experimental Botany. OHYAMA, T. (2010), Nitrogen as a major essential element of plants, Nitrogen Assimilation in Plants. PATTERSON, K., CAKMAK, T., COOPER, A., LAGER, I., RASMUSSON, A. G. & ESCOBAR, M. A. (2010), "Distinct signalling pathways and transcriptome response signatures differentiate ammonium- and nitrate-supplied plants", Plant Cell Environ, Vol. 33 No. 9, pp. 1486-501. RUBIO-ASENSIO, J. S. & BLOOM, A. J. (2017), "Inorganic nitrogen form: a major player in wheat and Arabidopsis responses to elevated CO 2 .", Journal of experimental botany, Vol. 68 No. 10, pp. SANTOS, L. A., SOUZA, S. & FERNANDES, M. S. (2012), "OsDof25 expression alters carbon and nitrogen metabolism in Arabidopsis under high N-supply", Plant Biotechnology Reports, Vol. 6 No. 4, pp. p.327-337. SCHIEFELBEIN, J., HUANG, L. & ZHENG, X. (2014), "Regulation of epidermal cell fate in Arabidopsis roots: the importance of multiple feedback loops", Front Plant Sci, Vol. 547. SOHLENKAMP, C., WOOD, C. C., ROEB, G. W. & UDVARDI, M. K. (2002), "Characterization of Arabidopsis AtAMT2, a high-affinity ammonium transporter of the plasma membrane", Plant Physiol, Vol. 130 No. 4, pp. 1788-96. UNTERGASSER, A., NIJVEEN, H., RAO, X., BISSELING, T., GEURTS, R. & LEUNISSEN, J. A. (2007), "Primer3Plus, an enhanced web interface to Primer3", Nucleic Acids Res, Vol. 35 No. Web Server issue, pp. W71-4. VIKTOR, A. & CRAMER, M. D. (2005), "The influence of root assimilated inorganic carbon on nitrogen acquisition/assimilation and carbon partitioning", New Phytol, Vol. 165 No. 1, pp. 157-69. VON WITTGENSTEIN, N. J., Le CH, HAWKINS, B. J. & EHLTING, J. (2014), "Evolutionary classification of ammonium, nitrate, and peptide transporters in land plants", BMC Evol Biol, Vol. 1411. WANG, Y. Y., HSU, P. K. & TSAY, Y. F. (2012), "Uptake, allocation and signaling of nitrate", Trends Plant Sci, Vol. 17 No. 8, pp. 458-67. WASZCZAK, C., CARMODY, M. & KANGASJARVI, J. (2018), "Reactive Oxygen Species in Plant Signaling", Annu Rev Plant Biol, Vol. 69209-236. WEI, X., LINA, Z., WENZHONG, Z., JIPING, G., JUN, Y., XIAOXI, Z., MING, D., YANZE, Z. & LIQIANG, C. (2019), "An Integrated Analysis of the Rice Transcriptome and Metabolome Reveals Root Growth Regulation Mechanisms in Response to Nitrogen Availability", International Journal of Molecular Sciences, Vol. 20 No. 23, pp. WU, Y., YANG, W., WEI, J., YOON, H. & AN, G. (2017), "Transcription Factor OsDOF18 Controls Ammonium Uptake by Inducing Ammonium Transporters in Rice Roots", Mol Cells, Vol. 40 No. 3, pp. 178-185. XU, G., FAN, X. & MILLER, A. J. (2012), "Plant nitrogen assimilation and use efficiency", Annu Rev Plant Biol, Vol. 63153-82. YANAGISAWA, S. (2000), "Dof1 and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize", Plant J, Vol. 21 No. 3, pp. 281-8. YANAGISAWA, S., AKIYAMA, A., KISAKA, H., UCHIMIYA, H. & MIWA, T. (2004), "Metabolic engineering with Dof1 transcription factor in plants: Improved nitrogen assimilation and growth under low-nitrogen conditions", Proc Natl Acad Sci U S A, Vol. 101 No. 20, pp. 7833-8. YANG, Y. Y., ZHENG, P. F., REN, Y. R., YAO, Y. X., YOU, C. X., WANG, X. F. & HAO, Y. J. (2021), "Apple MdSAT1 encodes a bHLHm1 transcription factor involved in salinity and drought responses", Planta, Vol. 253 No. 2, pp. YANG, Y. Z., DING, S., WANG, Y., LI, C. L., SHEN, Y., MEELEY, R., MCCARTY, D. R. & TAN, B. C. (2017), "Small kernel2 Encodes a Glutaminase in Vitamin B6 Biosynthesis Essential for Maize Seed Development", Plant Physiol, Vol. 174 No. 2, pp. 1127-1138. YUAN, L., LOQUE, D., KOJIMA, S., RAUCH, S., ISHIYAMA, K., INOUE, E., TAKAHASHI, H. & von WIREN, N. (2007), "The organization of high-affinity ammonium uptake in Arabidopsis roots depends on the spatial arrangement and biochemical properties of AMT1-type transporters", Plant Cell, Vol. 19 No. 8, pp. 2636-52. ZHAO C., L. Q. (2009), "Growth and physiological responses of Picea asperata seedlings to elevated temperature and to nitrogen fertilization", Acta Physiol. Plant, Vol. 1 No. 31, pp. 163. Supplementary Files Supplementarydata.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1520208","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":102119125,"identity":"24fe9136-53c4-4a1a-8cc6-eb0cbde930e0","order_by":0,"name":"Tong Li","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Li","suffix":""},{"id":102119126,"identity":"050d40c5-46cd-44c4-9cf0-db86e0890d5b","order_by":1,"name":"Zi-Quan Feng","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zi-Quan","middleName":"","lastName":"Feng","suffix":""},{"id":102119127,"identity":"8c8b1f18-d6a4-4e84-8201-cd54cb8aa8dc","order_by":2,"name":"Bai-Hui Zhu","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bai-Hui","middleName":"","lastName":"Zhu","suffix":""},{"id":102119128,"identity":"bbfeecb6-1862-4965-946c-3a49856e70d2","order_by":3,"name":"Ming-Li Li","email":"","orcid":"","institution":"Shandong Agricultural Technology Extension Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ming-Li","middleName":"","lastName":"Li","suffix":""},{"id":102119129,"identity":"53c6c429-5948-4fcf-b5e9-be056580f82e","order_by":4,"name":"Guo-Dong Li","email":"","orcid":"","institution":"Shandong Agricultural Technology Extension Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guo-Dong","middleName":"","lastName":"Li","suffix":""},{"id":102119130,"identity":"ae6694c4-0169-4db8-a945-7c1374301ebd","order_by":5,"name":"Wen-Sheng Gao","email":"","orcid":"","institution":"Shandong Agricultural Technology Extension Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wen-Sheng","middleName":"","lastName":"Gao","suffix":""},{"id":102119131,"identity":"0637b713-0b15-45ea-a35f-d299add7fd61","order_by":6,"name":"Chun-Xiang You","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chun-Xiang","middleName":"","lastName":"You","suffix":""},{"id":102119132,"identity":"eded986a-25c3-4331-ad8b-5ed8a5e674e7","order_by":7,"name":"Xiao‐Fei Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie3RMQrCMBSA4SdCcEjIJi2F9gopXQQ9TIPg7BEKQqfqbPESAcG5EshUd8FBwdWhIEjRgrYOjjajYL7hkeH9QxIAw/hF3ayZI4xAZllR6iQobObEpR3Ft2min8jAnuWB7CGNginsX6f3LhcqLyRg8Gg/+57YMQ6cdIG4yHdCTgfgp6vwe0K9uXBIgrnY18kSQ8gOLQlCZP0gicXF8XKS9bu1JxSRjYNLFthRDnqJHdPbkEShS0Gx+pGt9rswhcYHXD2brzwXRTnyqNOSvHXiz9HSWH+rdBcNwzD+0gvWrkmu4VNbbQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-4601-9781","institution":"Shandong Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiao‐Fei","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2022-04-04 03:21:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1520208/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1520208/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":21073592,"identity":"e1c84a24-030d-47f3-a5cc-d9ac0bfaafe8","added_by":"auto","created_at":"2022-05-04 16:12:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5435329,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationships, multiple sequence alignment, and protein structure of \u003cem\u003eMdSAT1\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(A) Phylogenetic tree of SAT1 sequences; the number on each branch represents the genetic distance. (B) Multiple sequence alignment for above 13 proteins. (C) Predicted protein secondary of MdSAT1 structural domain. (D) Predicted protein 3D structure of MdSAT1.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1520208/v1/ceab763e5c33b2542295ff34.jpg"},{"id":21071678,"identity":"7473a60b-0c4c-4346-b2b0-89db2f0e01a5","added_by":"auto","created_at":"2022-05-04 15:52:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":691203,"visible":true,"origin":"","legend":"\u003cp\u003eNitrogen response of \u003cem\u003eMdSAT1\u003c/em\u003e\u003c/p\u003e\u003cp\u003eShoot (A) and root (B) response of \u003cem\u003eMdSAT1\u003c/em\u003e to KCl (represents 0 N), KNO\u003csub\u003e3\u003c/sub\u003e (represents nitrate), and NH\u003csub\u003e4\u003c/sub\u003eCl (represents ammonium). (C) GUS staining of\u003cem\u003e ProMdSAT1::GUS \u003c/em\u003etransgenic \u003cem\u003eArabidopsis\u003c/em\u003e under above treatments. (D) GUS activity of \u003cem\u003eMdSAT1\u003c/em\u003e under above treatments. Error bars represent standard deviation (n≥3). Different letters above the bars indicate significantly different values (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1520208/v1/4623463e4e59adb1bfeceb28.jpg"},{"id":21071679,"identity":"c78fa67d-c3f0-449b-bc01-1f6692d39f5f","added_by":"auto","created_at":"2022-05-04 15:52:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2742192,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMdSAT1\u003c/em\u003e regulates ammonium uptake and plant growth\u003c/p\u003e\u003cp\u003e\u003cem\u003eMdSAT1-OE\u003c/em\u003e and Col plants grown for four weeks under Low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (0.5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) or High NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) conditions. Morphological changes (A), Fresh weight (B), and Ammonium content (C) are presented. (D) qRT-PCR analysis of \u003cem\u003eAtAMF1;1\u003c/em\u003e, \u003cem\u003eAtAMF1;2\u003c/em\u003e, \u003cem\u003eAtAMF1;3\u003c/em\u003e, \u003cem\u003eAtAMT1;1\u003c/em\u003e, \u003cem\u003eAtAMT1;3\u003c/em\u003e and \u003cem\u003eAtAMT1;5\u003c/em\u003e\u0026nbsp;expression in \u003cem\u003eMdSAT1-OE \u003c/em\u003eand Col\u003cem\u003e Arabidopsis.\u003c/em\u003e Error bars represent the standard deviation (n≥3). Different letters above the bars indicate significantly different values (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1520208/v1/501f296a5414718bc60aab8b.jpg"},{"id":21071685,"identity":"942ae4aa-11c2-42d7-9e5b-b585983dffec","added_by":"auto","created_at":"2022-05-04 15:52:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":903397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMdSAT1 \u003c/em\u003eaffects ammonium uptake through enzymatic activity\u003c/p\u003e\u003cp\u003e\u003cem\u003eMdSAT1-OE\u003c/em\u003e and Col plants grown for four weeks under Low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (0.5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) or High NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) conditions. Relative GDH activity (A and B), Relative ASN activity (C and D), and Relative GLS activity (E and F) are presented. Error bars represent the standard deviation (n≥3). Different letters above the bars indicate significantly different values (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1520208/v1/9585b8bbdad59acb837557a9.jpg"},{"id":21072775,"identity":"c30d42b4-9823-4395-bf48-9334cf96debe","added_by":"auto","created_at":"2022-05-04 16:02:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":191265,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMdSAT1\u003c/em\u003e regulates root system conformation\u003c/p\u003e\u003cp\u003e\u003cem\u003eMdSAT1-OE\u003c/em\u003e and Col\u003cem\u003e \u003c/em\u003eplants grown for seven days under Low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (0.5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) or High NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (1.5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) conditions. Morphological changes (A and B), Primary root length (C) and Lateral root number (D) are presented. Error bars represent the standard deviation (n≥3). Different letters above the bars indicate significantly different values (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1520208/v1/7a49fd7f3b4511bc08e5ccf6.jpg"},{"id":21071681,"identity":"59b7111b-3855-4c77-b2fc-90122f0c81e8","added_by":"auto","created_at":"2022-05-04 15:52:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":917916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMdSAT1\u003c/em\u003e regulates root hair growth and development\u003c/p\u003e\u003cp\u003e\u003cem\u003eMdSAT1-OE\u003c/em\u003e and Col plants grown for three days under Low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (0.5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) or High NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (1.5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) conditions. Morphological changes (A and B), Root hair number (C) and Root hair length (D) are presented. Error bars represent the standard deviation (n≥3). Different letters above the bars indicate significantly different values (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1520208/v1/d9b30cf980403f12af4b300b.jpg"},{"id":21073332,"identity":"262c7579-7a95-4984-a8ff-c129e962c347","added_by":"auto","created_at":"2022-05-04 16:07:41","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":935370,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMdSAT1 \u003c/em\u003eregulates the expression of genes related to root hair development\u003c/p\u003e\u003cp\u003eAnalysis of root hair development-related gene expression by qRT-PCR under Low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (0.5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) or High NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (1.5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) conditions: \u003cem\u003eAtEGL3\u003c/em\u003e (A), \u003cem\u003eAtTTG1\u003c/em\u003e (B), \u003cem\u003eAtGL3\u003c/em\u003e (C), and \u003cem\u003eAtSCM\u003c/em\u003e (D) are presented. Error bars represent the standard deviation (n≥3). Different letters above the bars indicate significantly different values (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1520208/v1/67fae4badae4c27a2ee74dc9.jpg"},{"id":21071961,"identity":"26fa8121-2dd2-42a7-8785-9f33501c2de0","added_by":"auto","created_at":"2022-05-04 15:57:41","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":153944,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMdSAT1\u003c/em\u003e regulates ROS accumulation\u003c/p\u003e\u003cp\u003eCol and \u003cem\u003eMdSAT1-OE\u003c/em\u003e plants grown for four weeks under Low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (0.5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) or High NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) conditions. NBT staining (A), MDA content (B), OFR concent (C), POD activity (D), CAT activity (E), and SOD activity (F) are presented. Error bars represent the standard deviation (n≥3). Different letters above the bars indicate significantly different values (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1520208/v1/e7f9725116349c80494ba423.jpg"},{"id":22056761,"identity":"c72c303e-d6f6-47ea-b880-1f60fe47d356","added_by":"auto","created_at":"2022-05-31 08:54:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1298781,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1520208/v1/27da21c7-a448-41a1-9b58-7a8b079f4b3f.pdf"},{"id":21071686,"identity":"7aed6a39-230b-4117-be78-26518142f177","added_by":"auto","created_at":"2022-05-04 15:52:41","extension":"docx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":1902683,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-1520208/v1/d659ae765dd39462eddf1307.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eFunctional identification of bHLH transcription factor MdSAT1 in the ammonium response in apple\u003c/p\u003e","fulltext":[{"header":"Key message","content":"\u003cp\u003eThe bHLH transcription factor MdSAT1 can enhance ammonium uptake and utilization in apple.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eNitrogen 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.\u003c/p\u003e \u003cp\u003eSoils 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 \u003cem\u003eet al.\u003c/em\u003e, 2008, Patterson \u003cem\u003eet al.\u003c/em\u003e, 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 \u003cem\u003eet al.\u003c/em\u003e, 1998).\u003c/p\u003e \u003cp\u003eInorganic 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 \u003cem\u003eet al.\u003c/em\u003e, 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 \u003cem\u003eet al.\u003c/em\u003e, 2015). Asparaginase (ASN) catalyzes the hydrolysis of asparagine to produce aspartate and ammonia, and participates in nitrogen fixation (Lopes et al., 2015, Atkins \u003cem\u003eet al.\u003c/em\u003e, 1975). Glutaminase (GLS) catalyzes the formation of glutamate from glutamine as part of amino acid catabolism (Yang \u003cem\u003eet al.\u003c/em\u003e, 2017). These enzymes play important roles in the process of amino acid cycling in plants, indirectly facilitating the uptake and fixation of ammonium.\u003c/p\u003e \u003cp\u003eAmmonium 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 \u003cem\u003eet al.\u003c/em\u003e, 1997). Two major groups of ammonium transporter proteins have been identified in plants: the AMT1 and AMT2 subfamilies (Couturier \u003cem\u003eet al.\u003c/em\u003e, 2007). The plant AMT2 subfamily is more distantly related to the plant AMT1 subfamily (Guether \u003cem\u003eet al.\u003c/em\u003e, 2009), and in Arabidopsis, AtAMT2 is likely to play a significant role in moving ammonium (Sohlenkamp \u003cem\u003eet al.\u003c/em\u003e, 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 \u003cem\u003eet al.\u003c/em\u003e, 2006, Yuan \u003cem\u003eet al.\u003c/em\u003e, 2007), and AtAMT1;2 exhibits a relatively low affinity for ammonium (Neuhauser \u003cem\u003eet al.\u003c/em\u003e, 2007). A plasma membrane NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e channel Ammonium Facilitator 1 (AMF1) has also been found to regulate plasma membrane permeability to NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake indirectly through AMT/MEP/Rh (Mazurkiewicz, 2013).\u003c/p\u003e \u003cp\u003eThe 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 \u003cem\u003ecis\u003c/em\u003e-element motif present in the promoter region of \u003cem\u003eOsAMT1;2\u003c/em\u003e to specifically activate expression. In \u003cem\u003eArabidipsis\u003c/em\u003e, transcription factor Long Hypocotyles 5 (HY5) negatively regulates the expression of \u003cem\u003eAtAMT1;2\u003c/em\u003e, an orthologous gene of \u003cem\u003eOsAMT1;2\u003c/em\u003e (Huang \u003cem\u003eet al.\u003c/em\u003e, 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 \u003cem\u003eOsAMTs\u003c/em\u003e (Yanagisawa \u003cem\u003eet al.\u003c/em\u003e, 2004, Santos \u003cem\u003eet al.\u003c/em\u003e, 2012, Wu \u003cem\u003eet al.\u003c/em\u003e, 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 \u003cem\u003eOsGS1;2\u003c/em\u003e and related genes (El-Kereamy \u003cem\u003eet al.\u003c/em\u003e, 2012).\u003c/p\u003e \u003cp\u003eA 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 \u003cem\u003eet al.\u003c/em\u003e, 2014). \u003cem\u003eGmSAT1\u003c/em\u003e 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 NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e channel \u003cem\u003eScAMF1\u003c/em\u003e, which indirectly enhances NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e permeability and finally promotes ammonium uptake (Chiasson et al., 2014, Mazurkiewicz, 2013).\u003c/p\u003e \u003cp\u003eThe 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 \u003cem\u003eet al.\u003c/em\u003e, 2011). The over application of ammonium fertilizer presents a significant burden to both soil and plants (Dawar \u003cem\u003eet al.\u003c/em\u003e, 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 \u003cem\u003eet al.\u003c/em\u003e, 2019). In this study, we identified an ammonium-responsive \u003cem\u003eMdSAT1\u003c/em\u003e 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.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant materials and growth conditions\u003c/h2\u003e \u003cp\u003eApple seedlings (\u003cem\u003eMalus domestica\u003c/em\u003e) were cultured in a plant growth chamber under 25\u0026deg;C/22\u0026deg;C, 14 h/8 h temperature, and photoperiod. Apple group culture seedlings were grown in Murashige \u0026amp; Skoog (MS) medium (pH\u0026thinsp;=\u0026thinsp;6.0) containing 6-Benzylamino Purine (6-BA, 0.5 mg/L), Naphthaleneacetic Acid (NAA, 0.1 mg/L), and Gibberellin (GA, 0.5 mg/L) for succession every 30 days. For the nitrogen treatment experiment, 1 month-old apple seedlings were selected for rooting in \u0026frac12;MS rooting medium containing 1 mg/L 3-Indoleacetic acid (IAA). When rooting was completed, the seedlings were transferred to a nutrient bowl and cultured for about 30 days. Seedlings of uniform growth were selected and pre-treated in hydroponic conditions with ddH\u003csub\u003e2\u003c/sub\u003eO for 1 week. The seedlings were then treated with 2 mM KCl (represents 0 N), KNO\u003csub\u003e3\u003c/sub\u003e (represents nitrate), or NH\u003csub\u003e4\u003c/sub\u003eCl (represents ammonium), and sampled after 0, 3, 6, 9, 12, and 24 h of treatment.\u003c/p\u003e \u003cp\u003e \u003cem\u003eArabidopsis\u003c/em\u003e seeds were disinfected with 75% alcohol and 3% sodium hypochlorite, and then sown on \u0026frac12; MS medium solid culture plates (15 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sucrose and 8.0 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e agar powder, pH adjusted to 5.9 with 1.0 M sodium hydroxide). The plates were incubated at 4\u0026deg;C with dark vernalization for 4 d. Seeds were germinated and grown at 22\u0026deg;C with a 16 h/8 h light/dark cycle.\u003c/p\u003e \u003cp\u003eDifferent types of \u003cem\u003eArabidopsis\u003c/em\u003e (\u003cem\u003eMdSAT1-OE\u003c/em\u003e, Col) seedlings used for gene expression analysis by RT-PCR were germinated on \u0026frac12; MS medium solid culture plates for 7 days before being transplanted to vermiculite, irrigated with tap water, and then watered weekly with a modified Hoagland\u0026rsquo;s nutrient solution with either Low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (0.5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) or High NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (5 mM NH\u003csub\u003e4\u003c/sub\u003eCl). The basic nutrient solution contained 1.0 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1.0 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 1.0 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.1 mM FeNa\u003csub\u003e2\u003c/sub\u003eEDTA, 50 \u0026micro;M MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO, 50 \u0026micro;M H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, 0.05 \u0026micro;M CuSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO, 0.5 \u0026micro;M Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 15 \u0026micro;M ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 2.5 \u0026micro;M KI, and 0.05 \u0026micro;M CoCl\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO with low or high concentration of NH\u003csub\u003e4\u003c/sub\u003eCl, and the pH was adjusted to 5.9 with 1.0 M sodium hydroxide. The final K\u003csup\u003e+\u003c/sup\u003e concentration was adjusted to be the same in both solutions by addition of K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. After growing for four weeks, the \u003cem\u003eArabidopsis\u003c/em\u003e (\u003cem\u003eMdSAT1-OE\u003c/em\u003e, Col) seedlings were subjected to phenotype observation and physiological and biochemical analysis.\u003c/p\u003e \u003cp\u003eOne or two days after germination, different types of \u003cem\u003eArabidopsis\u003c/em\u003e (\u003cem\u003eMdSAT1-OE\u003c/em\u003e, Col) seedlings were transplanted and grown on Low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (0.5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) or High NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (1.5 mM NH\u003csub\u003e4\u003c/sub\u003eCl) modified solid medium containing the above modified nutrient solution, plus organic matter (2 \u0026micro;M C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 0.02 \u0026micro;M NC\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003eCOOH, 0.001 \u0026micro;M C\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eClN\u003csub\u003e4\u003c/sub\u003eOS\u0026middot;HCl, 0.01 \u0026micro;M C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eN\u0026middot;HCl, 0.1 \u0026micro;M NH\u003csub\u003e2\u003c/sub\u003eCN\u003csub\u003e2\u003c/sub\u003e\u0026middot;COOH), 30 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sucrose, and 8.0 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e agar powder, with the pH adjusted to 5.9 with addition of 1.0 M sodium hydroxide. After three days, the root hairs of the seedlings grown on the above treatment medium were observed, and after seven days, the primary and lateral roots were observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Transgenic materials\u003c/h2\u003e \u003cp\u003e\u003cem\u003eMdSAT1-OE\u003c/em\u003e and \u003cem\u003eProMdSAT1::GUS Arabidopsis\u003c/em\u003e seeds were obtained as described (Fig. S1) (Yang \u003cem\u003eet al.\u003c/em\u003e, 2021).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Bioinformatics analysis\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Multiple sequence alignment and phylogenetic tree construction\u003c/h2\u003e \u003cp\u003eThe protein sequences of SAT1 from different species were obtained using blastp at the NCBI website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The obtained sequences were used to construct a neighbor-joining phylogenetic tree with 1000 bootstrap replicates in MEGA-X (Kumar \u003cem\u003eet al.\u003c/em\u003e, 2018) using the built-in ClustalW algorithm, Poisson model, and parameter settings for partial deletion (95%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Prediction of conserved domains\u003c/h2\u003e \u003cp\u003eThe conserved domains of the SAT1 protein were predicted using Phyre\u003csup\u003e2\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?\u003c/span\u003e\u003cspan address=\"http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e id\u0026thinsp;=\u0026thinsp;index) (Kelley \u003cem\u003eet al.\u003c/em\u003e, 2015).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Extraction of plant genomic DNA and RNA\u003c/h2\u003e \u003cp\u003eGenomic DNA Reagent Kit and Omni Plant RNA Kit (tDNase I) were used to extract plant DNA and RNA (Tiangen, Beijing, China), respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Real-time quantitative RT-PCR analysis of gene expression\u003c/h2\u003e \u003cp\u003eFrom the extracted RNA, cDNA required for quantitative PCR was synthesized using the PrimeScript First Chain cDNA Synthesis Kit (Takara, Dalian, China). These synthesized products were used as templates for real-time quantitative RT-PCR to detect the expression levels of selected genes. Apple 18S rRNA and Arabidopsis actin rRNA genes were used as controls. PCR analysis was performed using specific primer sequences designed using Primer3Plus (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://primer3plus.com/cgi-bin/dev/primer3plus.cgi\u003c/span\u003e\u003cspan address=\"http://primer3plus.com/cgi-bin/dev/primer3plus.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Untergasser \u003cem\u003eet al.\u003c/em\u003e, 2007) and listed in Supplemental Table S1. The qRT-PCR analysis performed in triplicate, and relative gene expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Physiological measurements\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1 Determination of substance content\u003c/h2\u003e \u003cp\u003eAmmoninum, Oxygen-derived free radicals (OFR), and Malondialdehyde (MDA) levels were measured by UV spectrophotometry as described below.\u003c/p\u003e \u003cp\u003eAmmonium interacts with hypochlorite and phenol in a strong alkaline medium to produce the water-soluble dye indophenol blue. Indophenol blue has a characteristic absorption peak at 625 nm and the absorbance value is proportional to the ammonium nitrogen content.\u003c/p\u003e \u003cp\u003eOFR react with hydroxylamine hydrochloride to form NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, which in the presence of p-aminobenzenesulfonic acid and α-naphthylamine produces a red azo compound with a characteristic absorption peak at 530 nm. The content of OFR in the sample can be calculated by measuring the change in absorbance at 530 nm.\u003c/p\u003e \u003cp\u003eMDA condenses with thiobarbituric acid (TBA) to produce a red product with a maximum absorption peak at 532 nm that can be used to estimate the amount of lipid peroxide in the sample. The absorbance at 600 nm was also measured, and the difference between the absorbance at 532 nm and 600 nm was used to calculate the amount of MDA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2 Determination of enzymatic activities\u003c/h2\u003e \u003cp\u003eWeigh 0.1g of plant material, GDH, ASN, GLS, Peroxidase (POD), Catalase (CAT), and Superoxide dismutase (SOD) activities were measured using activity assay kits (Comin, Suzhou, China) based on the below principles.\u003c/p\u003e \u003cp\u003eGDH catalyzes the formation of glutamate and NAD\u003csup\u003e+\u003c/sup\u003e from NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, α-ketoglutarate and NADH. This causes a decrease in absorbance at 340 nm, so GDH activity can be calculated by measuring the rate of decrease in absorbance at 340 nm.\u003c/p\u003e \u003cp\u003eASN catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia, and its enzymatic activity can be calculated by detecting the rate of ammonia increase using Nessler's reagent.\u003c/p\u003e \u003cp\u003eGLS catalyzes the hydrolysis of glutamine to L-glutamate and ammonia, and the rate of increase of ammonia was measured by using Nessler's reagent to calculate the enzymatic activity.\u003c/p\u003e \u003cp\u003ePOD catalyzes the oxidation of specific substrates by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and exhibits characteristic light absorption at 470 nm.\u003c/p\u003e \u003cp\u003eCAT catalyzes the decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by CAT with characteristic light absorption at 405 nm.\u003c/p\u003e \u003cp\u003eSOD can scavenge OFR, and OFR can reduce azotetrazolium to produce blue methanamine, which exhibits absorption at 560 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3 Nitroblue tetrazolium staining\u003c/h2\u003e \u003cp\u003eNitroblue tetrazolium (NBT) staining was performed according to existing methods (CORPAS, 2004).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.7 GUS staining and enzyme activity assay\u003c/h2\u003e \u003cp\u003eTransgenic \u003cem\u003eArabidopsis\u003c/em\u003e (\u003cem\u003eProMdSAT1::GUS\u003c/em\u003e) seedlings were immersed in GUS staining buffer consisting of 1 mM 5-bromo-4-chloro-3-indolyl-β-glutamic acid, 100 mM sodium phosphate (pH 7.0), 0.1 mM EDTA, 0.5 mM ferricyanide, and 0.1% (v/v) Triton X-100 37\u0026deg;C for 1 h in the dark. To quantify GUS activity, proteins were extracted from the seedlings with 1 mL of extraction buffer (50 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e/NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e [pH 7.0], 10 mM β-mercaptoethanol, 10 mM Na\u003csub\u003e2\u003c/sub\u003e-EDTA, 0.1% (v/v) Triton X-100), and 1 mL of RIPA lysis buffer. A protein assay kit (Bio-Rad) was used to determine the total protein concentration. To measure GUS, 100 \u0026micro;L of the protein extract was added to 900 \u0026micro;L of GUS reaction buffer containing 1 mm 4-methylumbelliferone glucuronide and the mixture was incubated at 37\u0026deg;C for 0, 5, 10, 15, 30, and 60 min. Then, 100 \u0026micro;L of the reaction mixture was added to 900 \u0026micro;L of the termination solution (1 M sodium carbonate). Fluorescence values were measured using a VersaFluor Spectrofluorometer (Bio-Rad) at an excitation wavelength of 365 nm and an emission wavelength of 455 nm. \u003cem\u003eArabidopsis\u003c/em\u003e (\u003cem\u003eProMdSAT1::GUS\u003c/em\u003e) seedlings (seven days-old) were pre-treated with ddH\u003csub\u003e2\u003c/sub\u003eO for two days, treated with 1 mM KCl, KNO\u003csub\u003e3\u003c/sub\u003e, or NH\u003csub\u003e4\u003c/sub\u003eCl for different time periods, immersed in the GUS staining solution, and photographed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Root system analysis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eArabidopsis\u003c/em\u003e taproots were observed and photographed under a body view microscope. Digimizer software was used to measure and calculate the number and length of root hairs in a 4-mm area starting 2 mm from the root tip.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Data analysis\u003c/h2\u003e \u003cp\u003eAll experiments were repeated independently three times, unless otherwise indicated. The data are expressed as mean and standard deviation. Data were analyzed by one-way analysis of variance, and means were compared using Duncan's multiple range test. Different letters indicate significant differences at the p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 level.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Phylogenetic relationships, multiple sequence alignment, and protein structure analysis of \u003cem\u003eMdSAT1\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eMdSAT1\u003c/em\u003e (MD10G1115500) gene was identified from the NCBI website according to the GmSAT1 sequence of soybean (\u003cem\u003eGlycine max\u003c/em\u003e). A phylogenetic tree was constructed, and apple MdSAT1 was most closest related to pear PbSAT1 (Rosaceae) (Fig.\u0026nbsp;1A), indicating that these genes diverged recently in evolution. We compared the SAT1 protein sequences of apple with those of other plant species, and the results showed that all 13 proteins had high sequence similarity and belonged to the plant bHLH transcription factor superfamily, members of which contain a bHLH domain and an H-E-R DNA binding region (Fig.\u0026nbsp;1B-C). The high-level structure of the MdSAT1 protein was predicted by homology model, and the results indicated that the secondary and tertiary structures of MdSAT1 match those of the core conserved domain (Fig.\u0026nbsp;1C-D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 \u003cem\u003eMdSAT1\u003c/em\u003e is an ammonium-responsive gene\u003c/h2\u003e \u003cp\u003eMdSAT1 is homologous to GmSAT1, which is involved in ammonium uptake (Chiasson et al., 2014), and qRT-PCR was next used to detect the expression of \u003cem\u003eMdSAT1\u003c/em\u003e in response to different nitrogen forms (KCl, KNO\u003csub\u003e3\u003c/sub\u003e, and NH\u003csub\u003e4\u003c/sub\u003eCl). The expression of \u003cem\u003eMdSAT1\u003c/em\u003e was significantly induced by NH\u003csub\u003e4\u003c/sub\u003eCl both in shoots and roots, however, the transcript level of \u003cem\u003eMdSAT1\u003c/em\u003e showed little change in response to nitrate (Fig.\u0026nbsp;2A-B), suggesting that \u003cem\u003eMdSAT1\u003c/em\u003e was specifically responsive to ammonium.\u003c/p\u003e \u003cp\u003e \u003cem\u003eProMdSAT1::GUS\u003c/em\u003e transgenic \u003cem\u003eArabidopsis\u003c/em\u003e seedlings were treated with different forms of nitrogen, and GUS staining results suggested that the highest GUS activity was observed under NH\u003csub\u003e4\u003c/sub\u003eCl treatment (Fig.\u0026nbsp;2C-D). With increasing time of different treatments, the expression activity of \u003cem\u003eProMdSAT1::GUS\u003c/em\u003e was specifically induced by NH\u003csub\u003e4\u003c/sub\u003eCl (Fig. S2). Taken together, these results suggest that \u003cem\u003eMdSAT1\u003c/em\u003e is specifically responsive to ammonium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Overexpression of \u003cem\u003eMdSAT1\u003c/em\u003e regulates ammonium uptake\u003c/h2\u003e \u003cp\u003eGiven that \u003cem\u003eMdSAT1\u003c/em\u003e is an ammonium-responsive gene, we next treated \u003cem\u003eMdSAT1-OE\u003c/em\u003e and wild type (Col) in MS medium containing 0.5 mM NH\u003csub\u003e4\u003c/sub\u003eCl (Low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) or 5 mM NH\u003csub\u003e4\u003c/sub\u003eCl (High NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) for four weeks and then assessed the effects on plant growth and ammonium content. Under low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e conditions, ectopic expression of \u003cem\u003eMdSAT1\u003c/em\u003e promoted seedling growth compared with Col, and \u003cem\u003eMdSAT1-OE\u003c/em\u003e showed greater fresh weight and increased ammonium content (Fig.\u0026nbsp;3A-C). In contrast, under high NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e conditions, ectopic expression of \u003cem\u003eMdSAT1\u003c/em\u003e reduced fresh weight and accumulated higher ammonium (Fig.\u0026nbsp;3A-C). These results indicate that \u003cem\u003eMdSAT1\u003c/em\u003e promotes ammonium uptake to regulate plant growth.\u003c/p\u003e \u003cp\u003eTo further evaluate the role of MdSAT1 in ammonium uptake, the effects of MdSAT1 on the expression of genes related to ammonium uptake were analyzed. The result showed that transcript levels of \u003cem\u003eAtAMTs\u003c/em\u003e were not increased in \u003cem\u003eMdSAT1-OE\u003c/em\u003e lines, however, expression of \u003cem\u003eAtAMF1;3\u003c/em\u003e was significantly induced in the \u003cem\u003eMdSAT1-OE\u003c/em\u003e lines (Fig.\u0026nbsp;3D). AMF proteins promote NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e permeable transport (Chiasson et al., 2014), so these results indicated that \u003cem\u003eMdSAT1\u003c/em\u003e promoted ammonium uptake by increasing the expression levels of genes related to ammonium uptake.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Overexpression of \u003cem\u003eMdSAT1\u003c/em\u003e affects the enzymatic activities of ammonium assimilation-related proteins\u003c/h2\u003e \u003cp\u003eAfter taken up by plant roots, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e is then assimilated to amino acids or amides through the action of GDH, ASN, and GLS (Yang et al., 2017, Lea, 2006, Lopes et al., 2015, Atkins et al., 1975). Therefore, we measured the activities of these ammonium assimilation-related enzymes in \u003cem\u003eMdSAT1\u003c/em\u003e transgenic \u003cem\u003eArabidopsis\u003c/em\u003e. The results showed that overexpression of \u003cem\u003eMdSAT1\u003c/em\u003e promoted GDH, ASN, and GLS activities \u003cem\u003ein vivo\u003c/em\u003e, independent of ammonium treatment concentration (Fig.\u0026nbsp;4). The expression levels of ammonium assimilation-related genes showed the same trend (Fig. S3). Therefore, the results demonstrate that \u003cem\u003eMdSAT1\u003c/em\u003e increases the activities of ammonium assimilation-related enzymes to influence ammonium assimilation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Overexpression of \u003cem\u003eMdSAT1\u003c/em\u003e promotes lateral root development\u003c/h2\u003e \u003cp\u003eAmmonium in the soil is actively taken up by the roots mainly by ammonium ion transporters (von Wittgenstein \u003cem\u003eet al.\u003c/em\u003e, 2014). The tissue-specific localization of \u003cem\u003eMdSAT1\u003c/em\u003e was detected using \u003cem\u003eProMdSAT1::GUS\u003c/em\u003e transgenic \u003cem\u003eArabidopsis\u003c/em\u003e. GUS staining results showed that \u003cem\u003eMdSAT1\u003c/em\u003e was differentially expressed during lateral root growth, with the highest expression observed at the time of lateral root primordium genesis (Fig. S4). There was no significant difference of primary root length, but the lateral root numbers were significantly increased in the \u003cem\u003eMdSAT1-OE\u003c/em\u003e lines compared with those of Col (Fig.\u0026nbsp;5A-D). These results suggest that \u003cem\u003eMdSAT1\u003c/em\u003e overexpression promotes lateral root growth and development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Overexpression of \u003cem\u003eMdSAT1\u003c/em\u003e regulates root hair growth and development\u003c/h2\u003e \u003cp\u003eRoot hairs play an important role in nutrient uptake (Moon \u003cem\u003eet al.\u003c/em\u003e, 2019), and we next observed the phenotypes of root hairs in \u003cem\u003eMdSAT1-OE\u003c/em\u003e and Col. \u003cem\u003eMdSAT1\u003c/em\u003e significantly increased the number and length of root hairs under low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e treatment (Fig.\u0026nbsp;6A, C-D), and the number and length of root hairs were inhibited under high NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e treatment (Fig.\u0026nbsp;6B-D). The expression levels of genes related to root hair development were also detected. The results showed significantly down-regulated transcript levels of \u003cem\u003eAtEGL3\u003c/em\u003e, \u003cem\u003eAtGL3\u003c/em\u003e, and \u003cem\u003eAtTTG1\u003c/em\u003e genes that inhibit root hair development under low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e treatment (Fig.\u0026nbsp;7A-C) (Bernhardt \u003cem\u003eet al.\u003c/em\u003e, 2003, Schiefelbein \u003cem\u003eet al.\u003c/em\u003e, 2014) and significantly up-regulated levels of \u003cem\u003eAtSCM\u003c/em\u003e, a positive regulator in root hair development (Fig.\u0026nbsp;7D) (Kwak and Schiefelbein, 2014); the expression patterns were reversed under high NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e treatment (Fig.\u0026nbsp;7A-D). These results suggest that \u003cem\u003eMdSAT1\u003c/em\u003e promotes the growth and development of root hairs by regulating the transcript levels of root hair development-related genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.7 \u003cem\u003eMdSAT1\u003c/em\u003e promotes the ROS accumulation\u003c/h2\u003e \u003cp\u003eROS plays a role in root hair development (Monshausen \u003cem\u003eet al.\u003c/em\u003e, 2007), so we next measured ROS content by NBT staining. The results showed that overexpression of \u003cem\u003eMdSAT1\u003c/em\u003e increased ROS accumulation of leaves compared with the level in Col (Fig.\u0026nbsp;8A). OFR content was promoted in the \u003cem\u003eMdSAT1-OE\u003c/em\u003e lines, both in high and low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e treatment (Fig.\u0026nbsp;8C), while the MDA content was only promoted in high NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e treatment (Fig.\u0026nbsp;8B). CAT, SOD, and POD are important enzymes for ROS deconstruction (Waszczak \u003cem\u003eet al.\u003c/em\u003e, 2018, Miller \u003cem\u003eet al.\u003c/em\u003e, 2008) so the activities of these enzymes were measured. Under low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e treatment, overexpression of \u003cem\u003eMdSAT1\u003c/em\u003e resulted in higher CAT and SOD activities but significantly lower POD activity compared to Col (Fig.\u0026nbsp;8D-F). Overall, these results suggest that overexpression of \u003cem\u003eMdSAT1\u003c/em\u003e promotes OFR production by affecting the activity of ROS-deconstruction-related enzymes.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eWhen the soil nitrogen concentration is low, plants are more likely to take up ammonium (Bloom \u003cem\u003eet al.\u003c/em\u003e, 1992). However, the absorption and utilization efficiency of nitrogen by wild-type plants (Col) is not ideal when only ammonium is applied as nitrogen fertilizer. Moreover, the growth of plants is significantly inhibited at low concentration of single ammonium fertilizer, and the use of a higher concentration of single ammonium fertilizer will cause ammonium toxicity to plants. These trends will guide the application of single ammonium as nitrogen fertilizer in agricultural production. Determination of the appropriate intermediate concentration of single ammonium for use as nitrogen fertilizer is essential to ensure the effective utilization of plants and also avoid the waste of resources and environmental pollution caused by excessive fertilization. Alternatively, combined application with nitrate nitrogen may be more appropriate. Therefore, it is important to study the law and mechanism of ammonium absorption by crops to optimize nitrogen utilization. GmSAT1 was functionally identified in soybean root nodule development (Dehcheshmeh, 2013). In this study, phylogenetic and conserved domains analysis indicated that the MdSAT1 protein may be similar in function to GmSAT1 (Fig.\u0026nbsp;1). The function of MdSAT1 was characterized and the results showed that it is highly expressed, mainly in nutrient organs (Fig. S5), and plays a key role in ammonium uptake and assimilation (Fig.\u0026nbsp;2; Fig.\u0026nbsp;3D; Fig.\u0026nbsp;4). MdSAT1 is phenotypically similar to GmSAT1, further confirming the genetic relationship of MdSAT1 with GmSAT1. MdSAT1 also regulates the accumulation of ROS and ultimately plant growth (Fig.\u0026nbsp;3A-C; Fig.\u0026nbsp;5\u0026ndash;8).\u003c/p\u003e\n\u003cp\u003eSeveral studies have shown that GmSAT1 is important for the symbiosis of soybean rhizobia and acts in NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake during soybean rhizome development (Chiasson et al., 2014, Dehcheshmeh, 2013). The Gmsat1 mutant negatively regulates nitrogen deficiency-induced genes to reduce nitrogen uptake (Dehcheshmeh, 2013). Given that \u003cem\u003eMdSAT1\u003c/em\u003e is an ammonium-responsive gene that is induced by NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e expression (Fig. 2; Fig. S2), we evaluated the role of \u003cem\u003eMdSAT1\u003c/em\u003e in ammonium uptake and found that ectopic expression of \u003cem\u003eMdSAT1\u003c/em\u003e promoted seedling growth under low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e conditions compared with the wild type (Fig. 3A-C). Previous study showed that \u003cem\u003eSAT1\u003c/em\u003e can regulate the nitrogen starvation response and coordinates related signaling regulatory networks (Dehcheshmeh, 2013). SAT1 transcriptionally activates a unique plasma membrane NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e channel AMF1, indirectly enhancing NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e permeability, which also affects the regulation of MEP by SAT1 (Mazurkiewicz, 2013). Interestingly, SAT1 was unable to enhance the expression of MEP3 in the absence of AMF1, and SAT1 affects NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e uptake by indirect regulation of the AMT/MEP/Rh family through activation of AMF1 (Mazurkiewicz, 2013). Given this, we analyzed the expression of genes related to ammonium uptake and found that overexpression of \u003cem\u003eMdSAT1\u003c/em\u003e significantly promoted the expression of \u003cem\u003eAtAMF1;3\u003c/em\u003e (Fig. 3D), suggesting that \u003cem\u003eMdSAT1\u003c/em\u003e may promote ammonium uptake by affecting the expression levels of \u003cem\u003eAMF1;3\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eMost of the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e absorbed by plant roots is enzymatically assimilated to glutamate in the root system, and glutamate is then converted to other amino acids and subsequently transported to various parts of the plant organs and tissues via the xylem (Lea PJ, 2006, Comadira \u003cem\u003eet al.\u003c/em\u003e, 2015). ASN is a amide hydrolase that participates in amino acid metabolism and GDH and GLS catabolize ammonia by deamination (Yang et al., 2017, Lopes et al., 2015, Atkins et al., 1975). Our experiments showed that independent of the external NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration, overexpression of \u003cem\u003eMdSAT1\u003c/em\u003e promoted the activities of GDH, ASN, and GLS, consistent with the observed changes in transcript levels of the corresponding genes (Fig. 4; Fig. S3). In this way, \u003cem\u003eMdSAT1\u003c/em\u003e can help regulate the free ammonia level \u003cem\u003ein vivo\u003c/em\u003e and promote the plant\u0026apos;s own ammonia cycle.\u003c/p\u003e\n\u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in soil is actively taken up by the root system mainly through ammonium ion transporters (Wang \u003cem\u003eet al.\u003c/em\u003e, 2012). NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e can affect root system conformation, including primary roots, lateral roots, and root hairs. Previous work found that the primary root length, lateral root length, and root surface area gradually decreased with increasing NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration (Li and Sun, 2007) and addition of 0.1\u0026ndash;10 mM of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e could promote the number and elongation of lateral roots (Yang, 2010). In this study, we found that the primary root lengths of \u003cem\u003eArabidopsis\u003c/em\u003e seedlings overexpressing \u003cem\u003eMdSAT1\u003c/em\u003e were not significantly different from those of Col (Fig. 5C), but the number of lateral roots increased significantly compared with Col (Fig. 5D). We also found that GUS activity of \u003cem\u003eProMdSAT1::GUS\u003c/em\u003e transgenic \u003cem\u003eArabidopsis\u003c/em\u003e seedlings was expressed at the highest level at the beginning of lateral root primordia (Fig. S4). The above results suggest that \u003cem\u003eMdSAT1\u003c/em\u003e is involved in the process of lateral root genesis and growth. A treatment of 0.1 mM NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e promotes the increase of root hair density and root hair number, but at NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentrations higher than 1 mM, the root hair density and root hair number gradually decrease with increasing NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentration (Yang, 2010). Our results showed that under low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e treatment, \u003cem\u003eMdSAT1\u003c/em\u003e significantly promoted root hair development (Fig. 6A, C-D), but under high NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e treatment, it showed the opposite inhibitory effect (Fig. 6B-D). \u003cem\u003eAtEGL3\u003c/em\u003e, \u003cem\u003eAtGL3\u003c/em\u003e, and \u003cem\u003eAtTTG1\u003c/em\u003e, which inhibit root hair development, were significantly down-regulated in \u003cem\u003eMdSAT1-OE Arabidopsis\u003c/em\u003e under low NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e treatment (Fig. 7A-C), but the \u003cem\u003eAtSCM\u003c/em\u003e genes were significantly up-regulated (Fig. 7D). These results suggest that \u003cem\u003eMdSAT1\u003c/em\u003e regulates root development by regulating the transcript levels of root hair-related genes in an ammonium dosage-dependent manner. A role for ROS in root hair growth and formation mechanisms was previously reported (Monshausen et al., 2007). To ask if \u003cem\u003eMdSAT1\u003c/em\u003e regulates root hair development through the ROS pathway, we performed NBT staining and measured MDA and OFR content. The results suggested that overexpression of \u003cem\u003eMdSAT1\u003c/em\u003e accumulated more OFR (Fig. 8A-C). Therefore, we speculate that OFR may regulate root hair development by ROS, a process that requires the involvement of the ammonium-responsive gene \u003cem\u003eMdSAT1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the results of this study showed that overexpression of \u003cem\u003eMdSAT1\u003c/em\u003e promotes plant growth and biomass accumulation. These findings provide theoretical guidance to resolve the mechanisms by which MdSAT1 regulates ammonium uptake and plant growth and provide a reference for future selection of superior germplasm with more efficient nitrogen uptake.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiao-Fei Wang and Wen-Sheng Gao designed the experiments. Tong Li, Zi-Quan Feng, Bai-Hui Zhu, Ming-Li Li and Guo-Dong Li performed the research. Tong Li, Zi-Quan Feng, Bai-Hui Zhu and Chun-Xiang You analyzed the data. Tong Li, Xiao-Fei Wang and Wen-Sheng Gao wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (31972378), Agricultural Variety Improvement Project of Shandong Province (2019LZGC007), China Agriculture Research System of MOF and MARA (CARS-27), Shandong Province Key R\u0026amp;D Program (2021CXGC010802) and Taishan scholar project (LJNY2020266).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank our team leader Dr. Yu-Jin Hao, who will be remembered for his great achievement and for the support and help in our work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eATKINS, C. A., PATE, J. S. \u0026amp; SHARKEY, P. J. (1975), \u0026quot;Asparagine metabolism-key to the nitrogen nutrition of developing legume seeds.\u0026quot;, Plant Physiology, Vol. 56 No. 6, pp. 807-812.\u003c/li\u003e\n \u003cli\u003eBAOHAI, L. \u0026amp; WEIMING, S. (2007), \u0026quot;EFFECTS OF ELEVATED NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e ON \u003cem\u003eARABIDOPSIS\u003c/em\u003e SEEDLINGS DIFFERENT IN ACCESSIONS\u0026quot;, Journal of Soil, Vol. 44 No. 3, pp. 508-515.\u003c/li\u003e\n \u003cli\u003eBERNHARDT, C., LEE, M. M., GONZALEZ, A., ZHANG, F., LLOYD, A. \u0026amp; SCHIEFELBEIN, J. (2003), \u0026quot;The bHLH genes GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the \u003cem\u003eArabidopsis\u003c/em\u003e root\u0026quot;, Development, Vol. 130 No. 26, pp. 6431-9.\u003c/li\u003e\n \u003cli\u003eBLOOM, A. J. (1997), \u0026quot;Nitrogen as a Limiting Factor: Crop Acquisition of Ammonium and Nitrate\u0026quot;, Ecology in Agriculture, 145-172.\u003c/li\u003e\n \u003cli\u003eBLOOM, A. J., SUKRAPANNA, S. S. \u0026amp; WARNER, R. L. (1992), \u0026quot;Root respiration associated with ammonium and nitrate absorption and assimilation by barley\u0026quot;, Plant Physiol, Vol. 99 No. 4, pp. 1294-301.\u003c/li\u003e\n \u003cli\u003eCHIASSON, D. M., LOUGHLIN, P. C., MAZURKIEWICZ, D., MOHAMMADIDEHCHESHMEH, M., FEDOROVA, E. E., OKAMOTO, M., MCLEAN, E., GLASS, A. D., SMITH, S. E., BISSELING, T., TYERMAN, S. D., DAY, D. A. \u0026amp; KAISER, B. N. (2014), \u0026quot;Soybean SAT1 (Symbiotic Ammonium Transporter 1) encodes a bHLH transcription factor involved in nodule growth and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e transport\u0026quot;, Proc Natl Acad Sci U S A, Vol. 111 No. 13, pp. 4814-9.\u003c/li\u003e\n \u003cli\u003eCOMADIRA, G., RASOOL, B., KARPINSKA, B., MORRIS, J., VERRALL, S. R., HEDLEY, P. E., FOYER, C. H. \u0026amp; HANCOCK, R. D. (2015), \u0026quot;Nitrogen deficiency in barley (Hordeum vulgare) seedlings induces molecular and metabolic adjustments that trigger aphid resistance.\u0026quot;, Journal of experimental botany, Vol. 66 No. 12, pp.\u003c/li\u003e\n \u003cli\u003eCORPAS, F. J. S. L. (2004), \u0026quot;Cadmium-induced subcellular accumulation of O\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003eand H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in pea leaves-ROMERO-PUERTAS \u0026quot;, Plant, Cell \u0026amp; Environment, Vol. 271122-1134.\u003c/li\u003e\n \u003cli\u003eCOUTURIER, J., MONTANINI, B., MARTIN, F., BRUN, A., BLAUDEZ, D. \u0026amp; CHALOT, M. (2007), \u0026quot;The expanded family of ammonium transporters in the perennial poplar plant\u0026quot;, New Phytol, Vol. 174 No. 1, pp. 137-150.\u003c/li\u003e\n \u003cli\u003eDAWAR, K., SAIF-UR-RAHMAN, FAHAD, S., ALAM, S. S., KHAN, S. A., DAWAR, A., YOUNIS, U., DANISH, S., DATTA, R. \u0026amp; DICK, R. P. (2021), \u0026quot;Influence of variable biochar concentration on yield-scaled nitrous oxide emissions, Wheat yield and nitrogen use efficiency\u0026quot;, Sci Rep, Vol. 11 No. 1, pp. 16774.\u003c/li\u003e\n \u003cli\u003eDEHCHESHMEH, M. M. (2013) Regulatory control of the symbiotic enhanced soybean bHLH transcription factor, GmSAT1.\u0026nbsp;Adelaide; 2013.\u003c/li\u003e\n \u003cli\u003eEL-KEREAMY, A., BI, Y. M., RANATHUNGE, K., BEATTY, P. H., GOOD, A. G. \u0026amp; ROTHSTEIN, S. J. (2012), \u0026quot;The rice R2R3-MYB transcription factor OsMYB55 is involved in the tolerance to high temperature and modulates amino acid metabolism\u0026quot;, PLoS One, Vol. 7 No. 12, pp. e52030.\u003c/li\u003e\n \u003cli\u003eGUETHER, M., NEUHAUSER, B., BALESTRINI, R., DYNOWSKI, M., LUDEWIG, U. \u0026amp; BONFANTE, P. (2009), \u0026quot;A mycorrhizal-specific ammonium transporter from Lotus japonicus acquires nitrogen released by arbuscular mycorrhizal fungi\u0026quot;, Plant Physiol, Vol. 150 No. 1, pp. 73-83.\u003c/li\u003e\n \u003cli\u003eHUANG, L., ZHANG, H., ZHANG, H., DENG, X. W. \u0026amp; WEI, N. (2015), \u0026quot;HY5 regulates nitrite reductase 1 (NIR1) and ammonium transporter1;2 (AMT1;2) in \u0026nbsp;\u003cem\u003eArabidopsis\u003c/em\u003e seedlings\u0026quot;, Plant Sci, Vol. 238330-9.\u003c/li\u003e\n \u003cli\u003eJACKSON, L. E., BURGER, M. \u0026amp; CAVAGNARO, T. R. (2008), \u0026quot;Roots, nitrogen transformations, and ecosystem services\u0026quot;, Annu Rev Plant Biol, Vol. 59341-63.\u003c/li\u003e\n \u003cli\u003eKELLEY, L. A., MEZULIS, S., YATES, C. M., WASS, M. N. \u0026amp; STERNBERG, M. J. (2015), \u0026quot;The Phyre2 web portal for protein modeling, prediction and analysis\u0026quot;, Nat Protoc, Vol. 10 No. 6, pp. 845-58.\u003c/li\u003e\n \u003cli\u003eKUMAR, S., STECHER, G., LI, M., KNYAZ, C. \u0026amp; TAMURA, K. (2018), \u0026quot;MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms\u0026quot;, Mol Biol Evol, Vol. 35 No. 6, pp. 1547-1549.\u003c/li\u003e\n \u003cli\u003eKWAK, S. H. \u0026amp; SCHIEFELBEIN, J. (2014), \u0026quot;TRIPTYCHON, not CAPRICE, participates in feedback regulation of SCM expression in the \u003cem\u003eArabidopsis\u003c/em\u003e root epidermis\u0026quot;, Plant Signal Behav, Vol. 9 No. 11, pp. e973815.\u003c/li\u003e\n \u003cli\u003eLAWLOR D. W., L. G. G. F. (2001), Plant Nitrogen. Springe, Berlin/Heidelberg, Germany.\u003c/li\u003e\n \u003cli\u003eLEA PJ, A. R. (2006), \u0026quot;Nitrogen use efficiency. 1. Uptake of nitrogen from the soil\u0026quot;, Annals of Applied Biology, Vol. 149243-347.\u003c/li\u003e\n \u003cli\u003eLOPES, A. M., OLIVEIRA-NASCIMENTO, L. D., RIBEIRO, A., TAIRUM, C. A., BREYER, C. A., OLIVEIRA, M. A. D., MONTEIRO, G., SOUZA-MOTTA, C. M. D., MAGALHES, P. D. O. \u0026amp; AVENDAO, J. G. F. (2015), \u0026quot;Therapeutic l-asparaginase: upstream, downstream and beyond\u0026quot;, Critical Reviews in Biotechnology, 82-99.\u003c/li\u003e\n \u003cli\u003eLOPEZ-PEDROSA, A., GONZALEZ-GUERRERO, M., VALDERAS, A., AZCON-AGUILAR, C. \u0026amp; FERROL, N. (2006), \u0026quot;GintAMT1 encodes a functional high-affinity ammonium transporter that is expressed in the extraradical mycelium of Glomus intraradices\u0026quot;, Fungal Genet Biol, Vol. 43 No. 2, pp. 102-10.\u003c/li\u003e\n \u003cli\u003eMARINI, A. M., SOUSSI-BOUDEKOU, S., VISSERS, S. \u0026amp; ANDRE, B. (1997), \u0026quot;A family of ammonium transporters in Saccharomyces cerevisiae\u0026quot;, Mol Cell Biol, Vol. 17 No. 8, pp. 4282-93.\u003c/li\u003e\n \u003cli\u003eMAZURKIEWICZ, D. (2013) Characterisation of a Novel Family of Eukaryotic Ammonium Transport Proteins.\u0026nbsp;Adelaide; 2013.\u003c/li\u003e\n \u003cli\u003eMIAO, Y., STEWART, B. A. \u0026amp; ZHANG, F. (2011), \u0026quot;Long-term experiments for sustainable nutrient management in China. A review\u0026quot;, Agronomy for Sustainable Development, Vol. 31 No. 2, pp. 397-414.\u003c/li\u003e\n \u003cli\u003eMILLER, G., SHULAEV, V. \u0026amp; MITTLER, R. (2008), \u0026quot;Reactive oxygen signaling and abiotic stress\u0026quot;, Physiol Plant, Vol. 133 No. 3, pp. 481-9.\u003c/li\u003e\n \u003cli\u003eMONSHAUSEN, G. B., BIBIKOVA, T. N., MESSERLI, M. A., SHI, C. \u0026amp; GILROY, S. (2007), \u0026quot;Oscillations in extracellular pH and reactive oxygen species modulate tip growth of \u003cem\u003eArabidopsis\u003c/em\u003e root hairs\u0026quot;, Proc Natl Acad Sci U S A, Vol. 104 No. 52, pp. 20996-1001.\u003c/li\u003e\n \u003cli\u003eMOON, S., CHO, L. H., KIM, Y. J., GHO, Y. S., JEONG, H. Y., HONG, W. J., LEE, C., PARK, H., JWA, N. S., DANGOL, S., CHEN, Y., PARK, H., CHO, H. S., AN, G. \u0026amp; JUNG, K. H. (2019), \u0026quot;RSL Class II Transcription Factors Guide the Nuclear Localization of RHL1 to Regulate Root Hair Development\u0026quot;, Plant Physiol, Vol. 179 No. 2, pp. 558-568.\u003c/li\u003e\n \u003cli\u003eNA, Y. (2010) Interactions between nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e,NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e) and phytohormones in the regulation of root conformation in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e., Nanjing Agricultural University.\u003c/li\u003e\n \u003cli\u003eNEUHAUSER, B., DYNOWSKI, M., MAYER, M. \u0026amp; LUDEWIG, U. (2007), \u0026quot;Regulation of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e transport by essential cross talk between AMT monomers through the carboxyl tails\u0026quot;, Plant Physiol, Vol. 143 No. 4, pp. 1651-9.\u003c/li\u003e\n \u003cli\u003eNOCTOR, G, FOYER \u0026amp; CH (1998), \u0026quot;Review article. A re-evaluation of the ATP:NADPH budget during Cphotosynthesis: a contribution from nitrate assimilation and its associated respiratory activity?\u0026quot;, Journal of Experimental Botany.\u003c/li\u003e\n \u003cli\u003eOHYAMA, T. (2010), Nitrogen as a major essential element of plants, Nitrogen Assimilation in Plants.\u003c/li\u003e\n \u003cli\u003ePATTERSON, K., CAKMAK, T., COOPER, A., LAGER, I., RASMUSSON, A. G. \u0026amp; ESCOBAR, M. A. (2010), \u0026quot;Distinct signalling pathways and transcriptome response signatures differentiate ammonium- and nitrate-supplied plants\u0026quot;, Plant Cell Environ, Vol. 33 No. 9, pp. 1486-501.\u003c/li\u003e\n \u003cli\u003eRUBIO-ASENSIO, J. S. \u0026amp; BLOOM, A. J. (2017), \u0026quot;Inorganic nitrogen form: a major player in wheat and \u003cem\u003eArabidopsis\u0026nbsp;\u003c/em\u003eresponses to elevated CO\u003csub\u003e2\u003c/sub\u003e.\u0026quot;, Journal of experimental botany, Vol. 68 No. 10, pp.\u003c/li\u003e\n \u003cli\u003eSANTOS, L. A., SOUZA, S. \u0026amp; FERNANDES, M. S. (2012), \u0026quot;OsDof25 expression alters carbon and nitrogen metabolism in \u003cem\u003eArabidopsis\u003c/em\u003e under high N-supply\u0026quot;, Plant Biotechnology Reports, Vol. 6 No. 4, pp. p.327-337.\u003c/li\u003e\n \u003cli\u003eSCHIEFELBEIN, J., HUANG, L. \u0026amp; ZHENG, X. (2014), \u0026quot;Regulation of epidermal cell fate in \u003cem\u003eArabidopsis\u003c/em\u003e roots: the importance of multiple feedback loops\u0026quot;, Front Plant Sci, Vol. 547.\u003c/li\u003e\n \u003cli\u003eSOHLENKAMP, C., WOOD, C. C., ROEB, G. W. \u0026amp; UDVARDI, M. K. (2002), \u0026quot;Characterization of \u003cem\u003eArabidopsis\u003c/em\u003e AtAMT2, a high-affinity ammonium transporter of the plasma membrane\u0026quot;, Plant Physiol, Vol. 130 No. 4, pp. 1788-96.\u003c/li\u003e\n \u003cli\u003eUNTERGASSER, A., NIJVEEN, H., RAO, X., BISSELING, T., GEURTS, R. \u0026amp; LEUNISSEN, J. A. (2007), \u0026quot;Primer3Plus, an enhanced web interface to Primer3\u0026quot;, Nucleic Acids Res, Vol. 35 No. Web Server issue, pp. W71-4.\u003c/li\u003e\n \u003cli\u003eVIKTOR, A. \u0026amp; CRAMER, M. D. (2005), \u0026quot;The influence of root assimilated inorganic carbon on nitrogen acquisition/assimilation and carbon partitioning\u0026quot;, New Phytol, Vol. 165 No. 1, pp. 157-69.\u003c/li\u003e\n \u003cli\u003eVON WITTGENSTEIN, N. J., Le CH, HAWKINS, B. J. \u0026amp; EHLTING, J. (2014), \u0026quot;Evolutionary classification of ammonium, nitrate, and peptide transporters in land plants\u0026quot;, BMC Evol Biol, Vol. 1411.\u003c/li\u003e\n \u003cli\u003eWANG, Y. Y., HSU, P. K. \u0026amp; TSAY, Y. F. (2012), \u0026quot;Uptake, allocation and signaling of nitrate\u0026quot;, Trends Plant Sci, Vol. 17 No. 8, pp. 458-67.\u003c/li\u003e\n \u003cli\u003eWASZCZAK, C., CARMODY, M. \u0026amp; KANGASJARVI, J. (2018), \u0026quot;Reactive Oxygen Species in Plant Signaling\u0026quot;, Annu Rev Plant Biol, Vol. 69209-236.\u003c/li\u003e\n \u003cli\u003eWEI, X., LINA, Z., WENZHONG, Z., JIPING, G., JUN, Y., XIAOXI, Z., MING, D., YANZE, Z. \u0026amp; LIQIANG, C. (2019), \u0026quot;An Integrated Analysis of the Rice Transcriptome and Metabolome Reveals Root Growth Regulation Mechanisms in Response to Nitrogen Availability\u0026quot;, International Journal of Molecular Sciences, Vol. 20 No. 23, pp.\u003c/li\u003e\n \u003cli\u003eWU, Y., YANG, W., WEI, J., YOON, H. \u0026amp; AN, G. (2017), \u0026quot;Transcription Factor OsDOF18 Controls Ammonium Uptake by Inducing Ammonium Transporters in Rice Roots\u0026quot;, Mol Cells, Vol. 40 No. 3, pp. 178-185.\u003c/li\u003e\n \u003cli\u003eXU, G., FAN, X. \u0026amp; MILLER, A. J. (2012), \u0026quot;Plant nitrogen assimilation and use efficiency\u0026quot;, Annu Rev Plant Biol, Vol. 63153-82.\u003c/li\u003e\n \u003cli\u003eYANAGISAWA, S. (2000), \u0026quot;Dof1 and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize\u0026quot;, Plant J, Vol. 21 No. 3, pp. 281-8.\u003c/li\u003e\n \u003cli\u003eYANAGISAWA, S., AKIYAMA, A., KISAKA, H., UCHIMIYA, H. \u0026amp; MIWA, T. (2004), \u0026quot;Metabolic engineering with Dof1 transcription factor in plants: Improved nitrogen assimilation and growth under low-nitrogen conditions\u0026quot;, Proc Natl Acad Sci U S A, Vol. 101 No. 20, pp. 7833-8.\u003c/li\u003e\n \u003cli\u003eYANG, Y. Y., ZHENG, P. F., REN, Y. R., YAO, Y. X., YOU, C. X., WANG, X. F. \u0026amp; HAO, Y. J. (2021), \u0026quot;Apple MdSAT1 encodes a bHLHm1 transcription factor involved in salinity and drought responses\u0026quot;, Planta, Vol. 253 No. 2, pp.\u003c/li\u003e\n \u003cli\u003eYANG, Y. Z., DING, S., WANG, Y., LI, C. L., SHEN, Y., MEELEY, R., MCCARTY, D. R. \u0026amp; TAN, B. C. (2017), \u0026quot;Small kernel2 Encodes a Glutaminase in Vitamin B6 Biosynthesis Essential for Maize Seed Development\u0026quot;, Plant Physiol, Vol. 174 No. 2, pp. 1127-1138.\u003c/li\u003e\n \u003cli\u003eYUAN, L., LOQUE, D., KOJIMA, S., RAUCH, S., ISHIYAMA, K., INOUE, E., TAKAHASHI, H. \u0026amp; von WIREN, N. (2007), \u0026quot;The organization of high-affinity ammonium uptake in \u003cem\u003eArabidopsis\u0026nbsp;\u003c/em\u003eroots depends on the spatial arrangement and biochemical properties of AMT1-type transporters\u0026quot;, Plant Cell, Vol. 19 No. 8, pp. 2636-52.\u003c/li\u003e\n \u003cli\u003eZHAO C., L. Q. (2009), \u0026quot;Growth and physiological responses of Picea asperata seedlings to elevated temperature and to nitrogen fertilization\u0026quot;, Acta Physiol. Plant, Vol. 1 No. 31, pp. 163.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Apple, Ammonium, MdSAT1, Functional identification","lastPublishedDoi":"10.21203/rs.3.rs-1520208/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1520208/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlants mainly uptake inorganic nitrogen from soil as ammonium and nitrate. Less energy is required to assimilate ammonium compared to nitrates, and plants prefer to take up ammonium when the external nitrogen concentration is low. Investigating the patterns and mechanisms of ammonium absorption can help improve crop nitrogen utilization. In this study of apple, we isolated \u003cem\u003eMdSAT1\u003c/em\u003e, a gene encoding an ammonium-responsive bHLH transcription factor. MdSAT1 promoted the growth and development of lateral roots and root hairs. Overexpression of MdSAT1 increased the transcript levels of genes related to ammonium uptake and assimilation and promoted the activities of ammonium assimilation-related enzymes, indicating that MdSAT1 can enhance ammonium uptake and utilization. \u003cem\u003eMdSAT1\u003c/em\u003e also can modulate ROS accumulation to ultimately regulate plant growth. Taken together, these findings provide insight into the mechanisms by which \u003cem\u003eMdSAT1 \u003c/em\u003econtrols ammoniun utilization as well as plant growth and development in apple.\u003c/p\u003e","manuscriptTitle":"Functional identification of bHLH transcription factor MdSAT1 in the ammonium response in apple","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-04 15:52:38","doi":"10.21203/rs.3.rs-1520208/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ae754297-e42f-43cd-8556-9012a9c3d4bf","owner":[],"postedDate":"May 4th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-05-31T08:53:57+00:00","versionOfRecord":[],"versionCreatedAt":"2022-05-04 15:52:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1520208","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1520208","identity":"rs-1520208","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}