Genome-wide Identification of Torreya grandis AMT Family Genes Revealed the Function and Regulation of the Nitrogen Stress Responsive Gene TgAMT5

Genome-wide Identification of Torreya grandis AMT Family Genes Revealed the Function and Regulation of the Nitrogen Stress Responsive Gene TgAMT5 | 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 Genome-wide Identification of Torreya grandis AMT Family Genes Revealed the Function and Regulation of the Nitrogen Stress Responsive Gene TgAMT5 Yuanyuan Guan, Yi Li, Yao Wei, Xu Li, Weijie Chen, Chenliang Yu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4957530/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 Background Ammonium transporters (AMTs) are a class of transmembrane proteins widely found in plants, bacteria, fungi, and other organisms, mediating transmembrane ammonium nitrogen (NH 4 + ) transport, which is one of the crucial pathways for plants to obtain nitrogen from resources. AMTs have been studied in many plants but have not been systematically analyzed in Torreya grandis . Results This study first used bioinformatics to identify members of the T . grandis AMT family and then real time quantitative PCR to explore their tissue expression patterns and abiotic stress responses. The physical and chemical properties, secondary structure, and evolutionary relationships of the encoded proteins were ascertained. There were ten members of the gene family, named TgAMT1 – TgAMT10 , which were located on six chromosomes, with coding sequence lengths of 975–1629 bp. Subcellular localization predicted all members to be located on the plasma membrane. Phylogenetic analysis divided the TgAMTs into two subfamilies, AMT1 and AMT2. There were significant differences in gene structure and conserved motifs among the subfamilies, but Motif 1, Motif 3, and Motif 4 were common to all. The expression of TgAMTs was histologically specific. Additionally, nitrogen morphology also affected TgAMTs expression. TgAMT5 was identified as a potential member involved in the response to NH 4 + -induced stress. The gene function of TgAMT5 was verified in transgenic A . thaliana and was found to promote plant growth and development, especially root growth, by absorbing ammonium salt through roots. In addition, dual-luciferase and yeast one-hybrid assays showed that the transcription factor TgWRKY2 could directly bind to the TgAMT5 promoter and enhance its expression. Conclusion This study can provide theoretical basis for the efficient use of nitrogen in Torreya grandis , and lay a foundation for exploring nitrogen uptake and utilization in gymnosperms. Torreya grandis Ammonium transporter Expression pattern Molecular regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Nitrogen (N) is one of the macrotrophic elements indispensable for plant growth and development and also plays a crucial role in flowering and fruit setting[ 1 ]. Insufficient nitrogen levels can result in stunted plant growth, reduced yields, and compromised quality [ 2 ]. Plants generally absorb inorganic nitrogen in the forms of nitrate (NO 3 − ) and ammonium (NH 4 + ), with a tendency of most plant species to favor NH 4 + [ 3 ]. This preference may be due to the lower energy consumption involved in the absorption process [ 4 , 5 ]. The AMT family encodes carrier proteins that mainly transport ammonium nitrogen [ 6 ]. The plant AMTs belong to the AMT/MEP/Rh family, which is primarily composed of the AMT1 and AMT2 two subfamilies [ 7 , 8 ]. These subfamilies are structurally similar, each containing 9 ~ 11 transmembrane (TM) domains [ 9 ]. These domains enable the AMT protein to form channels in the cell membrane, facilitating the transmembrane transport of ammonium ions [ 10 ]. AMTs play an essential role in plants by regulating the absorption and utilization of ammonium nitrogen in the soil [ 11 ]. Among plants, AMTs were first characterized in Arabidopsis [ 12 ], with a total of six AMTs, of which five and one belonged to the AMT1 and AMT2 subfamilies, respectively [ 8 , 13 ]. Examining the AMT family among diverse taxa such as Chlamydomonas reinhardtii [ 14 ], Physcomitrella patens [ 15 ], Selaginella moellendorffii [ 15 ], Zea mays [ 16 ], and Glycine max [ 17 ], illustrated the phenotypic diversity of AMTs across distinct evolutionary branches in plants. In response to the diverse distribution of NH 4 + concentrations in different soil environments, plants have evolved two distinct absorption systems to optimize the utilization of nitrogen: the low-affinity (LATs) and high-affinity transporter systems (HATs) [ 18 ]. Of these, AMT1-type transporters are mainly responsible for mediating the high-affinity uptake of ammonium by roots [ 19 ]. In the presence of high-affinity conditions, mutations in AtAMT1;1 and AtAMT1;3 reduced the absorption of NH 4 + in Arabidopsis roots [ 20 ]. Similarly, ZmAMT1;1a and ZmAMT1;3 are members of the HATs in maize roots, enhancing body mass [ 16 ]. In rice, elevating the expression levels of OsAMT1.1 bolstered the root absorption of NH 4 + upon exposure to high-ammonium conditions [ 21 ]. In contrast to AMT1, AMT2 demonstrates intricate gene structures and protein profiles, along with lower similarity to homologs from fungi and bacteria [ 22 , 23 ]. AMT2 did not participate in root ammonium uptake in Arabidopsis , but evidence from various plant species has shown that it can be involved in nitrogen transport [ 24 – 26 ]. Plants are capable of adapting to varying nitrogen supplies and growth environments by regulating the expression and activity of AMTs [ 27 ]. For example, in Arabidopsis , IDD10 enhances the root development function of AMT1;2 by activating its transcription [ 28 ]. Likewise, OsDOF18 and OsBZR1 regulate ammonium absorption in rice roots by stimulating the expression of AMTs [ 29 , 30 ]. Moreover, CIPK23 phosphorylates AtAMT1;1 and AtAMT1;2 , rendering them inactive, thereby regulating ammonia absorption [ 31 ]. Similarly, rice plants avoid ammonia toxicity by phosphorylating OsAMT1;2 via OsACTPK1 , leading to its inactivation [ 32 ]. In this study, the AMT family of Torreya grandis was identified, and its evolutionary relationship was analyzed. Further, AMT functional analysis was performed in this economically important subtropical Chinese tree, known for its delicious and nutritionally rich nuts [ 33 ]. The chromosome distribution, phylogenetic relationships, and conserved domains of the AMT family in this plant were comprehensively analyzed. Subsequently, real time quantitative PCR(RT-qPCR) revealed the expression patterns of the AMT family under different nitrogen conditions and identified TgAMT5 as a potential factor involved in ammonium nitrogen response. Multiple protein–DNA interaction analyses suggested that TgWRKY2 enhanced the activity of the TgAMT5 promoter. Materials and methods Plant materials We cultivated 12-month-old seedlings of T . grandis via hydroponic culture, utilizing a climate-controlled chamber placed in the greenhouse of the Zhejiang A&F University, Hangzhou, Zhejiang, China. The voucher specimens were identified byProf. Daiwensheng and deposited in the Zhejiang A&F University Intelligent Experimental Building Plant Specimen Room N205. The treatments included growing plants in normal Hoagland's solution (HB8870-1, Hopebio), nitrogen-deficient Hoagland's solution(HB8870-9, Hopebio), nitrogen-deficient Hoagland's solution supplemented with ammonium sulfate (50 mmol/L (NH 4 ) 2 SO 4 ), and nitrogen-deficient Hoagland's solution supplemented with calcium nitrate (50 mmol/L Ca(NO 3 ) 2 . The pH of all was 6.5. All materials were treated for 7 days and 14 days, and each sample was stored in the ultra-low temperature refrigerator (-80℃) of the State Key Laboratory of Subtropical Forest Cultivation in Zhejiang A & F University. Bioinformatic analysis The amino acid and nucleotide sequences of Ginkgo biloba , Populus trichocarpa , Oryza sativa , Lycopersicum esculentum , Arabidopsis thaliana (National Center for Biotechnology Information (nih.gov)), and T. grandis ( https://doi.org/10.6084/m9.figshare.21089869 ). All AMTs were downloaded from the database. First, based on the threshold value of e-value ≤ e − 5 , the AMT gene family was identified using hmmer v3.2.1 with Pfam ID (PF00909) as the target dataset. Subsequently, NCBI CDD analysis was utilized to identify potential domains within the AMTs [ 34 , 35 ]. Expasy ProtParam ( https://web.expasy.org/protparam/ ) was used to analyze the amino acid number, molecular weight, isoelectric point, and the physical and chemical properties of the AMTs. TMHMM Server 2.0 ( http://www.cbs.dtu.dk/services/TMHMM/ ) was utilized for transmembrane protein structure prediction. Wolfpsort ( http://wolfpsort.hgc.jp/ ) was applied to predict protein subcellular localization. SOPMA and SWISS-MODEL ( https://swissmodel.expasy.org/ ) were used for protein secondary structure prediction. Construction of a phylogenetic evolutionary tree and Cis-acting element analysis The AMTs prontein sequences of Oryza sativa [ 36 ], L . esculentum [ 37 ], Populus trichocarpa [ 38 ], Ginkgo biloba [ 39 ], Arabidopsis thaliana [ 40 ], and T . grandis were imported into MEGA 11. MUSCLE was used for multi-sequence comparison, and the NJ method was selected as the conformable tree method. The tree was visualized by applying Evolview v2. The promoter sequence of the target gene was extracted and predicted using Plantcare. The cis-acting elements were identified and imported into TBtools for visualization. Structural and chromosomal location analysis of the AMT gene family in T. grandis The protein sequence of TgAMT gene family was examined utilizing the MEME Suite (version 5.5.4) to analyze their conserved motifs and assess their functional importance, with reference to the NCBI-CDD database [ 32 , 34 ]. Subsequently, the gene structure and chromosomal positions of the TgAMTs family were analyzed using TB-tools-II [ 41 ]. Analysis of gene expression patterns Quantitative primers were designed using Primer3 ( https://primer3.ut.ee/ ) based on the T. grandis AMT sequence ( Table S1 ). RNA was extracted from the roots and leaves of plants cultivated under different nitrogen conditions as described in 2.1 by the non-Trizol method (Invitrogen). cDNA was synthesized by the HiScript Ⅲ RT Super Mix reverse transcription kit and RT-qPCR was performed using the Cham Q SYBR qPCR Master Mix. RT-qPCR was performed per the procedure: predenaturation at 95 ℃ for 30 min, 10 s at 95 ℃, 30 s at 60 ℃, and 40 reaction cycles. Annealing was performed by applying the default dissolution curve of the instrument. TgACTIN was used as the internal reference gene, and the relative expression of TgAMTs was estimated by the 2 −ΔΔCt method [ 42 ]. Statistical and significant differences in TgAMTs expression were analyzed, and GraphPad Prism10 was used for mapping. Cultivation of transgenic Arabidopsis plants The coding sequence (CDS) of TgAMT5 was cloned into pCAMBIA1300. Arabidopsis flowers were infected by Agrobacterium tumefaciens and obtain T2 generation transgenic seeds. The transgenic Arabidopsis seeds was washed twice with 5% sodium hypochlorite for 10 min each and then washed five times with sterilized water for 2 min each. The sterile seeds were planted on half strength of Hoagland's medium, wrapped in tin foil in a Petri dish, unfoiled after three days, and placed in an incubator (16/8 h light/dark; 65% RH). After three days of vertical culture, the seedlings with consistent growth (~ 1 cm root length) were transferred to an ammonium salt-containing solid medium (pH = 5.8) and cultured until an obvious phenotype appeared. Dual-luciferase assay The 2000 bp promoter of TgAMT5 was cloned into the pGreen II 0800-LUC vector for the dual-luciferase assay. Similarly, the CDS of the candidate TF genes was cloned into the pGreen II 62-SK vector to generate the effectors. The A.tumefaciens (GV3101: pSoup) cells carrying the reporter and effector plasmids were injected into the young leaves of Nicotiana benthamiana . Luciferase activity (LUC and REN) was assessed using a dual luciferase assay kit (RG027, Beyotime), and fluorescence was observed using a Tanon 5200 automatic chemiluminescence image analyzer. Yeast one-hybrid assay The W-box is a cis-acting element of the TgAMT5 promoter. This promoter was divided into six segments, and each was cloned into the pLacZi vecto. Simultaneously, the TgWRKY2 CDS was cloned into the pB42AD vector. The EGY48 yeast strain was transformed with these constructs and then cultured on a dropout/-Trp/-Ura/Gal/Raf/X-Gal (80 µg/mL) plate. The cultures were then incubated for three days to observe if they turned blue. The relevant primers designed are detailed in Table S2 . Results Identification of the TgAMTs family members and analysis of their fundamental characteristics This study identified ten members of the AMT gene family from the Torreya grandis genome, which were numbered TgAMT1 – TgAMT10 (Table 1 ). TgAMTs were found to be located on chromosomes 1, 2, 5, 6, 7, and 8, with two tandem repeating genes, namely TgAMT4 and TgAMT5 (Fig. 1 ). The length of the CDSs ranged from 975–1629 bp (Table 1 ). Table 1 Physicochemical properties of the AMT genes family in Torreya grandis. Gene Gene ID Chromosome location CDS Length Protein size Molecular mass pI GRAVY Trans- membrane Subcellular location TgAMT1 PTG007578L.5 Chr01 1431 476 51628.55 8.16 0.526 11 plas vacu E.R. TgAMT2 PTG006090L.35 Chr01 1518 505 54464.56 6.39 0.410 11 plas E.R nucl TgAMT3 PTG006814L.93 Chr02 1521 506 54574.42 6.29 0.376 11 plas chlo nucl vacu E.R. TgAMT4 PTG002221L.98 Chr05 1413 470 50896.98 6.36 0.624 11 plas vacu TgAMT5 PTG002221L.99 Chr05 1431 476 51681.85 6.79 0.567 11 plas vacu TgAMT6 PTG006706L.7 Chr05 975 324 35056.80 9.80 0.714 11 plas vacu E.R. TgAMT7 PTG015048L.1 Chr06 1629 542 59003.83 6.96 0.261 11 plas TgAMT8 PTG013339L.7 Chr06 1470 489 52987.02 8.16 0.482 9 plas TgAMT9 PTG004123L.21 Chr07 1443 480 53067.92 6.10 0.490 10 plas vacu E.R. TgAMT10 PTG027016L.5 Chr08 1467 488 52682.75 6.41 0.545 11 plas The physical and chemical properties of the encoded proteins were also analyzed. The number of encoded amino acids ranged from 324–542, and the isoelectric point range of the protein was 6.10–9.80. The hydrophilic coefficients of all were positive. The number of transmembrane domains encoded by TgAMT8 was nine, that by TgAMT9 was ten, and that by the others was 11. In addition, the prediction of subcellular localization revealed that TgAMTs was mainly confined to membrane structures and was also distributed in the vacuole, endoplasmic reticulum, and nuclear membranes. They may be related to cellular ammonium transport. The protein sequences are compared in Fig. S1 . They are dominated by α coils, followed by random coils, and a relatively small proportion of extension chains and β-angles (Table 2 ). Table 2 Secondary structure prediction of AMT gene in Torreya grandis . Gene Alpha helix/% Beta turn/% Random coil/% Extended strand/% TgAMT1 44.54 5.46 31.51 18.49 TgAMT2 44.75 3.76 31.68 19.8 TgAMT3 42.69 5.14 27.87 24.31 TgAMT4 42.55 6.17 33.40 17.87 TgAMT5 41.60 5.04 33.40 19.96 TgAMT6 45.37 5.25 26.85 22.53 TgAMT7 39.67 6.64 34.32 19.37 TgAMT8 43.15 7.16 31.08 18.61 TgAMT9 42.71 5.00 32.50 19.79 TgAMT10 43.85 5.74 33.61 16.80 Phylogenetic evolutionary tree of the TgAMTs A phylogenetic tree was constructed based on the protein sequences encoded by the AMTs and the TgAMTs families from Arabidopsis , Oryza sativa , Ginkgo biloba , Populus trichocarpa , and L. esculentum (Fig. 2 ). Based on the results, the TgAMTs family was divided into three evolutionary branches: TgAMT1, TgAMT2a, and TgAMT2b. Among the ten TgAMTs, only TgAMT2 and TgAMT3 belonged to the TgAMT1 subfamily, while four each belonged to the TgAMT2a and TgAMT2b subfamilies. TgAMTs were most closely related to poplar. Within the AMT2a subfamily, TgAMT7 was most highly correlated to the other three AMT families. TgAMT6 , TgAMT 8, and TgAMT 10 clustered with OsAMT3.1 , PtrAMT3.1 , and PbAMT3 , respectively. Analyses of the TgAMTs gene family structure, conserved motifs, and cis-acting promoter elements The structure and conserved motifs of the TgAMTs family were analyzed to understand the sequence characteristics (Fig. 3 A). The results revealed structural variations among the different TgAMTs. The TgAMT family genes were within the same cluster, with insignificant structural differences between them. Conserved motif analysis of TgAMTs was performed to understand the protein sequence structure (Fig. 3 B). The results showed that Motif 1, Motif 3, and Motif 4 were conserved, while Motif 10 was unique to TgAMT6 , TgAMT8 , and TgAMT10 . TgAMTs were highly conserved structurally; Motif 4 was present at the 5' terminals of TgAMT2 , TgAMT3 , and TgAMT6 , while Motif 7 was at the 5' terminals of the other TgAMTs. Thus, it can be inferred that the TgAMTs family plays a vital role in the stable transport of ammonium salts. The upstream promoter sequences of TgAMTs were analyzed (Fig. 3 C) and found to contain multiple functional elements. In addition to GAAT- and TATA-boxes, methyl jasmonate-induced, light-responsive, and multiple MYB binding elements were present, which play an essential role in the tolerance to adversity and abiotic stresses. In addition, the binding site of W-box element was also identified on the 2000bp promoter upstream of TgAMT5 , which is closely related to biological processes such as plant growth, development and signal transduction Tissue-specific expression patterns of TgAMTs RT-qPCR experiments were conducted to ascertain the expression patterns of TgAMTs in different tissues (Fig. 4 ). The expression levels of TgAMTs were relatively high in leaves and low in fruits. The expression of TgAMT5 was elevated in stems, leaves, and roots than in fruits. In addition, the levels of TgAMT4 , TgAMT7 , and TgAMT9 were enhanced in specific tissues. For example, that of TgAMT4 was comparatively more in leaves and roots but lower in stems and roots. Response of TgAMTs to Nitrogen stress An analysis of the cis-acting promoter elements of this gene family indicated the presence of abiotic stress-related elements. RNA was extracted from the roots of plants grown at different nitrogen levels for 7 and 14 days to clarify the expression patterns of TgAMTs under nitrogen stress (Fig. 5 A). The results showed that the relative expression levels of TgAMT1 , TgAMT4 , TgAMT5 , and TgAMT7 in T. grandis roots were elevated after 7 and 14 days of nitrate nitrogen treatment; those of TgAMT10 were higher after 7 and 14 days of ammonium nitrogen treatment; and those of TgAMT3 were higher after seven days of nitrogen deficiency treatment. However, those of TgAMT2 and TgAMT8 declined post-treatment with varying nitrogen concentrations. The expression level of TgAMT6 did not alter. As shown in Fig. 5 B, under different nitrogen conditions, the expression of TgAMTs in Torreya grandis leaves and roots was similar but was relatively enhanced in leaves. Thus, different nitrogen level stresses had varying effects on the expression patterns of TgAMTs . Most TgAMTs were up-regulated, most remarkably under nitrate nitrogen stress. Allogeneic expression of TgAMT5 The expression of TgAMT5 in T. grandis was high and relatively stable under different nitrogen stress conditions. Therefore, it may have a high affinity for the absorption and transport of ammonium. First, the secondary and tertiary structures of TgAMT5 were predicted ( Fig. S2 ). Arabidopsis plants were allogeneously transformed with TgAMT5 to investigate its function. As shown in Fig. 6 A, semi-quantitative PCR assays showed that TgAMT5 was independently expressed in three different Arabidopsis lines, while no TgAMT5 -related transcripts were detected in the wild-type (WT). As shown in Fig. 6 B, without NH 4 addition, the growth trends of the wild-type and overexpression lines were roughly identical. However, under the exogenous addition of 1 mM NH 4 , the root lengths of the WT and overexpression lines were markedly elevated compared to those without NH 4 . However, there were no remarkable differences between the root lengths of the WT and overexpression lines. Under the exogenous addition of 10 mM NH 4 , the root lengths of the WT and overexpression lines varied conspicuously; those of the latter were significantly longer, which may be due to the robust NH 4 absorption capacity of TgAMT5 , which promoted plant growth. The root length and fresh weight reflected more directly the differences between the wild type and overexpression plants, with those of the latter being higher (Fig. 6 C). Identification of candidate TFs regulating TgAMT5 Analysis of the cis-acting elements of the TgAMTs gene family showed that the promoters could interact with MYB, W-box, and other transcription factors to enhance gene expression. Therefore, the WRKY and MYB family genes were selected for the dual-luciferase assay. The 2000 bp promoter of TgAMT5 was cloned and fused to the DNA fragment encoding the N-terminal of the firefly luciferase protein (FLUC), which also contains the renilla luciferase (REN). As shown in Fig. 7 B, only TgWRKY2 enhanced the activity of the TgAMT5 promoter, while other transcription factors had no remarkable regulatory effects on the promoters of the candidate genes. Similarly, the fluorescence image of the tobacco experimental group plants was indeed brighter than that of the control group plants (Fig. 7 C). Next, the yeast single hybridization technique was used to detect whether TgWRKY2 could interact with the TgAMT5 promoter. It was found that TgWRKY2 directly interacted with A1 (Fig. 7 D). The growth of the transformants in SD/-Trp/-Ura solid medium is shown in Fig. S3 . Discussion Ammonium transporters play an essential role in the absorption and utilization of ammonium nitrogen. To date, multiple AMT gene families have been identified in many plant species, including Arabidopsis [ 20 ], Capsicum annuum [ 43 ], Populus [ 44 ], and wheat [ 45 ]. However, there is little research on this family in T. grandis . In this study, we identified ten AMT gene family members in T. grandis . These genes showed tissue-specific expression in plants, and their expression patterns varied under nitrogen stress conditions. Thus, the fine regulation of nitrogen absorption can be better achieved via modulating the expression of these genes. This study also comprehensively identified and characterized the TgAMT gene family of their chromosomal location and distribution, as well as the physical and chemical properties of the encoded proteins, and predicted their secondary structure (Fig. 1 ). The AMT gene family of T. grandis was distributed on six chromosomes, which was consistent with that of A. thaliana [ 46 ]. Subcellular localization predicted all TgAMTs to be located on the plasma membrane, which was similar to all plant AMT proteins studied so far [ 47 ]. The phylogenetic evolutionary tree of AMT proteins from multiple species was constructed, and they were clustered into two subfamilies, AMT1 and AMT2. However, only two of the ten Torreya AMTs belonged to AMT1, and the rest to AMT2. This finding was similar to the AMT family proteins of rice (Fig. 2 ) [ 48 ] The structure of the TgAMTs affected the biological function of T. grandis AMTs. Analysis shows varied exon–intron structures between the two subfamilies, suggesting different functions in the subfamilies (Fig. 3 A). Meanwhile, a study of the conserved motifs of TgAMTs further confirmed the results of evolutionary branching (Fig. 3 B), which suggested that members of the same subfamily have identical or similar conserved motifs, while members of different subfamilies contain unique conserved motifs. The TgAMT promoters contain the elements that bind to W-box, MYB, and other transcription factors, which indicates that transcriptional regulation plays a vital role in nitrogen absorption and assimilation. Ammonium transporters perform tissue-specific functions. For example, in Arabidopsis , AtAMT1 ; 3 promotes lateral root development [ 49 ], while AtAMT2;1 plays a role in the transport and redistribution of ammonium [ 13 ]. This study used RT-qPCR to investigate the expression of AMT genes in the different tissues of T. grandis . Most TgAMTs were highly expressed in leaves, stems, and roots but marginally in fruits, suggesting that they played an essential role in the growth and development of roots and leaves during plant growth. This observation was similar to the expression patterns in plants such as A. thaliana and O.sativa [ 50 ]. The expression of TgAMTs under N stress was studied by employing RT-qPCR. Among them TgAMT2 , TgAMT6 , TgAMT8 , and TgAMT9 were fundamentally not or negligibly expressed, while TgAMT1 , TgAMT3 , TgAMT4 , and TgAMT5 were highly expressed (Fig. 5 ). These results indicated that these genes exert their biological functions under nitrogen stress conditions, improve plant tolerance, and regulate the absorption and transport of ammonium to facilitate the adaptation to changes. The functional analysis of TgAMT5 was carried out by expressing it in Arabidopsis . As shown in Fig. 6 B, under a certain concentration, with the increase of NH 4 concentration, the root length of Arabidopsis plants increased significantly. At 10 mM NH 4 , the roots of the overexpression lines were significantly longer than those of the wild type, indicating that TgAMT5 improved the absorption efficiency of ammonium and promoted root growth. TgAMT5 was identified to belong to the TgAMT2 subfamily, which was consistent with previous studies reporting that the AMT2 subfamily in A. thaliana was mainly expressed in the roots and promoted root elongation [ 13 ]. Transcription factors promote the expression of candidate genes at the transcriptional level by binding to their promoters [ 51 ]. They play an important role in many biological processes, such as stress response, defense regulation, and the induction of development [ 52 ]. Previous studies have shown that WRKY1 can bind to the promoters of multiple nitrogen transporter coding genes in A. thaliana and activate their expression [ 53 ]. This study verified the binding of TgWRKY2 to the promoter of TgAMT5 and promoted the expression of TgAMT5 through yeast monohybrid and dual luciferase tests. Conclusions This report is the most comprehensive study of the Torreya AMT gene family to date. It identified ten TgAMTs gene families from the T. grandis genome, which were located on six chromosomes. Based on their evolutionary relationships and structural characteristics, TgAMTs were divided into two subfamilies. The expression of TgAMTs was tissue-specific and regulated by abiotic stress, of which TgAMT1 , TgAMT3 , TgAMT4 , and TgAMT5 were most responsive. Functional analysis of TgAMT5 indicated that it promoted the absorption and utilization of ammonium and then enhanced root growth. In addition, the transcriptional regulation of genes also plays a crucial role in plant response to environmental changes. The critical role of TgWRKY2 in the regulation of TgAMT5 by binding to its promoter was revealed through dual-luciferase and yeast single-hybridization assays. These findings provide a basis for studying the molecular mechanism of ammonium absorption and utilization in Torreya . Declarations Conflict of Interests The authors have no conflicts of interest to declare. Funding This work was supported by the “Pioneer” and “Leading Goose” R&D Program of Zhejiang (2022C02061); the National Natural Science Foundation of China (Grant no. 32171830); the cooperative forestry science and technology project of Zhejiang Provincial Academy (2022SY14); the Breeding of New Varieties of Torreya grandis Program (2021C02066-11). Author Contribution Y.Y.G. and Y.L. conceptualized the initial research; Y.Y.G., Y.L. and Y.W. participated in the experimental arrangement and carried out laboratory experiments; X.L. and Y.Y.G. drafted the original article; Y.Y.G. and W.J.C. performed sequence analysis. W.J.C. , C.L.Y and J.S.W supervised and revised the manuscript. All authors read and revised the manuscript, and approved the final manuscript. References Fu YF, Yang XY, Zhang ZW, Yuan S. Synergistic effects of nitrogen metabolites on auxin regulating plant growth and development. Front Plant Sci. 2022;13:1098787. Yun Y, Kim G, Cho G, Lee Y, Yun T, Kim H. 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Wu X, Yang H, Qu C, Xu Z, Li W, Hao B, Yang C, Sun G, Liu G. Sequence and expression analysis of the AMT gene family in poplar. Front Plant Sci. 2015;6:337. Li T, Liao K, Xu X, Gao Y, Wang Z, Zhu X, Jia B, Xuan Y. Wheat Ammonium Transporter (AMT) Gene Family: Diversity and Possible Role in Host-Pathogen Interaction with Stem Rust. Front Plant Sci. 2017;8:1637. Yuan L, Graff L, Loqué D, Kojima S, Tsuchiya YN, Takahashi H. von Wirén N: AtAMT1;4, a pollen-specific high-affinity ammonium transporter of the plasma membrane in Arabidopsis. Plant Cell Physiol. 2009;50(1):13–25. Yuan L, Loqué D, Ye F, Frommer WB, von Wirén N. Nitrogen-dependent posttranscriptional regulation of the ammonium transporter AtAMT1;1. Plant Physiol. 2007;143(2):732–44. Suenaga A, Moriya K, Sonoda Y, Ikeda A, Von Wirén N, Hayakawa T, Yamaguchi J, Yamaya T. Constitutive expression of a novel-type ammonium transporter OsAMT2 in rice plants. Plant Cell Physiol. 2003;44(2):206–11. Lima JE, Kojima S, Takahashi H, von Wirén N. Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANSPORTER1;3-dependent manner. Plant Cell. 2010;22(11):3621–33. Loqué D, Lalonde S, Looger LL, von Wirén N, Frommer WB. A cytosolic trans-activation domain essential for ammonium uptake. Nature. 2007;446(7132):195–8. Zeng D, Teixeira da Silva JA, Zhang M, Yu Z, Si C, Zhao C, Dai G, He C, Duan J. Genome-Wide Identification and Analysis of the APETALA2 (AP2) Transcription Factor in Dendrobium officinale. Int J Mol Sci. 2021;22(10):5221. Chen W, Zhang J, Zheng S, Wang Z, Xu C, Zhang Q, Wu J, Lou H. Metabolite profiling and transcriptome analyses reveal novel regulatory mechanisms of melatonin biosynthesis in hickory. Hortic Res. 2021;8(1):196. Zhang W, Tang S, Li X, Chen Y, Li J, Wang Y, Bian R, Jin Y, Zhu X, Zhang K. Arabidopsis WRKY1 promotes monocarpic senescence by integrative regulation of flowering, leaf senescence and nitrogen remobilization. Mol Plant. 2024;S1674–2052(24):00224–7. Additional Declarations No competing interests reported. Supplementary Files Fig.S1.tif FIg.S2.tif Fig.S3.tif Supplementaryinformation.docx Table.S1.xlsx Table.S2.xlsx 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4957530","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":347317003,"identity":"565ffbda-35b0-4fbb-b3d7-28a04270ea71","order_by":0,"name":"Yuanyuan Guan","email":"","orcid":"","institution":"Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Guan","suffix":""},{"id":347317004,"identity":"17841280-c2e0-489a-b9af-c0028bf3bf01","order_by":1,"name":"Yi Li","email":"","orcid":"","institution":"Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Li","suffix":""},{"id":347317005,"identity":"df34317d-d831-4399-9263-2ce60c05d9de","order_by":2,"name":"Yao Wei","email":"","orcid":"","institution":"Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Wei","suffix":""},{"id":347317006,"identity":"a7ce3d5c-5e5a-4b9d-8995-c18a353d4e3e","order_by":3,"name":"Xu Li","email":"","orcid":"","institution":"Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Li","suffix":""},{"id":347317007,"identity":"7b9a7376-e71a-4502-9864-3bf8971ae046","order_by":4,"name":"Weijie Chen","email":"","orcid":"","institution":"Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Weijie","middleName":"","lastName":"Chen","suffix":""},{"id":347317008,"identity":"4e899008-266c-4509-8bac-63c222bc30e2","order_by":5,"name":"Chenliang Yu","email":"","orcid":"","institution":"Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Chenliang","middleName":"","lastName":"Yu","suffix":""},{"id":347317009,"identity":"962b5f08-59bd-4c3a-b51d-423760cfb313","order_by":6,"name":"Jiasheng Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYHCCBIYPDAz8IJYE0VoYZzAwSDaQooWBmYckLQY3Eh5/tvljJ2FwgPngbR4GuzxitKRJ5/AkA7WwJVvzMCQXE9RiBtTCnCPBXGdwgMdMmofhQGIDEVqSP1sY1ANt4f9GtJYEaYaEw0AtPGzEabE/8yBNsufAcQnJw2zGlnMMkglrkWzPSf7w40+1BN/x5oc33lTYEdbCwMCTAKGZQYQBYfVAwH6AKGWjYBSMglEwggEANs04Xutq1fEAAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang A\u0026F University","correspondingAuthor":true,"prefix":"","firstName":"Jiasheng","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-08-22 11:07:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4957530/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4957530/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65636876,"identity":"2d3d37d0-6047-4268-a193-5ee02b2b5d38","added_by":"auto","created_at":"2024-09-30 18:34:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":490432,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal localization of the \u003cem\u003eTorreya grandis\u003c/em\u003e AMT gene family.\u003c/p\u003e","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/eef5b8b21f69d2d2816f4551.png"},{"id":65636872,"identity":"d03321d4-63eb-4cbe-b72b-7eed755b9bd3","added_by":"auto","created_at":"2024-09-30 18:34:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":849860,"visible":true,"origin":"","legend":"\u003cp\u003eA\u003cstrong\u003e \u003c/strong\u003ephylogenetic tree of the AMTs from \u003cem\u003eOryza sativa L\u003c/em\u003e,\u003cem\u003e Lycopersicum esculentum\u003c/em\u003e, \u003cem\u003ePopulus trichocarpa\u003c/em\u003e, \u003cem\u003eGinkgo biloba\u003c/em\u003e,\u003cem\u003e Arabidopsis thaliana\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eTorreya grandis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/1a308f5b9a150990ed1ba8c5.png"},{"id":65636881,"identity":"832a065c-2363-46a5-94e7-2354f15abe0f","added_by":"auto","created_at":"2024-09-30 18:34:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1104319,"visible":true,"origin":"","legend":"\u003cp\u003eAnalyses of the\u003cstrong\u003e \u003c/strong\u003eTgAMTs family structure, conserved motifs, and cis-acting promoter elements. (A) In the analysis of TgAMTs gene structure, the green box represents exons and the black line represents introns. (B) TgAMTs conserved motif analysis. (C) Analysis of cis-acting elements of the promoter of TgAMTs gene family.\u003c/p\u003e","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/a1b080a1a2fabfc67b83eff0.png"},{"id":65636875,"identity":"129a99d3-0709-4bf4-8438-863d3102bad7","added_by":"auto","created_at":"2024-09-30 18:34:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":354950,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of the AMT gene family in different tissues of\u003cem\u003e Torreya grandis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/c672d49a905c07841c0753af.png"},{"id":65637583,"identity":"55aab6a9-c255-408b-8055-57eaec7ec53d","added_by":"auto","created_at":"2024-09-30 18:42:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":723326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe relative expression level of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTgAMTs \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene family under nitrogen stress was analyzed by RT-qPCR. \u003c/strong\u003e(A) Levels of \u003cem\u003eTgAMTs\u003c/em\u003e in roots after 7(left) and 14 (right) days of nitrogen stress. (B) \u003cem\u003eTgAMTs \u003c/em\u003elevels in leaves after nitrogen stress 7(left) and 14 days (right). CK, plants growing in normal Hoagland's solution; -N, plants growing in nitrogen-deficient Hoagland's solution; NH, plants growing in nitrogen-deficient Hoagland's solution supplemented with ammonium sulfate (50 mmol/L (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e); NO, plants growing in nitrogen-deficient Hoagland's solution supplemented with calcium nitrate (50 mmol/L Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/8318a9ae6bde0ee5b795f4f9.png"},{"id":65636877,"identity":"9431eb53-a0c5-4210-a683-a84c68e6615f","added_by":"auto","created_at":"2024-09-30 18:34:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1349290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTgAMT5.\u003c/strong\u003e\u003c/em\u003e (A) Identification of the transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e plants expressing \u003cem\u003eTgAMT5\u003c/em\u003e. (B) Growth of transgenic \u003cem\u003eArabidopsis \u003c/em\u003etreated with different concentrations of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. (C) The root length (left) and fresh weight (right) of the transgenics cultured on a solid medium for ten days. ***significant differences (P \u0026lt;0.001), ****significant differences (P \u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/c91acc51d723b1e50db93a6f.png"},{"id":65637582,"identity":"9fbe52bb-eea1-40ea-8b46-fc20fffd6b15","added_by":"auto","created_at":"2024-09-30 18:42:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":746264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the transcription factors interacting with the\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e TgAMT5 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epromoters.\u003c/strong\u003e (A) The \u003cem\u003eTgAMT5\u003c/em\u003epromoter and effector model. (B) The candidate TFs regulated the \u003cem\u003eTgAMT5\u003c/em\u003e promoter; the LUC/REN of the SK-empty vector was compared with the value of the \u003cem\u003eTgAMT5\u003c/em\u003e promoter set to 1.0; the LUC/REN values of the other transcription factors and the \u003cem\u003eTgAMT5\u003c/em\u003epromoter were recorded in three biological replicates. (C) An image of the \u003cem\u003eTgAMT5\u003c/em\u003epromoter (left) with the\u003cem\u003e TgWRKY2\u003c/em\u003e transcription factor and with the empty SK vector (right)\u003cem\u003e \u003c/em\u003ein tobacco. (D) The binding of TgWRKY2 to the \u003cem\u003eTgAMT5\u003c/em\u003e promoter was verified was ascertained by yeast single hybridization. The empty pB42AD vector was compared with the pLacZi vector linked to a single fragment of the \u003cem\u003eTgAMT5\u003c/em\u003epromoter.\u003c/p\u003e","description":"","filename":"OnlineFig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/d7f8bca91167dbab1b93ac09.png"},{"id":69504579,"identity":"be009f3d-24bf-41d9-bae4-de1be2a8949b","added_by":"auto","created_at":"2024-11-21 06:17:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9373320,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/99b2e75b-8e41-4929-af85-a5c45b3da6f7.pdf"},{"id":65636873,"identity":"9fa1df0e-7592-4d90-9bd8-82753ccc8e09","added_by":"auto","created_at":"2024-09-30 18:34:26","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13259736,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/b8ff171f1037b15f21d0ff31.tif"},{"id":65637581,"identity":"a2ae82bc-aab3-470a-bcce-668bcfa5c9d7","added_by":"auto","created_at":"2024-09-30 18:42:26","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5726712,"visible":true,"origin":"","legend":"","description":"","filename":"FIg.S2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/39072eac39b498218c0fb3e0.tif"},{"id":65636879,"identity":"04e39e63-8f2a-4c73-9f99-22d50b213c61","added_by":"auto","created_at":"2024-09-30 18:34:27","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":7491576,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S3.tif","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/d66b3e990b4015f5f73be6cd.tif"},{"id":65636880,"identity":"59e0bab1-1cc0-44d0-9c93-91d8ee6d1165","added_by":"auto","created_at":"2024-09-30 18:34:27","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11172,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/a5327124464112195284059f.docx"},{"id":65636884,"identity":"55bce191-87eb-4bcd-bdef-0df45692d2e4","added_by":"auto","created_at":"2024-09-30 18:34:29","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10318,"visible":true,"origin":"","legend":"","description":"","filename":"Table.S1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/8be257b683d7766c5fa0548d.xlsx"},{"id":65636878,"identity":"26043c18-3b24-41f9-9973-323a27a99763","added_by":"auto","created_at":"2024-09-30 18:34:26","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":11836,"visible":true,"origin":"","legend":"","description":"","filename":"Table.S2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4957530/v1/b41a5c4636b3aaae5fa1896c.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide Identification of Torreya grandis AMT Family Genes Revealed the Function and Regulation of the Nitrogen Stress Responsive Gene TgAMT5","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNitrogen (N) is one of the macrotrophic elements indispensable for plant growth and development and also plays a crucial role in flowering and fruit setting[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Insufficient nitrogen levels can result in stunted plant growth, reduced yields, and compromised quality [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Plants generally absorb inorganic nitrogen in the forms of nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) and ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e), with a tendency of most plant species to favor NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This preference may be due to the lower energy consumption involved in the absorption process [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe AMT family encodes carrier proteins that mainly transport ammonium nitrogen [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The plant AMTs belong to the AMT/MEP/Rh family, which is primarily composed of the AMT1 and AMT2 two subfamilies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These subfamilies are structurally similar, each containing 9\u0026thinsp;~\u0026thinsp;11 transmembrane (TM) domains [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These domains enable the AMT protein to form channels in the cell membrane, facilitating the transmembrane transport of ammonium ions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. AMTs play an essential role in plants by regulating the absorption and utilization of ammonium nitrogen in the soil [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Among plants, AMTs were first characterized in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], with a total of six AMTs, of which five and one belonged to the AMT1 and AMT2 subfamilies, respectively [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Examining the AMT family among diverse taxa such as \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], \u003cem\u003ePhyscomitrella patens\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], \u003cem\u003eSelaginella moellendorffii\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], \u003cem\u003eZea mays\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and \u003cem\u003eGlycine max\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], illustrated the phenotypic diversity of AMTs across distinct evolutionary branches in plants.\u003c/p\u003e \u003cp\u003eIn response to the diverse distribution of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e concentrations in different soil environments, plants have evolved two distinct absorption systems to optimize the utilization of nitrogen: the low-affinity (LATs) and high-affinity transporter systems (HATs) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Of these, AMT1-type transporters are mainly responsible for mediating the high-affinity uptake of ammonium by roots [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In the presence of high-affinity conditions, mutations in \u003cem\u003eAtAMT1;1\u003c/em\u003e and \u003cem\u003eAtAMT1;3\u003c/em\u003e reduced the absorption of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e roots [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Similarly, \u003cem\u003eZmAMT1;1a\u003c/em\u003e and \u003cem\u003eZmAMT1;3\u003c/em\u003e are members of the HATs in maize roots, enhancing body mass [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In rice, elevating the expression levels of \u003cem\u003eOsAMT1.1\u003c/em\u003e bolstered the root absorption of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e upon exposure to high-ammonium conditions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In contrast to AMT1, AMT2 demonstrates intricate gene structures and protein profiles, along with lower similarity to homologs from fungi and bacteria [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. AMT2 did not participate in root ammonium uptake in \u003cem\u003eArabidopsis\u003c/em\u003e, but evidence from various plant species has shown that it can be involved in nitrogen transport [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Plants are capable of adapting to varying nitrogen supplies and growth environments by regulating the expression and activity of AMTs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For example, in \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eIDD10\u003c/em\u003e enhances the root development function of \u003cem\u003eAMT1;2\u003c/em\u003e by activating its transcription [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Likewise, \u003cem\u003eOsDOF18\u003c/em\u003e and \u003cem\u003eOsBZR1\u003c/em\u003e regulate ammonium absorption in rice roots by stimulating the expression of AMTs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Moreover, CIPK23 phosphorylates \u003cem\u003eAtAMT1;1\u003c/em\u003e and \u003cem\u003eAtAMT1;2\u003c/em\u003e, rendering them inactive, thereby regulating ammonia absorption [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Similarly, rice plants avoid ammonia toxicity by phosphorylating \u003cem\u003eOsAMT1;2\u003c/em\u003e via \u003cem\u003eOsACTPK1\u003c/em\u003e, leading to its inactivation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, the AMT family of \u003cem\u003eTorreya grandis\u003c/em\u003e was identified, and its evolutionary relationship was analyzed. Further, AMT functional analysis was performed in this economically important subtropical Chinese tree, known for its delicious and nutritionally rich nuts [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The chromosome distribution, phylogenetic relationships, and conserved domains of the AMT family in this plant were comprehensively analyzed. Subsequently, real time quantitative PCR(RT-qPCR) revealed the expression patterns of the AMT family under different nitrogen conditions and identified \u003cem\u003eTgAMT5\u003c/em\u003e as a potential factor involved in ammonium nitrogen response. Multiple protein\u0026ndash;DNA interaction analyses suggested that \u003cem\u003eTgWRKY2\u003c/em\u003e enhanced the activity of the \u003cem\u003eTgAMT5\u003c/em\u003e promoter.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eWe cultivated 12-month-old seedlings of \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egrandis\u003c/em\u003e via hydroponic culture, utilizing a climate-controlled chamber placed in the greenhouse of the Zhejiang A\u0026amp;F University, Hangzhou, Zhejiang, China. The voucher specimens were identified byProf. Daiwensheng and deposited in the Zhejiang A\u0026amp;F University Intelligent Experimental Building Plant Specimen Room N205. The treatments included growing plants in normal Hoagland's solution (HB8870-1, Hopebio), nitrogen-deficient Hoagland's solution(HB8870-9, Hopebio), nitrogen-deficient Hoagland's solution supplemented with ammonium sulfate (50 mmol/L (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e), and nitrogen-deficient Hoagland's solution supplemented with calcium nitrate (50 mmol/L Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. The pH of all was 6.5. All materials were treated for 7 days and 14 days, and each sample was stored in the ultra-low temperature refrigerator (-80℃) of the State Key Laboratory of Subtropical Forest Cultivation in Zhejiang A \u0026amp; F University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic analysis\u003c/h2\u003e \u003cp\u003eThe amino acid and nucleotide sequences of \u003cem\u003eGinkgo biloba\u003c/em\u003e, \u003cem\u003ePopulus trichocarpa\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eLycopersicum esculentum\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (National Center for Biotechnology Information (nih.gov)), and \u003cem\u003eT. grandis\u003c/em\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.6084/m9.figshare.21089869\u003c/span\u003e\u003cspan address=\"10.6084/m9.figshare.21089869\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All AMTs were downloaded from the database. First, based on the threshold value of e-value\u0026thinsp;\u0026le;\u0026thinsp;e\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, the AMT gene family was identified using hmmer v3.2.1 with Pfam ID (PF00909) as the target dataset. Subsequently, NCBI CDD analysis was utilized to identify potential domains within the AMTs [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Expasy ProtParam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze the amino acid number, molecular weight, isoelectric point, and the physical and chemical properties of the AMTs. TMHMM Server 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbs.dtu.dk/services/TMHMM/\u003c/span\u003e\u003cspan address=\"http://www.cbs.dtu.dk/services/TMHMM/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized for transmembrane protein structure prediction. Wolfpsort (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://wolfpsort.hgc.jp/\u003c/span\u003e\u003cspan address=\"http://wolfpsort.hgc.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was applied to predict protein subcellular localization. SOPMA and SWISS-MODEL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used for protein secondary structure prediction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of a phylogenetic evolutionary tree and Cis-acting element analysis\u003c/h2\u003e \u003cp\u003eThe AMTs prontein sequences of \u003cem\u003eOryza sativa\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], \u003cem\u003eL\u003c/em\u003e. \u003cem\u003eesculentum\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], \u003cem\u003ePopulus trichocarpa\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], \u003cem\u003eGinkgo biloba\u003c/em\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], \u003cem\u003eArabidopsis thaliana\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egrandis\u003c/em\u003e were imported into MEGA 11. MUSCLE was used for multi-sequence comparison, and the NJ method was selected as the conformable tree method. The tree was visualized by applying Evolview v2. The promoter sequence of the target gene was extracted and predicted using Plantcare. The cis-acting elements were identified and imported into TBtools for visualization.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStructural and chromosomal location analysis of the AMT gene family in\u003c/b\u003e \u003cb\u003eT. grandis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe protein sequence of \u003cem\u003eTgAMT\u003c/em\u003e gene family was examined utilizing the MEME Suite (version 5.5.4) to analyze their conserved motifs and assess their functional importance, with reference to the NCBI-CDD database [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Subsequently, the gene structure and chromosomal positions of the TgAMTs family were analyzed using TB-tools-II [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of gene expression patterns\u003c/h2\u003e \u003cp\u003eQuantitative primers were designed using Primer3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://primer3.ut.ee/\u003c/span\u003e\u003cspan address=\"https://primer3.ut.ee/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) based on the \u003cem\u003eT. grandis\u003c/em\u003e AMT sequence (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). RNA was extracted from the roots and leaves of plants cultivated under different nitrogen conditions as described in 2.1 by the non-Trizol method (Invitrogen). cDNA was synthesized by the HiScript Ⅲ RT Super Mix reverse transcription kit and RT-qPCR was performed using the Cham\u003csup\u003eQ\u003c/sup\u003e SYBR qPCR Master Mix. RT-qPCR was performed per the procedure: predenaturation at 95 ℃ for 30 min, 10 s at 95 ℃, 30 s at 60 ℃, and 40 reaction cycles. Annealing was performed by applying the default dissolution curve of the instrument. \u003cem\u003eTgACTIN\u003c/em\u003e was used as the internal reference gene, and the relative expression of \u003cem\u003eTgAMTs\u003c/em\u003e was estimated by the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Statistical and significant differences in \u003cem\u003eTgAMTs\u003c/em\u003e expression were analyzed, and GraphPad Prism10 was used for mapping.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCultivation of transgenic Arabidopsis plants\u003c/h2\u003e \u003cp\u003eThe coding sequence (CDS) of \u003cem\u003eTgAMT5\u003c/em\u003e was cloned into pCAMBIA1300. \u003cem\u003eArabidopsis\u003c/em\u003e flowers were infected by \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e and obtain T2 generation transgenic seeds. The transgenic \u003cem\u003eArabidopsis\u003c/em\u003e seeds was washed twice with 5% sodium hypochlorite for 10 min each and then washed five times with sterilized water for 2 min each. The sterile seeds were planted on half strength of Hoagland's medium, wrapped in tin foil in a Petri dish, unfoiled after three days, and placed in an incubator (16/8 h light/dark; 65% RH). After three days of vertical culture, the seedlings with consistent growth (~\u0026thinsp;1 cm root length) were transferred to an ammonium salt-containing solid medium (pH\u0026thinsp;=\u0026thinsp;5.8) and cultured until an obvious phenotype appeared.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDual-luciferase assay\u003c/h2\u003e \u003cp\u003eThe 2000 bp promoter of \u003cem\u003eTgAMT5\u003c/em\u003e was cloned into the pGreen II 0800-LUC vector for the dual-luciferase assay. Similarly, the CDS of the candidate TF genes was cloned into the pGreen II 62-SK vector to generate the effectors. The \u003cem\u003eA.tumefaciens\u003c/em\u003e (GV3101: pSoup) cells carrying the reporter and effector plasmids were injected into the young leaves of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e. Luciferase activity (LUC and REN) was assessed using a dual luciferase assay kit (RG027, Beyotime), and fluorescence was observed using a Tanon 5200 automatic chemiluminescence image analyzer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eYeast one-hybrid assay\u003c/h2\u003e \u003cp\u003eThe W-box is a cis-acting element of the \u003cem\u003eTgAMT5\u003c/em\u003e promoter. This promoter was divided into six segments, and each was cloned into the pLacZi vecto. Simultaneously, the \u003cem\u003eTgWRKY2\u003c/em\u003e CDS was cloned into the pB42AD vector. The EGY48 yeast strain was transformed with these constructs and then cultured on a dropout/-Trp/-Ura/Gal/Raf/X-Gal (80 \u0026micro;g/mL) plate. The cultures were then incubated for three days to observe if they turned blue. The relevant primers designed are detailed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification of the\u003c/b\u003e \u003cb\u003eTgAMTs\u003c/b\u003e \u003cb\u003efamily members and analysis of their fundamental characteristics\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study identified ten members of the AMT gene family from the \u003cem\u003eTorreya grandis\u003c/em\u003e genome, which were numbered \u003cem\u003eTgAMT1\u003c/em\u003e\u0026ndash;\u003cem\u003eTgAMT10\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). TgAMTs were found to be located on chromosomes 1, 2, 5, 6, 7, and 8, with two tandem repeating genes, namely \u003cem\u003eTgAMT4\u003c/em\u003e and \u003cem\u003eTgAMT5\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The length of the CDSs ranged from 975\u0026ndash;1629 bp (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical properties of the AMT genes family in Torreya grandis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene\u0026nbsp;ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChromosome\u0026nbsp;location\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCDS\u0026nbsp;Length\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProtein\u0026nbsp;size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMolecular\u0026nbsp;mass\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003epI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGRAVY\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTrans-\u003c/p\u003e \u003cp\u003emembrane\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSubcellular\u0026nbsp;location\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePTG007578L.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1431\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e476\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e51628.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.526\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eplas\u0026nbsp;vacu\u003c/p\u003e \u003cp\u003eE.R.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePTG006090L.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e54464.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eplas\u0026nbsp;E.R\u003c/p\u003e \u003cp\u003enucl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePTG006814L.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e506\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e54574.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.376\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eplas\u0026nbsp;chlo\u003c/p\u003e \u003cp\u003enucl\u0026nbsp;vacu\u003c/p\u003e \u003cp\u003eE.R.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePTG002221L.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1413\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e50896.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.624\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eplas\u0026nbsp;vacu\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePTG002221L.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1431\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e476\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e51681.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eplas\u0026nbsp;vacu\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePTG006706L.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e975\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e324\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e35056.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.714\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eplas\u0026nbsp;vacu\u003c/p\u003e \u003cp\u003eE.R.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePTG015048L.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1629\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e59003.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eplas\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePTG013339L.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e489\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e52987.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.482\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eplas\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePTG004123L.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1443\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e480\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e53067.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eplas\u0026nbsp;vacu\u003c/p\u003e \u003cp\u003eE.R.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePTG027016L.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1467\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e488\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e52682.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.545\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eplas\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe physical and chemical properties of the encoded proteins were also analyzed. The number of encoded amino acids ranged from 324\u0026ndash;542, and the isoelectric point range of the protein was 6.10\u0026ndash;9.80. The hydrophilic coefficients of all were positive. The number of transmembrane domains encoded by \u003cem\u003eTgAMT8\u003c/em\u003e was nine, that by \u003cem\u003eTgAMT9\u003c/em\u003e was ten, and that by the others was 11. In addition, the prediction of subcellular localization revealed that TgAMTs was mainly confined to membrane structures and was also distributed in the vacuole, endoplasmic reticulum, and nuclear membranes. They may be related to cellular ammonium transport. The protein sequences are compared in \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. They are dominated by α coils, followed by random coils, and a relatively small proportion of extension chains and β-angles (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSecondary structure prediction of AMT gene in \u003cem\u003eTorreya grandis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlpha\u0026nbsp;helix/%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBeta\u0026nbsp;turn/%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRandom\u0026nbsp;coil/%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtended\u0026nbsp;strand/%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e44.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e44.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e41.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e32.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTgAMT10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic evolutionary tree of the TgAMTs\u003c/h2\u003e \u003cp\u003eA phylogenetic tree was constructed based on the protein sequences encoded by the AMTs and the TgAMTs families from \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eGinkgo biloba\u003c/em\u003e, \u003cem\u003ePopulus trichocarpa\u003c/em\u003e, and \u003cem\u003eL. esculentum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Based on the results, the TgAMTs family was divided into three evolutionary branches: TgAMT1, TgAMT2a, and TgAMT2b. Among the ten TgAMTs, only \u003cem\u003eTgAMT2\u003c/em\u003e and \u003cem\u003eTgAMT3\u003c/em\u003e belonged to the TgAMT1 subfamily, while four each belonged to the TgAMT2a and TgAMT2b subfamilies. TgAMTs were most closely related to poplar. Within the AMT2a subfamily, \u003cem\u003eTgAMT7\u003c/em\u003e was most highly correlated to the other three AMT families. \u003cem\u003eTgAMT6\u003c/em\u003e, \u003cem\u003eTgAMT\u003c/em\u003e8, and \u003cem\u003eTgAMT\u003c/em\u003e10 clustered with \u003cem\u003eOsAMT3.1\u003c/em\u003e, \u003cem\u003ePtrAMT3.1\u003c/em\u003e, and \u003cem\u003ePbAMT3\u003c/em\u003e, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalyses of the TgAMTs gene family structure, conserved motifs, and cis-acting promoter elements\u003c/h2\u003e \u003cp\u003eThe structure and conserved motifs of the TgAMTs family were analyzed to understand the sequence characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The results revealed structural variations among the different TgAMTs. The TgAMT family genes were within the same cluster, with insignificant structural differences between them. Conserved motif analysis of TgAMTs was performed to understand the protein sequence structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The results showed that Motif 1, Motif 3, and Motif 4 were conserved, while Motif 10 was unique to \u003cem\u003eTgAMT6\u003c/em\u003e, \u003cem\u003eTgAMT8\u003c/em\u003e, and \u003cem\u003eTgAMT10\u003c/em\u003e. TgAMTs were highly conserved structurally; Motif 4 was present at the 5' terminals of \u003cem\u003eTgAMT2\u003c/em\u003e, \u003cem\u003eTgAMT3\u003c/em\u003e, and \u003cem\u003eTgAMT6\u003c/em\u003e, while Motif 7 was at the 5' terminals of the other TgAMTs. Thus, it can be inferred that the TgAMTs family plays a vital role in the stable transport of ammonium salts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe upstream promoter sequences of TgAMTs were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and found to contain multiple functional elements. In addition to GAAT- and TATA-boxes, methyl jasmonate-induced, light-responsive, and multiple MYB binding elements were present, which play an essential role in the tolerance to adversity and abiotic stresses. In addition, the binding site of W-box element was also identified on the 2000bp promoter upstream of \u003cem\u003eTgAMT5\u003c/em\u003e, which is closely related to biological processes such as plant growth, development and signal transduction\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTissue-specific expression patterns of TgAMTs\u003c/h2\u003e \u003cp\u003eRT-qPCR experiments were conducted to ascertain the expression patterns of TgAMTs in different tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The expression levels of \u003cem\u003eTgAMTs\u003c/em\u003e were relatively high in leaves and low in fruits. The expression of \u003cem\u003eTgAMT5\u003c/em\u003e was elevated in stems, leaves, and roots than in fruits. In addition, the levels of \u003cem\u003eTgAMT4\u003c/em\u003e, \u003cem\u003eTgAMT7\u003c/em\u003e, and \u003cem\u003eTgAMT9\u003c/em\u003e were enhanced in specific tissues. For example, that of \u003cem\u003eTgAMT4\u003c/em\u003e was comparatively more in leaves and roots but lower in stems and roots.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eResponse of\u003c/b\u003e \u003cb\u003eTgAMTs\u003c/b\u003e \u003cb\u003eto Nitrogen stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAn analysis of the cis-acting promoter elements of this gene family indicated the presence of abiotic stress-related elements. RNA was extracted from the roots of plants grown at different nitrogen levels for 7 and 14 days to clarify the expression patterns of \u003cem\u003eTgAMTs\u003c/em\u003e under nitrogen stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The results showed that the relative expression levels of \u003cem\u003eTgAMT1\u003c/em\u003e, \u003cem\u003eTgAMT4\u003c/em\u003e, \u003cem\u003eTgAMT5\u003c/em\u003e, and \u003cem\u003eTgAMT7\u003c/em\u003e in \u003cem\u003eT. grandis\u003c/em\u003e roots were elevated after 7 and 14 days of nitrate nitrogen treatment; those of \u003cem\u003eTgAMT10\u003c/em\u003e were higher after 7 and 14 days of ammonium nitrogen treatment; and those of \u003cem\u003eTgAMT3\u003c/em\u003e were higher after seven days of nitrogen deficiency treatment. However, those of \u003cem\u003eTgAMT2\u003c/em\u003e and \u003cem\u003eTgAMT8\u003c/em\u003e declined post-treatment with varying nitrogen concentrations. The expression level of \u003cem\u003eTgAMT6\u003c/em\u003e did not alter. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, under different nitrogen conditions, the expression of \u003cem\u003eTgAMTs\u003c/em\u003e in \u003cem\u003eTorreya grandis\u003c/em\u003e leaves and roots was similar but was relatively enhanced in leaves. Thus, different nitrogen level stresses had varying effects on the expression patterns of \u003cem\u003eTgAMTs\u003c/em\u003e. Most \u003cem\u003eTgAMTs\u003c/em\u003e were up-regulated, most remarkably under nitrate nitrogen stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAllogeneic expression of\u003c/b\u003e \u003cb\u003eTgAMT5\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe expression of \u003cem\u003eTgAMT5\u003c/em\u003e in \u003cem\u003eT. grandis\u003c/em\u003e was high and relatively stable under different nitrogen stress conditions. Therefore, it may have a high affinity for the absorption and transport of ammonium. First, the secondary and tertiary structures of \u003cem\u003eTgAMT5\u003c/em\u003e were predicted (\u003cb\u003eFig.\u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). \u003cem\u003eArabidopsis\u003c/em\u003e plants were allogeneously transformed with \u003cem\u003eTgAMT5\u003c/em\u003e to investigate its function. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, semi-quantitative PCR assays showed that \u003cem\u003eTgAMT5\u003c/em\u003e was independently expressed in three different \u003cem\u003eArabidopsis\u003c/em\u003e lines, while no \u003cem\u003eTgAMT5\u003c/em\u003e-related transcripts were detected in the wild-type (WT). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, without NH\u003csub\u003e4\u003c/sub\u003e addition, the growth trends of the wild-type and overexpression lines were roughly identical. However, under the exogenous addition of 1 mM NH\u003csub\u003e4\u003c/sub\u003e, the root lengths of the WT and overexpression lines were markedly elevated compared to those without NH\u003csub\u003e4\u003c/sub\u003e. However, there were no remarkable differences between the root lengths of the WT and overexpression lines. Under the exogenous addition of 10 mM NH\u003csub\u003e4\u003c/sub\u003e, the root lengths of the WT and overexpression lines varied conspicuously; those of the latter were significantly longer, which may be due to the robust NH\u003csub\u003e4\u003c/sub\u003e absorption capacity of \u003cem\u003eTgAMT5\u003c/em\u003e, which promoted plant growth. The root length and fresh weight reflected more directly the differences between the wild type and overexpression plants, with those of the latter being higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of candidate TFs regulating\u003c/b\u003e \u003cb\u003eTgAMT5\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnalysis of the cis-acting elements of the \u003cem\u003eTgAMTs\u003c/em\u003e gene family showed that the promoters could interact with MYB, W-box, and other transcription factors to enhance gene expression. Therefore, the WRKY and MYB family genes were selected for the dual-luciferase assay. The 2000 bp promoter of \u003cem\u003eTgAMT5\u003c/em\u003e was cloned and fused to the DNA fragment encoding the N-terminal of the firefly luciferase protein (FLUC), which also contains the renilla luciferase (REN). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, only \u003cem\u003eTgWRKY2\u003c/em\u003e enhanced the activity of the \u003cem\u003eTgAMT5\u003c/em\u003e promoter, while other transcription factors had no remarkable regulatory effects on the promoters of the candidate genes. Similarly, the fluorescence image of the tobacco experimental group plants was indeed brighter than that of the control group plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Next, the yeast single hybridization technique was used to detect whether \u003cem\u003eTgWRKY2\u003c/em\u003e could interact with the \u003cem\u003eTgAMT5\u003c/em\u003e promoter. It was found that \u003cem\u003eTgWRKY2\u003c/em\u003e directly interacted with A1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The growth of the transformants in SD/-Trp/-Ura solid medium is shown in \u003cb\u003eFig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAmmonium transporters play an essential role in the absorption and utilization of ammonium nitrogen. To date, multiple AMT gene families have been identified in many plant species, including \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], \u003cem\u003eCapsicum annuum\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], \u003cem\u003ePopulus\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and \u003cem\u003ewheat\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. However, there is little research on this family in \u003cem\u003eT. grandis\u003c/em\u003e. In this study, we identified ten AMT gene family members in \u003cem\u003eT. grandis\u003c/em\u003e. These genes showed tissue-specific expression in plants, and their expression patterns varied under nitrogen stress conditions. Thus, the fine regulation of nitrogen absorption can be better achieved via modulating the expression of these genes.\u003c/p\u003e \u003cp\u003eThis study also comprehensively identified and characterized the \u003cem\u003eTgAMT\u003c/em\u003e gene family of their chromosomal location and distribution, as well as the physical and chemical properties of the encoded proteins, and predicted their secondary structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The AMT gene family of \u003cem\u003eT. grandis\u003c/em\u003e was distributed on six chromosomes, which was consistent with that of \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Subcellular localization predicted all TgAMTs to be located on the plasma membrane, which was similar to all plant AMT proteins studied so far [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The phylogenetic evolutionary tree of AMT proteins from multiple species was constructed, and they were clustered into two subfamilies, AMT1 and AMT2. However, only two of the ten \u003cem\u003eTorreya\u003c/em\u003e AMTs belonged to AMT1, and the rest to AMT2. This finding was similar to the AMT family proteins of rice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe structure of the TgAMTs affected the biological function of \u003cem\u003eT. grandis\u003c/em\u003e AMTs. Analysis shows varied exon\u0026ndash;intron structures between the two subfamilies, suggesting different functions in the subfamilies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Meanwhile, a study of the conserved motifs of TgAMTs further confirmed the results of evolutionary branching (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), which suggested that members of the same subfamily have identical or similar conserved motifs, while members of different subfamilies contain unique conserved motifs. The \u003cem\u003eTgAMT\u003c/em\u003e promoters contain the elements that bind to W-box, MYB, and other transcription factors, which indicates that transcriptional regulation plays a vital role in nitrogen absorption and assimilation. Ammonium transporters perform tissue-specific functions. For example, in \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eAtAMT1\u003c/em\u003e; \u003cem\u003e3\u003c/em\u003e promotes lateral root development [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], while \u003cem\u003eAtAMT2;1\u003c/em\u003e plays a role in the transport and redistribution of ammonium [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study used RT-qPCR to investigate the expression of AMT genes in the different tissues of \u003cem\u003eT. grandis\u003c/em\u003e. Most TgAMTs were highly expressed in leaves, stems, and roots but marginally in fruits, suggesting that they played an essential role in the growth and development of roots and leaves during plant growth. This observation was similar to the expression patterns in plants such as \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eO.sativa\u003c/em\u003e [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe expression of \u003cem\u003eTgAMTs\u003c/em\u003e under N stress was studied by employing RT-qPCR. Among them \u003cem\u003eTgAMT2\u003c/em\u003e, \u003cem\u003eTgAMT6\u003c/em\u003e, \u003cem\u003eTgAMT8\u003c/em\u003e, and \u003cem\u003eTgAMT9\u003c/em\u003e were fundamentally not or negligibly expressed, while \u003cem\u003eTgAMT1\u003c/em\u003e, \u003cem\u003eTgAMT3\u003c/em\u003e, \u003cem\u003eTgAMT4\u003c/em\u003e, and \u003cem\u003eTgAMT5\u003c/em\u003e were highly expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results indicated that these genes exert their biological functions under nitrogen stress conditions, improve plant tolerance, and regulate the absorption and transport of ammonium to facilitate the adaptation to changes. The functional analysis of \u003cem\u003eTgAMT5\u003c/em\u003e was carried out by expressing it in \u003cem\u003eArabidopsis\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, under a certain concentration, with the increase of NH\u003csub\u003e4\u003c/sub\u003e concentration, the root length of \u003cem\u003eArabidopsis\u003c/em\u003e plants increased significantly. At 10 mM NH\u003csub\u003e4\u003c/sub\u003e, the roots of the overexpression lines were significantly longer than those of the wild type, indicating that \u003cem\u003eTgAMT5\u003c/em\u003e improved the absorption efficiency of ammonium and promoted root growth. \u003cem\u003eTgAMT5\u003c/em\u003e was identified to belong to the TgAMT2 subfamily, which was consistent with previous studies reporting that the AMT2 subfamily in \u003cem\u003eA. thaliana\u003c/em\u003e was mainly expressed in the roots and promoted root elongation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Transcription factors promote the expression of candidate genes at the transcriptional level by binding to their promoters [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. They play an important role in many biological processes, such as stress response, defense regulation, and the induction of development [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Previous studies have shown that \u003cem\u003eWRKY1\u003c/em\u003e can bind to the promoters of multiple nitrogen transporter coding genes in \u003cem\u003eA. thaliana\u003c/em\u003e and activate their expression [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This study verified the binding of \u003cem\u003eTgWRKY2\u003c/em\u003e to the promoter of \u003cem\u003eTgAMT5\u003c/em\u003e and promoted the expression of \u003cem\u003eTgAMT5\u003c/em\u003e through yeast monohybrid and dual luciferase tests.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis report is the most comprehensive study of the \u003cem\u003eTorreya\u003c/em\u003e AMT gene family to date. It identified ten TgAMTs gene families from the \u003cem\u003eT. grandis\u003c/em\u003e genome, which were located on six chromosomes. Based on their evolutionary relationships and structural characteristics, TgAMTs were divided into two subfamilies. The expression of TgAMTs was tissue-specific and regulated by abiotic stress, of which \u003cem\u003eTgAMT1\u003c/em\u003e, \u003cem\u003eTgAMT3\u003c/em\u003e, \u003cem\u003eTgAMT4\u003c/em\u003e, and \u003cem\u003eTgAMT5\u003c/em\u003e were most responsive. Functional analysis of \u003cem\u003eTgAMT5\u003c/em\u003e indicated that it promoted the absorption and utilization of ammonium and then enhanced root growth. In addition, the transcriptional regulation of genes also plays a crucial role in plant response to environmental changes. The critical role of \u003cem\u003eTgWRKY2\u003c/em\u003e in the regulation of \u003cem\u003eTgAMT5\u003c/em\u003e by binding to its promoter was revealed through dual-luciferase and yeast single-hybridization assays. These findings provide a basis for studying the molecular mechanism of ammonium absorption and utilization in \u003cem\u003eTorreya\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eConflict of Interests\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the \u0026ldquo;Pioneer\u0026rdquo; and \u0026ldquo;Leading Goose\u0026rdquo; R\u0026amp;D Program of Zhejiang (2022C02061); the National Natural Science Foundation of China (Grant no. 32171830); the cooperative forestry science and technology project of Zhejiang Provincial Academy (2022SY14); the Breeding of New Varieties of Torreya grandis Program (2021C02066-11).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.Y.G. and Y.L. conceptualized the initial research; Y.Y.G., Y.L. and Y.W. participated in the experimental arrangement and carried out laboratory experiments; X.L. and Y.Y.G. drafted the original article; Y.Y.G. and W.J.C. performed sequence analysis. W.J.C. , C.L.Y and J.S.W supervised and revised the manuscript. All authors read and revised the manuscript, and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFu YF, Yang XY, Zhang ZW, Yuan S. Synergistic effects of nitrogen metabolites on auxin regulating plant growth and development. Front Plant Sci. 2022;13:1098787.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYun Y, Kim G, Cho G, Lee Y, Yun T, Kim H. Effect of Nitrogen Application Methods on Yield and Grain Quality of an Extremely Early Maturing Rice Variety. Agriculture. 2023;13:832.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui J, Yu C, Qiao N, Xu X, Tian Y, Ouyang H. Plant preference for NH4\u0026thinsp;+\u0026thinsp;versus NO3\u0026thinsp;\u0026ndash;\u0026thinsp;at different growth stages in an alpine agroecosystem. 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Mol Plant. 2024;S1674\u0026ndash;2052(24):00224\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\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":"Torreya grandis, Ammonium transporter, Expression pattern, Molecular regulation","lastPublishedDoi":"10.21203/rs.3.rs-4957530/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4957530/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAmmonium transporters (AMTs) are a class of transmembrane proteins widely found in plants, bacteria, fungi, and other organisms, mediating transmembrane ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) transport, which is one of the crucial pathways for plants to obtain nitrogen from resources. AMTs have been studied in many plants but have not been systematically analyzed in \u003cem\u003eTorreya grandis\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThis study first used bioinformatics to identify members of the \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egrandis\u003c/em\u003e AMT family and then real time quantitative PCR to explore their tissue expression patterns and abiotic stress responses. The physical and chemical properties, secondary structure, and evolutionary relationships of the encoded proteins were ascertained. There were ten members of the gene family, named \u003cem\u003eTgAMT1\u003c/em\u003e\u0026ndash;\u003cem\u003eTgAMT10\u003c/em\u003e, which were located on six chromosomes, with coding sequence lengths of 975\u0026ndash;1629 bp. Subcellular localization predicted all members to be located on the plasma membrane. Phylogenetic analysis divided the TgAMTs into two subfamilies, AMT1 and AMT2. There were significant differences in gene structure and conserved motifs among the subfamilies, but Motif 1, Motif 3, and Motif 4 were common to all. The expression of TgAMTs was histologically specific. Additionally, nitrogen morphology also affected TgAMTs expression. \u003cem\u003eTgAMT5\u003c/em\u003e was identified as a potential member involved in the response to NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-induced stress. The gene function of \u003cem\u003eTgAMT5\u003c/em\u003e was verified in transgenic \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ethaliana\u003c/em\u003e and was found to promote plant growth and development, especially root growth, by absorbing ammonium salt through roots. In addition, dual-luciferase and yeast one-hybrid assays showed that the transcription factor \u003cem\u003eTgWRKY2\u003c/em\u003e could directly bind to the \u003cem\u003eTgAMT5\u003c/em\u003e promoter and enhance its expression.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study can provide theoretical basis for the efficient use of nitrogen in \u003cem\u003eTorreya grandis\u003c/em\u003e, and lay a foundation for exploring nitrogen uptake and utilization in gymnosperms.\u003c/p\u003e","manuscriptTitle":"Genome-wide Identification of Torreya grandis AMT Family Genes Revealed the Function and Regulation of the Nitrogen Stress Responsive Gene TgAMT5","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-30 18:34:21","doi":"10.21203/rs.3.rs-4957530/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":"7eff9f0c-52f4-4210-82d0-71976e6a5d9e","owner":[],"postedDate":"September 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-21T06:08:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-30 18:34:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4957530","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4957530","identity":"rs-4957530","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}