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

doi:10.21203/rs.3.rs-4957530/v1

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

https://doi.org/10.21203/rs.3.rs-4957530/v1

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Background

Ammonium transporters (AMTs) are a class of transmembrane proteins widely found in plants, bacteria, fungi, and other organisms, mediating transmembrane ammonium nitrogen (NH₄⁺) 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 NH₄⁺-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

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 (NO₃⁻) and ammonium (NH₄⁺), with a tendency of most plant species to favor NH₄⁺ [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 NH₄⁺ 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 NH₄⁺ 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 NH₄⁺ 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.

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 (NH₄)₂SO₄), and nitrogen-deficient Hoagland's solution supplemented with calcium nitrate (50 mmol/L Ca(NO₃)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.

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, TgAMT8, and TgAMT10 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. 3A). 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. 3B). 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. 3C) 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. 5A). 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. 5B, 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. 6A, 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. 6B, without NH₄ addition, the growth trends of the wild-type and overexpression lines were roughly identical. However, under the exogenous addition of 1 mM NH₄, the root lengths of the WT and overexpression lines were markedly elevated compared to those without NH₄. However, there were no remarkable differences between the root lengths of the WT and overexpression lines. Under the exogenous addition of 10 mM NH₄, 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 NH₄ 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. 6C).

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. 7B, 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. 7C). 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. 7D). The growth of the transformants in SD/-Trp/-Ura solid medium is shown in Fig. S3.

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. 3A). Meanwhile, a study of the conserved motifs of TgAMTs further confirmed the results of evolutionary branching (Fig. 3B), 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. 6B, under a certain concentration, with the increase of NH₄ concentration, the root length of Arabidopsis plants increased significantly. At 10 mM NH₄, 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.

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.

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.

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

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Abstract

Background

Results

Conclusion

Figures

Introduction

Materials and methods

Plant materials

Bioinformatic analysis

Construction of a phylogenetic evolutionary tree and Cis-acting element analysis

Analysis of gene expression patterns

Cultivation of transgenic Arabidopsis plants

Dual-luciferase assay

Yeast one-hybrid assay

Results

Phylogenetic evolutionary tree of the TgAMTs

Analyses of the TgAMTs gene family structure, conserved motifs, and cis-acting promoter elements

Tissue-specific expression patterns of TgAMTs

Discussion

Conclusions

Declarations

Conflict of Interests

Funding

Author Contribution

References

Additional Declarations

Supplementary Files

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