Cloning and Functional Determination of Ammonium Transporter PpeAMT3;4 in Peach

You, Shuanghong; Wang, Yuqing; Li, Yanju; Li, Yuhe; Tan, Ping; Wu, Zheng; Shi, Wenjing; Song, Zhizhong

doi:https://doi.org/10.1155/2020/2147367

BioMed Research International

On this page

Abstract Introduction Materials and Methods Results Discussion Conclusions Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2020 | Article ID 2147367 | https://doi.org/10.1155/2020/2147367

Cloning and Functional Determination of Ammonium Transporter PpeAMT3;4 in Peach

Shuanghong You,^1,2Yuqing Wang,^2,3Yanju Li,^2,4Yuhe Li,^2,3Ping Tan,^1,2Zheng Wu,^1,2Wenjing Shi,^1,2and Zhizhong Song^2,3

Academic Editor: Hamid Mukhtar

Received20 Jul 2020

Revised15 Nov 2020

Accepted20 Nov 2020

Published04 Dec 2020

Abstract

Ammonium (NH₄⁺) plays key roles in plant growth, development, fruit quality, and yield. In plants, NH₄⁺ uptake and transport are facilitated by NH₄⁺ transporters (AMT). However, molecular mechanisms and physiological functions of type-II AMT (AMT2) transporters in fruit trees are still unclear, especially in peach. In this study, we cloned and characterized an AMT2 family gene from peach, PpeAMT3;4, and determined its function in yeast mutant. Expression analysis showed that PpeAMT3;4 was majorly expressed in peach roots and significantly decreased by NH₄⁺ excess but had no response to NH₄⁺ deficiency. Functional determination and ¹⁵nitrogen-labeled NH₄⁺ uptake assay in yeast cells implied that PpeAMT3;4 was a typical high-affinity transporter, with a value of 86.3 μM, that can uptake external NH₄⁺ in yeast cells. This study provides gene resources to uncover the biological function of AMT2 transporters and reveals molecular basis for NH₄⁺ uptake and nitrogen (N) nutrition mechanisms in fruit trees.

1. Introduction

Ammonium (NH₄⁺) is the preferred form of nitrogen (N) source absorbed by both annual and perennial plant species, especially in N-deficient soils [1, 2]. However, with the increasing soil N input and atmospheric deposition, plants have to deal with NH₄⁺ stress from sources below and above the ground [1–6]. Thus, an optimal amount of NH₄⁺ should be effectively absorbed from the soil via plant roots, to sustain basic growth demands.

Extensive studies in angiosperms [3–5] and in basal land plant liverwort [6] indicated that NH₄⁺ acquisition and uptake were performed by NH₄⁺ transporters (AMT) through the plasma membrane of root cells. The first plant AMT family gene was observed in Arabidopsis [7], and AtAMT1;1, AtAMT1;2, and AtAMT1;3 successfully restored the normal growth of the yeast mutant that is deficient in NH₄⁺ uptake systems. Subsequent studies of AMT transporters have been identified in diverse plant species, including Lotus japonicus (L. japonicus) [8, 9], Lycopersicon esculentum (L. esculentum) [10–12], Populus trichocarpa (P. trichocarpa) [13], Oryza sativa (O. sativa) [14, 15], Triticum aestivum (T. aestivum) [16], Alternanthera philoxeroides (A. philoxeroides) [17], and so on, which have been characterized in Xenopus oocytes or yeast mutant. The AMT family transporters can be divided into two subfamilies: AMT1 and AMT2 [3–5]. In particular, the phosphorylation of amino acid residues of AMT proteins is a recognized means, by which NH₄⁺ uptake activities were regulated [18, 19]. In Arabidopsis, the phosphorylation of the threonine (Thr) residue in the C-terminal tail region of AtAMT1.1 (Thr⁴⁶⁰), AtAMT1.2 (Thr⁴⁷²), and AtAMT1.3 (Thr⁴⁶⁴) led to the loss of NH₄⁺ transport activity in response to increasing external NH₄⁺ supplies [18–21], providing a novel regulatory mechanism that NH₄⁺ transport can be rapidly shut-off under high NH₄⁺ supply conditions.

Functional studies of plant AMT members were mainly focused on the AMT1 family, especially of annual model plants. However, molecular mechanisms towards NH₄⁺ uptake and transport in fruit trees are largely rare but were just observed in citrus [22] and pear [23, 24]. The biological and physiological functions of AMT2 members remain unknown. Favorably, the knowledge on NH₄⁺ uptake and transport in model plants provides valuable insights into the investigation of their roles in fruit trees.

As one of the most popular Rosaceae fruit crops, peach (Prunus persica) has its genome sequenced [25, 26]. In this study, we isolated and characterized a putative AMT2 family gene from peach (entitled as PpeAMT3;4) and determined that PpeAMT3;4 is a typical HATS-mediated AMT member responsible for NH₄⁺ uptake in peach roots. Nonetheless, our findings provide a molecular basis of NH₄⁺ uptake for fruit trees.

2. Materials and Methods

2.1. Plant Material and Growth Condition

‘Feicheng’ peach seedlings grown in the Key Laboratory of Molecular Module-Based Breeding of High Yield and Abiotic Resistant Plants in Universities of Shandong in Yantai, China, were used throughout this study. Germinated seedlings with similar growth status were transferred into 1/2 Murashige and Skoog (MS) liquid solution ([17, 27], containing approximately 1 mM NH4Cl), cultivated in a climate-controlled growth cabinet that was maintained under 28°C/23°C and 12/12 h light/dark, with 60% relative humidity.

For the NH₄⁺ deficiency treatments, NH₄⁺ was omitted from the 1/2 MS medium. For the NH₄⁺ excess treatments, germinated seedlings were grown in 1/2 MS solution containing 20 mM NH₄Cl (pH 5.8). Seedlings were exposed to treatment for 72 h before quantitative real-time PCR (qRT-PCR) determination.

2.2. Cloning of PpeAMT3; 4 Gene in Peach

Protein sequences of poplar AMT genes [13] were used as query to BLAST the Phytozome Peach Genome Database (http://www.phytozome.net) to identify putative homologues in peach. Information of PpeAMT genes were listed in Supplemental Table 1. Coding sequence (CDS) of the PpeAMT3;4 gene was downloaded from the database, and the first 18 bp sequence from the ATG codon and the last 19 bp sequence from the TAA codon were chosen as the forward primer and reverse primer, respectively (Table 1). Total RNA of ‘Feicheng’ peach seedling was extracted using MiniBEST Plant RNA Extraction Kit (TaKaRa, Dalian, China) and reverse transcribed into the 1st cDNA using PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China). Normal gene amplification was carried out to obtain the CDS region of PpeAMT3;4 and then sequenced in Sanon Biotech (Shanghai, China).

2.3. Phylogenetic Analysis of PpeAMT Genes in Peach

Full-length amino acid sequences of PpeAMT transporters were aligned by ClustalX2.1 and imported into the Molecular Evolutionary Genetics Analysis (MEGA) package version 4.1. Phylogenetic analyses were conducted according to the description of Liang et al. [28]. Branch lengths indicate the corresponding phylogenetic distances. Sequence logos of AMT1 and AMT2 subfamilies were shown on the right, generated using WEBLOGO (http://weblogo.berkeley.edu/logo.cgi). The grand average of hydropathicity (GRAVY) and aliphatic index analyses of PpeAMT3;4 were calculated using the ProtParam tool (http://web.expasy.org/protparam/), as described by Liang et al. [28].

2.4. qRT-PCR Assays

Specific expression primers for PpeAMT3;4 and Ubiquitin (control gene) were designed using the NCBI/Primer-BLAST online server. Primer sequences were listed in Table 1. According to the description of Liang et al. [28], qRT-PCR analyses were carried out on 7500 Real-Time PCR System (Applied Biosystems, New York, USA), using SYBR Premix Ex Taq reaction kit (TaKaRa, Dalian, China). To calculate RT-qPCR efficiency and the starting template concentration for each sample, the linear regression of the log (fluorescence) per cycle number data was used according to the description of Gazzarrini et al. [29]. The relative expression levels were presented after normalization to the internal control Ubiquitin from three independent biological repeats.

2.5. Functional Determination of PpeAMT3;4 in Yeast Mutant 31019b

The recombinant plasmid pYES2-AMT3;4 was constructed by cloning the CDS region of the PpeAMT3;4 gene into the pYES2 expression vector [17], using the primer pairs (Table 1, Kpn I site was introduced and underlined in the forward primer, Not I site was introduced and underlined in the reverse primer). The yeast strain 31019b (MATa mepl△ mep2△::LEU2 mep3△::KanMX2 ura3, [7, 13, 17]) was transformed with pYES2 harboring the CDS of PpeAMT3;4. Yeast complementation tests were carried out as described in previous reports [7, 13, 17]. Yeast strain 31019b was transformed with pYES2 or pYES2-AMT3;4, respectively. The growth of yeast cells were determined in the yeast nitrogen base (YNB) liquid medium, supplemented with 0.02, 0.2, or 2 mM NH₄⁺ as the sole N source (pH 5.8). Pictures were taken at the 3rd day after incubation at 30°C. The growth of transformed yeast cells in the YNB liquid medium was checked by determining absorbance at 600 nm [29].

2.6. ¹⁵N Uptake Kinetics Assay

¹⁵N labeled NH₄⁺ uptake assays were carried out according to the description of Guo et al. [17]. Yeast cells transformed with pYES2-MT3;4 or pYES2 were grown in the YNB liquid medium, supplemented with ¹⁵N labeled NH₄Cl for 10 min. NH₄⁺ content was determined by Flash-2000 Delta VADVADTAGE Mass Spectrometer (Thermo Fisher, Waltham, Massachusetts, USA). Michaelis-Menten kinetic constants ( and ) were calculated as described by Guo et al. [17].

2.7. Statistical Analysis

All data were statistically analyzed using independent samples’ -test in SPSS 13.0 software (SPSS Chicago, Ilinois, USA). Details are described in figure legends. Asterisks indicate statistical differences between plants under control and stress treatment (, ).

3. Results

3.1. Characteristics and Phylogenetic Tree Construction of PpeAMT Transporters in Peach

By BLAST searching of the Phytozome Peach Genome Database, 14 putative PpeAMT genes were screened and identified [27]. Pr7otein domain verification analyses showed that all PpeAMT transporters contain the NH₄⁺ transporter transmembrane domain (PF00909). Information of these PpeAMT genes, including gene ID, gene location, CDS (coding sequence) length, and peptide length, are listed in Supplemental Table 1. Notably, the amino acid sequences of PpeAMT proteins shared an overall identity of 51.09% (Figure 1). To confirm the evolutionary relationships of PpeAMT proteins, a maximum likelihood (ML) phylogenetic tree was generated based on the alignment of the amino acid sequences of PpeAMT proteins. All peach AMT transporters were classified into 2 major subgroups: AMT1 and AMT2 (Figure 2), each with 5 and 9 members, respectively. The AMT2 subgroup in peach was further divided into three subclades, including PpeAMT2 (1 member), PpeAMT3 (5 members), and PpeAMT4 (3 members) (Figure 2).

3.2. Cloning and Expression Analysis of PpeAMT3;4

As a member of the AMT2 subgroup in peach, the nucleotide sequence of PpeAMT3;4 CDS was amplified from ‘Feicheng’ peach seedlings, using the specific primer pairs and the 1st cDNA template. According to the sequencing results, PpeAMT3;4 possessed a coding sequence of 1065 bp, encoding a polypeptide of 355 amino acids with the deduced molecular weight of 39.09 kDa. Instability index assay implicated that PpeAMT3;4 was stable and alkalescent, with the theoretical PI value of 8.55. Moreover, both the GRAVY and aliphatic index analysis indicated that PpeAMT3;4 was a hydrophilic protein.

3.3. Expression Profiles of PpeAMT3;4

We further performed reverse-transcribed PCR to determine the expression profiles of PpeAMT3;4 in different tissues of 7-year-old ‘Feicheng’ trees. Results showed that PpeAMT3;4 was majorly expressed in roots, followed by leaves, and hardly observed in stems, flowers, and fruits (Figure 3).

To investigate the role of PpeAMT3;4 in maintaining NH₄⁺ homeostasis in peach, we analyzed the expression profiles of PpeAMT3;4 via the qRT-PCR analysis. Results showed that the expression of PpeAMT3;4 was significantly decreased by NH₄⁺ excess in roots of ‘Feicheng’ seedlings but had no response to NH₄⁺ deficiency (Figure 4).

3.4. Functional Determination of PpeAMT1;1 in Yeast Mutant

We further carried out functional determination of PpeAMT3;4 via complementation of the yeast mutant 31019b [6, 13, 17, 30]. Results showed that yeast cells harboring pYES2 or pYES2-PpeAMT3;4 grew well, with similar growth status, on the YNB medium that contains 2 mM Arg as the sole N source (Figure 5). Yeast cells harboring pYES2 could not grow well on the YNB medium containing 0.02, 0.2, or 2 mM NH₄Cl under pH 5.8, while yeast cells harboring pYES2-PpeAMT3;4 grew normally on the YNB medium supplemented under 0.2 and 2 mM NH₄Cl (Figure 5). These findings imply that PpeAMT3;4 is involved in NH₄⁺ in yeast, which may be responsible for NH₄⁺ uptake in peach roots.

3.5. ¹⁵N Uptake Kinetics Assay of ApAMT3;4 in Yeast Cells

To verify the proposition that PpeAMT3;4 really functions as an active NH₄⁺ transporter, we determined its NH₄⁺ uptake activity and the exact amount of ¹⁵N-labeled NH₄⁺ uptake in yeast cells via the ¹⁵N isotope labeling method. Yeast cells were grown in the NYB liquid medium labeled with ¹⁵N-labeled NH₄Cl, ranging from 0.02 mM to 2 mM. Affinity constant analysis showed that PpeAMT3;4 exhibited a value of 86.3 μM and a value of 3.69 μmol min⁻¹ μg⁻¹, respectively (Figure 6). These findings indicate that PpeAMT3;4 was primarily involved in HATS-mediated NH₄⁺ uptake in peach roots.

4. Discussion

NH₄⁺ is the preferred form of N transport for lower energetic cost in assimilation [3–5, 29], while excess NH₄⁺ is toxic to plant growth and development [30, 31]. In higher plants, high-affinity AMT1 subgroup genes encode NH₄⁺ transporters with a value of micromolar grade involved in the HATS for NH₄⁺ uptake [12]. However, the molecular mechanism towards the AMT2 subgroup genes is largely rare. In this present study, we initially isolated and determined a high-affinity AMT2 subgroup transporter gene in peach, which helps to investigate the biological role of AMT2 family transporters in fruit trees.

Transcript levels of AMT1 genes are closely related to the N nutrition status of the plant, and NH₄⁺ starvation mainly induced the expression levels of most AMT1 genes, may it be a clue of ‘starvation response’ for plants to grow under suboptimal N-source conditions [3, 16, 22]. However, PpeAMT3;4 was mainly expressed in roots and was nonresponsive to NH₄⁺ starvation in peach roots in this study but reduced significantly by excessive NH₄⁺ stress (Figures 3 and 4). These findings implies that PpeAMT3;4 may be an active NH₄⁺ transporter at normal NH₄⁺ supplies, even at extremely low NH₄⁺ conditions, which is an indispensable AMT2 transporter responsible for NH₄⁺ uptake in peach roots. If the NH₄⁺ supply reached an excessive state, PpeAMT3;4 began to stop its NH₄⁺ uptake and transport capacity that are sufficient enough for internal N metabolism and usage.

Heterologous expression studies using NH₄⁺ uptake defective yeast mutant or Xenopus oocytes indicate that AMT1 family genes encode high-affinity transporters [3, 4, 15], which play key roles in high-affinity NH₄⁺ uptake from soils via the roots. While molecular mechanisms or biological functions of AMT2 subgroup members are extremely rare, in this present study, PpeAMT3;4 could utilize the external NH₄⁺ in yeast cells (Figure 5), and ¹⁵N labeled NH₄⁺ uptake kinetics analysis exhibited a value of 86.3 μM (Figure 6). Nonetheless, PpeAMT3;4 is a functional AMT2 transporter responsible for NH₄⁺ uptake in peach roots, especially under normal and low external NH₄⁺ conditions.

5. Conclusions

In this study, we isolated and characterized PpeAMT3;4, an AMT2 family gene from peach, and determined its function in yeast mutant. The PpeAMT3;4 gene was majorly expressed in peach roots, whose expression was decreased under NH₄⁺ excess but had no response to NH₄⁺ deficiency. Functional determination and ¹⁵N-labeled NH₄⁺ uptake kinetics assay in yeast cells indicate that ApAMT3;4 was a typical high-affinity transporter, with a value of 86.3 μM and a value of 3.69 μmol min⁻¹ μg⁻¹, which can uptake external NH₄⁺ in yeast cells. Nonetheless, PpeAMT3;4 might be a NH₄⁺ transporter involved in NH₄⁺ uptake in peach roots. This study not only provides a technological system to uncover the biological function of AMT2 transporters in fruit trees but also reveals molecular basis for NH₄⁺ uptake and N nutrition mechanisms.

Data Availability

The data used to support the findings of this study are included within the article.

Conflicts of Interest

We declare that we do not have any commercial or associative interest that represents a conflict of interest with the work submitted.

Authors’ Contributions

Shuanghong You, Yuqing Wang, and Yanju Li contributed equally to this work.

Acknowledgments

This work was jointly supported by the following grants: the National Key R & D Program of China (2019YFD1000500), the Sci-Tech Innovation Special Project for Social Undertakings and People’s Livelihood Guarantee of Chongqing Science and Technology Bureau (cstc2019jscx-gksbX0138), the Improved Variety Innovation Project for Characteristic Benefit Agriculture of Chongqing (NKY20190030), the Agricultural Variety Improvement Project of Shandong Province (2019LZGC009), the Performance Incentive & Guidance Project Special for the Scientific Research Institutions of Chongqing (cstc2019jxjl80012), and the Key R&D Program of Shandong Province (2019GSF107095, 2018JHZ006).

Supplementary Materials

Supplemental Table 1: information of PpeAMT family genes in peach. (Supplementary Materials)

References

P. J. Syrett and I. Morris, “The inhibition of nitrate assimilation by ammonium in Chlorella,” Biochimica et Biophysica Acta, vol. 67, pp. 566–575, 1963.
View at: Publisher Site | Google Scholar
Q. Dortch, “The interaction between ammonium and nitrate uptake in phytoplankton,” Marine Ecology Progress Series, vol. 61, pp. 183–201, 1990.
View at: Publisher Site | Google Scholar
U. Ludewig, B. Neuhauser, and M. Dynowski, “Molecular mechanisms of ammonium transport and accumulation in plants,” FEBS Letters, vol. 581, no. 12, pp. 2301–2308, 2007.
View at: Publisher Site | Google Scholar
Y. Liu and N. von Wirén, “Ammonium as a signal for physiological and morphological responses in plants,” Journal of Experimental Botany, vol. 68, no. 10, pp. 2581–2592, 2017.
View at: Publisher Site | Google Scholar
T. R. McDonald and J. M. Ward, “Evolution of electrogenic ammonium transporters (AMTs),” Frontiers in Plant Science, vol. 7, p. 352, 2016.
View at: Publisher Site | Google Scholar
H. Guo, N. Wang, T. R. McDonald, A. Reinders, and J. M. Ward, “MpAMT1;2 from Marchantia polymorpha is a high-affinity, plasma membrane ammonium transporter,” Plant Cell Physiology, vol. 59, no. 5, pp. 997–1005, 2018.
View at: Publisher Site | Google Scholar
O. Ninnemann, J. C. Jauniaux, and W. B. Frommer, “Identification of a high affinity NH₄⁺ transporter from plants,” EMBO Journal, vol. 13, no. 15, pp. 3464–3471, 1994.
View at: Publisher Site | Google Scholar
F. Salvemini, A. M. Marini, A. Riccio, E. J. Patriarca, and M. Chiurazzi, “Functional characterization of an ammonium transporter gene from Lotus japonicus,” Gene, vol. 270, no. 1-2, pp. 237–243, 2001.
View at: Publisher Site | Google Scholar
E. D'Apuzzo, A. Rogato, U. Simon-Rosin et al., “Characterization of three functional high-affinity ammonium transporters in Lotus japonicus with differential transcriptional regulation and spatial expression,” Plant Physiology, vol. 134, no. 4, pp. 1763–1774, 2004.
View at: Publisher Site | Google Scholar
F. R. Lauter, O. Ninnemann, M. Bucher, J. W. Riesmeier, and W. B. Frommer, “Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato,” Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 15, pp. 8139–8144, 1996.
View at: Publisher Site | Google Scholar
U. Ludewig, N. von Wirén, and W. B. Frommer, “Uniport of ^NH by the root hair plasma membrane ammonium transporter LeAMT1;1,” Journal of Biological Chemistry, vol. 277, no. 16, pp. 13548–13555, 2002.
View at: Publisher Site | Google Scholar
N. von Wirén, S. Gazzarrini, A. Gojon, and W. B. Frommer, “The molecular physiology of ammonium uptake and retrieval,” Current Opinion in Plant Biology, vol. 3, no. 3, pp. 254–261, 2000.
View at: Publisher Site | Google Scholar
J. Couturier, B. Montanini, F. Martin, A. Brun, D. Blaudez, and M. Chalot, “The expanded family of ammonium transporters in the perennial poplar plant,” New Phytologist, vol. 174, no. 1, pp. 137–150, 2007.
View at: Publisher Site | Google Scholar
Y. Sonoda, A. Ikeda, S. Saiki, N. . Wirén, T. Yamaya, and J. Yamaguchi, “Distinct expression and function of three ammonium transporter genes (OsAMT1; 1–1; 3) in rice,” Plant Cell Physiology, vol. 44, no. 7, pp. 726–734, 2003.
View at: Publisher Site | Google Scholar
A. Suenaga, K. Moriya, Y. Sonoda et al., “Constitutive expression of a novel-type ammonium transporter OsAMT2 in rice plants,” Plant Cell Physiology, vol. 44, no. 2, pp. 206–211, 2003.
View at: Publisher Site | Google Scholar
T. Li, K. Liao, X. Xu et al., “Wheat ammonium transporter (AMT) gene family: diversity and possible role in host-pathogen interaction with stem rust,” Frontiers in Plant Science, vol. 8, p. 1637, 2017.
View at: Publisher Site | Google Scholar
X. T. Guo, Y. T. Sheng, S. Y. Yang et al., “Isolation and characterization of a high-affinity ammonium transporter ApAMT1;1 in alligatorweed,” Plant Growth Regulation, vol. 89, no. 3, pp. 321–330, 2019.
View at: Publisher Site | Google Scholar
L. Yuan, R. Gu, Y. Xuan et al., “Allosteric regulation of transport activity by heterotrimerization of Arabidopsis ammonium transporter complexes in vivo,” Plant Cell, vol. 25, no. 3, pp. 974–984, 2013.
View at: Publisher Site | Google Scholar
T. Straub, U. Ludewig, and B. Neuhäuser, “The kinase CIPK23 inhibits ammonium transport inArabidopsis thaliana,” Plant Cell, vol. 29, no. 2, pp. 409–422, 2017.
View at: Publisher Site | Google Scholar
D. Loqué, S. Lalonde, L. L. Looger, N. von Wirén, and W. B. Frommer, “A cytosolic trans-activation domain essential for ammonium uptake,” Nature, vol. 446, no. 7132, pp. 195–198, 2007.
View at: Publisher Site | Google Scholar
L. Yuan, D. Loqué, S. Kojima et al., “The organization of high-affinity ammonium uptake in Arabidopsis roots depends on the spatial arrangement and biochemical properties of AMT1-type transporters,” Plant Cell, vol. 19, no. 8, pp. 2636–2652, 2007.
View at: Publisher Site | Google Scholar
G. Camañes, M. Cerezo, E. Primo-Millo, A. Gojon, and P. García-Agustín, “Ammonium transport and CitAMT1 expression are regulated by N in citrus plants,” Planta, vol. 229, no. 2, pp. 331–342, 2009.
View at: Publisher Site | Google Scholar
H. Li, J. L. Han, Y. H. Chang, J. Lin, and Q. S. Yang, “Gene characterization and transcription analysis of two new ammonium transporters in pear rootstock (Pyrus betulaefolia),” Journal of Plant Research, vol. 129, no. 4, pp. 737–748, 2016.
View at: Publisher Site | Google Scholar
H. Li, Y. Cong, Y. H. Chang, and J. Lin, “Two AMT2-type ammonium transporters from Pyrus betulaefolia demonstrate distinct expression characteristics,” Plant Molecular Biology Reporter, vol. 34, no. 4, pp. 707–719, 2016.
View at: Publisher Site | Google Scholar
D. R. Layne and D. Bassi, The Peach: Botany, Production and Uses, CABI, London, 2008.
S. Jung, M. Staton, T. Lee et al., “GDR (Genome Database for Rosaceae): integrated web-database for Rosaceae genomics and genetics data,” Nucleic Acids Research, vol. 36, no. Database, pp. D1034–D1040, 2007.
View at: Publisher Site | Google Scholar
M. Tang, Y. Li, Y. Chen, L. Han, H. Zhang, and Z. Song, “Characterization and expression of ammonium transporter in peach (Prunus persica) and regulation analysis in response to external ammonium supply,” Phyton-International Journal of Experimental Botany, vol. 89, no. 4, pp. 925–941, 2020.
View at: Publisher Site | Google Scholar
M. Liang, Y. Gao, T. Mao et al., “Characterization and expression of KT/HAK/KUP transporter family genes in willow under potassium deficiency, drought, and salt stresses,” BioMed Research International, vol. 2020, Article ID 2690760, 12 pages, 2020.
View at: Publisher Site | Google Scholar
S. Gazzarrini, L. Lejay, A. Gojon, O. Ninnemann, W. B. Frommer, and N. von Wirén, “Three functional transporters for constitutive, diurnally regulated and starvation-induced uptake of ammonium into Arabidopsis roots,” Plant Cell, vol. 11, no. 5, pp. 937–947, 1999.
View at: Publisher Site | Google Scholar
D. T. Britto, M. Y. Siddiqi, A. D. M. Glass, and H. J. Kronzucker, “Futile transmembrane NH₄⁺ cycling: a cellular hypothesis to explain ammonium toxicity in plants,” Proceedings of the National. Academy of Sciences of the United States of America, vol. 98, no. 7, pp. 4255–4258, 2001.
View at: Publisher Site | Google Scholar
D. T. Britto and H. J. Kronzucker, “NH₄⁺ toxicity in higher plants: a critical review,” Journal of Plant Physiology, vol. 159, no. 6, pp. 567–584, 2002.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2020 Shuanghong You et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

380

Downloads

985

Citations

BioMed Research International

Cloning and Functional Determination of Ammonium Transporter PpeAMT3;4 in Peach

Abstract

1. Introduction

2. Materials and Methods

2.1. Plant Material and Growth Condition

2.2. Cloning of PpeAMT3; 4 Gene in Peach

2.3. Phylogenetic Analysis of PpeAMT Genes in Peach

2.4. qRT-PCR Assays

2.5. Functional Determination of PpeAMT3;4 in Yeast Mutant 31019b

2.6. 15N Uptake Kinetics Assay

2.7. Statistical Analysis

3. Results

3.1. Characteristics and Phylogenetic Tree Construction of PpeAMT Transporters in Peach

3.2. Cloning and Expression Analysis of PpeAMT3;4

3.3. Expression Profiles of PpeAMT3;4

3.4. Functional Determination of PpeAMT1;1 in Yeast Mutant

3.5. 15N Uptake Kinetics Assay of ApAMT3;4 in Yeast Cells

4. Discussion

5. Conclusions

Data Availability

Conflicts of Interest

Authors’ Contributions

Acknowledgments

Supplementary Materials

References

Copyright

2.6. ¹⁵N Uptake Kinetics Assay

3.5. ¹⁵N Uptake Kinetics Assay of ApAMT3;4 in Yeast Cells