Cloning of chrysanthemum high-affinity nitrate transporter family (CmNRT2) and characterization of CmNRT2.1

The family of NITRATE TRANSPORTER 2 (NRT2) proteins belongs to the high affinity transport system (HATS) proteins which acts at low nitrate concentrations. The relevant gene content of the chrysanthemum genome was explored here by isolating the full length sequences of six distinct CmNRT2 genes. One of these (CmNRT2.1) was investigated at the functional level. Its transcription level was inducible by low concentrations of both nitrate and ammonium. A yeast two hybrid assay showed that CmNRT2.1 interacts with CmNAR2, while a BiFC assay demonstrated that the interaction occurs at the plasma membrane. Arabidopsis thaliana plants heterologously expressing CmNRT2.1 displayed an enhanced rate of labeled nitrogen uptake, suggesting that CmNRT2.1 represents a high affinity root nitrate transporter.

Scientific RepoRts | 6:23462 | DOI: 10.1038/srep23462 Finally, a demonstration is given that the heterologous expression of CmNRT2.1 in A. thaliana resulted in an increased rate of nitrate uptake into the root.

Results
The CmNRT2s and their predicted products. A peptide level sequence comparison between the set of AtNRT2s and the single previously known CmNRT2 (CmNRT2. 4 (Fig. 1A,B). Based on these motifs, the two degenerate primers A1f and A2-2f (Table 1) were designed to amplify the 3′ end of the CmNRT2s using 3′ RACE. The outcome of the procedure was the successful identification of seven distinct 3′ untranslated regions (3′ UTRs), including that of CmNRT2.4 (Fig. 1C), which implied that the chrysanthemum genome housed seven CmNRT2 genes. The full length cDNAs of CmNRT2.1 through 2.3 and 2.5 through 2.7 were subsequently obtained by means of 5′ RACE-PCR, following Gu et al. 19 . The resulting predicted polypeptide sequences were then aligned (Fig. 1D, Table 2). The greatest extent of sequence identity obtained between CmNRT2.1/2.2 and 2.2/2.4 (94.3%), followed by the 2.1/2.4 pair (93.0%), but only a low level of identity (41.5%) existed between 2.1 and 2.7. The DNA sequences were used to determine the genes' intron-exon structure 3 and a schematic representation of the inferred structures is given as Fig. S1. Five of the genes (2.1, 2.2, 2.3, 2.4 and 2.6) share two conserved introns, whereas 2.5 harbors two introns sited in a different part of the gene, and 2.7 lacks any introns. A phylogenetic tree based on peptide sequences is shown in Fig. 2; this indicated that five of the gene products (CmNRT2.1, 2.2, 2.3, 2.4 and 2.6) cluster within a single clade with a highly supportive bootstrap value, while 2.5 and 2.7 are outliers. CmNRT2.4 has previously been shown to be a strong candidate as a nitrate uptake protein 18 , so the strong level of peptide similarity and gene structure between it and CmNRT2.1 was suggestive of the latter protein sharing a similar functionality.

, AB921547) revealed the presence of two conserved regions, namely A1 [FGMRGRLW(N/T/A/I/G)(L/W)W] and A2 [(H/Y)FPQWG(S/G)M(F/C)]
CmNRT2.1 is inducible by nitrate and ammonium. The topological profiling of CmNRT2.1 transcription showed that the gene was constitutively transcribed throughout the plant except in the flower, with the root being the site where its transcript was most abundant (Fig. S2). When the plants were provided with various concentrations of either nitrate or ammonium, the abundance of CmNRT2.1 transcript in the root was enhanced. The nitrate response peaked after 4 h irrespective of the nitrate concentration ( Fig. 3A-C), and similarly, the provision of ammonium provoked a transcriptional peak after 4 h (Fig. 3D).
In vivo interaction between CmNRT2.1 and CmNAR2. A BiFC analysis was conducted to characterize the interaction between CmNRT2.1 and CmNAR2, based on the transient expression of split YFP-labelled CmNRT2.1 and CmNAR2 in onion epidermal cells. The CmNRT2.1 and CmNAR2 fusion proteins were engineered to have complementary N terminal and C terminal EYFP fragments. The epidermal cells expressing CmNAR2-cEYFP and CmNRT2.1-nEYFP showed strong YFP complementation. In contrast, cells transformed with either CmNRT2.1-nYFP/cYFP, nYFP/CmNAR2.1-cYFP or nYFP/cYFP emitted no fluorescence (Fig. 4). The interpretation of these observations was that CmNRT2.1 can interact with CmNAR2 in vivo. As a follow-up experiment, a split-ubiquitin membrane two hybrid system experiment was performed. This confirmed that CmNRT2.1 and CmNAR2 interact, as shown by the ability of the yeast cells to grow on the quadruple dropout medium, while cells carrying either pPR3-N/pBT3-C-NAR2.1 or pPR3-N/pBT3-C-NRT2.1 were unable to grow (Fig. 5A). The three combinations used as a positive control all promoted growth on the double-dropout medium (Fig. 5B). When the colonies were re-streaked onto a plate containing X-α -Gal, only cells harboring both CmNRT2.1 and CmNAR2 were able to produce a signal (Fig. 5C).  6B) demonstrated that the transgene was successfully incorporated and transcribed. Two independent transgene homozygous T 3 selections (RT-2 and -19) were used to study the transgene's impact on plant growth and nitrate uptake. Both root and shoot fresh weight (FW) of the two lines were significantly higher than those of the wild type (WT) control and vector-transformed lines when the growing medium contained 0.25 mM nitrate. With respect to root FW, there was no significant difference in performance between the transgenics and the controls when the medium contained 10 mM nitrate, but shoot FW was enhanced in RT-2 (Fig. S3). When nitrate uptake was assessed using labeled nitrate, both transgenics out-performed both WT and the empty vector control (Fig. 6C), consistent with the suggestion that CmNRT2.1 provides an improved capacity to take up nitrate.

Discussion
NRT2s have been isolated to date largely using degenerate primers; presently, seven members are known in A. thaliana 12 , six in poplar 20 and four in Lotus japonicus 21 . Here, the same approach has been used to identify and isolate six further CmNRT2s to add to the one isolated previously 18 . At the peptide level, AtNRT2.1, 2.2 and 2.4 share homology with one another, as do AtNRT2.3 and 2.6. Similarly, a group of four CmNRTs (2.1, 2.4, 2.2 and 2.3) shared closely related sequences (which aligned well with that of AtNRT2.6) ( Table 2). It has been shown previously that CmNRT2.4 interacts with CmNAR2 to promote nitrate uptake 18 .
The extensive homology between CmNRT2.1 and 2.4, as well as the nitrate inducibility of CmNRT2.1 ( Table 2) 22 . In contrast to the transcriptional behavior of CmNRT2.4 18 and the A. thaliana genes AtNRT2.3, 2.6 and 2.7 12 , CmNRT2.1 transcript was not detectable when the plants were deprived of either nitrate or ammonium (Fig. 3). Moreover, the increased folds of highest transcription level of CmNRT2.1 compared to the control is larger than that in CmNRT2.4 under 4 h nitrate exposure.
The yeast two hybrid and BiFC analyses confirmed that CmNRT2.1 and CmNAR2 interacted with one another in vivo, as do a number of the A. thaliana NRT2s with AtNAR2.1 23,24 , rice NRT2s with OsNAR2.1 16 and barley NRT2s with HvNAR2.3 10 . Further experiments will be needed to establish whether any of the CmNRT2s other than CmNRT2.1 and 2.4 are able to likewise interact with CmNAR2. The AtNRT2.1/NAR2.1 interaction has been shown to take place in the plasma membrane, forming a 150 kDa complex, thought to act as a high affinity nitrate transporter 24 . The present data were consistent with the CmNRT2.1/NAR2 complex similarly localizing to the plasma membrane (Fig. 4). Transgenic A. thaliana plants constitutively expressing CmNRT2.4 display an enhanced rate of nitrate uptake compared to the WT and the empty vector controls 18 , and the present experiments have shown that CmNRT2.1 activity can also contribute to nitrate uptake (Fig. 6), as expected given the high degree of sequence similarity ( > 93%) existing between CmNRT2.1 and 2.4 ( Table 2). The conclusion is that CmNRT2.1 is a nitrate inducible gene, the product of which is a high affinity nitrate transporter. As such, it represents a suitable candidate for the engineering of nitrate uptake efficiency in chrysanthemum.

Methods
Plant materials and growth conditions. The experiments were based on the chrysanthemum cultivar 'Nannongxuefeng' , maintained at the Nanjing Agricultural University Chrysanthemum Germplasm Resource Preserving Centre (Nanjing, China). Phenotypically uniform seedlings at the eight leaf stage were grown in a pH 6.5 medium containing 5 mM NH 4    Isolation and sequencing of CmNRT2 full-length cDNAs. For the gene isolation experiment, seedlings were grown in the solution (as described in the part of plant materials and growing conditions) for four weeks, then starved of nitrogen by removing them to a nitrogen-free version of the same medium for one week. After exposing the seedlings to 5 mM KNO 3 for 4 h, RNA was extracted from the roots using the RNAiso reagent (TaKaRa, Tokyo, Japan), following the manufacturer's protocol, then treated with RNase-free DNaseI (TaKaRa, Tokyo, Japan). The concentration and the integrity of the extract were assessed following Gu et al. 25 . The first cDNA strand was synthesized using Reverse Transcriptase M-MLV (RNase H − ) (TaKaRa, Tokyo, Japan), following the manufacturer's protocol. Two degenerate primers A1f and A2-2f (sequences given in Table 1) were designed based on regions conserved between the CmNRT2.4 and the AtNRT2 sequences. The remainder of the cDNA sequence was acquired using RACE-PCR, following Liu et al. 26 . Open reading frames (ORFs) were identified from the resulting sequences using ORF finder software (www.ncbi.nlm.nih.gov). The genes' deduced polypeptide sequences were used in a BLASTp search to identify homologs. An alignment of the seven derived CmNRT2s was performed using DNAman v 5.   Table 1). CmPsaA-F (5′ -CCAATAACCACGACCGCTAA-3′ ) and CmPsaA-R (5′ -GGCACAGTCCTCCCAAGTAA-3′ ) were used to detect the expression level of reference gene of CmPsaA (Table 1). The 2 −△△C T method was used to calculate relative changes in transcript abundance 28 . Each derived relative transcript abundance was based on the mean of three biological replicates.
The performance of transgenic A. thaliana expressing CmNRT2.1. Seedlings were raised on vertical MS agar plates two weeks, then transferred to a growth culture conditions medium 29 , containing either 0.25 mM nitrate (0.125 mM KNO 3 , 0.0625 mM Ca(NO 3 ) 2 ) or 10 mM nitrate (5 mM KNO 3 and 2.5 mM Ca(NO 3 ) 2 ). The nitrates were replaced by the same molarity of chloride salts to produce a nitrogen deficient medium. Each of three replicated treatments comprised a set of 50 plants. Shoot and root FW was measured after 14 days. The uptake of labeled nitrate ( 15 NO 3 ) was assayed as described elsewhere 18 . Briefly, the plants were exposed to 0.1 mM CaSO 4 for 1 min, then to a complete nutrient solution containing 0.2 mM 15 NO 3 -for 5 min and finally to 0.1 mM CaSO 4 for 1 min. The root homogenate was dried overnight at 80 °C. The content of labeled nitrate was analyzed using a PDZ Europa ANCA-MS device (Northwich, UK). The recorded measurements represent the mean of three biological repeats.
Statistical analysis. Statistical analysis was performed by the one-way analysis of variance (ANOVA) using SPSS 11.5 software (SPSS Inc., Champaign, IL), and Duncan's multiple range test was employed to detect differences between means.