Two short sequences in OsNAR2.1 promoter are necessary for fully activating the nitrate induced gene expression in rice roots

Nitrate is an essential nitrogen source and serves as a signal to control growth and gene expression in plants. In rice, OsNAR2.1 is an essential partner of multiple OsNRT2 nitrate transporters for nitrate uptake over low and high concentration range. Previously, we have reported that −311 bp upstream fragment from the translational start site in the promoter of OsNAR2.1 gene is the nitrate responsive region. To identify the cis-acting DNA elements necessary for nitrate induced gene expression, we detected the expression of beta-glucuronidase (GUS) reporter in the transgenic rice driven by the OsNAR2.1 promoter with different lengths and site mutations of the 311 bp region. We found that −129 to −1 bp region is necessary for the nitrate-induced full activation of OsNAR2.1. Besides, the site mutations showed that the 20 bp fragment between −191 and −172 bp contains an enhancer binding site necessary to fully drive the OsNAR2.1 expression. Part of the 20 bp fragment is commonly presented in the sequences of different promoters of both the nitrate induced NAR2 genes and nitrite reductase NIR1 genes from various higher plants. These findings thus reveal the presence of conserved cis-acting element for mediating nitrate responses in plants.

There might be multiple cis-elements involved in different response of the genes to nitrate and nitrogen (N) supplies in plants 27 .
The cis-acting regulatory components for sensing nitrate in rice were scarcely reported. Previously, we have shown that a region from the position -311 to -1 bp, relative to the translation start site in the promoter of OsNAR2.1, was found to contain the nitrate responsive cis-element(s), while no similar cis-element(s) is presented in the promoters of OsNAR2.1 and OsNRT2s 32 . In this study, we revealed that the 20 bp sequence between -191 and -172 bp in addition to -129-1 bp region contains the nitrate enhancer element which is required to drive OsNAR2.1 expression. Our results demonstrated that both a minimal fragment and conserved cis-acting element are necessary for mediating the nitrate induced gene expression in rice.

Results
A 192 bp region at the upstream from translational start codon of OsNAR2.1 gene is sufficient for fully mediating the nitrate induced expression. Previously, we identified that the -311/-1 bp region of OsNAR2.1 promoter contains the nitrate regulated element(s) 32 . To further dissect the nitrate response cis-acting element(s), we first made different deletions from the upstream of TATA-box region (-129/-123 bp) and generated three fragments of -284/-1 bp, -192/-1 bp and -129/-1 bp of OsNAR2.1 promoter (Fig. 1a,b). These truncated promoter regions were respectively fused with beta-glucuronidase (GUS) reporter gene and transformed into rice (cv. Nipponbare). We generated twenty independent transgenic lines for each of the constructs harboring the different lengths of OsNAR2.1 promoter (Fig. S1), and nearly all of these lines had the responses of the GUS reporter to nitrate in their roots (Fig. 1c). The histochemical staining of GUS reporter in the transgenic lines showed that these promoters were not activated by exogenously supplied ammonium ( Fig. 1d-f). In contrast, the nitrate induced GUS expression controlled by all these promoters and the expression pattern in both the root and root-shoot junction were similar for the lines transformed with -311p::GUS, -284p::GUS, and -192p::GUS (Fig. 1d,e). However, the 129p::GUS transgenic lines showed a remarkably suppression of GUS activity compared with other transgenic lines (Fig. 1d,e). Quantitative analysis of GUS reporter enzyme activity in the transgenic rice roots confirmed the visible difference (Fig. 1f). Furthermore, qRT-PCR analysis using the roots of WT and the transgenic lines revealed that supply of nitrate in comparison to ammonium strongly elevated the levels of endogenous OsNAR2.1 mRNAs (Fig. S2). No significant difference of the abundance of OsNAR2.1 transcripts was observed between WT and the transgenic lines (Fig. S2), indicating that transforming the GUS report construct into rice did not affect the expression of endogenous OsNAR2.1 gene. Although the -129/-1 bp promoter drove the GUS activity only about 40% of that by -192/-1 bp promoter, their expression patterns in both roots and root-shoot junction were similar to that obtained with the -311/-1 bp promoter (Fig. 1d,e; Fig. S1). The GUS expression patterns indicate that the cis-regulatory elements involved in nitrate induced gene expression locate in the -192/-1 bp region of the promoter, and the -192/-129 bp region might contain the nitrate cis-element(s) which are required for enhancing OsNAR2.1 expression.
A 129 bp region at the upstream from translational start codon of OsNAR2.1 gene is essential for mediating basic nitrate response. To further characterize if the -129/-1 bp region is critical for OsNAR2.1 to sensing nitrate supply, we generated three -129 bp deleted promoters with different lengths (-311/-129 bp, -284/-129 bp, -192/-129 bp) and the -129 bp promoter in which both CaMV 35S minimal promoter (min) and GUS reporter gene were fused in the constructs (Fig. 2a). Twenty independent transgenic lines for harboring each of the constructs were tested for detecting the GUS expression under either nitrate or ammonium supply condition (Fig. S1). It showed that all the three -129 bp deleted promoters of OsNAR2.1 gene spanning -311/-129 bp, -284/-129 bp and -192/-129 bp region, respectively, lost the function in driving the nitrate induced GUS activity in both the roots and root-shoot junction ( Fig. 2b-d). The quantitative GUS activity measurement confirmed the visible results (Fig. 2e). The insertion of the GUS construct with the different promoters did not affect the response of endogenous OsNAR2.1 expression to nitrate (Fig. S2). In contrast, the transgenic lines expressing -129/-1::min::GUS containing the TATA-box (-129/-123 bp) showed nitrate induced GUS activity, even  5′ -GCCTCTT(GAATCCAACG)AAG-3′ at the region between -191 bp and -172 bp of the OsNAR2.1 promoter showed a high similarity with the motif 5′ -GACTCTTN 10 AAG-3′ in the AtNIR1 promoter which is critical for nitrate inducibility 27 . In addition, we found that the 20 bp sequence in OsNAR2.1 promoter is relatively conserved in the putative promoters of NAR2 genes from different plant species including Arabidopsis, bean, birch and tobacco (Fig. 3).
To test if this 20 bp-sequence functions as the putative nitrate enhancer element in the -192/-129 bp region, the effects of mutating this fragment on the promoter activity were examined. We generated four -192/-1 bp promoters with the 20 bp-sequence mutations of M1 (6 bp), M2 (8 bp), M3 (17 bp), M4 (19 bp) and one synthetic promoter with the fusion of four copies of the 20 bp sequence (4 × 20 bp) to the 35S minimal promoter (Fig. 4a,b). These point or site mutated or synthetic promoters were further  Figure S1. Both histochemical staining and GUS activity measurement showed that the mutations in comparison to the native promoter did not change the very faint basal activity of the promoter under ammonium supply condition (Fig. 4c-f). Randomly point mutation of total 6 bp (M1) and 8 bps (M2) which are not completely conserved in the 20 bp-fragment of NAR2.1 promoters from different species (Fig. 3) did not significantly alter the promoter activity in response to nitrate to drive GUS reporter (Fig. 4d-f). However, mutating most of the base pairs of the 20 bp fragment (M3 and M4) drastically decreased the promoter activity to drive GUS reporter under the same nitrate treatment (Fig. 4d-f). Interestingly, the M3 and M4 mutations resulted in the same activity of -192/-1 bp and native -129/-1 bp promoter of OsNAR2.1 in responses to nitrate (Fig. 4d-f), indicating that the 20 bp sequence contains essential cis-element for enhancing the nitrate response of OsNAR2.1 in rice. However, 4 × 20 bp::mini::GUS transgenic lines had no GUS activities under the same nitrate treatment (Fig. 4d-f), which implied that the 20 bp sequence itself is not enough for conferring the nitrate signal to induce the transcriptional expression.

Discussion
Nitrate supply can trigger the rapid change of expression of the genes involved in nitrate uptake and assimilation, as well as their associated carbon and energy metabolism 20,28,46 . Although there are common responses of the genes encoding two components of high affinity nitrate transporters (NAR2.1 and its associated NRT2s) as well as a number of the genes encoding nitrate and nitrite reductase to different N forms, the identity of the nitrate regulatory factor(s) and conserved cis-acting element(s) were uncertain in plants 36 . In our current study, we analyzed the OsNAR2.1 promoter and identified that both -129/-1 bp and 20 bp (-191/-172 bp) fragments upstream from the translational start site are necessary to mediate the nitrate induced gene activation in rice. The 20 bp sequence is relatively conserved among NAR2 and NIR1 genes from various plant genomes (Fig. 3).
OsNAR2.1 in rice might be a key gene not only for nitrate uptake but also for the early sensing nitrate supply and transferring this signal to other nitrate responsive genes 32,37 . In this study, we detected that a short fragment of -192/-1 bp in the OsNAR2.1 promoter contains the necessary cis-elements for responding to nitrate supply, while the -311/-193 bp fragment did not contribute to sense nitrate signal in mediating the gene expression (Fig. 1). Konishi and Yanagisawa (2010) compared the sequences of several nitrite reductase gene promoters from various higher plants and identified a conserved sequence motif 5′ -GCCcCTTN 10 AAG-3′ as the putative nitrate responsive element (NRE) 27 . We found that the 20 bp sequence at -191/-172 bp region of the OsNAR2.1 promoter, 5′ -GCCTCTT(GAATCCAACG)AAG-3′ , showed a high similarity with the NRE. However, this fragment is not presented in any of the putative promoters of nitrate responsive OsNRT2 genes 32 . Since OsNAR2.1 interacts with multiple OsNRT2 members in both transcriptional and translational levels 44,45 , lack of the conserved motif in the promoters of OsNRT2 genes indicates that either OsNAR2.1 and these OsNRT2 are regulated by different transcription factors or OsNAR2.1 transfers the nitrate signal for upregulating the expression of OsNRT2 members.
Mutations of the conserved sequence in the 20 bp motif of NAR2 promoters (M3 and M4) from different plant species (Fig. 3) resulted in markedly suppression of the nitrate-responsive activity (Fig. 4), indicating that this motif might be a key sequence for the transcriptional enhancing and the sensing the nitrate supply. Konishi and Yanagisawa (2010) have defined that the 43 bp sequence containing the 20 bp conserved sequence is necessary for the full activation of the native NIR1 promoter by nitrate. However in this study, its four copies fused to 35S minimum promoter did not have the function in mediating the nitrate induced gene expression (Fig. 4). In general, promoter activity is determined by the combined effects of many cis-elements 27,28 . Some of these binding sites mediate particular intercellular or intracellular stimuli, or developmental signals, and others function simply as an enhancer that is independent of a particular signaling pathway 27,28 . In this way, the 20 bp conserved sequence of OsNAR2.1 promoter functions simply as an enhancer which is necessary for nitrate-responsive transcription (Fig. 4), while  the 20 bp fragment itself is not enough for conferring the nitrate signal to induce the transcriptional expression. Interestingly, point mutations in not completely conserved positions (M1 and M2) did not affect the promoter activity in driving GUS expression (Fig. 4d-f). The expression pattern implies that the discontinuous 12 bp region in the motif (Figs 3 and 4) maybe the key biding site of transcription enhancer for mediating the nitrate regulation. Since the 12 bp are highly conserved among the promoters of known plant NAR2 members (Fig. 3), it will be interesting to explore whether they perform a similar function for other NAR2s genes in different plant species.
Within the 20 bp of OsNAR2.1 promoter (Fig. 3), the highly conserved sequence 5′ -AATCCAAC-3′ has been reported to be specifically binding site with a GBF factor isolated from nuclear extracts of tomato and Arabidopsis 46 . In addition, the sequence 5′ -CTCTT-3′ in the 20 bp region is putative nodulin consensus sequences as a cis-acting elements controlling expression of the root nodule-specific soybean leghemoglobin gene 47,48 . To date, no trans-acting factor that directly regulates the nitrate-responsive transcription have been identified in rice. Our identification of the relative conserved sequence will facilitate a search for a novel-type of transcription factor in sensing nitrate signaling.
The -129/-1 bp fragment of OsNAR2.1 promoter could drive the nitrate induced gene expression (Fig. 1), while the fragment between -311/-129 bp was not able to cis-activate the transcription (Fig. 2), implicating that the 129 bp region also have the cis-regulatory elements in controlling the promoter activity. The GATA transcription factors have been predicted to be involved in regulating nitrate acquisition pathways [49][50][51] , while R2R3-MYB is involved in nitrate signaling 52 . Interestingly, the 129 bp cis-acting sequence contains the motif potentially being able to interact with GATA transcription factors and a binding site of the transcription factor R2R3-MYB (Table S1).
Some transcription factors bind multiple recognition sequences [53][54][55] . Multiple transcription factors function as a hub to perceive phosphate and mycorrhiza signals in plants have been well characterized 56,57 . For example, two conserved cis-acting elements, MYCS and P1BS, are involved in the regulation of mycorrhiza-activated phosphate transporters in eudicot species 57 . A single pair of the core motif in a large number of nitrate responsive genes is neither specific to nitrate responsive genes, nor common to all nitrate responsive genes and is randomly distributed throughout the genomes in both Arabidopsis and rice 52 . So, we deduced that the relative conserved 20 bp sequence in -192/-129 bp region is required to allow the enhancement of the GUS expression via the -129 bp fragment as combining sites of nitrate signal factor(s). However, whether these motifs are sufficient to confer the transcriptional regulation or need to interact with other elements remain unknown.  Table S2. The obtained DNA fragment for the OsNAR2.1 promoter was digested with NcoI and HindIII. These cloned fragments were used to replace the 35S-promoter which was inserted at upstream of the 5′ end of the GUS reporter gene in the pCB302-35S-GUS vector 27 .

Materials and Methods
The 35S minimal promoter (min) is a 62 bp fragment with HindIII and Xho I sites located respectively at the 5′ and 3′ ends. Four copies of 20 bp (4 × 20 bp) were commercially synthesized by GenScript company. Chimaeric promoter constructs ( Table S3.
Reporter constructs with mutated OsNAR2.1 promoters were generated by PCR using 192::GUS plasmid of OsNAR2.1. For the M1, M2, M3 and M4 mutation, we got the 6 bp, 8 bp, 17 bp and 19 bp nucleotides mutation on the basis of 20 bp sequence using primers in Table S4. The cloned fragments containing the intended mutations were recovered from the resultant plasmid, and used to replace the 35S-promoter of the pCB302-35S-GUS vector.
Rice Transformation. The constructs were obtained and transformed into callus initiated from the seeds of rice (Nipponbare) by Agrobacterium tumefaciens (strain EHA105)-mediated transformation 58 . Rice embryonic calli were induced on N 6 media and transformation was performed by Agrobacterium-mediated co-cultivation 58 . Transgenic plants were selected on a medium containing 50 mg/L glyphosate (Roche, Indianapolis, IN, USA).
Plant material growth conditions. Both WT and the transgenic seeds of rice (cv. Nipponbare) were surface-sterilized with 10% (v/v) H 2 O 2 for 30 min and rinsed thoroughly with deionized water. The sterilized seeds were germinated on a plastic support netting (mesh 1 mm2) mounted in plastic containers for one week. Uniform seedlings were selected and then transferred to a tank containing 8 L of IRRI nutrient solution for 4 weeks at pH 5.5. After N starved for 4 days, seedlings were grown for seven days in the culture solution for nitrate or ammonium treatment with solution refreshed every 2 days.
Quantitative measurement of GUS activity. Histochemical GUS staining was performed as described previously 27 , and quantification of the extractable GUS enzymatic activity using fluorescent substrate was carried out according to the method described by Jefferson et al. 60 . Samples (1-10 mg of root tissues) frozen in liquid N were disrupted for 1 min, then suspended into 1 mL GUS extraction buffer (50 mM Na 3 PO 4 , pH 7.4, 10 mM EDTA, 0.1% Triton X-100 (Sigma, St-Louis, MD, USA), 0.1% sodium lauryl sarcosine, 10 mM b-mercaptethanol). Reactions were initiated by mixing 50 mL of protein extract with 120 mL of 1 mM p-nitrophenyl-b-Dglucuronide (Sigma-Aldrich) at 37 °C for 1 to 4 h (GUS activity stayed linear for up to 16 h), and were stopped by adding 800 mL 125 mM Na 2 CO 3 , then measured with a Wallac Victor 2 spectrofluorimeter (Perkin Elmer, Waltham, MA, USA) at 355 nm excitation and 460 nm emission. Protein concentration was quantified using the Protein Assay reagent (Bio-Rad Laboratories, http://www.bio-rad.com).