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

Liu X, Feng H, Huang D, Song M, Fan X, Xu G - Sci Rep (2015)

Bottom Line: We found that -129 to -1 bp region is necessary for the nitrate-induced full activation of OsNAR2.1.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.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Crop Genetics and Germplasm Enhancement [2] MOA Key Laboratory of Plant Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze River, Nanjing Agricultural University, Nanjing 210095, China.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Deletion analysis of the OsNAR2.1 311 bp promoter fragment in rice transgenic lines for nitrate enhancer elements.(a) TATABOX position in OsNAR2.1 promoter. (b) Schematic representation of the diagram of binary cassettes fused the OsNAR2.1 promoter fragments with GUS reporter gene. –311::GUS, –284::GUS, –192::GUS, and –129::GUS, represent the binary cassette with GUS under the control of the flanking region upstream of the translation start codon (ATG) of 311 bp, 284 bp, 192 bp, and 129 bp, respectively. (c) Analysis was performed on 10 and 20 independent lines transformed with each construct. (d,e) Histochemical analysis of GUS activity in the roots (d) and root-shoot junction (e) of the representative transgenic line grown in the nutrient solution containing 0.2 mM NH4+ or 0.2 mM NO3− for seven days. Bars: 1 mm (d) and 0.5 mm (e). (f) Quantification of the root GUS activity. Analysis was performed on six independent transgenic lines grown in either ammonium or nitrate solution. Each GUS activity assay was performed for each line as described in “Materials and Methods”. a, b and c indicate the significant difference at p < 0.05 between the four lengths of OsNAR2.1 promoter treated with different forms of N. Values are mean ± SE of six biological replicates.
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f1: Deletion analysis of the OsNAR2.1 311 bp promoter fragment in rice transgenic lines for nitrate enhancer elements.(a) TATABOX position in OsNAR2.1 promoter. (b) Schematic representation of the diagram of binary cassettes fused the OsNAR2.1 promoter fragments with GUS reporter gene. –311::GUS, –284::GUS, –192::GUS, and –129::GUS, represent the binary cassette with GUS under the control of the flanking region upstream of the translation start codon (ATG) of 311 bp, 284 bp, 192 bp, and 129 bp, respectively. (c) Analysis was performed on 10 and 20 independent lines transformed with each construct. (d,e) Histochemical analysis of GUS activity in the roots (d) and root-shoot junction (e) of the representative transgenic line grown in the nutrient solution containing 0.2 mM NH4+ or 0.2 mM NO3− for seven days. Bars: 1 mm (d) and 0.5 mm (e). (f) Quantification of the root GUS activity. Analysis was performed on six independent transgenic lines grown in either ammonium or nitrate solution. Each GUS activity assay was performed for each line as described in “Materials and Methods”. a, b and c indicate the significant difference at p < 0.05 between the four lengths of OsNAR2.1 promoter treated with different forms of N. Values are mean ± SE of six biological replicates.

Mentions: 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.


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

Liu X, Feng H, Huang D, Song M, Fan X, Xu G - Sci Rep (2015)

Deletion analysis of the OsNAR2.1 311 bp promoter fragment in rice transgenic lines for nitrate enhancer elements.(a) TATABOX position in OsNAR2.1 promoter. (b) Schematic representation of the diagram of binary cassettes fused the OsNAR2.1 promoter fragments with GUS reporter gene. –311::GUS, –284::GUS, –192::GUS, and –129::GUS, represent the binary cassette with GUS under the control of the flanking region upstream of the translation start codon (ATG) of 311 bp, 284 bp, 192 bp, and 129 bp, respectively. (c) Analysis was performed on 10 and 20 independent lines transformed with each construct. (d,e) Histochemical analysis of GUS activity in the roots (d) and root-shoot junction (e) of the representative transgenic line grown in the nutrient solution containing 0.2 mM NH4+ or 0.2 mM NO3− for seven days. Bars: 1 mm (d) and 0.5 mm (e). (f) Quantification of the root GUS activity. Analysis was performed on six independent transgenic lines grown in either ammonium or nitrate solution. Each GUS activity assay was performed for each line as described in “Materials and Methods”. a, b and c indicate the significant difference at p < 0.05 between the four lengths of OsNAR2.1 promoter treated with different forms of N. Values are mean ± SE of six biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493634&req=5

f1: Deletion analysis of the OsNAR2.1 311 bp promoter fragment in rice transgenic lines for nitrate enhancer elements.(a) TATABOX position in OsNAR2.1 promoter. (b) Schematic representation of the diagram of binary cassettes fused the OsNAR2.1 promoter fragments with GUS reporter gene. –311::GUS, –284::GUS, –192::GUS, and –129::GUS, represent the binary cassette with GUS under the control of the flanking region upstream of the translation start codon (ATG) of 311 bp, 284 bp, 192 bp, and 129 bp, respectively. (c) Analysis was performed on 10 and 20 independent lines transformed with each construct. (d,e) Histochemical analysis of GUS activity in the roots (d) and root-shoot junction (e) of the representative transgenic line grown in the nutrient solution containing 0.2 mM NH4+ or 0.2 mM NO3− for seven days. Bars: 1 mm (d) and 0.5 mm (e). (f) Quantification of the root GUS activity. Analysis was performed on six independent transgenic lines grown in either ammonium or nitrate solution. Each GUS activity assay was performed for each line as described in “Materials and Methods”. a, b and c indicate the significant difference at p < 0.05 between the four lengths of OsNAR2.1 promoter treated with different forms of N. Values are mean ± SE of six biological replicates.
Mentions: 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.

Bottom Line: We found that -129 to -1 bp region is necessary for the nitrate-induced full activation of OsNAR2.1.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.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Crop Genetics and Germplasm Enhancement [2] MOA Key Laboratory of Plant Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze River, Nanjing Agricultural University, Nanjing 210095, China.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus