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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.


Identification of the region required for the nitrate response using the CaMV 35S minimal promoter.(a) Schematic representation of cis-activation of the CaMV 35S minimal promoter by sequences located upstream of the TATA box of OsNAR2.1 in the transgenic lines. Constructs of –311/–129::min::GUS, –284/–129::min::GUS, –192/–129::min::GUS and –129/–1::min::GUS are the different promoter fragments fused with 35S minimal promoter and GUS reporter gene. (b) Analysis was performed on 20 independent transgenic lines for each construct. (c,d) GUS expression pattern by histochemical staining in the roots (c, Bars: 1 mm) and root-shoot junction (d, Bars: 0.5 mm). (e) 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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f2: Identification of the region required for the nitrate response using the CaMV 35S minimal promoter.(a) Schematic representation of cis-activation of the CaMV 35S minimal promoter by sequences located upstream of the TATA box of OsNAR2.1 in the transgenic lines. Constructs of –311/–129::min::GUS, –284/–129::min::GUS, –192/–129::min::GUS and –129/–1::min::GUS are the different promoter fragments fused with 35S minimal promoter and GUS reporter gene. (b) Analysis was performed on 20 independent transgenic lines for each construct. (c,d) GUS expression pattern by histochemical staining in the roots (c, Bars: 1 mm) and root-shoot junction (d, Bars: 0.5 mm). (e) 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: 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 though the activity was much less stronger than that driven by the –311::GUS (Fig. 2c–e). These results suggest the sequence of –129/–1 bp indeed be required for the nitrate regulated expression of OsNAR2.1.


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)

Identification of the region required for the nitrate response using the CaMV 35S minimal promoter.(a) Schematic representation of cis-activation of the CaMV 35S minimal promoter by sequences located upstream of the TATA box of OsNAR2.1 in the transgenic lines. Constructs of –311/–129::min::GUS, –284/–129::min::GUS, –192/–129::min::GUS and –129/–1::min::GUS are the different promoter fragments fused with 35S minimal promoter and GUS reporter gene. (b) Analysis was performed on 20 independent transgenic lines for each construct. (c,d) GUS expression pattern by histochemical staining in the roots (c, Bars: 1 mm) and root-shoot junction (d, Bars: 0.5 mm). (e) 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

License
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getmorefigures.php?uid=PMC4493634&req=5

f2: Identification of the region required for the nitrate response using the CaMV 35S minimal promoter.(a) Schematic representation of cis-activation of the CaMV 35S minimal promoter by sequences located upstream of the TATA box of OsNAR2.1 in the transgenic lines. Constructs of –311/–129::min::GUS, –284/–129::min::GUS, –192/–129::min::GUS and –129/–1::min::GUS are the different promoter fragments fused with 35S minimal promoter and GUS reporter gene. (b) Analysis was performed on 20 independent transgenic lines for each construct. (c,d) GUS expression pattern by histochemical staining in the roots (c, Bars: 1 mm) and root-shoot junction (d, Bars: 0.5 mm). (e) 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: 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 though the activity was much less stronger than that driven by the –311::GUS (Fig. 2c–e). These results suggest the sequence of –129/–1 bp indeed be required for the nitrate regulated expression of OsNAR2.1.

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.