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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.


Analysis of the –191 bp to –172 bp sequence as a conserved transcription enhancer element in OsNAR2.1 promoter.(a) Comparison of the essential nitrate enhancer element in AtNIR1 gene and the 20 bp sequence with different mutations between –191 bp and –172 bp of OsNAR2.1 promoter. M1, 6 bp mutation; M2, 8 bp mutation; M3, 17 bp mutation; M4, 19 bp mutation. The highly conserved nucleotides and mutated nucleotides are labeled with red and blue color, respectively. (b) The diagram of binary cassettes of the 4 × 20 bp::min::GUS, representation of a reporter construct with a synthetic promoter in which four copies of the 20-bp sequence are placed upstream of the 35S minimal promoter. (c) Analysis was performed on 20 independent transgenic lines for each construct. (d,e) GUS expression pattern by histochemical staining in the roots (c, Bars: 1 mm) and root-shoot junction (d, Bars: 0.5 mm) of M1::GUS, M2:GUS, M3::GUS, M4::GUS and 4 × 20 bp::min::GUS transgenic lines. (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 site mutations and 4 × 20 bp::min::GUS of OsNAR2.1 promoter treated with different forms of N. Values are mean ± SE of six biological replicates.
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f4: Analysis of the –191 bp to –172 bp sequence as a conserved transcription enhancer element in OsNAR2.1 promoter.(a) Comparison of the essential nitrate enhancer element in AtNIR1 gene and the 20 bp sequence with different mutations between –191 bp and –172 bp of OsNAR2.1 promoter. M1, 6 bp mutation; M2, 8 bp mutation; M3, 17 bp mutation; M4, 19 bp mutation. The highly conserved nucleotides and mutated nucleotides are labeled with red and blue color, respectively. (b) The diagram of binary cassettes of the 4 × 20 bp::min::GUS, representation of a reporter construct with a synthetic promoter in which four copies of the 20-bp sequence are placed upstream of the 35S minimal promoter. (c) Analysis was performed on 20 independent transgenic lines for each construct. (d,e) GUS expression pattern by histochemical staining in the roots (c, Bars: 1 mm) and root-shoot junction (d, Bars: 0.5 mm) of M1::GUS, M2:GUS, M3::GUS, M4::GUS and 4 × 20 bp::min::GUS transgenic lines. (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 site mutations and 4 × 20 bp::min::GUS of OsNAR2.1 promoter treated with different forms of N. Values are mean ± SE of six biological replicates.

Mentions: 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 fused with GUS reporter and transformed into rice. The GUS expression patterns of the transgenic lines are shown in 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.


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)

Analysis of the –191 bp to –172 bp sequence as a conserved transcription enhancer element in OsNAR2.1 promoter.(a) Comparison of the essential nitrate enhancer element in AtNIR1 gene and the 20 bp sequence with different mutations between –191 bp and –172 bp of OsNAR2.1 promoter. M1, 6 bp mutation; M2, 8 bp mutation; M3, 17 bp mutation; M4, 19 bp mutation. The highly conserved nucleotides and mutated nucleotides are labeled with red and blue color, respectively. (b) The diagram of binary cassettes of the 4 × 20 bp::min::GUS, representation of a reporter construct with a synthetic promoter in which four copies of the 20-bp sequence are placed upstream of the 35S minimal promoter. (c) Analysis was performed on 20 independent transgenic lines for each construct. (d,e) GUS expression pattern by histochemical staining in the roots (c, Bars: 1 mm) and root-shoot junction (d, Bars: 0.5 mm) of M1::GUS, M2:GUS, M3::GUS, M4::GUS and 4 × 20 bp::min::GUS transgenic lines. (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 site mutations and 4 × 20 bp::min::GUS 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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f4: Analysis of the –191 bp to –172 bp sequence as a conserved transcription enhancer element in OsNAR2.1 promoter.(a) Comparison of the essential nitrate enhancer element in AtNIR1 gene and the 20 bp sequence with different mutations between –191 bp and –172 bp of OsNAR2.1 promoter. M1, 6 bp mutation; M2, 8 bp mutation; M3, 17 bp mutation; M4, 19 bp mutation. The highly conserved nucleotides and mutated nucleotides are labeled with red and blue color, respectively. (b) The diagram of binary cassettes of the 4 × 20 bp::min::GUS, representation of a reporter construct with a synthetic promoter in which four copies of the 20-bp sequence are placed upstream of the 35S minimal promoter. (c) Analysis was performed on 20 independent transgenic lines for each construct. (d,e) GUS expression pattern by histochemical staining in the roots (c, Bars: 1 mm) and root-shoot junction (d, Bars: 0.5 mm) of M1::GUS, M2:GUS, M3::GUS, M4::GUS and 4 × 20 bp::min::GUS transgenic lines. (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 site mutations and 4 × 20 bp::min::GUS of OsNAR2.1 promoter treated with different forms of N. Values are mean ± SE of six biological replicates.
Mentions: 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 fused with GUS reporter and transformed into rice. The GUS expression patterns of the transgenic lines are shown in 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.

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.