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Conserved cis-regulatory modules in promoters of genes encoding wheat high-molecular-weight glutenin subunits.

Ravel C, Fiquet S, Boudet J, Dardevet M, Vincent J, Merlino M, Michard R, Martre P - Front Plant Sci (2014)

Bottom Line: The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors.However, the differences in annotation between promoter alleles could not be related to their level of expression.In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Recherche Agronomique, UMR1095, Genetics, Diversity and Ecophysiology of Cereals Clermont-Ferrand, France ; UMR1095, Genetics, Diversity and Ecophysiology of Cereals, Department of Biology, Blaise Pascal University Aubière, France.

ABSTRACT
The concentration and composition of the gliadin and glutenin seed storage proteins (SSPs) in wheat flour are the most important determinants of its end-use value. In cereals, the synthesis of SSPs is predominantly regulated at the transcriptional level by a complex network involving at least five cis-elements in gene promoters. The high-molecular-weight glutenin subunits (HMW-GS) are encoded by two tightly linked genes located on the long arms of group 1 chromosomes. Here, we sequenced and annotated the HMW-GS gene promoters of 22 electrophoretic wheat alleles to identify putative cis-regulatory motifs. We focused on 24 motifs known to be involved in SSP gene regulation. Most of them were identified in at least one HMW-GS gene promoter sequence. A common regulatory framework was observed in all the HMW-GS gene promoters, as they shared conserved cis-regulatory modules (CCRMs) including all the five motifs known to regulate the transcription of SSP genes. This common regulatory framework comprises a composite box made of the GATA motifs and GCN4-like Motifs (GLMs) and was shown to be functional as the GLMs are able to bind a bZIP transcriptional factor SPA (Storage Protein Activator). In addition to this regulatory framework, each HMW-GS gene promoter had additional motifs organized differently. The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors. However, the differences in annotation between promoter alleles could not be related to their level of expression. In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression.

No MeSH data available.


Related in: MedlinePlus

Binding of recombinant SPA protein with the probes derived from the Glu-B1-1 gene promoter. (A) Expression and purification of recombinant His-SPA protein. Crude extracts from uninduced and induced bacteria harboring the pHis-SPA expression vector and the eluted protein were resolved on an SDS-polyacrylamide gel. The molecular mass markers are indicated at left in kilodaltons. (B) EMSA of the recombinant SPA protein with the 25-bp biotin-labeled, GLM1 (−647), GLM2 (−626), and G-box (−227) probes derived from the Glu-B1-1 gene promoter and their mutated versions glm1, glm2, and G-box. The sequences of the oligonucleotides used as probes are shown with the GLM1, GLM2, and G-box in bold; identical residues are represented by dots, and mutated bases are shown in lowercase.
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Figure 7: Binding of recombinant SPA protein with the probes derived from the Glu-B1-1 gene promoter. (A) Expression and purification of recombinant His-SPA protein. Crude extracts from uninduced and induced bacteria harboring the pHis-SPA expression vector and the eluted protein were resolved on an SDS-polyacrylamide gel. The molecular mass markers are indicated at left in kilodaltons. (B) EMSA of the recombinant SPA protein with the 25-bp biotin-labeled, GLM1 (−647), GLM2 (−626), and G-box (−227) probes derived from the Glu-B1-1 gene promoter and their mutated versions glm1, glm2, and G-box. The sequences of the oligonucleotides used as probes are shown with the GLM1, GLM2, and G-box in bold; identical residues are represented by dots, and mutated bases are shown in lowercase.

Mentions: To verify the potential binding activity of the two GLMs (GLM1 and GLM2 at positions −647 and −626, respectively) present in the GLM-GATA box of the Glu-B1-1 gene promoter, we performed EMSAs with synthetic oligonucleotides and a recombinant SPA protein expressed as a His fusion in E. coli (Figure 7). We also determined the in vitro binding of SPA to the G-box motif, which was previously shown to bind bZIP proteins (Norre et al., 2002). As shown in Figure 7A, arabinose treatment induced expression of a protein of 50–75 kDa that was not present in uninduced cell extracts. The apparent size of the recombinant protein determined by SDS-PAGE was larger than the expected 48 kDa molecular mass of the His-tagged SPA fusion protein. A similar apparent increase in size on SDS gels was already reported by Albani et al. (1997) in their study of SPA. The recombinant His-SPA protein was purified to near homogeneity and used for binding assays. A DNA-protein complex was clearly observed with the GLM2 motif, while the shifted band detected for the GLM1 and the G-box was considerably fainter (Figure 7B). No shifted band was observed when incubation was carried out with the mutated probes (glm1, glm2, and G-box). DNA-binding affinity of the recombinant protein seems to be greater with the GLM2 probe than the other probes tested.


Conserved cis-regulatory modules in promoters of genes encoding wheat high-molecular-weight glutenin subunits.

Ravel C, Fiquet S, Boudet J, Dardevet M, Vincent J, Merlino M, Michard R, Martre P - Front Plant Sci (2014)

Binding of recombinant SPA protein with the probes derived from the Glu-B1-1 gene promoter. (A) Expression and purification of recombinant His-SPA protein. Crude extracts from uninduced and induced bacteria harboring the pHis-SPA expression vector and the eluted protein were resolved on an SDS-polyacrylamide gel. The molecular mass markers are indicated at left in kilodaltons. (B) EMSA of the recombinant SPA protein with the 25-bp biotin-labeled, GLM1 (−647), GLM2 (−626), and G-box (−227) probes derived from the Glu-B1-1 gene promoter and their mutated versions glm1, glm2, and G-box. The sequences of the oligonucleotides used as probes are shown with the GLM1, GLM2, and G-box in bold; identical residues are represented by dots, and mutated bases are shown in lowercase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4228979&req=5

Figure 7: Binding of recombinant SPA protein with the probes derived from the Glu-B1-1 gene promoter. (A) Expression and purification of recombinant His-SPA protein. Crude extracts from uninduced and induced bacteria harboring the pHis-SPA expression vector and the eluted protein were resolved on an SDS-polyacrylamide gel. The molecular mass markers are indicated at left in kilodaltons. (B) EMSA of the recombinant SPA protein with the 25-bp biotin-labeled, GLM1 (−647), GLM2 (−626), and G-box (−227) probes derived from the Glu-B1-1 gene promoter and their mutated versions glm1, glm2, and G-box. The sequences of the oligonucleotides used as probes are shown with the GLM1, GLM2, and G-box in bold; identical residues are represented by dots, and mutated bases are shown in lowercase.
Mentions: To verify the potential binding activity of the two GLMs (GLM1 and GLM2 at positions −647 and −626, respectively) present in the GLM-GATA box of the Glu-B1-1 gene promoter, we performed EMSAs with synthetic oligonucleotides and a recombinant SPA protein expressed as a His fusion in E. coli (Figure 7). We also determined the in vitro binding of SPA to the G-box motif, which was previously shown to bind bZIP proteins (Norre et al., 2002). As shown in Figure 7A, arabinose treatment induced expression of a protein of 50–75 kDa that was not present in uninduced cell extracts. The apparent size of the recombinant protein determined by SDS-PAGE was larger than the expected 48 kDa molecular mass of the His-tagged SPA fusion protein. A similar apparent increase in size on SDS gels was already reported by Albani et al. (1997) in their study of SPA. The recombinant His-SPA protein was purified to near homogeneity and used for binding assays. A DNA-protein complex was clearly observed with the GLM2 motif, while the shifted band detected for the GLM1 and the G-box was considerably fainter (Figure 7B). No shifted band was observed when incubation was carried out with the mutated probes (glm1, glm2, and G-box). DNA-binding affinity of the recombinant protein seems to be greater with the GLM2 probe than the other probes tested.

Bottom Line: The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors.However, the differences in annotation between promoter alleles could not be related to their level of expression.In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Recherche Agronomique, UMR1095, Genetics, Diversity and Ecophysiology of Cereals Clermont-Ferrand, France ; UMR1095, Genetics, Diversity and Ecophysiology of Cereals, Department of Biology, Blaise Pascal University Aubière, France.

ABSTRACT
The concentration and composition of the gliadin and glutenin seed storage proteins (SSPs) in wheat flour are the most important determinants of its end-use value. In cereals, the synthesis of SSPs is predominantly regulated at the transcriptional level by a complex network involving at least five cis-elements in gene promoters. The high-molecular-weight glutenin subunits (HMW-GS) are encoded by two tightly linked genes located on the long arms of group 1 chromosomes. Here, we sequenced and annotated the HMW-GS gene promoters of 22 electrophoretic wheat alleles to identify putative cis-regulatory motifs. We focused on 24 motifs known to be involved in SSP gene regulation. Most of them were identified in at least one HMW-GS gene promoter sequence. A common regulatory framework was observed in all the HMW-GS gene promoters, as they shared conserved cis-regulatory modules (CCRMs) including all the five motifs known to regulate the transcription of SSP genes. This common regulatory framework comprises a composite box made of the GATA motifs and GCN4-like Motifs (GLMs) and was shown to be functional as the GLMs are able to bind a bZIP transcriptional factor SPA (Storage Protein Activator). In addition to this regulatory framework, each HMW-GS gene promoter had additional motifs organized differently. The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors. However, the differences in annotation between promoter alleles could not be related to their level of expression. In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression.

No MeSH data available.


Related in: MedlinePlus