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

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.

Three Rice NAC Transcription Factors Heteromerize and Are Associated with Seed Size

“…For the promoter of ONAC026 , SN and NB have an additional stretch of 677 bp, holding multiple copies of DOFCOREZM (AAAG) and EBOXBNNAPA (CANNTG, Figure 3B ), a probable explanation for the higher expression levels in these two accessions ( Figures 2A,D ). EBOXBNNAPA, conserved in many seed storage protein promoters is critical for directing seed-specific expression (Ellerstrom et al, 1996; Ravel et al, 2014), while DOFCOREZM is the recognition core of DOF proteins, reported to activate several storage protein genes (Yamamoto et al, 2006; Abraham et al, 2016). For ONAC023 , the promoter sequences for SN and NB are similar, though there are SNPs amongst the two as well, and hence a similar expression pattern though levels are different.…”

Chromosome variation and HMW glutenins in synthetic hexaploid wheats (Triticum turgidumssp.dicoccum/Aegilops tauschii)

“…Five subunits, reported in this study, including the three pairs in the Glu-D t 1, were not detected by the authors. Ribeiro et al (2013) studied the Portuguese wheat Barbela and reported a new x-type HMW-GS encoded at the Glu-A1 locus that was named 1Ax1.1., while Ravel et al (2014) discovered Glu-B1-1 protein 22 in Fruh-weizen (13310) wheat accession. We investigated nine SHW and reported 11 subunits (3 at the Glu-D t 1 locus) as compared to Rasheed et al (2012) analyzing ninety-five Elite-1 synthetic hexaploid wheats (Triticum turgidum/Aegilops tauschii) with 22 glutenins (13 at the Glu-D t 1 locus).…”

Multiple elements controlling the expression of wheat high molecular weight glutenin paralogs

“…Transformation of endosperm was based on a method described by Ravel and co-workers (Ravel et al 2014) with the modification that 1 g l -1 casein hydrolysate was included in the medium after the transformation, instead of separately adding all the 20 proteinogenic amino acids. Biolistic bombardment of endosperms was carried out identically to the leaves.…”

“…Since then the first draft genome of Triticim aestivum has been published (Mayer et al 2014a) and it gave us a new opportunity to improve the experiment by calculating the global overrepresentations of binding sites and binding site clusters. In a parallel study, Ravel et coworkers reported conserved cis-regulatory modules in the promoters of HMW-GS promoters that they identified by a simple and non-statistical method (Ravel et al 2014). …”

“…In this study, we aimed to decipher the regulatory logic of Glu-1 genes by a co-expressional network analysis and a high-throughput cis-regulatory module detection study as well as experimental studies. The function and strength of both wild-type and mutant Glu-1x type promoters were studied in reporter gene assays using particle bombardment similarly to earlier studies (Basu et al 2003;Ravel et al 2014). In conclusion, a possible regulatory logic of combinatorial cis-regulation of Glu-1 genes is detailed, which was formulated by a reverse approach described in Werner and co-workers (Werner et al 2003).…”