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We recently identified a novel R1 MYB transcription factor, GmMYB176, which regulates the CHS8 gene expression and influences isoflavonoid biosynthesis in soybeans. GmMYB176 recognizes a unique sequence motif [TAGT(T/A)(A/T)] in CHS8 promoter and binds with it. The in planta role of GmMYB176 was established by RNAi silencing of GmMYB176 in soybean hairy roots. Silencing of GmMYB176 reduced the expression of CHS8 gene expression and isoflavonoid accumulation in hairy roots. However, the overexpression of GmMYB176 did not lead to increase in both CHS8 expression and isoflavonoid level in hairy roots suggesting that GmMYB176 is essential but not sufficient for CHS8 gene activation.1
As anticipated for its function as a transcription factor GmMYB176 was localized in nucleus. The nuclear localization of GmMYB176 was confirmed by producing a translational fusion of GmMYB176 with smGFP, followed by its transient expression in tobacco epidermal cells and visualization by confocal laser scanning microscopy. The in silico analysis of GmMYB176 predicted no potential nuclear localization signal within the sequence. Instead, the analysis revealed the presence of a potential pST binding site in GmMYB176 at amino acid residues between positions 132 to 146, consisting of the sequence NRRRRRSSLFDITTD where phosphoserine motif at Ser139 may act as a binding site for 14-3-3 family proteins. 14-3-3s are highly conserved eukaryotic proteins that interact with other phosphorylated proteins. Two consensus 14-3-3 binding phosphopeptide motifs, RSXpSXP (mode I) and RXY/FXpSXP (mode II), where X is any amino acid and pS is the phosphoserine, have been reported.2 However, many 14-3-3-binding sites do not conform to these consensus motifs.3 The predicted 14-3-3 binding site in GmMYB176 is similar to mode I; however, the match is not exact. In GmMYB176, the amino acid residue between the non-phosphorylated and phosphorylated serine is missing and proline at position P + 2 is substituted by phenylalanine.1 It is possible that the GmMYB176 and 14-3-3 protein interaction may have evolved in a different way, or other structural features may contribute to this interaction. Plant 14-3-3 proteins have been shown to play important roles in several regulatory processes, such as metabolic pathways, gene expression and signal transduction.4 Two extensively studied examples of proteins that are regulated by 14-3-3s and involved in plant primary metabolic activity are nitrate reductase5,6 and ATPases.7 However, the involvement of 14-3-3s in the regulation of plant secondary metabolism has not been reported yet. Binding of a 14-3-3 protein to its target can affect localization of the target protein within the cell.8,9 A telomerase protein, TERT, is transported to the nucleus upon 14-3-3 interaction,10 while histone deacetylases, HDAC4 and HDAC5, are sequestered in the cytoplasm upon binding to a 14-3-3 protein.11 Binding by a 14-3-3 protein also reduced the nuclear localization of OsBZR1.12 Deletion of a predicted pST binding site from GmMYB176 affected its subcellular localization where GmMYB176 was not restricted to nucleus but also dispersed in the cytoplasm.1 This observation confirmed the role of the pST binding site within GmMYB176 in its subcellular localization. In this report, we demonstrate the interaction between the GmMYB176, a SHAQKYF motif containing R1 MYB transcription factor and a soybean 14-3-3 protein in vivo. Furthermore, we show that GmMYB176 monomers can interact with each other and form a homodimer.
To demonstrate if GmMYB176 monomers can interact with each other, we performed Bimolecular fluorescence complementation (BiFC) assay where translational fusion of GmMYB176 was created with N-terminal (YN) and C-terminal (YC) half of YFP under the control of CaMV35S promoter. The GmMYB176-YN and GmMYB176-YC constructs were coexpressed transiently in tobacco epidermal cells and YFP expression was monitored by confocal laser scanning microscopy. Control experiments were performed by expressing YN and YC fusion alone for GmMYB176 or coexpressing vector only YN and YC. As indicated by the YFP signal, the results confirmed its nuclear localization and demonstrated the ability of GmMYB176 monomers to interact with each other and form a dimer (Fig. 1A).
A search for genes encoding soybean 14-3-3 proteins in the National Center for Biotechnology Information database identified SGF14d (accession no. U70536). A conserved domain for 14-3-3 superfamily was detected in SGF14d by in silico analysis.13 The sequence information for SGF14d was used to design primers to isolate cDNA encoding SGF14d. To establish whether SGF14d protein interacts with GmMYB176, we performed targeted yeast two hybrid (Y2H) assay using Gateway-compatible vectors14 where GmMYB176 and SGF14d full length cDNAs were introduced into the vectors pGBKT7-DEST (bait) and pGADT7-DEST (prey), respectively. The interaction between GmMYB176 and 14-3-3 SGF14d was determined by colony growth on selective media (Fig. 2).
The interaction between SGF14d and GmMYB176 was further validated by using BiFC assay where translational fusion of SGF14d and GmMYB176 were created with YN and YC half of YFP separately under the control of CaMV35S promoter. Both SGF14d-YN and GmMYB176-YC constructs were coexpressed transiently in tobacco epidermal cells and protein expression was monitored by confocal laser scanning microscopy. 14-3-3s are phosphoprotein binding proteins which in its homo- or heterodimeric form interact with phosphorylated client proteins. Dimerization is essential for the stability and function of 14-3-3 in vivo.15 The dimeric structure allows 14-3-3s to dock on to phosphorylated serine or threonine residues present in their client proteins.16 As shown in Figure 1B, SGF14d forms a homodimer and is localized in both nucleus and cytoplasm. In addition, the results validated the Y2H observation that SGF14d interacts with GmMYB176 and produces YFP signal (Fig. 1C). Similar results were obtained in a reciprocal interaction experiment coexpressing SGF14d-YC and GmMYB176-YN followed by YFP visualization. The YFP signal in SGF14d-YC and GmMYB176-YN coexpression is stronger in nucleus compared to expression of SGF14d-YC and SGF14d-YN. In conclusion, our findings confirmed the interaction between GmMYB176 and SGF14d and established GmMYB176 as the first R1 MYB client protein of 14-3-3 protein subfamily.
Finally, we have demonstrated the unique interaction between SGF14d and GmMYB176 from soybean and that this interaction affects subcellular location of GmMYB176. Since GmMYB176 has been implicated to play a role in isoflavonoid biosynthesis,1 the interaction between SGF14d and GmMYB176 and its impact on GmMYB176 localization suggests the possibility that soybean 14-3-3s may directly or indirectly play a role in isoflavonoid biosynthesis in soybean. However, the molecular mechanism of 14-3-3 function in isoflavonoid biosynthesis remains to be studied.
Addendum to: Yi J, Derynck M, Li X, Telmer P, Marsolais F, Dhaubhadel S. A single repeat MYB transcription factor, GmMYB176, regulates CHS8 gene expression and affects isoflavonoid biosynthesis in soybeanPlant J20106210191034 doi: 10.1111/j.1365-313X.2010.04214.x.
Previously published online: www.landesbioscience.com/journals/psb/article/12133