Abstract
The end-use quality of wheat, including its unique rheology and viscoelastic properties, is predominantly determined by the composition and concentration of gluten proteins. While, the mechanism regulating expression of the seed storage protein (SSP) genes and other related genes in wheat remains unclear. In this study, we report on the cloning and functional identification of TaFUSCA3, a B3-superfamily transcription factor (TF) gene in wheat. Sequence alignment indicated that wheat and barley FUSCA3 genes are highly conserved. Quantitative reverse-transcription (qRT)-PCR analysis showed that the transcript of TaFUSCA3 was accumulated mostly in the stamens and the endosperms of immature wheat seeds. Yeast-one-hybrid results proved that the full-length TaFUSCA3 and its C-terminal region had transcriptional activities. Yeast-two-hybrid and bimolecular fluorescence complementation assays indicated that TaFUSCA3 could activate the expression of the high molecular weight glutenin subunit gene Glu-1Bx7 and interact with the seed-specific bZIP protein TaSPA. DNA-protein-interaction enzyme-linked immunosorbent assay demonstrated that TaFUSCA3 specifically recognizes the RY-box of the Glu-1Bx7 promoter region. Transient expression results showed that TaFUSCA3 could trans-activate the Glu-1Bx7 promoter, which contains eight RY-box sequences. TaFUSCA3 was unable to activate the downstream transcription when the RY-box was fully mutated. TaFUSCA3 could activate the transcription of the At2S3 gene promoter in a complementation of loss-of-function experiment using the Arabidopsis thaliana line fus3-3, which is a FUSCA3 mutant, demonstrating the evolutionary conservation of the TaFUSCA3 gene. In conclusion, the wheat B3-type TF, TaFUSCA3, is functional conserved between monocot and dicot, and could regulate SSP gene expression by interacting specifically with TaSPA.
Highlights
Wheat (Triticum aestivum L.) is a very important cereal crop throughout the world, which can be made into diverse edible products for human beings because of the distinct viscoelastic characteristics of its cohesive dough conferred by the seed storage proteins (SSPs) in the endosperm (Shewry, 2009)
According to the Multiple EM for Motif Elicitation tool5, TaFUSCA3 had seven motifs, including a typical conserved B3 domain, which could recognize and bind to the specific sequence (RY-boxes), and a transcription activating domain (TAD) containing 115 amino acid resides in the C-terminal region
When the open reading frame (ORF) of TaFUSCA3 was transformed into A. thaliana line fus3-3 with AtFUSCSA3 mutated, we found that characteristics were recovered in transgenic plants, such as activated expression of the promoter from the SSP gene At2S3 (Figure 9A)
Summary
Wheat (Triticum aestivum L.) is a very important cereal crop throughout the world, which can be made into diverse edible products for human beings because of the distinct viscoelastic characteristics of its cohesive dough conferred by the SSPs in the endosperm (Shewry, 2009). The HMW-GSs of SSPs play important roles in wheat gluten as the skeletal network that determines its structure and formation (Shewry and Tatham, 1990). A group of important TFs, the AFL subfamily containing a plantspecific B3 DNA binding domain that takes part in this process, has been identified. Members of this family include ABSCISIC ACID-INSENSITIVE3 (ABI3), FUSCA3 (FUS3), LEAFY COTYLEDON1 (LEC1), and LEAFY COTYLEDON1 (LEC2), which regulate the desiccation tolerance and dormancy of the maturation stage of Arabidopsis thaliana (Stone et al, 2001; Swaminathan et al, 2008; Roscoe et al, 2015). The TFs are interlocked in an intricate stratified reticular regulatory network by virtue of mutual interconnections (To et al, 2006)
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