Conserved cis-acting motifs and localization of transcripts and proteins of MFT2 in barley and rice

MFT is an important regulator of seed dormancy in flowering plants. A natural mutation in the A-box motif in the promoter of wheat (Triticum aestivum) MFT2 on chromosome 3A (TaMFT2-3A) has been used to prevent pre-harvest sprouting (PHS) in wheat cultivars in East Asia. Previous research using in situ hybridization showed that TaMFT2-3A is primarily expressed in the seed scutellum. In this study, we analyzed the localization of transcripts and encoded proteins for the TaMFT2 homologs from barley (Hordeum vulgare) and rice (Oryza sativa), HvMFT2 and OsMFT2, respectively. RNA in situ hybridization showed that, like the wheat genes, the rice and barley homologs are primarily expressed in the scutellum, indicating that these three MFT2 genes have a common expression pattern during seed development. Analysis of the cis-acting regulatory elements of their promoter sequences showed that the three MFT2 promoters share eight seed-specific cis-acting RY motifs, which are binding sites for B3-domain transcription factors of the AFL-B3 and VIVIPAROUS1/ABI3-LIKE (VAL) families. In addition, we detected tandemly repeated and partially overlapping A-box motifs in the promoters of HvMFT2, TaMFT2-3B, and TaMFT2-3D, possibly explaining why the natural allele of TaMFT2-3A has been employed in breeding. We generated transgenic rice plants expressing nuclear-localized green fluorescent protein (NLS-2xGFP) and OsMFT2-GFP under the control of a 3-kb fragment of the OsMFT2 promoter. Immunohistochemical staining using anti-GFP antibodies mainly detected GFP in the scutellum and scutellar epithelium, which is an important tissue for initiating germination upon seed hydration. We confirmed these results by confocal microscopy of GFP fluorescence in seeds. Our results suggest that MFT2 expression in rice, barley, and wheat might be regulated by a similar network of transcription factors through multi-RY motifs in the MFT2 promoters, with possible roles in scutellar epithelium development.