Conserved cis-acting motifs and localization of transcripts and proteins ofMFT2in barley and rice

Abstract

AbstractMFTis an important regulator of seed dormancy in flowering plants. A natural mutation in the A-box motif in the promoter of wheat (Triticum aestivum) MFT2on chromosome 3A (TaMFT2-3A) has been used to prevent pre-harvest sprouting (PHS) in wheat cultivars in East Asia. Previous research usingin situhybridization showed thatTaMFT2-3Ais primarily expressed in the seed scutellum. In this study, we analyzed the localization of transcripts and encoded proteins for theTaMFT2homologs from barley (Hordeum vulgare) and rice (Oryza sativa), HvMFT2andOsMFT2, respectively. RNAin situhybridization showed that, like the wheat genes, the rice and barley homologs are primarily expressed in the scutellum, indicating that these threeMFT2genes have a common expression pattern during seed development. Analysis of thecis-acting regulatory elements of their promoter sequences showed that the threeMFT2promoters share eight seed-specificcis-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 ofHvMFT2, TaMFT2-3B, andTaMFT2-3D, possibly explaining why the natural allele ofTaMFT2-3Ahas 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 theOsMFT2promoter. 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 thatMFT2expression in rice, barley, and wheat might be regulated by a similar network of transcription factors through multi-RY motifs in theMFT2promoters, with possible roles in scutellar epithelium development.