Expression pattern and subcellular localization of the ovate protein family in rice

Abstract

The Arabidopsis ovate family proteins (AtOFPs) have been shown to function as transcriptional repressors and regulate multiple aspects of plant growth and development. There are 31 genes that encode the full-length OVATE-domain containing proteins in the rice genome. In this study, the gene structure analysis revealed that OsOFPs are intron poor. Phylogenetic analysis suggested that OVATE proteins from rice, Arabidopsis and tomato can be divided into 4 groups (I-IV). Real-time quantitative polymerase chain reaction (RT-qPCR) analysis identified OsOFPs with different tissue-specific expression patterns at all stages of development in the rice plant. Interestingly, nearly half of the total number of OsOFP family was more highly expressed during the seed developmental stage. In addition, seed developmental cis-elements were found in the promoter region of the OsOFPs. Subcellular localization analysis revealed that YFP-OsOFP fusion proteins predominantly localized in the nucleus. Our results suggest that OsOFPs may act as regulatory proteins and play pivotal roles in the growth and development of rice.

Fig 1. Genome distribution of the OsOFPs.

Fig 2. Relative proportions of intron-containing OFPs in Physcomitrella patens, Selaginella moellendorffii, Populus trichocarpa, Prunus persica, Sorghum bicolor, Zea mays, Oryza sativa, Solanum lycopersicum, Arabidopsis thaliana and Cucumis melo.

Proportions are colored as: no introns (white), 1 intron (light gray), 2–5 introns (dark gray), and ≥6 (black).

Fig 3. Multiple sequence alignment and phylogenetic tree of the OVATE domains of OFP proteins from rice, Arabidopsis and tomato.

(A) The phylogenetic trees based on the multiple sequence alignments of the OVATE domains of OFP proteins. Bootstrap values from 1000 replicates are indicated at each node. The proteins on the tree can be divided into four distinct subfamilies (I–IV). Bootstrap values under 50% was not reported. (B) Multiple sequence alignments of the OVATE domains of rice, Arabidopsis and tomato are shown. The black region represents identical amino acids, and the grey region represents similar amino acids.

Fig 4. Expression profiles of the OsOFPs in various rice tissues at different developmental stages.

The tissue-specific expression profiles were performed by qRT-PCR. The expression profiles of genes from OsOFP01 to OsOFP08 (A), The expression profiles of genes from OsOFP09 to OsOFP16 (B), The expression profiles of genes from OsOFP17 to OsOFP24 (C), The expression profiles of genes from OsOFP25 to OsOFP31 (D).

Fig 5. Expression profiles of OsOFPs under phytohormone treatments.

(A) Expression profile of OsOFP29 responding to ABA (50 μM; 0, 2, 4, 8, 16, 24, 36, and 48 h). (B, C) Expression profiles of OsOFP03 and OsOFP15 responding to BR, respectively (1 μM; 0, 2, 4, 8, 16, 24, 36, and 48 h). The expression profiles of OsOFPs responding to ABA and BL were determined by qRT-PCR. Actin was used as an internal control. Error bars indicate the SD based on 3 biological replicates.

Fig 6. Subcellular localization of the OsOFPs.

Protoplast transient expression using YFP-OsOFP fusion constructs were used to determine the subcellular localization. AHL22 fused with CFP was used as a nuclear marker. The 35S: YFP-OsOFP and 35S: CFP-AHL22 constructs were transformed into an Arabidopsis protoplast cell. Fluorescence images of CFP, YFP, and chlorophyll autofluorescence (Chl) were captured with confocal laser scanning microscopy and are shown in cyan, yellow, and red, respectively (scale bars, 10 μm).