Os05g0445900

Please input one-sentence summary here.

Annotated Information

Function

Cytosine DNA methylation, which occurs in the CG, CHG(H = A, C or T) and CHH sequence contexts, is an epigenetic modification in plants. As well, there are some proteins coded for DNA demethylation. The genes and the corresponding encoded enzymes that mediate DNA methylation and demethylation have been characterized mainly in Arabidopsis. The Arabidopsis enzymes that mediate 5-methylcytosine (5-meC) DNA demethylation are DEMETER (DME), REPRESSOR OFSILENCING 1 (ROS1),DEMETER-LIKE 2 (DML2) and DEMETER-LIKE 3 (DML3). Phylogenetic analysis revealed that the rice (Oryza sativa)genome encodes six putative bi-functional DNA glycosylases that mediate cytosine DNA demethylation: four ROS1orthologs and twoDML3orthologs, but no DME orthologs.

1.It has been demonstrated that rice ROS1a protein is a bi-functional DNA glycosylase/lyase for 5-meC DNA demethylation, although biochemical characterization of the ROS1a enzyme remains to be performed to confirm this.Rice ROS1a is toxic to E. coli containing 5-meC in its genome. When ROS1a cDNA was expressed under the control of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter, ROS1a was toxic to an E. coli dcm+ strain containing 5-meC in an IPTG-dependent manner, and was less toxic to a dcm− mutant without 5-meC.

2. ROS1a is the most abundantly expressed gene in tissues. It was recently reported that null mutants of ROS1c, which encodes a 5-meC DNA glycosylase/lyase, show no effects on transmission of the null alleles and produce a small portion of wrinkled seeds.And the null mutation, ros1a-GUS1, was hardly ever transmitted to progeny.Even in the presence of the wild-type paternal ROS1a allele, the maternal nullros1a-GUS1allele caused failure of early stage endosperm development, indicating non-equivalent contribution of maternal and paternal ROS1a to endosperm development.

Figure.  Histochemical GUS staining patterns in ROS1a/ros1a-GUS1 plants. (a) Longitudinal section through basal shoot meristem region of mature plants. Insets are magnified views of shoot apical and lateral (upper) and inflorescence (lower) meristems. (b) Flowers. (c) Anthers. Approximately half of the pollen present showed GUS staining (see also Figure S7b). The inset is a magnified view. (d) Pistils. Insets are ovules before pollination exposed by removing the carpel. The upper and lower insets show a GUS-stained ovule and its sibling, which displayed no GUS staining, respectively. Before pollination, ovules containing gametes with the wild-type ROS1a allele were morphologically indistinguishable from those with ros1a-GUS1 allele. (e) Schematic representation of a rice female gametophyte enclosed by the maternal tissues of the ovule: ovary tissue (light brown), integument (dark brown), and nucellus (gray). The haploid female gametophyte consists of the egg apparatus (green), including the egg cell and two synergids, the central cell (white), which contains large vacuoles and thin lines of cytoplasm (pink), and antipodals (blue). (f,g) Differential interference contrast micrographs of GUS-stained female gametophytes of ROS1a/ros1a-GUS1 plants. To make the borders of different tissues clear, white lines are drawn on the image in (f), which corresponds to the area enclosed by the red line in (e). (h) Differential interference contrast micrograph of a sibling female gametophyte displaying no GUS staining. Arrows indicate GUS-stained meristems (a), pollen (b) and the probable egg apparatus (d). In (b), the arrowhead indicates the GUS-stained lodicule. AP, antipodals; PN, polar nuclei. The images in (g,h) correspond to the area enclosed by the blue line in (e). Scale bars = 1 mm [a,b, inset in (a)], 500 μm [c,d], 100 μm [inset in (d)] and 50 μm [inset in (c) and f-h].

3.Rare transmission of the ros1a-GUS1 allele to progeny.To obtain homozygous knock-in plants, we self-pollinated the isolated T0 plants. Of the 250 fully grown seeds selected, 232 germinated, and the resulting T1 seedlings were genotyped by PCR analysis. Surprisingly, only two were ROS1a/ros1a-GUS1 plants, and the remaining 230 seedlings were homozygous for the wild-type allele (ROS1a/ROS1a) (Table 1). Moreover, the ros1a-GUS1 allele in the two heterozygous T1 plants was not transmitted to progeny, confirming that the ros1a-GUS1 allele is rarely transmittable to progeny. Close inspection revealed that the T0 panicles comprised three types of grain: empty grains with only infertile flower remnants , grains with normal-shaped seeds , and grains with deformed seeds containing severely under-developed and non-starch-producing endosperm . Approximately equal numbers of normal-shaped and deformed seeds were observed (54:56, 1:1, χ2 = 0.036, P > 0.8; Table 2). Embryos in the deformed seeds always displayed GUS-positive staining, but none of the normal-shaped seeds showed GUS-positive patterns, indicating that the ros1a-GUS1 allele co-segregated with the deformed seed phenotype and that the normal-shaped seeds were ROS1a/ROS1a (Table 2 and Figure 5b,d). In the analysis of the segregation ratio of ros1a-GUS1 in seedlings (Table 1), deformed seeds bearing the ros1a-GUS1 allele were probably excluded from the analysis because only fully grown seeds were used.

Expression

Rice contains four ROS1 orthologs and two DML3 orthologs tentatively named ROS1a–d and DML3a and DML3b, that contain characteristic DNA glycosylase domains flanked by conserved domains of unknown functions. Of these, ROS1ais the longest gene, comprising 17 exons that encode a protein with 1952 amino acids, and 5¢ and 3¢ UTRs of 73 and 607 bp, respectively.RT-PCR analysis revealed thatROS1awas expressed in all vegetative and reproductive tissues tested . Quantitative RT-PCR analysis revealed that ROS1ais the most extensively expressed gene among the four genes(ROS1a, ROS1c, ROS1d and DML3a) expressed in five selected tissues examined, including anthers and pistils,whereas ROS1b and DML3bare scarcely expressed in these tissues. Interestingly, moderate levels of transcripts for ROS1c, ROS1dandDML3awere detected in pistils and immature seeds 2 days after pollination.

Evolution

Phylogenetic analysis revealed that the rice (Oryza sativa) genome encodes six putative bi-functional DNA glycosylases that mediate cytosine DNA demethylation: four ROS1 orthologs and two DML3 orthologs, but no DME orthologs (Zemach et al., 2010). Rice endosperm DNA is hypomethylated in all sequence contexts, implying that hypomethylation in rice endosperm relies on some of these DNA glycosylases or alternative biochemical mechanisms (Zemach et al., 2010). In this study, to characterize the function of one of the four rice ROS1 orthologs, tentatively named ROS1a (LOC_Os01g11900.1), that resides on chromosome 1, we used homologous recombination-promoted knock-in targeting with positive/negative selection (Yamauchi et al., 2009) to obtain a mutant that disrupts ROS1a by fusion of its endogenous promoter with the GUS reporter gene encoding β-glucuronidase. We reproducibly obtained T0 plants with the null knock-in allele, ros1a-GUS1, in the heterozygous condition, and detected GUS expression in the T0 plants in the shoot apical, lateral and inflorescence meristems, as well as in both female and male gametophytes before fertilization. The ros1a-GUS1 allele was hardly transmitted to the next generation; neither the maternal nor the paternal ros1a-GUS1 allele was virtually found in the progeny. The results indicate that ROS1a, presumably through DNA demethylation, is indispensable in both gametophytes, and that the null allele of ROS1a is difficult to isolate by conventional mutagenesis techniques, in which mutants are usually obtained as segregants in the progeny population.

Labs working on this gene

1.National Institute for Basic Biology, Okazaki 444-8585, Japan 2.Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan 3.Graduate School of Nutritional and Environmental Sciences, Graduate School of Pharmaceutical Sciences, and Global Center of Excellence Program, University of Shizuoka, Shizuoka 422-8526, Japan

References

1.A. Zemach et al., Local DNA hypomethylation activates genes in rice endosperm. Proc Natl Acad Sci U S A 107, 18729 (Oct 26, 2010).

2.A. Ono et al., A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny. Plant J 71, 564 (Aug, 2012).

Structured Information

Gene Name	Os05g0445900
Description	Similar to ROS1 (Fragment)
Version	NM_001062220.1 GI:115464170 GeneID:4338940
Length	5779 bp
Definition	Oryza sativa Japonica Group Os05g0445900, complete gene.
Source	Oryza sativa Japonica Group ORGANISM Oryza sativa Japonica Group Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP clade; Ehrhartoideae; Oryzeae; Oryza.
Chromosome	Chromosome 5
Location	Chromosome 5:21889157..21894935
Sequence Coding Region	21889581..21889708,21890545..21890583,21890664..21890778,21890883..21890948,21892020..21892159 ,21892239..21892302,21892380..21892416,21892564..21893001,21893713..21893795 ,21893897..21893933,21894150..21894218,21894322..21894413,21894487..21894571 ,21894906..21894934
Expression	GEO Profiles:Os05g0445900
Genome Context	<gbrowseImage1> name=NC_008398:21889157..21894935 source=RiceChromosome05 preset=GeneLocation </gbrowseImage1>
Gene Structure	<gbrowseImage2> name=NC_008398:21889157..21894935 source=RiceChromosome05 preset=GeneLocation </gbrowseImage2>
Coding Sequence	<cdnaseq>ttaagagatgttcctccagactcagcaaaggactatctgcttagtatacgtggattggggctcaaaagtgttgagtgtgtccgccttttgacattacatcatcttgcattcccagttgatactaatgttggtcgtatatgtgtacgattgggatgggtgccaattcaacccctccctgaatctcttcagttacaccttctggagctataccctgtcttggagactatacaaaagtacctctggcctcgtctgtgtaaacttgatcaacaaacactgtatgagttacattatcagatgattacttttggaaaggtgttctgtaccaaaagcaagccgaattgcaatgcatgtccaatgaggagtgaatgcaggcattttgcaagtgcctttgcaagtgcaagacttgcacttccttctcctcaggacaaaaggttggtgaatctgagcaatcaatttgctttccataatggcacaatgcccacaccaaattcaactcctctgcctcagctcgaggggagtatccatgcaagggatgttcatgctaacaacacaaatccaataattgaggagccagcaagtccaagagaggaagaatgccgagaacttttagagaatgatattgaagattttgatgaagatactgatgaaatcccaataataaaacttaacatggaagctttttctcaaaacttggaaaattgcataaaagaaagcaataaggatttccaatctgatgatattacaaaagcattggttgctatcagcaatgaagcagcttcaattcctgtacctaaactaaagaatgtgcatagacttcggacagaacactatgtttacgaacttccagattcacatcccctcatgcaacagctagcactcgaccaacgggagcctgatgatccaagtccttacctgttggccatatggacaccagatgaactaaaggacacaagggaggcaccaaaaccgtgctgcaatcctcaaactgaaggtggcttatgcagcaatgagatgtgccacaactgtgtatctgaacgagaaaaccaatatagatacgtcagaggcacggttctggttccttgccgaacagccatgagaggtagttttccacttaatggcacttactttcaagttaatgaggtttttgctgatcacagttctagccacaatcccataaatatcccaagggagcagttatggaacttgcataggcgtatggtttactttgggacttcagtgccaaccatattcaaaggtctaacaactgaagaaatacagcactgcttctggagaggatttgtctgtgtgagaggattcaacatggaaactagggcaccaaggcctctatgcccccatttccaccttgcagcaagcaaactgcgaagatcctctaaaaaagcagcaactgagcaaacacactga</cdnaseq>
Protein Sequence	<aaseq>LRDVPPDSAKDYLLSIRGLGLKSVECVRLLTLHHLAFPVDTNVG RICVRLGWVPIQPLPESLQLHLLELYPVLETIQKYLWPRLCKLDQQTLYELHYQMITF GKVFCTKSKPNCNACPMRSECRHFASAFASARLALPSPQDKRLVNLSNQFAFHNGTMP TPNSTPLPQLEGSIHARDVHANNTNPIIEEPASPREEECRELLENDIEDFDEDTDEIP IIKLNMEAFSQNLENCIKESNKDFQSDDITKALVAISNEAASIPVPKLKNVHRLRTEH YVYELPDSHPLMQQLALDQREPDDPSPYLLAIWTPDELKDTREAPKPCCNPQTEGGLC SNEMCHNCVSERENQYRYVRGTVLVPCRTAMRGSFPLNGTYFQVNEVFADHSSSHNPI NIPREQLWNLHRRMVYFGTSVPTIFKGLTTEEIQHCFWRGFVCVRGFNMETRAPRPLC PHFHLAASKLRRSSKKAATEQTH</aaseq>
Gene Sequence	<dnaseqindica>5228..5355#4353..4391#4158..4272#3988..4053#2777..2916#2634..2697#2520..2556#1935..2372#1141..1223#1003..1039#718..786#523..614#365..449#2..30#gttaagagatgttcctccagactcagcaaagtaagatactacacctgtattcaatatttacaaatcccattgccctgctgatgagatttttctagttctggaaatgataaatcaagtatcaaagaagtttgtgaaccaattaaatggagcaagctgatccaagaacttgccagttaatatctagaattaaatactatgagcaaagttctatgaaatgggcacgcagaacagaaaaggctgtataatgtatgacacatgatcgaagcagtgaaggaatttttttttaggaaagaaaattccatgaaccctatttatttcacaggcgagaagtatgtcaattacaatgtgttcgaattattttcagggactatctgcttagtatacgtggattggggctcaaaagtgttgagtgtgtccgccttttgacattacatcatcttgcattcccagtaagtttctgttggattaattctccttcacctgcagccaatttgaaatttacactacattgttgcaactcaggttgatactaatgttggtcgtatatgtgtacgattgggatgggtgccaattcaacccctccctgaatctcttcagttacaccttctggagctgtaagaatgttgcattgtctactacagttgtataaatactctctgcaattttatacgatgatgtttcaatttgaatatattgattttgtttgtgccttcaaagataccctgtcttggagactatacaaaagtacctctggcctcgtctgtgtaaacttgatcaacaaacactgtgagttataaccaattaaccatacccactcatagttgcccttcttattagtttttttttacgcaagcccttcttattagtttgtgtaacttctaggttatttctttttcatgcaaattacagtatttggaatcatgaataattaaggcaataaggagtaaataacataatttataactataaggaattattaacagagtttcatggttttcacaggtatgagttacattatcagatgattacttttggaaaggtacttcattagcattaaggagtttggtctcttcattagcatctaccgacgcatttgttctcttttgtttgagtaaagataaattatcataacaaatgcaggtgttctgtaccaaaagcaagccgaattgcaatgcatgtccaatgaggagtgaatgcaggcattttgcaagtgcctttgcaaggtattatggcaagcagaaaatgggagccttctggctgcaaggagacttacaaaataaacactatttgcttcagtagtcacttttttccgataaaggaagctttattaaaactcagtcaaatacaccaagatgatacattctaactgagccactcccggcctctgcatgaaatgcacaccgccacaaaacaggatctaactagaccctcaacacaaaacaaataaattagtgactatcaagccgtagactatatcgccacccatgctccagggtaaaaaactcctgtgccacctgatagtgcacagcaaaaaccacgagaaggatgtgctttcgttggtggagagaccgaattagctccattaacataatatgaccactaagagcgcaaaatagtgcttttgaagaataaaagggaaaatatgctaggagaaaaatcaatatatggttctagtacaccgccatggcactttgtgtagtctctgtctgcaacaaaatatatttgccgctttcacttatcttacctgggaattttgttatcacgtatttcctcgttccactatctttcttgttttgtttgtgcgcgcgcgcatgcctgatatatcttgctctttcagttgaccttacctgggagccttcattttgcatatttcttcatttgttattttttccttattaactctgttatattttgtttgacttttttaaatgaagtgcaagacttgcacttccttctcctcaggacaaaaggttggtgaatctgagcaatcaatttgctttccataatggcacaatgcccacaccaaattcaactcctctgcctcagctcgaggggagtatccatgcaagggatgttcatgctaacaacacaaatccaataattgaggagccagcaagtccaagagaggaagaatgccgagaacttttagagaatgatattgaagattttgatgaagatactgatgaaatcccaataataaaacttaacatggaagctttttctcaaaacttggaaaattgcataaaagaaagcaataaggatttccaatctgatgatattacaaaagcattggttgctatcagcaatgaagcagcttcaattcctgtacctaaactaaagaatgtgcatagacttcggacagaacactatgtgtaagtgttggactgatattttatttagcttgaacatgtacctgtttaaacttacatttattatagagcccaccatctaagtaattctatacatcctgaacaaaatattttttaatttctattactgacttaaggttctgctcgcagttacgaacttccagattcacatcccctcatgcaacaggtgagaccatgagaaattgtgccatctttctttaaaacacttcaagatattatttctgattgcaaatgatcttacagctagcactcgaccaacgggagcctgatgatccaagtccttacctgttggccatatggacaccaggcaagtgcatttttctttaaatttataactgtccgtgtgtgtgtgctggttttcgtcgagtcatgtggctactgtgcagatgaactaaaggacacaagggaggcaccaaaaccgtgctgcaatcctcaaactgaaggtggcttatgcagcaatgagatgtgccacaactgtgtatctgaacgagaaaaccaatatagatacgtcagaggcacggttctggtaaatcaaccaacattatgtagcaatcatcaacattgaagagctgaacctctgcattagtcagctagtaaacaaaatattcattataatccaaacattggaatgaattcaggaattcaagaaaatgttgaagataacttttaatgaaaactagtgcttacaaagtgccaagcaaagctattctgttgtctgtgtaatctcttttcttgcatgaaatagcccactgtcttcactttaaacaataagaatagcatagattttgggtttccggctatacaacacatcttaatatgaacttttgtggaagtctaaaagagaacctctctgaacctttaaactgtacactatttaaccaacaatcaaagccgatcacttctcaacgtcattctgaattagctttgggcgatgtttcttaaccaggagatgtggtctgagattttagattttttttttcatctcctttatattagtagacgacttaaaattttctggaataatcttacttcgctgtgaagtatttataacccagataaatcggtaagattgaagtaaagtattattgtcccacgaaatttagtgtgtgaaagtagacaacatttttttctactgtccgattaaactttatttaactctgccccattttatctaggctataaatggtgatttcttctaggctatacggtttttcaatccaattttaccttattttgctttaccaaatttgtcaacattataatactttgtagtgcaagtgagattttttttttcaattttgttatgtcatttactaattttaaaattgaaaacctggttcagccttgggccattttgccacattagggacacatagtccaaagccactttaaatttgagacaaggttgaaaagcgcatggttttgatacttcaggttctgggttgaattgtgtccagttttctgctttggggttgtctctggactaacagaaaagttcagtattgcaaaacagactgtttcctatacaatcatgcattgttggtttgtgaagttgcaagtattgcttgactaatgacggttactggaacatgaacaggttccttgccgaacagccatgagaggtagttttccacttaatggcacttactttcaagttaatgaggtatgctgagttatatctgtcaaagtatgagtaggatagtggagagttgctatccatgggcctttatagagttgactttacgatttctggatttgtttttgcaggtttttgctgatcacagttctagccacaatcccataaatatcccaagggagcagttatggaacttgcataggcgtatggtttactttgggacttcagtgccaaccatattcaaaggttaccaccattcacccatacaatgactcaaagaaatcttatctgcaactttaccgacaactgttattttcattctttaggtctaacaactgaagaaatacagcactgcttctggagaggtattaacagtatttttttcactcgctgtttcagcgcattctcctctgggacatatcttgtttcctcatatgatcaaacaatttgcagaatgcagatgccatcatataatcacaatatgtttctacagttatatagcattcgtgtgattgtgtcaaggtgtccacacacttttcacaaaaacttactccccctgtcccataatataagggattttgagcttttgtttgcactgtttgaccattcgtcttattcaaaaaaatttagaattatattttttttctttgtgacttactttattatccaaaatactttaagcacaacttttcgttttttatatttgcacaaattttttgaataagacgagtggtcaaacattataatcaaaaaactcaaaatcccttgtattatgggacggagggagtagtattaagggtctaaaaggagcctgaatatttggttcagttagagtctatatctgagtccattttaattcctagtgctatggataaaggtgtcctccttcaataaataaataaaggtctctccagttattagtcaccagcatccaatataactgcctgataatatatgtggtaagttatatggtgccaaagaggcaaagatactgctaccatgactaaccccctttttttttctgagttaataagggtgttagcatacatatagcttccaaattggtttagttaggtggaaaaagtcgtcatttcaggatgcagatataagttagggcatatcctcttgttggggttttcatttcagctgtgtcttatactttatatggtgtctcattaacatgcactaaaatgcaatgcaggatttgtctgtgtgagaggattcaacatggaaactagggcaccaaggcctctatgcccccatttccaccttgcagcaagcaaactgcgaagatcctctaaaaaagcagcaactgagcaaacacactgatttcaacagggacatatcaattctataaaataatgttgatggtgattacatcagttctcgatctgtgcatgggtcatattagttcctgcaggacgatagccattcttctaggaaataaacagtggagttggaaacagttaactttaagatgtgggagccaattgggcacgagaaagcaccaatatttcatatgcacctctggggcaggtcggatgcggatcatcatcatcagttaacatgggcgtagaatatcattccgttttttttttctgtggctttggtgtatcttattcacttttcatttgttttatccattcaactagcttgccaagagaccttaccatctgttctcatgacatgttagtttttcagaacttatagcaggtagtaccttcatattgatgatatgactttgattttcatt</dnaseqindica>
External Link(s)	NCBI Gene:Os05g0445900, RefSeq:Os05g0445900