Os05g0445900 - Revision history

192.168.72.52: /* Structured Information */

2015-06-12T07:39:38Z

‎Structured Information

Lina at 07:14, 10 June 2014

2014-06-10T07:14:12Z

Jc0407: /* summary */

2014-06-10T07:11:49Z

‎summary

Jc0407 at 04:37, 10 June 2014

2014-06-10T04:37:38Z

Jc0407 at 04:36, 10 June 2014

2014-06-10T04:36:54Z

Jc0407: /* summary */

2014-06-10T04:36:08Z

‎summary

Jc0407 at 04:34, 10 June 2014

2014-06-10T04:34:54Z

Bzwangjie: /* Evolution */

2014-06-10T04:26:15Z

‎Evolution

Bzwangjie: /* Labs working on this gene */

2014-06-10T04:25:44Z

‎Labs working on this gene

Bzwangjie at 04:25, 10 June 2014

2014-06-10T04:25:13Z

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 ==Structured Information==
 [[Category:Genes]]
 [[Category:Japonica mRNA]]

← Older revision		Revision as of 07:14, 10 June 2014
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	===summary===		===summary===

		+	DME is preferentially expressed in the homodiploid central cell of the female gametophyte before fertilization, and promotes maternal allele-specific global hypomethylation, leading to maternal allele-specific expression of imprinted genes in the endosperm, including the Polycomb Repressive Complex 2 (PRC2) genes MEDEA (MEA) and FERTILIZATION INDEPENDENT SEED 2 , and to maternal allele-specific expression of transposons in the endosperm. Expression of these imprinted genes is prerequisite for normal endosperm development. mea and fis2 mutants exhibited characteristic maternal defects in the endosperm similar to the dme mutant, because PRC2 represses autonomous growth of unfertilized endosperm and overgrowth of fertilized endosperm . In rice, GUS staining was detected in the central cells of ROS1a/ros1a-GUS1 plants , indicating that ROS1a is expressed in the central cell of the female gametophyte. Although many maternally expressed imprinted genes were identified in the rice endosperm, very few were in common with those in Arabidopsis . Expression of FERTILIZATION INDEPENDENT ENDOSPERM 1 (FIE1) (LOC_Os08g04290), one such imprinted gene encoding a component of PRC2 in rice, appears to correlate with DNA demethylation at its 5′ region, but an fie1 disruptant showed no defects in endosperm development . The results imply that certain roles of PRC2 in rice endosperm development differ from those of Arabidopsis. Although we could not demonstrate ROS1a-promoted DNA demethylation, ROS1a is postulated to activate the imprinted genes through DNA demethylation in order to facilitate normal endosperm development. GUS staining was also detected in the antipodals and the egg apparatus comprising two synergids and an egg cell whose genetic information is transmittable to progeny (Figure 4f,g). The molecular mechanisms of ROS1a function in these cells remain to be elucidated.

		+	Although DME in Arabidopsis is predominantly expressed in the central cell of the female gametophyte, and is only marginally expressed to demethylate some imprinted genes and transposons in the vegetative cell of the male gametophyte , comparable amounts of ROS1a were expressed in both the female and male gametophytes , and the paternal ros1a-GUS1 allele could not be transmitted to progeny . Although the molecular mechanisms for the inability to transmit the paternal ros1a-GUS1 allele are unknown, ros1a-GUS1 pollen germinated less efficiently than did ROS1a pollen. Reduced germination and transmission of dme pollen were also observed in certain Arabidopsis ecotypes (Col-gl and Col-0) . The vegetative nucleus in pollen supports the sperm cell before fertilization and shows reduced DNA methylation in Arabidopsis, resulting in transient reactivation of diverse transposons and generation of small interfering RNAs that may ensure the genomic integrity of sperm cells by silencing transposons in the sperm cells . Whether similar DNA demethylation occurs in the vegetative nucleus in rice pollen, and whether the ROS1a protein promotes such demethylation, remain to be determined. If such DNA demethylation occurs in the vegetative nucleus, it will be interesting to determine whether such demethylation directly correlates with the intransmittable nature of the paternal ros1a-GUS1 allele .

	Quantitative RT-PCR analysis revealed that ROS1a, ROS1c, ROS1d and DML3a are expressed in five selected tissues tested, including anthers and pistils, and ROS1a is the most abundantly expressed gene in these tissues . The same analysis showed that ROS1c, ROS1d and DML3a are moderately expressed in both pistils and immature seeds 2 days after pollination. 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 . Their results are in sharp contrast to those for the null allele of ROS1a described here, and indicate that moderate expression of ROS1c, ROS1d and DML3a in pistils could not compensate for the loss of ROS1a. It would be interesting to determine whether ROS1a and any of the other three genes are expressed simultaneously in the same cells in the pistil. If ROS1a and ROS1c are expressed in the same cells, it would then be interesting to determine whether the different transmittability of their null alleles may be explained simply by their expression levels or whether it is due to intrinsic differences in their molecular functions.		Quantitative RT-PCR analysis revealed that ROS1a, ROS1c, ROS1d and DML3a are expressed in five selected tissues tested, including anthers and pistils, and ROS1a is the most abundantly expressed gene in these tissues . The same analysis showed that ROS1c, ROS1d and DML3a are moderately expressed in both pistils and immature seeds 2 days after pollination. 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 . Their results are in sharp contrast to those for the null allele of ROS1a described here, and indicate that moderate expression of ROS1c, ROS1d and DML3a in pistils could not compensate for the loss of ROS1a. It would be interesting to determine whether ROS1a and any of the other three genes are expressed simultaneously in the same cells in the pistil. If ROS1a and ROS1c are expressed in the same cells, it would then be interesting to determine whether the different transmittability of their null alleles may be explained simply by their expression levels or whether it is due to intrinsic differences in their molecular functions.

@@ Line 36: / Line 36: @@
 ===summary===
 Quantitative RT-PCR analysis revealed that ROS1a, ROS1c, ROS1d and DML3a are expressed in five selected tissues tested, including anthers and pistils, and ROS1a is the most abundantly expressed gene in these tissues . The same analysis showed that ROS1c, ROS1d and DML3a are moderately expressed in both pistils and immature seeds 2 days after pollination. 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 . Their results are in sharp contrast to those for the null allele of ROS1a described here, and indicate that moderate expression of ROS1c, ROS1d and DML3a in pistils could not compensate for the loss of ROS1a. It would be interesting to determine whether ROS1a and any of the other three genes are expressed simultaneously in the same cells in the pistil. If ROS1a and ROS1c are expressed in the same cells, it would then be interesting to determine whether the different transmittability of their null alleles may be explained simply by their expression levels or whether it is due to intrinsic differences in their molecular functions.
 The null ros1a-GUS1 allele was not transmitted to progeny . In conventional mutagenesis procedures, mutants are usually identified and isolated as segregants in the progeny population. Such procedures would not have enabled isolation of the ros1a-GUS1 allele. In contrast, our homologous recombination-promoted gene targeting procedure allowed T0 transgenic plants to be reproducibly obtained with the exactly anticipated structure of the targeted gene in the heterozygous condition. Moreover, the knock-in targeting strategy allowed detection of spatio-temporal ROS1a expression and tracking of the transmitted ros1a allele. However, because GUS staining was necessary to detect ROS1a expression, it was difficult to analyze in vivo and/or to isolate viable tissues that express GUS for detailed examination. Utilization of other markers such as green fluorescent protein, in combination with other techniques including laser micro-dissection and/or fluorescence-activated cell sorting, would facilitate further characterization of ROS1a function in endosperm development and pollen gametophytic transmission.
 ==Labs working on this gene==

@@ Line 20: / Line 20: @@
 [[File:360截图20140610120515955.jpg]]
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 The null ros1a-GUS1 allele was not transmitted to progeny . In conventional mutagenesis procedures, mutants are usually identified and isolated as segregants in the progeny population. Such procedures would not have enabled isolation of the ros1a-GUS1 allele. In contrast, our homologous recombination-promoted gene targeting procedure allowed T0 transgenic plants to be reproducibly obtained with the exactly anticipated structure of the targeted gene in the heterozygous condition. Moreover, the knock-in targeting strategy allowed detection of spatio-temporal ROS1a expression and tracking of the transmitted ros1a allele. However, because GUS staining was necessary to detect ROS1a expression, it was difficult to analyze in vivo and/or to isolate viable tissues that express GUS for detailed examination. Utilization of other markers such as green fluorescent protein, in combination with other techniques including laser micro-dissection and/or fluorescence-activated cell sorting, would facilitate further characterization of ROS1a function in endosperm development and pollen gametophytic transmission.
-===Expression===
 ==Labs working on this gene==

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 [[File:360截图20140610120515955.jpg]]
 ===summary===
@@ Line 34: / Line 44: @@
 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.
 [[File:TPJ_5009_f1.gif]]
 ==Labs working on this gene==

← Older revision		Revision as of 04:34, 10 June 2014
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	[[File:360截图20140610120515955.jpg]]		[[File:360截图20140610120515955.jpg]]
		+
		+
		+	===summary===
		+
		+	Using homologous recombination-promoted knock-in gene targeting, whereby GUS was fused to the endogenous promoter of a target gene , we reproducibly generated an identical structure of a null mutation for the rice ROS1a gene encoding a putative cytosine DNA demethylase, whose activity was implied by its toxic effects on E. coli containing 5-meC in their genome . The obtained null mutation, ros1a-GUS1, was hardly ever transmitted to progeny . The knock-in T0 mutants (ROS1a/ros1a-GUS1) were subsequently crossed with Nipponbare (ROS1a/ROS1a), and the results clearly showed that both paternal and maternal ros1a-GUS1 alleles are not transmitted to progeny , indicating that DNA demethylation must play important roles in both male and female gametophytic generation. Indeed, the GUS staining patterns of the knock-in T0 plants indicated that ROS1a was expressed in pollen and unfertilized ovules as well as in meristematic cells .
		+
		+	Even in the presence of the wild-type paternal ROS1a allele, the maternal null ros1a-GUS1 allele caused failure of early stage endosperm development, indicating non-equivalent contribution of maternal and paternal ROS1a to endosperm development. Thus, ros1a-GUS in rice appears to exert effects comparable to those of dme in Arabidopsis in female gametophytes. Both ros1a-GUS and dme cause maternal allele-specific endosperm defects: ros1a-GUS causes severely under-developed endosperm , whereas dme causes an enlarged endosperm that then collaps. Althoes ugh phylogenetic analysis indicated that the rice genome contains no DME orthologs , their maternal allele-specific defects in endosperm development imply that ROS1a and DME play some analogous roles.
		+
		+	DME is preferentially expressed in the homodiploid central cell of the female gametophyte before fertilization, and promotes maternal allele-specific global hypomethylation, leading to maternal allele-specific expression of imprinted genes in the endosperm, including the Polycomb Repressive Complex 2 (PRC2) genes MEDEA (MEA) and FERTILIZATION INDEPENDENT SEED 2 , and to maternal allele-specific expression of transposons in the endosperm. Expression of these imprinted genes is prerequisite for normal endosperm development. mea and fis2 mutants exhibited characteristic maternal defects in the endosperm similar to the dme mutant, because PRC2 represses autonomous growth of unfertilized endosperm and overgrowth of fertilized endosperm . In rice, GUS staining was detected in the central cells of ROS1a/ros1a-GUS1 plants , indicating that ROS1a is expressed in the central cell of the female gametophyte. Although many maternally expressed imprinted genes were identified in the rice endosperm, very few were in common with those in Arabidopsis . Expression of FERTILIZATION INDEPENDENT ENDOSPERM 1 (FIE1) (LOC_Os08g04290), one such imprinted gene encoding a component of PRC2 in rice, appears to correlate with DNA demethylation at its 5′ region, but an fie1 disruptant showed no defects in endosperm development . The results imply that certain roles of PRC2 in rice endosperm development differ from those of Arabidopsis. Although we could not demonstrate ROS1a-promoted DNA demethylation, ROS1a is postulated to activate the imprinted genes through DNA demethylation in order to facilitate normal endosperm development. GUS staining was also detected in the antipodals and the egg apparatus comprising two synergids and an egg cell whose genetic information is transmittable to progeny (Figure 4f,g). The molecular mechanisms of ROS1a function in these cells remain to be elucidated.
		+
		+	Although DME in Arabidopsis is predominantly expressed in the central cell of the female gametophyte, and is only marginally expressed to demethylate some imprinted genes and transposons in the vegetative cell of the male gametophyte , comparable amounts of ROS1a were expressed in both the female and male gametophytes , and the paternal ros1a-GUS1 allele could not be transmitted to progeny . Although the molecular mechanisms for the inability to transmit the paternal ros1a-GUS1 allele are unknown, ros1a-GUS1 pollen germinated less efficiently than did ROS1a pollen. Reduced germination and transmission of dme pollen were also observed in certain Arabidopsis ecotypes (Col-gl and Col-0) . The vegetative nucleus in pollen supports the sperm cell before fertilization and shows reduced DNA methylation in Arabidopsis, resulting in transient reactivation of diverse transposons and generation of small interfering RNAs that may ensure the genomic integrity of sperm cells by silencing transposons in the sperm cells . Whether similar DNA demethylation occurs in the vegetative nucleus in rice pollen, and whether the ROS1a protein promotes such demethylation, remain to be determined. If such DNA demethylation occurs in the vegetative nucleus, it will be interesting to determine whether such demethylation directly correlates with the intransmittable nature of the paternal ros1a-GUS1 allele .
		+
		+	Quantitative RT-PCR analysis revealed that ROS1a, ROS1c, ROS1d and DML3a are expressed in five selected tissues tested, including anthers and pistils, and ROS1a is the most abundantly expressed gene in these tissues . The same analysis showed that ROS1c, ROS1d and DML3a are moderately expressed in both pistils and immature seeds 2 days after pollination. 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 . Their results are in sharp contrast to those for the null allele of ROS1a described here, and indicate that moderate expression of ROS1c, ROS1d and DML3a in pistils could not compensate for the loss of ROS1a. It would be interesting to determine whether ROS1a and any of the other three genes are expressed simultaneously in the same cells in the pistil. If ROS1a and ROS1c are expressed in the same cells, it would then be interesting to determine whether the different transmittability of their null alleles may be explained simply by their expression levels or whether it is due to intrinsic differences in their molecular functions.
		+
		+	The null ros1a-GUS1 allele was not transmitted to progeny . In conventional mutagenesis procedures, mutants are usually identified and isolated as segregants in the progeny population. Such procedures would not have enabled isolation of the ros1a-GUS1 allele. In contrast, our homologous recombination-promoted gene targeting procedure allowed T0 transgenic plants to be reproducibly obtained with the exactly anticipated structure of the targeted gene in the heterozygous condition. Moreover, the knock-in targeting strategy allowed detection of spatio-temporal ROS1a expression and tracking of the transmitted ros1a allele. However, because GUS staining was necessary to detect ROS1a expression, it was difficult to analyze in vivo and/or to isolate viable tissues that express GUS for detailed examination. Utilization of other markers such as green fluorescent protein, in combination with other techniques including laser micro-dissection and/or fluorescence-activated cell sorting, would facilitate further characterization of ROS1a function in endosperm development and pollen gametophytic transmission.

@@ Line 30: / Line 30: @@
 ===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.
 [[File:pnas.1009695107fig05.jpg]]

@@ Line 34: / Line 34: @@
 ==Labs working on this gene==
 .National Institute for Basic Biology, Okazaki 444-8585, Japan
 .Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan
 .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

@@ Line 33: / Line 33: @@
 ==Labs working on this gene==
-Please input related labs here.
+.National Institute for Basic Biology, Okazaki 444-8585, Japan
 ==References==