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Inactivation of the UGPase1 gene causes genic male sterility and endosperm chalkiness in rice (Oryza sativa L.).

Woo MO, Ham TH, Ji HS, Choi MS, Jiang W, Chu SH, Piao R, Chin JH, Kim JA, Park BS, Seo HS, Jwa NS, McCouch S, Koh HJ - Plant J. (2008)

Bottom Line: A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm.After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3'-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 (UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing.In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T(1) plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Science and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea.

ABSTRACT
A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm. After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3'-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 (UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing. An in vitro functional assay showed that both proteins encoded by the two abnormal transcripts have no UGPase activity. The suppression of UGPase by the introduction of a UGPase1-RNAi construct in wild-type plants nearly eliminated seed set because of the male defect, with developmental retardation similar to the ms-h mutant phenotype, whereas overexpression of UGPase1 in ms-h mutant plants restored male fertility and the transformants produced T(1) seeds that segregated into normal and chalky endosperms. In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T(1) plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm. Our results suggest that UGPase1 plays a key role in pollen development as well as seed carbohydrate metabolism.

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Transgene constructs and phenotypes of transgenic plants.Schematic diagrams of the pUGP1RNAi construct for double-stranded RNA interference and the pUGP1COM construct used for the complementation test. In pUGP1RNAi, the 473-bp gene-specific fragment of the UGPase1 gene was linked with the intron in both antisense and sense orientations, such that the transcripts were expected to create a dsRNA stem with a single-stranded loop. Phenotype of UGPase1-RNAi plants (b–g).Hwacheong plants after ripening: containing the empty vector (left) and transformed by pUGP1RNAi (right).Photograph (c) is the double enlargement of a part of the photo (b).Panicles of a vector-transformed plant and a UGPase1-RNAi plant at anthesis (left) and after ripening (right).Flower and anther morphology of a vector-transformed plant (left) and UGPase1-RNAi plant (right) at the heading stage. To view the anthers, the lemma was ripped off.I2-KI staining of pollen grains from a vector-transformed plant at the heading stage, showing the presence of normal, round and starch-filled grains.I2-KI staining of pollen grains from a UGPase1-RNAi plant at the heading stage, showing the presence of abnormal, small and non-stained grains caused by the lack of starch. Phenotypic complementation by introduction of the UGPase1 gene (h–m).Phenotype of Hwacheong ms-h mutants after ripening: plants containing the empty vector (left) and complemented by the introduction of pUGP1COM (right).Photograph (i) is the triple enlargement of a part of the photo (h).Panicles of a vector-transformed plant and the complemented plant at anthesis (left) and after ripening (right).Flower and anther morphology of an empty vector-transformed plant (left) and the complemented plant (right) at heading stage.I2-KI staining of pollen grains from a empty vector-transformed plant at heading, showing the presence of abnormal and non-stained grains.I2-KI staining of pollen grains from the complemented plant at heading. This photograph shows the presence of normal grains, indicating the restoration of fertility. The scale bar corresponds to 100 μm.
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fig05: Transgene constructs and phenotypes of transgenic plants.Schematic diagrams of the pUGP1RNAi construct for double-stranded RNA interference and the pUGP1COM construct used for the complementation test. In pUGP1RNAi, the 473-bp gene-specific fragment of the UGPase1 gene was linked with the intron in both antisense and sense orientations, such that the transcripts were expected to create a dsRNA stem with a single-stranded loop. Phenotype of UGPase1-RNAi plants (b–g).Hwacheong plants after ripening: containing the empty vector (left) and transformed by pUGP1RNAi (right).Photograph (c) is the double enlargement of a part of the photo (b).Panicles of a vector-transformed plant and a UGPase1-RNAi plant at anthesis (left) and after ripening (right).Flower and anther morphology of a vector-transformed plant (left) and UGPase1-RNAi plant (right) at the heading stage. To view the anthers, the lemma was ripped off.I2-KI staining of pollen grains from a vector-transformed plant at the heading stage, showing the presence of normal, round and starch-filled grains.I2-KI staining of pollen grains from a UGPase1-RNAi plant at the heading stage, showing the presence of abnormal, small and non-stained grains caused by the lack of starch. Phenotypic complementation by introduction of the UGPase1 gene (h–m).Phenotype of Hwacheong ms-h mutants after ripening: plants containing the empty vector (left) and complemented by the introduction of pUGP1COM (right).Photograph (i) is the triple enlargement of a part of the photo (h).Panicles of a vector-transformed plant and the complemented plant at anthesis (left) and after ripening (right).Flower and anther morphology of an empty vector-transformed plant (left) and the complemented plant (right) at heading stage.I2-KI staining of pollen grains from a empty vector-transformed plant at heading, showing the presence of abnormal and non-stained grains.I2-KI staining of pollen grains from the complemented plant at heading. This photograph shows the presence of normal grains, indicating the restoration of fertility. The scale bar corresponds to 100 μm.

Mentions: To confirm that the UGPase1 gene is causally related to male fertility, we generated UGPase1-RNAi transgenic plants by exploiting double-stranded RNA (dsRNA)-mediated interference to silence the target gene (Baulcombe, 2002; Moritoh et al., 2005; Prasad and Vijayraghavan, 2003). The RNAi construct included 473-bp of the gene-specific sequence, corresponding to the full-length UGPase1 cDNA, which was linked with the intronic sequence in the antisense and sense configurations, and then placed under the control of the constitutive 35S promoter (Figure 5a). The UGPase1-RNAi construct was introduced into calli derived from wt Hwacheong immature embryos by Agrobacterium-mediated transformation, with an empty vector used as a control. Thirty-three independent transformants were regenerated, and the presence of the RNAi construct was confirmed by PCR using Bar-F and Bar-R primers (data not shown). At the spikelet ripening stage, five of the transformed lines displayed low fertility, and two lines were male sterile (Figure 5b–e). Moreover, these transformed lines showed pleiotropic developmental abnormalities similar to the Hwacheong ms-h mutant phenotype, including reduced culm length and retarded growth (Table 2).


Inactivation of the UGPase1 gene causes genic male sterility and endosperm chalkiness in rice (Oryza sativa L.).

Woo MO, Ham TH, Ji HS, Choi MS, Jiang W, Chu SH, Piao R, Chin JH, Kim JA, Park BS, Seo HS, Jwa NS, McCouch S, Koh HJ - Plant J. (2008)

Transgene constructs and phenotypes of transgenic plants.Schematic diagrams of the pUGP1RNAi construct for double-stranded RNA interference and the pUGP1COM construct used for the complementation test. In pUGP1RNAi, the 473-bp gene-specific fragment of the UGPase1 gene was linked with the intron in both antisense and sense orientations, such that the transcripts were expected to create a dsRNA stem with a single-stranded loop. Phenotype of UGPase1-RNAi plants (b–g).Hwacheong plants after ripening: containing the empty vector (left) and transformed by pUGP1RNAi (right).Photograph (c) is the double enlargement of a part of the photo (b).Panicles of a vector-transformed plant and a UGPase1-RNAi plant at anthesis (left) and after ripening (right).Flower and anther morphology of a vector-transformed plant (left) and UGPase1-RNAi plant (right) at the heading stage. To view the anthers, the lemma was ripped off.I2-KI staining of pollen grains from a vector-transformed plant at the heading stage, showing the presence of normal, round and starch-filled grains.I2-KI staining of pollen grains from a UGPase1-RNAi plant at the heading stage, showing the presence of abnormal, small and non-stained grains caused by the lack of starch. Phenotypic complementation by introduction of the UGPase1 gene (h–m).Phenotype of Hwacheong ms-h mutants after ripening: plants containing the empty vector (left) and complemented by the introduction of pUGP1COM (right).Photograph (i) is the triple enlargement of a part of the photo (h).Panicles of a vector-transformed plant and the complemented plant at anthesis (left) and after ripening (right).Flower and anther morphology of an empty vector-transformed plant (left) and the complemented plant (right) at heading stage.I2-KI staining of pollen grains from a empty vector-transformed plant at heading, showing the presence of abnormal and non-stained grains.I2-KI staining of pollen grains from the complemented plant at heading. This photograph shows the presence of normal grains, indicating the restoration of fertility. The scale bar corresponds to 100 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2327258&req=5

fig05: Transgene constructs and phenotypes of transgenic plants.Schematic diagrams of the pUGP1RNAi construct for double-stranded RNA interference and the pUGP1COM construct used for the complementation test. In pUGP1RNAi, the 473-bp gene-specific fragment of the UGPase1 gene was linked with the intron in both antisense and sense orientations, such that the transcripts were expected to create a dsRNA stem with a single-stranded loop. Phenotype of UGPase1-RNAi plants (b–g).Hwacheong plants after ripening: containing the empty vector (left) and transformed by pUGP1RNAi (right).Photograph (c) is the double enlargement of a part of the photo (b).Panicles of a vector-transformed plant and a UGPase1-RNAi plant at anthesis (left) and after ripening (right).Flower and anther morphology of a vector-transformed plant (left) and UGPase1-RNAi plant (right) at the heading stage. To view the anthers, the lemma was ripped off.I2-KI staining of pollen grains from a vector-transformed plant at the heading stage, showing the presence of normal, round and starch-filled grains.I2-KI staining of pollen grains from a UGPase1-RNAi plant at the heading stage, showing the presence of abnormal, small and non-stained grains caused by the lack of starch. Phenotypic complementation by introduction of the UGPase1 gene (h–m).Phenotype of Hwacheong ms-h mutants after ripening: plants containing the empty vector (left) and complemented by the introduction of pUGP1COM (right).Photograph (i) is the triple enlargement of a part of the photo (h).Panicles of a vector-transformed plant and the complemented plant at anthesis (left) and after ripening (right).Flower and anther morphology of an empty vector-transformed plant (left) and the complemented plant (right) at heading stage.I2-KI staining of pollen grains from a empty vector-transformed plant at heading, showing the presence of abnormal and non-stained grains.I2-KI staining of pollen grains from the complemented plant at heading. This photograph shows the presence of normal grains, indicating the restoration of fertility. The scale bar corresponds to 100 μm.
Mentions: To confirm that the UGPase1 gene is causally related to male fertility, we generated UGPase1-RNAi transgenic plants by exploiting double-stranded RNA (dsRNA)-mediated interference to silence the target gene (Baulcombe, 2002; Moritoh et al., 2005; Prasad and Vijayraghavan, 2003). The RNAi construct included 473-bp of the gene-specific sequence, corresponding to the full-length UGPase1 cDNA, which was linked with the intronic sequence in the antisense and sense configurations, and then placed under the control of the constitutive 35S promoter (Figure 5a). The UGPase1-RNAi construct was introduced into calli derived from wt Hwacheong immature embryos by Agrobacterium-mediated transformation, with an empty vector used as a control. Thirty-three independent transformants were regenerated, and the presence of the RNAi construct was confirmed by PCR using Bar-F and Bar-R primers (data not shown). At the spikelet ripening stage, five of the transformed lines displayed low fertility, and two lines were male sterile (Figure 5b–e). Moreover, these transformed lines showed pleiotropic developmental abnormalities similar to the Hwacheong ms-h mutant phenotype, including reduced culm length and retarded growth (Table 2).

Bottom Line: A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm.After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3'-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 (UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing.In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T(1) plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Science and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea.

ABSTRACT
A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm. After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3'-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 (UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing. An in vitro functional assay showed that both proteins encoded by the two abnormal transcripts have no UGPase activity. The suppression of UGPase by the introduction of a UGPase1-RNAi construct in wild-type plants nearly eliminated seed set because of the male defect, with developmental retardation similar to the ms-h mutant phenotype, whereas overexpression of UGPase1 in ms-h mutant plants restored male fertility and the transformants produced T(1) seeds that segregated into normal and chalky endosperms. In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T(1) plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm. Our results suggest that UGPase1 plays a key role in pollen development as well as seed carbohydrate metabolism.

Show MeSH
Related in: MedlinePlus