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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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RT-PCR analysis with UGPase1-specific primers. As a result of each RT-PCR using the UGP1-PRT primer set (left) and the UGP1-FRT primer set (right), wild-type (wt) Hwacheong displayed only the expected size fragment in both reactions; whereas the ms-h mutant contained two fragments, one similar to the wt and a second, longer fragment. The same banding pattern was observed in both leaf and panicle. The arrows pointing upwards indicate the fragment size of the wt Hwacheong, and the arrows pointing left represent the fragment size of the ms-h mutant. Abbreviations: FL, fertile leaf; ML: male-sterile leaf; FP, fertile panicle; MP, male-sterile panicle.
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fig03: RT-PCR analysis with UGPase1-specific primers. As a result of each RT-PCR using the UGP1-PRT primer set (left) and the UGP1-FRT primer set (right), wild-type (wt) Hwacheong displayed only the expected size fragment in both reactions; whereas the ms-h mutant contained two fragments, one similar to the wt and a second, longer fragment. The same banding pattern was observed in both leaf and panicle. The arrows pointing upwards indicate the fragment size of the wt Hwacheong, and the arrows pointing left represent the fragment size of the ms-h mutant. Abbreviations: FL, fertile leaf; ML: male-sterile leaf; FP, fertile panicle; MP, male-sterile panicle.

Mentions: As this point mutation occurred at the splice site between the 14th intron and 15th exon, we performed RT-PCR analysis of the ms-h mutant and wt Hwacheong using two sets of UGPase1-specific primers to investigate whether the pre-mRNA splice site was altered. One set of UGPase1-specific primers, UGP1-PRT primers, was designed from the sequences of the 14th and 15th exons, to fully span the 14th intron region, and the second set, UGP1-FRT primers, was used to amplify a full-length UGPase1 cDNA. As shown in Figure 3, wt Hwacheong displayed only a 108-bp fragment in RT-PCR with UGP1-PRT primers, whereas the ms-h mutant contained two fragments, a 182-bp fragment as well as a shorter fragment that appeared identical to the 108-bp fragment observed in the wt, Hwacheong. When the shorter fragment amplified from the ms-h mutant was cloned and sequenced, the RT-PCR product showed a 1-bp deletion in the spliced message, compared with the corresponding sequence of the wt RT-PCR product, although this 1-bp difference was not detectable on the PAGE gel. Moreover, the 182-bp fragment amplified from the ms-h mutant was revealed to contain the entire, unspliced 14th intron (74 bp).


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)

RT-PCR analysis with UGPase1-specific primers. As a result of each RT-PCR using the UGP1-PRT primer set (left) and the UGP1-FRT primer set (right), wild-type (wt) Hwacheong displayed only the expected size fragment in both reactions; whereas the ms-h mutant contained two fragments, one similar to the wt and a second, longer fragment. The same banding pattern was observed in both leaf and panicle. The arrows pointing upwards indicate the fragment size of the wt Hwacheong, and the arrows pointing left represent the fragment size of the ms-h mutant. Abbreviations: FL, fertile leaf; ML: male-sterile leaf; FP, fertile panicle; MP, male-sterile panicle.
© Copyright Policy
Related In: Results  -  Collection

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

fig03: RT-PCR analysis with UGPase1-specific primers. As a result of each RT-PCR using the UGP1-PRT primer set (left) and the UGP1-FRT primer set (right), wild-type (wt) Hwacheong displayed only the expected size fragment in both reactions; whereas the ms-h mutant contained two fragments, one similar to the wt and a second, longer fragment. The same banding pattern was observed in both leaf and panicle. The arrows pointing upwards indicate the fragment size of the wt Hwacheong, and the arrows pointing left represent the fragment size of the ms-h mutant. Abbreviations: FL, fertile leaf; ML: male-sterile leaf; FP, fertile panicle; MP, male-sterile panicle.
Mentions: As this point mutation occurred at the splice site between the 14th intron and 15th exon, we performed RT-PCR analysis of the ms-h mutant and wt Hwacheong using two sets of UGPase1-specific primers to investigate whether the pre-mRNA splice site was altered. One set of UGPase1-specific primers, UGP1-PRT primers, was designed from the sequences of the 14th and 15th exons, to fully span the 14th intron region, and the second set, UGP1-FRT primers, was used to amplify a full-length UGPase1 cDNA. As shown in Figure 3, wt Hwacheong displayed only a 108-bp fragment in RT-PCR with UGP1-PRT primers, whereas the ms-h mutant contained two fragments, a 182-bp fragment as well as a shorter fragment that appeared identical to the 108-bp fragment observed in the wt, Hwacheong. When the shorter fragment amplified from the ms-h mutant was cloned and sequenced, the RT-PCR product showed a 1-bp deletion in the spliced message, compared with the corresponding sequence of the wt RT-PCR product, although this 1-bp difference was not detectable on the PAGE gel. Moreover, the 182-bp fragment amplified from the ms-h mutant was revealed to contain the entire, unspliced 14th intron (74 bp).

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