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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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Expression level analysis of UGPase1 and UGPase2 genes in UGPase1-RNAi plants.Northern blot analysis of UGPase1 gene expression. The upper panel shows the RNA gel blot probed with the NotI fragments of UGP1 i pGEMT containing the 473-bp gene-specific region of the UGPase1 gene. The lower panel shows ethidium bromide stained rRNA as a loading control.Quantitative RT-PCR analysis of UGPase1 gene expression. The expression value was normalized with a ubiquitin control, and the results represent the average values of duplicate experiments shown as relative expression levels compared with empty vector-transformed plants.Semi-quantitative RT-PCR analysis of the expression of the UGPase2 gene. Amplification of the ubiquitin gene was used as a control. V, empty vector-transformed plant; r4, r11, r15, r23 and r29: UGPase1-RNAi plants.
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fig06: Expression level analysis of UGPase1 and UGPase2 genes in UGPase1-RNAi plants.Northern blot analysis of UGPase1 gene expression. The upper panel shows the RNA gel blot probed with the NotI fragments of UGP1 i pGEMT containing the 473-bp gene-specific region of the UGPase1 gene. The lower panel shows ethidium bromide stained rRNA as a loading control.Quantitative RT-PCR analysis of UGPase1 gene expression. The expression value was normalized with a ubiquitin control, and the results represent the average values of duplicate experiments shown as relative expression levels compared with empty vector-transformed plants.Semi-quantitative RT-PCR analysis of the expression of the UGPase2 gene. Amplification of the ubiquitin gene was used as a control. V, empty vector-transformed plant; r4, r11, r15, r23 and r29: UGPase1-RNAi plants.

Mentions: When pollen viability was compared in high- and low-fertility lines, by staining for starch with I2-KI solution, the five low-fertility transformants showed light pollen staining as compared with empty vector-transformed plants that displayed normal starch accumulation, and pollens from the two male-sterile transformants (r23 and r29) did not stain for starch (Figure 5f,g). If the low or no-staining phenotypes of UGPase1-RNAi transformants were caused by the introduced dsRNA, we would expect to see reduced UGPase1 transcription levels in these transgenic plants. When Northern blot analysis was used to examine UGPase1 expression levels in RNA samples harvested from spikelets at the booting stage in transgenic lines, it could be seen that transcription was most severely suppressed in the two male-sterile transgenic lines (r23 and r29), and was partially suppressed in the low-fertility transformants, compared with empty vector-transformed plants (Figure 6a). Subsequently, we examined the expression of UGPase1 transcripts in more detail in seven transgenic lines using real-time quantitative RT-PCR analysis. First-strand cDNAs that were reverse-transcribed with oligo (dT) were used as the template for the quantitative PCR analysis. Results were computed to show relative expression levels in UGPase1-RNAi transformants compared with a vector-transformed plant using ubiquitin as a standard. As can be seen in Figure 6b, UGPase1 transcriptional levels were slightly suppressed in the low-fertility lines (r4, r11 and r15), whereas the levels were severely reduced in the ms transformants, to 26% in r23 and 43% in r29. To determine whether the expression of a dsRNA interference construct towards UGPase1 affects the expression of UGPase2, we analyzed UGPase2 expression by semi-quantitative RT-PCR of the total RNA extracted from spikelets. As shown in Figure 6c, UGPase2 transcription in most RNAi transformants was just slightly suppressed, but UGPase2 transcripts of the r23 line were completely suppressed, similar to the suppression pattern of UGPase1 transcripts.


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)

Expression level analysis of UGPase1 and UGPase2 genes in UGPase1-RNAi plants.Northern blot analysis of UGPase1 gene expression. The upper panel shows the RNA gel blot probed with the NotI fragments of UGP1 i pGEMT containing the 473-bp gene-specific region of the UGPase1 gene. The lower panel shows ethidium bromide stained rRNA as a loading control.Quantitative RT-PCR analysis of UGPase1 gene expression. The expression value was normalized with a ubiquitin control, and the results represent the average values of duplicate experiments shown as relative expression levels compared with empty vector-transformed plants.Semi-quantitative RT-PCR analysis of the expression of the UGPase2 gene. Amplification of the ubiquitin gene was used as a control. V, empty vector-transformed plant; r4, r11, r15, r23 and r29: UGPase1-RNAi plants.
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Related In: Results  -  Collection

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fig06: Expression level analysis of UGPase1 and UGPase2 genes in UGPase1-RNAi plants.Northern blot analysis of UGPase1 gene expression. The upper panel shows the RNA gel blot probed with the NotI fragments of UGP1 i pGEMT containing the 473-bp gene-specific region of the UGPase1 gene. The lower panel shows ethidium bromide stained rRNA as a loading control.Quantitative RT-PCR analysis of UGPase1 gene expression. The expression value was normalized with a ubiquitin control, and the results represent the average values of duplicate experiments shown as relative expression levels compared with empty vector-transformed plants.Semi-quantitative RT-PCR analysis of the expression of the UGPase2 gene. Amplification of the ubiquitin gene was used as a control. V, empty vector-transformed plant; r4, r11, r15, r23 and r29: UGPase1-RNAi plants.
Mentions: When pollen viability was compared in high- and low-fertility lines, by staining for starch with I2-KI solution, the five low-fertility transformants showed light pollen staining as compared with empty vector-transformed plants that displayed normal starch accumulation, and pollens from the two male-sterile transformants (r23 and r29) did not stain for starch (Figure 5f,g). If the low or no-staining phenotypes of UGPase1-RNAi transformants were caused by the introduced dsRNA, we would expect to see reduced UGPase1 transcription levels in these transgenic plants. When Northern blot analysis was used to examine UGPase1 expression levels in RNA samples harvested from spikelets at the booting stage in transgenic lines, it could be seen that transcription was most severely suppressed in the two male-sterile transgenic lines (r23 and r29), and was partially suppressed in the low-fertility transformants, compared with empty vector-transformed plants (Figure 6a). Subsequently, we examined the expression of UGPase1 transcripts in more detail in seven transgenic lines using real-time quantitative RT-PCR analysis. First-strand cDNAs that were reverse-transcribed with oligo (dT) were used as the template for the quantitative PCR analysis. Results were computed to show relative expression levels in UGPase1-RNAi transformants compared with a vector-transformed plant using ubiquitin as a standard. As can be seen in Figure 6b, UGPase1 transcriptional levels were slightly suppressed in the low-fertility lines (r4, r11 and r15), whereas the levels were severely reduced in the ms transformants, to 26% in r23 and 43% in r29. To determine whether the expression of a dsRNA interference construct towards UGPase1 affects the expression of UGPase2, we analyzed UGPase2 expression by semi-quantitative RT-PCR of the total RNA extracted from spikelets. As shown in Figure 6c, UGPase2 transcription in most RNAi transformants was just slightly suppressed, but UGPase2 transcripts of the r23 line were completely suppressed, similar to the suppression pattern of UGPase1 transcripts.

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