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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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Nucleotide sequence of the UGPase1 open reading frame, and alignment of the nucleotide and deduced amino acid sequence of the 14th exon 3′-region derived from wild-type (wt) Hwacheong and from the ms-h mutant. Nucleotide numbering starts from the start of translation; the protein sequence is derived from the nucleotide sequence. Wild transcript denotes the UGPase1 transcript of wt Hwacheong, whereas mutant transcripts 1 and 2 represent the 1-bp deleted transcript and the 74-bp inserted transcript found in the ms-h mutant, respectively. The deduced amino acid sequences of the two ms-h mutant transcripts with abnormal sizes demonstrate that both the 1-bp deletion and the 74-bp insertion cause a frame shift and generate stop codons, resulting in truncated 299- and 298-aa proteins, instead of the full-length 469-aa protein. The start codon is set in bold with a row of dots above it. The underlined sequences and asterisk indicate termination codons. The 1-bp deletion site is marked by an arrowhead.SDS-PAGE of recombinant proteins induced with isopropyl-beta-D-thiogalactopyranoside (left) and purified-recombinant UGPase1 proteins (right). The molecular weight of glutathione S-transferase (GST) (23-kDa)-tagged recombinant UGPase1 proteins agrees with the estimated values of 74 and 56 kDa for the full-length UGPase1 protein (469 aa) and the two C-terminal deleted proteins (299 and 298 aa).Activity assays of three recombinant UGPase1 proteins. The formation of NADPH was calculated from the absorption changes at 340 nm monitored for 5 h using an NADPH molar extinction coefficient of 6.22 × 103 m−1 cm−1. •, GST-UGP, GST-tagged recombinant protein containing full-length UGPase1; ▴, GST-mUGP1, GST-tagged recombinant protein containing C-terminal deleted 299-aa UGPase1; ▪, GST-mUGP2, GST-tagged recombinant protein containing C-terminal deleted 298-aa UGPase1.
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fig04: Nucleotide sequence of the UGPase1 open reading frame, and alignment of the nucleotide and deduced amino acid sequence of the 14th exon 3′-region derived from wild-type (wt) Hwacheong and from the ms-h mutant. Nucleotide numbering starts from the start of translation; the protein sequence is derived from the nucleotide sequence. Wild transcript denotes the UGPase1 transcript of wt Hwacheong, whereas mutant transcripts 1 and 2 represent the 1-bp deleted transcript and the 74-bp inserted transcript found in the ms-h mutant, respectively. The deduced amino acid sequences of the two ms-h mutant transcripts with abnormal sizes demonstrate that both the 1-bp deletion and the 74-bp insertion cause a frame shift and generate stop codons, resulting in truncated 299- and 298-aa proteins, instead of the full-length 469-aa protein. The start codon is set in bold with a row of dots above it. The underlined sequences and asterisk indicate termination codons. The 1-bp deletion site is marked by an arrowhead.SDS-PAGE of recombinant proteins induced with isopropyl-beta-D-thiogalactopyranoside (left) and purified-recombinant UGPase1 proteins (right). The molecular weight of glutathione S-transferase (GST) (23-kDa)-tagged recombinant UGPase1 proteins agrees with the estimated values of 74 and 56 kDa for the full-length UGPase1 protein (469 aa) and the two C-terminal deleted proteins (299 and 298 aa).Activity assays of three recombinant UGPase1 proteins. The formation of NADPH was calculated from the absorption changes at 340 nm monitored for 5 h using an NADPH molar extinction coefficient of 6.22 × 103 m−1 cm−1. •, GST-UGP, GST-tagged recombinant protein containing full-length UGPase1; ▴, GST-mUGP1, GST-tagged recombinant protein containing C-terminal deleted 299-aa UGPase1; ▪, GST-mUGP2, GST-tagged recombinant protein containing C-terminal deleted 298-aa UGPase1.

Mentions: The deduced amino acid sequences of the ms-h mutant transcripts displaying two abnormal sizes suggests that both the 1-bp deletion and the 74-bp insertion cause frame shifts that generate two independent stop codons in the process of translation, resulting in truncated 299- and 298-aa proteins, instead of the 469-aa protein encoded by the wt UGPase1 transcript (Figure 4a). To further confirm whether two C-terminal deleted UGPase1 proteins of the ms-h mutant are nonfunctional, we performed enzyme activity assays in vitro. The UGPase1 cDNAs encoding full-length protein of the wt Hwacheong and two truncated proteins of the ms-h mutant were amplified by PCR with each specific primer, and were transformed into Escherichia coli after vector construction. Glutathione S-transferase (GST)-tagged three-recombinant proteins were purified and separated by SDS-PAGE (Figure 4b). UGPase activity assays were performed with recombinant proteins by monitoring the NADPH formation at 340 nm. As expected, enzyme activity of GST-UGP containing the full-length UGPase1 protein appeared, whereas both GST-mUGP1 and GST-mUGP2 did not show any enzyme activity, indicating that two C-terminal deleted proteins were nonfunctional (Figure 4c). Thus, this single base substitution in the splice site found in the ms-h mutant appears to cause unstable splicing, leading to the presence of two mature transcripts, both with abnormal sizes and, because of the corresponding stop codons, the mRNA transcripts are translated into a truncated and a nonfunctional UGPase1 protein, respectively.


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)

Nucleotide sequence of the UGPase1 open reading frame, and alignment of the nucleotide and deduced amino acid sequence of the 14th exon 3′-region derived from wild-type (wt) Hwacheong and from the ms-h mutant. Nucleotide numbering starts from the start of translation; the protein sequence is derived from the nucleotide sequence. Wild transcript denotes the UGPase1 transcript of wt Hwacheong, whereas mutant transcripts 1 and 2 represent the 1-bp deleted transcript and the 74-bp inserted transcript found in the ms-h mutant, respectively. The deduced amino acid sequences of the two ms-h mutant transcripts with abnormal sizes demonstrate that both the 1-bp deletion and the 74-bp insertion cause a frame shift and generate stop codons, resulting in truncated 299- and 298-aa proteins, instead of the full-length 469-aa protein. The start codon is set in bold with a row of dots above it. The underlined sequences and asterisk indicate termination codons. The 1-bp deletion site is marked by an arrowhead.SDS-PAGE of recombinant proteins induced with isopropyl-beta-D-thiogalactopyranoside (left) and purified-recombinant UGPase1 proteins (right). The molecular weight of glutathione S-transferase (GST) (23-kDa)-tagged recombinant UGPase1 proteins agrees with the estimated values of 74 and 56 kDa for the full-length UGPase1 protein (469 aa) and the two C-terminal deleted proteins (299 and 298 aa).Activity assays of three recombinant UGPase1 proteins. The formation of NADPH was calculated from the absorption changes at 340 nm monitored for 5 h using an NADPH molar extinction coefficient of 6.22 × 103 m−1 cm−1. •, GST-UGP, GST-tagged recombinant protein containing full-length UGPase1; ▴, GST-mUGP1, GST-tagged recombinant protein containing C-terminal deleted 299-aa UGPase1; ▪, GST-mUGP2, GST-tagged recombinant protein containing C-terminal deleted 298-aa UGPase1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2327258&req=5

fig04: Nucleotide sequence of the UGPase1 open reading frame, and alignment of the nucleotide and deduced amino acid sequence of the 14th exon 3′-region derived from wild-type (wt) Hwacheong and from the ms-h mutant. Nucleotide numbering starts from the start of translation; the protein sequence is derived from the nucleotide sequence. Wild transcript denotes the UGPase1 transcript of wt Hwacheong, whereas mutant transcripts 1 and 2 represent the 1-bp deleted transcript and the 74-bp inserted transcript found in the ms-h mutant, respectively. The deduced amino acid sequences of the two ms-h mutant transcripts with abnormal sizes demonstrate that both the 1-bp deletion and the 74-bp insertion cause a frame shift and generate stop codons, resulting in truncated 299- and 298-aa proteins, instead of the full-length 469-aa protein. The start codon is set in bold with a row of dots above it. The underlined sequences and asterisk indicate termination codons. The 1-bp deletion site is marked by an arrowhead.SDS-PAGE of recombinant proteins induced with isopropyl-beta-D-thiogalactopyranoside (left) and purified-recombinant UGPase1 proteins (right). The molecular weight of glutathione S-transferase (GST) (23-kDa)-tagged recombinant UGPase1 proteins agrees with the estimated values of 74 and 56 kDa for the full-length UGPase1 protein (469 aa) and the two C-terminal deleted proteins (299 and 298 aa).Activity assays of three recombinant UGPase1 proteins. The formation of NADPH was calculated from the absorption changes at 340 nm monitored for 5 h using an NADPH molar extinction coefficient of 6.22 × 103 m−1 cm−1. •, GST-UGP, GST-tagged recombinant protein containing full-length UGPase1; ▴, GST-mUGP1, GST-tagged recombinant protein containing C-terminal deleted 299-aa UGPase1; ▪, GST-mUGP2, GST-tagged recombinant protein containing C-terminal deleted 298-aa UGPase1.
Mentions: The deduced amino acid sequences of the ms-h mutant transcripts displaying two abnormal sizes suggests that both the 1-bp deletion and the 74-bp insertion cause frame shifts that generate two independent stop codons in the process of translation, resulting in truncated 299- and 298-aa proteins, instead of the 469-aa protein encoded by the wt UGPase1 transcript (Figure 4a). To further confirm whether two C-terminal deleted UGPase1 proteins of the ms-h mutant are nonfunctional, we performed enzyme activity assays in vitro. The UGPase1 cDNAs encoding full-length protein of the wt Hwacheong and two truncated proteins of the ms-h mutant were amplified by PCR with each specific primer, and were transformed into Escherichia coli after vector construction. Glutathione S-transferase (GST)-tagged three-recombinant proteins were purified and separated by SDS-PAGE (Figure 4b). UGPase activity assays were performed with recombinant proteins by monitoring the NADPH formation at 340 nm. As expected, enzyme activity of GST-UGP containing the full-length UGPase1 protein appeared, whereas both GST-mUGP1 and GST-mUGP2 did not show any enzyme activity, indicating that two C-terminal deleted proteins were nonfunctional (Figure 4c). Thus, this single base substitution in the splice site found in the ms-h mutant appears to cause unstable splicing, leading to the presence of two mature transcripts, both with abnormal sizes and, because of the corresponding stop codons, the mRNA transcripts are translated into a truncated and a nonfunctional UGPase1 protein, respectively.

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