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Transcripts of two ent-copalyl diphosphate synthase genes differentially localize in rice plants according to their distinct biological roles.

Toyomasu T, Usui M, Sugawara C, Kanno Y, Sakai A, Takahashi H, Nakazono M, Kuroda M, Miyamoto K, Morimoto Y, Mitsuhashi W, Okada K, Yamaguchi S, Yamane H - J. Exp. Bot. (2014)

Bottom Line: Here, experiments were performed to account for the non-redundant biological function of OsCPS1 and OsCPS2.Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that OsCPS2 transcript levels were drastically lower than those of OsCPS1 in the basal parts, including the meristem of the second-leaf sheaths of rice seedlings. qRT-PCR results using tissue samples prepared by laser microdissection suggested that OsCPS1 transcripts mainly localized in vascular bundle tissues, similar to Arabidopsis CPS, which is responsible for GA biosynthesis, whereas OsCPS2 transcripts mainly localized in epidermal cells that address environmental stressors such as pathogen attack.The results indicate that transcripts of the two ent-CDP synthase genes differentially localize in rice plants according to their distinct biological roles, OsCPS1 for growth and OsCPS2 for defence.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioresource Engineering, Yamagata University, Yamagata 997-8555, Japan toyomasu@tds1.tr.yamagata-u.ac.jp.

No MeSH data available.


Related in: MedlinePlus

Complementation experiment of a loss-of-function OsCPS1 mutant by ectopic expression of OsCPS2. T1 seeds were obtained from several lines of T0 plants, transgenic heterozygous oscps1-1 (NE3024) plants into which OsCPS1p::OsCPS2 cDNA (Fig. 5) was introduced. (A) Results of genotyping and images of T1 seedlings of line 2, the knockout line (no. 3), complemented line (no. 15), and the segregated wild-type line (no. 4). Tos-CPS1, bands from Tos17-inserted OsCPS1 genome DNA (Tos17-F and CPS1-WT-R in Supplementary Fig. S1 available at JXB online); WT-CPS1, bands from wild-type OsCPS1 genomic DNA (CPS1-WT-F and CPS1-WT-R in Supplementary Fig. S1); endoCPS2, bands from native OsCPS2 genomic DNA (with intron); transCPS2, bands from transgene OsCPS2 cDNA (no intron). The sense primer (CPS2-QPCR-F; Supplementary Table S) and antisense primer (CPS2-QPCR-R; Supplementary Table S1) for OsCPS2 genotyping were designed from the nucleotide sequences of exons 10 and 11, respectively. (B) Images of adult T1 rice plants of line 1, the segregated wild-type line (no. 8), and the complemented line (no. 2).
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Figure 6: Complementation experiment of a loss-of-function OsCPS1 mutant by ectopic expression of OsCPS2. T1 seeds were obtained from several lines of T0 plants, transgenic heterozygous oscps1-1 (NE3024) plants into which OsCPS1p::OsCPS2 cDNA (Fig. 5) was introduced. (A) Results of genotyping and images of T1 seedlings of line 2, the knockout line (no. 3), complemented line (no. 15), and the segregated wild-type line (no. 4). Tos-CPS1, bands from Tos17-inserted OsCPS1 genome DNA (Tos17-F and CPS1-WT-R in Supplementary Fig. S1 available at JXB online); WT-CPS1, bands from wild-type OsCPS1 genomic DNA (CPS1-WT-F and CPS1-WT-R in Supplementary Fig. S1); endoCPS2, bands from native OsCPS2 genomic DNA (with intron); transCPS2, bands from transgene OsCPS2 cDNA (no intron). The sense primer (CPS2-QPCR-F; Supplementary Table S) and antisense primer (CPS2-QPCR-R; Supplementary Table S1) for OsCPS2 genotyping were designed from the nucleotide sequences of exons 10 and 11, respectively. (B) Images of adult T1 rice plants of line 1, the segregated wild-type line (no. 8), and the complemented line (no. 2).

Mentions: The OsCPS1p fragment was accordingly fused to OsCPS2 ORF cDNA (Fig. 5) in the pZH2B vector and introduced into a heterozygous oscps1-1 mutant by Agrobacterium infection. oscps1-1 is a Tos17-inserted OsCPS1 mutant that displays a severe dwarf phenotype caused by GA deficiency (Sakamoto et al., 2004). Because the homozygous oscps1-1 mutant was incapable of dedifferentiation, a heterozygous mutant was transformed by Agrobacterium infection, and self-pollination of transgenic plants produced T1 seeds. Transgenic homozygous oscps1-1 (complemented line), non-transgenic homozygous oscps1-1 (knockout line), and non-transgenic wild-type (segregated wild-type line) plants were observed by genotyping the T1 seedlings (Fig. 6A). Four complemented line T1 plants were identified, and displayed almost the same height as that of the segregated wild-type lines (Fig. 6B). OsCPS1 and OsCPS2 expression patterns were analysed in the second-leaf sheath to check successful ectopic expression of OsCPS2. qRT–PCR showed that OsCPS2 transcripts accumulated in basal parts of the second-leaf sheath of T2 complemented line seedlings, similar to OsCPS1 transcripts in wild-type Nipponbare (Fig. 3B) and in segregated wild-type line seedlings, whereas wild-type OsCPS1 transcripts were not detected in complemented line plants (Fig. 7). These results indicate that OsCPS2 expression under the OsCPS1 promoter rescued the oscps1-1 mutant phenotype.


Transcripts of two ent-copalyl diphosphate synthase genes differentially localize in rice plants according to their distinct biological roles.

Toyomasu T, Usui M, Sugawara C, Kanno Y, Sakai A, Takahashi H, Nakazono M, Kuroda M, Miyamoto K, Morimoto Y, Mitsuhashi W, Okada K, Yamaguchi S, Yamane H - J. Exp. Bot. (2014)

Complementation experiment of a loss-of-function OsCPS1 mutant by ectopic expression of OsCPS2. T1 seeds were obtained from several lines of T0 plants, transgenic heterozygous oscps1-1 (NE3024) plants into which OsCPS1p::OsCPS2 cDNA (Fig. 5) was introduced. (A) Results of genotyping and images of T1 seedlings of line 2, the knockout line (no. 3), complemented line (no. 15), and the segregated wild-type line (no. 4). Tos-CPS1, bands from Tos17-inserted OsCPS1 genome DNA (Tos17-F and CPS1-WT-R in Supplementary Fig. S1 available at JXB online); WT-CPS1, bands from wild-type OsCPS1 genomic DNA (CPS1-WT-F and CPS1-WT-R in Supplementary Fig. S1); endoCPS2, bands from native OsCPS2 genomic DNA (with intron); transCPS2, bands from transgene OsCPS2 cDNA (no intron). The sense primer (CPS2-QPCR-F; Supplementary Table S) and antisense primer (CPS2-QPCR-R; Supplementary Table S1) for OsCPS2 genotyping were designed from the nucleotide sequences of exons 10 and 11, respectively. (B) Images of adult T1 rice plants of line 1, the segregated wild-type line (no. 8), and the complemented line (no. 2).
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Figure 6: Complementation experiment of a loss-of-function OsCPS1 mutant by ectopic expression of OsCPS2. T1 seeds were obtained from several lines of T0 plants, transgenic heterozygous oscps1-1 (NE3024) plants into which OsCPS1p::OsCPS2 cDNA (Fig. 5) was introduced. (A) Results of genotyping and images of T1 seedlings of line 2, the knockout line (no. 3), complemented line (no. 15), and the segregated wild-type line (no. 4). Tos-CPS1, bands from Tos17-inserted OsCPS1 genome DNA (Tos17-F and CPS1-WT-R in Supplementary Fig. S1 available at JXB online); WT-CPS1, bands from wild-type OsCPS1 genomic DNA (CPS1-WT-F and CPS1-WT-R in Supplementary Fig. S1); endoCPS2, bands from native OsCPS2 genomic DNA (with intron); transCPS2, bands from transgene OsCPS2 cDNA (no intron). The sense primer (CPS2-QPCR-F; Supplementary Table S) and antisense primer (CPS2-QPCR-R; Supplementary Table S1) for OsCPS2 genotyping were designed from the nucleotide sequences of exons 10 and 11, respectively. (B) Images of adult T1 rice plants of line 1, the segregated wild-type line (no. 8), and the complemented line (no. 2).
Mentions: The OsCPS1p fragment was accordingly fused to OsCPS2 ORF cDNA (Fig. 5) in the pZH2B vector and introduced into a heterozygous oscps1-1 mutant by Agrobacterium infection. oscps1-1 is a Tos17-inserted OsCPS1 mutant that displays a severe dwarf phenotype caused by GA deficiency (Sakamoto et al., 2004). Because the homozygous oscps1-1 mutant was incapable of dedifferentiation, a heterozygous mutant was transformed by Agrobacterium infection, and self-pollination of transgenic plants produced T1 seeds. Transgenic homozygous oscps1-1 (complemented line), non-transgenic homozygous oscps1-1 (knockout line), and non-transgenic wild-type (segregated wild-type line) plants were observed by genotyping the T1 seedlings (Fig. 6A). Four complemented line T1 plants were identified, and displayed almost the same height as that of the segregated wild-type lines (Fig. 6B). OsCPS1 and OsCPS2 expression patterns were analysed in the second-leaf sheath to check successful ectopic expression of OsCPS2. qRT–PCR showed that OsCPS2 transcripts accumulated in basal parts of the second-leaf sheath of T2 complemented line seedlings, similar to OsCPS1 transcripts in wild-type Nipponbare (Fig. 3B) and in segregated wild-type line seedlings, whereas wild-type OsCPS1 transcripts were not detected in complemented line plants (Fig. 7). These results indicate that OsCPS2 expression under the OsCPS1 promoter rescued the oscps1-1 mutant phenotype.

Bottom Line: Here, experiments were performed to account for the non-redundant biological function of OsCPS1 and OsCPS2.Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that OsCPS2 transcript levels were drastically lower than those of OsCPS1 in the basal parts, including the meristem of the second-leaf sheaths of rice seedlings. qRT-PCR results using tissue samples prepared by laser microdissection suggested that OsCPS1 transcripts mainly localized in vascular bundle tissues, similar to Arabidopsis CPS, which is responsible for GA biosynthesis, whereas OsCPS2 transcripts mainly localized in epidermal cells that address environmental stressors such as pathogen attack.The results indicate that transcripts of the two ent-CDP synthase genes differentially localize in rice plants according to their distinct biological roles, OsCPS1 for growth and OsCPS2 for defence.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioresource Engineering, Yamagata University, Yamagata 997-8555, Japan toyomasu@tds1.tr.yamagata-u.ac.jp.

No MeSH data available.


Related in: MedlinePlus