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Functional analysis of alternative splicing of the FLOWERING LOCUS T orthologous gene in Chrysanthemum morifolium

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ABSTRACT

As the junction of floral development pathways, the FLOWERING LOCUS T (FT) protein called ‘florigen’ plays an important role in the process of plant flowering through signal integration. We isolated four transcripts encoding different isoforms of a FT orthologous gene CmFTL1, from Chrysanthemum morifolium cultivar ‘Jimba’. Sequence alignments suggested that the four transcripts are related to the intron 1. Expression analysis showed that four alternative splicing (AS) forms of CmFTL1 varied depending on the developmental stage of the flower. The functional complement experiment using an Arabidopsis mutant ft-10 revealed that the archetypal and AS forms of CmFTL1 had the function of complementing late flower phenotype in different levels. In addition, transgenic confirmation at transcript level showed CmFTL1 and CmFTL1ast coexist in the same tissue type at the same developmental stage, indicating a post-transcriptional modification of CmFTL1 in Arabidopsis. Moreover, ectopic expression of different AS forms in chrysanthemum resulted in the development of multiple altered phenotypes, varying degrees of early flowering. We found that an alternative splicing form (CmFTL1-astE134) without the exon 2 lacked the ability causing the earlier flower phenotype. The evidence in this study indicates that complex alternative processing of CmFTL1 transcripts in C. morifolium may be associated with flowering regulation and hold some potential for biotechnical engineering to create early-flowering phenotypes in ornamental cultivars.

No MeSH data available.


Analysis of the amino-acid alignments of CmFTL1 and CmFTL1asts. Multiple alignment on GENEBANK: AtFT (AB027504.1), AtTFL1 (NM_120465.2), AtTSF (GQ884981.1), BvFT1 (HM448910.1), BvFT2 (HM448912.1), HaFT1 (GQ884981.1), HaFT2 (GQ884987.1), HaFT4 (GU985573.1), LsFT (BAK14368.1), OsHd3a (JX472280), OsRFT1 (AB426873.1). (a) Partial amino-acid sequence alignment of the phosphatidylethanol-amine-binding protein (PEBP) family members. Asterisks indicate the residues Tyr85 (Y)/Gln140 (Q) and His88 (H)/Asp144 (D) contributing to FT and TFL1 functioning, respectively.30, 31 The conserved segment region B in the fourth exon, corresponding to the external loop of the PEBP family proteins, is boxed. (b) Alignment of amino-acid sequence of CmFTL1 and its alternative splicing transcripts, the asterisks separate the exons into Exon1, Exon2, Exon3 and Exon4.
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fig2: Analysis of the amino-acid alignments of CmFTL1 and CmFTL1asts. Multiple alignment on GENEBANK: AtFT (AB027504.1), AtTFL1 (NM_120465.2), AtTSF (GQ884981.1), BvFT1 (HM448910.1), BvFT2 (HM448912.1), HaFT1 (GQ884981.1), HaFT2 (GQ884987.1), HaFT4 (GU985573.1), LsFT (BAK14368.1), OsHd3a (JX472280), OsRFT1 (AB426873.1). (a) Partial amino-acid sequence alignment of the phosphatidylethanol-amine-binding protein (PEBP) family members. Asterisks indicate the residues Tyr85 (Y)/Gln140 (Q) and His88 (H)/Asp144 (D) contributing to FT and TFL1 functioning, respectively.30, 31 The conserved segment region B in the fourth exon, corresponding to the external loop of the PEBP family proteins, is boxed. (b) Alignment of amino-acid sequence of CmFTL1 and its alternative splicing transcripts, the asterisks separate the exons into Exon1, Exon2, Exon3 and Exon4.

Mentions: The archetypal CmFTL1 transcript encodes a 174 residue protein which shares 72% sequence identity with the A. thaliana FT protein, 90% with lettuce LsFT and 89% with rice RFT1 (Table 1). The residues Tyr85 and Gln140, both of which are critical for the functionality of AtFT,30,31 were present in CmFTL1, at positions 84 and 139, respectively (Figure 2a). Those AS transcripts in which the number of nucleotides inserted/deleted was not a multiple of three are expected to encode a frame shift product, while those truncated by the creation of a premature termination codon are expected to have lost residues in exon 4 essential for FT-like activity30,31 (Figure 2b).


Functional analysis of alternative splicing of the FLOWERING LOCUS T orthologous gene in Chrysanthemum morifolium
Analysis of the amino-acid alignments of CmFTL1 and CmFTL1asts. Multiple alignment on GENEBANK: AtFT (AB027504.1), AtTFL1 (NM_120465.2), AtTSF (GQ884981.1), BvFT1 (HM448910.1), BvFT2 (HM448912.1), HaFT1 (GQ884981.1), HaFT2 (GQ884987.1), HaFT4 (GU985573.1), LsFT (BAK14368.1), OsHd3a (JX472280), OsRFT1 (AB426873.1). (a) Partial amino-acid sequence alignment of the phosphatidylethanol-amine-binding protein (PEBP) family members. Asterisks indicate the residues Tyr85 (Y)/Gln140 (Q) and His88 (H)/Asp144 (D) contributing to FT and TFL1 functioning, respectively.30, 31 The conserved segment region B in the fourth exon, corresponding to the external loop of the PEBP family proteins, is boxed. (b) Alignment of amino-acid sequence of CmFTL1 and its alternative splicing transcripts, the asterisks separate the exons into Exon1, Exon2, Exon3 and Exon4.
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Related In: Results  -  Collection

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fig2: Analysis of the amino-acid alignments of CmFTL1 and CmFTL1asts. Multiple alignment on GENEBANK: AtFT (AB027504.1), AtTFL1 (NM_120465.2), AtTSF (GQ884981.1), BvFT1 (HM448910.1), BvFT2 (HM448912.1), HaFT1 (GQ884981.1), HaFT2 (GQ884987.1), HaFT4 (GU985573.1), LsFT (BAK14368.1), OsHd3a (JX472280), OsRFT1 (AB426873.1). (a) Partial amino-acid sequence alignment of the phosphatidylethanol-amine-binding protein (PEBP) family members. Asterisks indicate the residues Tyr85 (Y)/Gln140 (Q) and His88 (H)/Asp144 (D) contributing to FT and TFL1 functioning, respectively.30, 31 The conserved segment region B in the fourth exon, corresponding to the external loop of the PEBP family proteins, is boxed. (b) Alignment of amino-acid sequence of CmFTL1 and its alternative splicing transcripts, the asterisks separate the exons into Exon1, Exon2, Exon3 and Exon4.
Mentions: The archetypal CmFTL1 transcript encodes a 174 residue protein which shares 72% sequence identity with the A. thaliana FT protein, 90% with lettuce LsFT and 89% with rice RFT1 (Table 1). The residues Tyr85 and Gln140, both of which are critical for the functionality of AtFT,30,31 were present in CmFTL1, at positions 84 and 139, respectively (Figure 2a). Those AS transcripts in which the number of nucleotides inserted/deleted was not a multiple of three are expected to encode a frame shift product, while those truncated by the creation of a premature termination codon are expected to have lost residues in exon 4 essential for FT-like activity30,31 (Figure 2b).

View Article: PubMed Central - PubMed

ABSTRACT

As the junction of floral development pathways, the FLOWERING LOCUS T (FT) protein called ‘florigen’ plays an important role in the process of plant flowering through signal integration. We isolated four transcripts encoding different isoforms of a FT orthologous gene CmFTL1, from Chrysanthemum morifolium cultivar ‘Jimba’. Sequence alignments suggested that the four transcripts are related to the intron 1. Expression analysis showed that four alternative splicing (AS) forms of CmFTL1 varied depending on the developmental stage of the flower. The functional complement experiment using an Arabidopsis mutant ft-10 revealed that the archetypal and AS forms of CmFTL1 had the function of complementing late flower phenotype in different levels. In addition, transgenic confirmation at transcript level showed CmFTL1 and CmFTL1ast coexist in the same tissue type at the same developmental stage, indicating a post-transcriptional modification of CmFTL1 in Arabidopsis. Moreover, ectopic expression of different AS forms in chrysanthemum resulted in the development of multiple altered phenotypes, varying degrees of early flowering. We found that an alternative splicing form (CmFTL1-astE134) without the exon 2 lacked the ability causing the earlier flower phenotype. The evidence in this study indicates that complex alternative processing of CmFTL1 transcripts in C. morifolium may be associated with flowering regulation and hold some potential for biotechnical engineering to create early-flowering phenotypes in ornamental cultivars.

No MeSH data available.