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Overexpression of a truncated CTF7 construct leads to pleiotropic defects in reproduction and vegetative growth in Arabidopsis.

Liu D, Makaroff CA - BMC Plant Biol. (2015)

Bottom Line: Inactivation of Arabidopsis CTF7 (AtCTF7) results in severe defects in reproduction and vegetative growth.Transgenic plants expressing 35S:AtCTF7∆B displayed similar vegetative defects, suggesting the defects in 35S:NTAP:AtCTF7∆B plants are caused by high-level expression of AtCTF7∆B.High level expression of AtCTF7∆B disrupts megasporogenesis, megagametogenesis and male meiosis, as well as causing a broad range of vegetative defects, including dwarfism that are inherited in a non-Mendelian fashion.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Eco1/Ctf7 is essential for the establishment of sister chromatid cohesion during S phase of the cell cycle. Inactivation of Ctf7/Eco1 leads to a lethal phenotype in most organisms. Altering Eco1/Ctf7 levels or point mutations in the gene can lead to alterations in nuclear division as well as a wide range of developmental defects. Inactivation of Arabidopsis CTF7 (AtCTF7) results in severe defects in reproduction and vegetative growth.

Results: To further investigate the function(s) of AtCTF7, a tagged version of AtCTF7 and several AtCTF7 deletion constructs were created and transformed into wild type or ctf7 +/- plants. Transgenic plants expressing 35S:NTAP:AtCTF7∆299-345 (AtCTF7∆B) displayed a wide range of phenotypic alterations in reproduction and vegetative growth. Male meiocytes exhibited chromosome fragmentation and uneven chromosome segregation. Mutant ovules contained abnormal megasporocyte-like cells during pre-meiosis, megaspores experienced elongated meiosis and megagametogenesis, and defective megaspores/embryo sacs were produced at various stages. The transgenic plants also exhibited a broad range of vegetative defects, including meristem disruption and dwarfism that were inherited in a non-Mendelian fashion. Transcripts for epigenetically regulated transposable elements (TEs) were elevated in transgenic plants. Transgenic plants expressing 35S:AtCTF7∆B displayed similar vegetative defects, suggesting the defects in 35S:NTAP:AtCTF7∆B plants are caused by high-level expression of AtCTF7∆B.

Conclusions: High level expression of AtCTF7∆B disrupts megasporogenesis, megagametogenesis and male meiosis, as well as causing a broad range of vegetative defects, including dwarfism that are inherited in a non-Mendelian fashion.

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Transcript levels of epigenetically regulated transposable elements and other select genes in 35S:NTAP:AtCTF7∆B plants. (A) Transcript levels of MU1, COPIA28 and soloLTR, are increased dramatically in 35S:NTAP:AtCTF7∆B plants. (B) Transcript levels of genes associated with epigenetic events are differently affected. HDA19 and RDM4 transcript levels are decreased, while MET1 and DMT7 transcripts are not altered. (C) Transcript levels of cell cycle genes, CYCB1.1, CYCA1.1 and RBR, and DNA repair genes, BRCA1 and BRCA2B, are increased. Buds of wild type and non-dwarf, reduced fertile 4th generation Line 11 plants were used. Data are shown as means ± SD (n = 3).
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Fig7: Transcript levels of epigenetically regulated transposable elements and other select genes in 35S:NTAP:AtCTF7∆B plants. (A) Transcript levels of MU1, COPIA28 and soloLTR, are increased dramatically in 35S:NTAP:AtCTF7∆B plants. (B) Transcript levels of genes associated with epigenetic events are differently affected. HDA19 and RDM4 transcript levels are decreased, while MET1 and DMT7 transcripts are not altered. (C) Transcript levels of cell cycle genes, CYCB1.1, CYCA1.1 and RBR, and DNA repair genes, BRCA1 and BRCA2B, are increased. Buds of wild type and non-dwarf, reduced fertile 4th generation Line 11 plants were used. Data are shown as means ± SD (n = 3).

Mentions: Rather, and most surprisingly, the dwarf phenotype was not inherited in a Mendelian fashion (Table 1). When dwarf plants were selfed they produced a mixture of dwarf and non-dwarf, reduced fertile plants. The frequency of dwarf plants produced from selfed dwarf plants was similar to the frequency of dwarf plants resulting from selfing a non-dwarf plant. The stochastic appearance of the dwarf phenotype and variation in phenotypes suggested that the alterations could be the result of epigenetic changes. In order to investigate this possibility, qRT-PCR was carried out to measure the expression levels of several epigenetically regulated transposable elements (TEs), including MU1, COPIA 28 and solo LTR [46], as well as several genes associated with epigenetic events [47-50]. Expression levels of MU1, COPIA 28 and solo LTR were increased between five (MU1) and 24 fold (COPIA 28) in 35S:NTAP:AtCTF7∆B plants (Figure 7A). Subtle changes were also observed in the transcript levels of several siRNA associated genes (Figure 7B). ARGONAUTE1 (AGO1), RDR2 and mir156 transcript levels were reduced approximately 40-60% while AGO4 transcripts were elevated slightly. Transcript levels of HDA19 and RDM4 were also decreased approximately 50% (Figure 7B), while transcript levels of the canonical DNA methylation genes, MET1 and DMT7, did not vary significantly (Figure 7B).Figure 7


Overexpression of a truncated CTF7 construct leads to pleiotropic defects in reproduction and vegetative growth in Arabidopsis.

Liu D, Makaroff CA - BMC Plant Biol. (2015)

Transcript levels of epigenetically regulated transposable elements and other select genes in 35S:NTAP:AtCTF7∆B plants. (A) Transcript levels of MU1, COPIA28 and soloLTR, are increased dramatically in 35S:NTAP:AtCTF7∆B plants. (B) Transcript levels of genes associated with epigenetic events are differently affected. HDA19 and RDM4 transcript levels are decreased, while MET1 and DMT7 transcripts are not altered. (C) Transcript levels of cell cycle genes, CYCB1.1, CYCA1.1 and RBR, and DNA repair genes, BRCA1 and BRCA2B, are increased. Buds of wild type and non-dwarf, reduced fertile 4th generation Line 11 plants were used. Data are shown as means ± SD (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359560&req=5

Fig7: Transcript levels of epigenetically regulated transposable elements and other select genes in 35S:NTAP:AtCTF7∆B plants. (A) Transcript levels of MU1, COPIA28 and soloLTR, are increased dramatically in 35S:NTAP:AtCTF7∆B plants. (B) Transcript levels of genes associated with epigenetic events are differently affected. HDA19 and RDM4 transcript levels are decreased, while MET1 and DMT7 transcripts are not altered. (C) Transcript levels of cell cycle genes, CYCB1.1, CYCA1.1 and RBR, and DNA repair genes, BRCA1 and BRCA2B, are increased. Buds of wild type and non-dwarf, reduced fertile 4th generation Line 11 plants were used. Data are shown as means ± SD (n = 3).
Mentions: Rather, and most surprisingly, the dwarf phenotype was not inherited in a Mendelian fashion (Table 1). When dwarf plants were selfed they produced a mixture of dwarf and non-dwarf, reduced fertile plants. The frequency of dwarf plants produced from selfed dwarf plants was similar to the frequency of dwarf plants resulting from selfing a non-dwarf plant. The stochastic appearance of the dwarf phenotype and variation in phenotypes suggested that the alterations could be the result of epigenetic changes. In order to investigate this possibility, qRT-PCR was carried out to measure the expression levels of several epigenetically regulated transposable elements (TEs), including MU1, COPIA 28 and solo LTR [46], as well as several genes associated with epigenetic events [47-50]. Expression levels of MU1, COPIA 28 and solo LTR were increased between five (MU1) and 24 fold (COPIA 28) in 35S:NTAP:AtCTF7∆B plants (Figure 7A). Subtle changes were also observed in the transcript levels of several siRNA associated genes (Figure 7B). ARGONAUTE1 (AGO1), RDR2 and mir156 transcript levels were reduced approximately 40-60% while AGO4 transcripts were elevated slightly. Transcript levels of HDA19 and RDM4 were also decreased approximately 50% (Figure 7B), while transcript levels of the canonical DNA methylation genes, MET1 and DMT7, did not vary significantly (Figure 7B).Figure 7

Bottom Line: Inactivation of Arabidopsis CTF7 (AtCTF7) results in severe defects in reproduction and vegetative growth.Transgenic plants expressing 35S:AtCTF7∆B displayed similar vegetative defects, suggesting the defects in 35S:NTAP:AtCTF7∆B plants are caused by high-level expression of AtCTF7∆B.High level expression of AtCTF7∆B disrupts megasporogenesis, megagametogenesis and male meiosis, as well as causing a broad range of vegetative defects, including dwarfism that are inherited in a non-Mendelian fashion.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Eco1/Ctf7 is essential for the establishment of sister chromatid cohesion during S phase of the cell cycle. Inactivation of Ctf7/Eco1 leads to a lethal phenotype in most organisms. Altering Eco1/Ctf7 levels or point mutations in the gene can lead to alterations in nuclear division as well as a wide range of developmental defects. Inactivation of Arabidopsis CTF7 (AtCTF7) results in severe defects in reproduction and vegetative growth.

Results: To further investigate the function(s) of AtCTF7, a tagged version of AtCTF7 and several AtCTF7 deletion constructs were created and transformed into wild type or ctf7 +/- plants. Transgenic plants expressing 35S:NTAP:AtCTF7∆299-345 (AtCTF7∆B) displayed a wide range of phenotypic alterations in reproduction and vegetative growth. Male meiocytes exhibited chromosome fragmentation and uneven chromosome segregation. Mutant ovules contained abnormal megasporocyte-like cells during pre-meiosis, megaspores experienced elongated meiosis and megagametogenesis, and defective megaspores/embryo sacs were produced at various stages. The transgenic plants also exhibited a broad range of vegetative defects, including meristem disruption and dwarfism that were inherited in a non-Mendelian fashion. Transcripts for epigenetically regulated transposable elements (TEs) were elevated in transgenic plants. Transgenic plants expressing 35S:AtCTF7∆B displayed similar vegetative defects, suggesting the defects in 35S:NTAP:AtCTF7∆B plants are caused by high-level expression of AtCTF7∆B.

Conclusions: High level expression of AtCTF7∆B disrupts megasporogenesis, megagametogenesis and male meiosis, as well as causing a broad range of vegetative defects, including dwarfism that are inherited in a non-Mendelian fashion.

Show MeSH
Related in: MedlinePlus