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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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Phenotypes of 35S:NTAP:AtCTF7∆B ovules from Line 15. (A-C) Pre-meiotic 35S:NTAP:AtCTF7∆B ovules containing abnormal enlarged cells (white arrows) adjacent to megaspore mother cell-like cells (MMC like; black arrows). (D) Pre-meiotic ovule containing a MMC with degenerating nucleus. (E-Q) Meiotic ovules containing abnormal enlarged cells (white arrows) adjacent to DMs (stars). Cells with functional megaspore-like (FML) characteristics are indicated by black arrows in F, G, H and Q. The abnormal cell in Q contains two nuclei (white arrows). (R-S) Post-meiotic ovules containing FMLs. (R) The FML is associated with abnormal cells, which are not degenerating (white arrows). (S) Extra cells/nuclei are present between the DM (star) and FML. (T) Ovule containing two abnormal cells (white arrows) adjacent to the DM (star). (U-V) Post-meiotic ovules containing female gametophytes with one nucleus. DM, degenerated megaspore are denoted by stars; FML, functional megaspore like; MMC, megaspore mother cell. Size bars, 10 μm.
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Fig5: Phenotypes of 35S:NTAP:AtCTF7∆B ovules from Line 15. (A-C) Pre-meiotic 35S:NTAP:AtCTF7∆B ovules containing abnormal enlarged cells (white arrows) adjacent to megaspore mother cell-like cells (MMC like; black arrows). (D) Pre-meiotic ovule containing a MMC with degenerating nucleus. (E-Q) Meiotic ovules containing abnormal enlarged cells (white arrows) adjacent to DMs (stars). Cells with functional megaspore-like (FML) characteristics are indicated by black arrows in F, G, H and Q. The abnormal cell in Q contains two nuclei (white arrows). (R-S) Post-meiotic ovules containing FMLs. (R) The FML is associated with abnormal cells, which are not degenerating (white arrows). (S) Extra cells/nuclei are present between the DM (star) and FML. (T) Ovule containing two abnormal cells (white arrows) adjacent to the DM (star). (U-V) Post-meiotic ovules containing female gametophytes with one nucleus. DM, degenerated megaspore are denoted by stars; FML, functional megaspore like; MMC, megaspore mother cell. Size bars, 10 μm.

Mentions: To determine if the alterations observed in Line 11 are representative of 35S:NTAP:AtCTF7∆B plants in general, ovules from other lines exhibiting severely reduced fertility (#13 and #15) were examined. Similar to the situation in Line11, pre-meiotic ovules from Line15 contained abnormally enlarged sub-epidermal cells adjacent to megaspore mother-like cells (Figures 5A-C). During meiosis, ovules contained abnormal cells adjacent to degenerated megaspores (Figures 5D-Q). Some ovules contained functional megaspore-like cells (Figure 5F-H,Q). Mature ovules contained differentiated functional megaspore-like cells (Figures 5R,S) and female gametophytes with various defects (Figures 5U,V). Similar to Line 11, female gametophytes from Lines 13 and 15 developed slowly and asynchronously; embryo sacs arrested at FG1 and FG3 (Additional file 1: Figure S6A,C). Additional alterations not observed in Line 11 were also identified, including some ovules that appeared to contain two functional megaspore-like cells (Additional file 1: Figure S6B). In some ovules the middle megaspores appeared to differentiate into functional megaspore-like cells while the megaspores at the chalazal-end degenerated (Additional file 1: Figure S6D), suggesting that ovule polarity was disrupted. All together, 30.4% (48/158) ovules examined in Line 15 plants displayed alterations at pre-meiosis; 66.0% (68/103) of the ovules were defective at meiosis and 79.3% (73/92) of the ovules were defective at FG1. Therefore, common defects associated with NTAP:AtCTF7∆B include a delay and alterations in both megasporogenesis and megagametogenesis with embryo sacs arresting at various stages of development.Figure 5


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)

Phenotypes of 35S:NTAP:AtCTF7∆B ovules from Line 15. (A-C) Pre-meiotic 35S:NTAP:AtCTF7∆B ovules containing abnormal enlarged cells (white arrows) adjacent to megaspore mother cell-like cells (MMC like; black arrows). (D) Pre-meiotic ovule containing a MMC with degenerating nucleus. (E-Q) Meiotic ovules containing abnormal enlarged cells (white arrows) adjacent to DMs (stars). Cells with functional megaspore-like (FML) characteristics are indicated by black arrows in F, G, H and Q. The abnormal cell in Q contains two nuclei (white arrows). (R-S) Post-meiotic ovules containing FMLs. (R) The FML is associated with abnormal cells, which are not degenerating (white arrows). (S) Extra cells/nuclei are present between the DM (star) and FML. (T) Ovule containing two abnormal cells (white arrows) adjacent to the DM (star). (U-V) Post-meiotic ovules containing female gametophytes with one nucleus. DM, degenerated megaspore are denoted by stars; FML, functional megaspore like; MMC, megaspore mother cell. Size bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: Phenotypes of 35S:NTAP:AtCTF7∆B ovules from Line 15. (A-C) Pre-meiotic 35S:NTAP:AtCTF7∆B ovules containing abnormal enlarged cells (white arrows) adjacent to megaspore mother cell-like cells (MMC like; black arrows). (D) Pre-meiotic ovule containing a MMC with degenerating nucleus. (E-Q) Meiotic ovules containing abnormal enlarged cells (white arrows) adjacent to DMs (stars). Cells with functional megaspore-like (FML) characteristics are indicated by black arrows in F, G, H and Q. The abnormal cell in Q contains two nuclei (white arrows). (R-S) Post-meiotic ovules containing FMLs. (R) The FML is associated with abnormal cells, which are not degenerating (white arrows). (S) Extra cells/nuclei are present between the DM (star) and FML. (T) Ovule containing two abnormal cells (white arrows) adjacent to the DM (star). (U-V) Post-meiotic ovules containing female gametophytes with one nucleus. DM, degenerated megaspore are denoted by stars; FML, functional megaspore like; MMC, megaspore mother cell. Size bars, 10 μm.
Mentions: To determine if the alterations observed in Line 11 are representative of 35S:NTAP:AtCTF7∆B plants in general, ovules from other lines exhibiting severely reduced fertility (#13 and #15) were examined. Similar to the situation in Line11, pre-meiotic ovules from Line15 contained abnormally enlarged sub-epidermal cells adjacent to megaspore mother-like cells (Figures 5A-C). During meiosis, ovules contained abnormal cells adjacent to degenerated megaspores (Figures 5D-Q). Some ovules contained functional megaspore-like cells (Figure 5F-H,Q). Mature ovules contained differentiated functional megaspore-like cells (Figures 5R,S) and female gametophytes with various defects (Figures 5U,V). Similar to Line 11, female gametophytes from Lines 13 and 15 developed slowly and asynchronously; embryo sacs arrested at FG1 and FG3 (Additional file 1: Figure S6A,C). Additional alterations not observed in Line 11 were also identified, including some ovules that appeared to contain two functional megaspore-like cells (Additional file 1: Figure S6B). In some ovules the middle megaspores appeared to differentiate into functional megaspore-like cells while the megaspores at the chalazal-end degenerated (Additional file 1: Figure S6D), suggesting that ovule polarity was disrupted. All together, 30.4% (48/158) ovules examined in Line 15 plants displayed alterations at pre-meiosis; 66.0% (68/103) of the ovules were defective at meiosis and 79.3% (73/92) of the ovules were defective at FG1. Therefore, common defects associated with NTAP:AtCTF7∆B include a delay and alterations in both megasporogenesis and megagametogenesis with embryo sacs arresting at various stages of development.Figure 5

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