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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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Embryo sac development is delayed and arrests early in 35S:NTAP:AtCTF7∆B ovules. (A-E) and (J-M) Wild type ovules. (F-I) and (N-P) 35S:NTAP:AtCTF7∆B ovules. (A) FG0 ovule. No FM is identified. (B) Early FG1 ovule showing the FM and DM. The nucellus is not surrounded by the integument. (C) FG1 ovule. The nucellus is surrounded by the outer integument but not the inner integument. (D) FG3 ovule containing a two-nucleate embryo sac. The nucellus is enclosed by the inner integument. (E) FG4 ovule containing a four-nucleate embryo sac. (F-I) Embryo sac development in 35S:NTAP:AtCTF7∆B ovules observed by CLSM. (F) Ovule containing megaspore(s). (G) Ovule containing a FG1 embryo sac. Chalazal end megaspore becomes functional megaspore like (FML) and the other megaspores are degrading. (H) FG1 embryo sac. FML locates at a more chalazal position. (I) FG3 embryo sac containing two nuclei with a vacuole between them. (J-M) Embryo sac development in WT ovules visualized by DIC (also see Additional file 1: Figure S3). (J) Meiotic ovule containing a dyad (stage 2-IV). (K) FG1 ovule. FM (arrow) is uni-nucleate. (L) FG3 ovule, containing an embryo sac with two nuclei and a vacuole. (M) FG4 ovule. (N-P) Embryo sac development in 35S:NTAP:AtCTF7∆B ovules observed by DIC. (N) FG1 embryo sac. The nucellus is surrounded by the outer integument and the inner integument. Extra cells are present between the FM (arrow) and L1 cells. (O-O’) Ovule containing a two-nucleate embryo sac. Non-degenerated L1 cells are present. (P) Ovule with a FG3 embryo sac. FMLs are identified as having distinctly bright nuclear autofluorescence and DMs contain a diffuse signal throughout the cells, but no clearly defined nucleus, defined according to Barrell and Grossniklaus [37]. Size bar = 10 μm. Developmental stages in CLSM and DIC are defined according to Christensen et al. [34,35] and Schneitz et al. [32], respectively.
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Fig4: Embryo sac development is delayed and arrests early in 35S:NTAP:AtCTF7∆B ovules. (A-E) and (J-M) Wild type ovules. (F-I) and (N-P) 35S:NTAP:AtCTF7∆B ovules. (A) FG0 ovule. No FM is identified. (B) Early FG1 ovule showing the FM and DM. The nucellus is not surrounded by the integument. (C) FG1 ovule. The nucellus is surrounded by the outer integument but not the inner integument. (D) FG3 ovule containing a two-nucleate embryo sac. The nucellus is enclosed by the inner integument. (E) FG4 ovule containing a four-nucleate embryo sac. (F-I) Embryo sac development in 35S:NTAP:AtCTF7∆B ovules observed by CLSM. (F) Ovule containing megaspore(s). (G) Ovule containing a FG1 embryo sac. Chalazal end megaspore becomes functional megaspore like (FML) and the other megaspores are degrading. (H) FG1 embryo sac. FML locates at a more chalazal position. (I) FG3 embryo sac containing two nuclei with a vacuole between them. (J-M) Embryo sac development in WT ovules visualized by DIC (also see Additional file 1: Figure S3). (J) Meiotic ovule containing a dyad (stage 2-IV). (K) FG1 ovule. FM (arrow) is uni-nucleate. (L) FG3 ovule, containing an embryo sac with two nuclei and a vacuole. (M) FG4 ovule. (N-P) Embryo sac development in 35S:NTAP:AtCTF7∆B ovules observed by DIC. (N) FG1 embryo sac. The nucellus is surrounded by the outer integument and the inner integument. Extra cells are present between the FM (arrow) and L1 cells. (O-O’) Ovule containing a two-nucleate embryo sac. Non-degenerated L1 cells are present. (P) Ovule with a FG3 embryo sac. FMLs are identified as having distinctly bright nuclear autofluorescence and DMs contain a diffuse signal throughout the cells, but no clearly defined nucleus, defined according to Barrell and Grossniklaus [37]. Size bar = 10 μm. Developmental stages in CLSM and DIC are defined according to Christensen et al. [34,35] and Schneitz et al. [32], respectively.

Mentions: The effects of 35S:NTAP:AtCTF7∆B on megagametogenesis were investigated by confocal laser scanning microscopy (CLSM) and DIC microscopy. During wild type megagametogenesis, the functional megaspore undergoes three rounds of mitosis accompanied by nuclear migration, fusion, degeneration and cellularization to form the final embryo sac (Additional file 1: Figures S3H-L; S4D-J). At FG1, the functional megaspore undergoes mitosis to produce a two-nucleate embryo sac (FG2; Additional file 1: Figure S3H). Formation of a vacuole between the two nuclei marks stage FG3 (Figure 4,D and L; Additional file 1: Figures S3I, S4D). During FG3 the ovule becomes curved and the inner integument embraces the nucellus. A second round of mitosis produces a four-nucleate embryo sac (FG4; Figure 4E,M; Additional file 1: Figures S3J, S4E). This is followed by migration of the two chalazal nuclei from an orthogonal orientation to a chalazal-micropylar orientation. After nuclear migration, a third round of mitosis gives rise to eight nuclei in a 4n + 4n configuration (FG5; Additional file 1: Figure S3K). The two polar nuclei, one from each side, meet at the embryo sac’s micropylar half and fuse to form the central cell, while the antipodal nuclei start to degenerate (Additional file 1: Figure S4H). The central cell has formed and the antipodal nuclei are completed degenerated by FG7 (Additional file 1: Figures S3K, S4I). Prior to fertilization, one synergid nucleus degenerates, such that the embryo sac consists of one egg cell, one central cell and one synergid nucleus (FG8; Additional file 1: Figures S3L, S4J).Figure 4


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)

Embryo sac development is delayed and arrests early in 35S:NTAP:AtCTF7∆B ovules. (A-E) and (J-M) Wild type ovules. (F-I) and (N-P) 35S:NTAP:AtCTF7∆B ovules. (A) FG0 ovule. No FM is identified. (B) Early FG1 ovule showing the FM and DM. The nucellus is not surrounded by the integument. (C) FG1 ovule. The nucellus is surrounded by the outer integument but not the inner integument. (D) FG3 ovule containing a two-nucleate embryo sac. The nucellus is enclosed by the inner integument. (E) FG4 ovule containing a four-nucleate embryo sac. (F-I) Embryo sac development in 35S:NTAP:AtCTF7∆B ovules observed by CLSM. (F) Ovule containing megaspore(s). (G) Ovule containing a FG1 embryo sac. Chalazal end megaspore becomes functional megaspore like (FML) and the other megaspores are degrading. (H) FG1 embryo sac. FML locates at a more chalazal position. (I) FG3 embryo sac containing two nuclei with a vacuole between them. (J-M) Embryo sac development in WT ovules visualized by DIC (also see Additional file 1: Figure S3). (J) Meiotic ovule containing a dyad (stage 2-IV). (K) FG1 ovule. FM (arrow) is uni-nucleate. (L) FG3 ovule, containing an embryo sac with two nuclei and a vacuole. (M) FG4 ovule. (N-P) Embryo sac development in 35S:NTAP:AtCTF7∆B ovules observed by DIC. (N) FG1 embryo sac. The nucellus is surrounded by the outer integument and the inner integument. Extra cells are present between the FM (arrow) and L1 cells. (O-O’) Ovule containing a two-nucleate embryo sac. Non-degenerated L1 cells are present. (P) Ovule with a FG3 embryo sac. FMLs are identified as having distinctly bright nuclear autofluorescence and DMs contain a diffuse signal throughout the cells, but no clearly defined nucleus, defined according to Barrell and Grossniklaus [37]. Size bar = 10 μm. Developmental stages in CLSM and DIC are defined according to Christensen et al. [34,35] and Schneitz et al. [32], respectively.
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Related In: Results  -  Collection

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Fig4: Embryo sac development is delayed and arrests early in 35S:NTAP:AtCTF7∆B ovules. (A-E) and (J-M) Wild type ovules. (F-I) and (N-P) 35S:NTAP:AtCTF7∆B ovules. (A) FG0 ovule. No FM is identified. (B) Early FG1 ovule showing the FM and DM. The nucellus is not surrounded by the integument. (C) FG1 ovule. The nucellus is surrounded by the outer integument but not the inner integument. (D) FG3 ovule containing a two-nucleate embryo sac. The nucellus is enclosed by the inner integument. (E) FG4 ovule containing a four-nucleate embryo sac. (F-I) Embryo sac development in 35S:NTAP:AtCTF7∆B ovules observed by CLSM. (F) Ovule containing megaspore(s). (G) Ovule containing a FG1 embryo sac. Chalazal end megaspore becomes functional megaspore like (FML) and the other megaspores are degrading. (H) FG1 embryo sac. FML locates at a more chalazal position. (I) FG3 embryo sac containing two nuclei with a vacuole between them. (J-M) Embryo sac development in WT ovules visualized by DIC (also see Additional file 1: Figure S3). (J) Meiotic ovule containing a dyad (stage 2-IV). (K) FG1 ovule. FM (arrow) is uni-nucleate. (L) FG3 ovule, containing an embryo sac with two nuclei and a vacuole. (M) FG4 ovule. (N-P) Embryo sac development in 35S:NTAP:AtCTF7∆B ovules observed by DIC. (N) FG1 embryo sac. The nucellus is surrounded by the outer integument and the inner integument. Extra cells are present between the FM (arrow) and L1 cells. (O-O’) Ovule containing a two-nucleate embryo sac. Non-degenerated L1 cells are present. (P) Ovule with a FG3 embryo sac. FMLs are identified as having distinctly bright nuclear autofluorescence and DMs contain a diffuse signal throughout the cells, but no clearly defined nucleus, defined according to Barrell and Grossniklaus [37]. Size bar = 10 μm. Developmental stages in CLSM and DIC are defined according to Christensen et al. [34,35] and Schneitz et al. [32], respectively.
Mentions: The effects of 35S:NTAP:AtCTF7∆B on megagametogenesis were investigated by confocal laser scanning microscopy (CLSM) and DIC microscopy. During wild type megagametogenesis, the functional megaspore undergoes three rounds of mitosis accompanied by nuclear migration, fusion, degeneration and cellularization to form the final embryo sac (Additional file 1: Figures S3H-L; S4D-J). At FG1, the functional megaspore undergoes mitosis to produce a two-nucleate embryo sac (FG2; Additional file 1: Figure S3H). Formation of a vacuole between the two nuclei marks stage FG3 (Figure 4,D and L; Additional file 1: Figures S3I, S4D). During FG3 the ovule becomes curved and the inner integument embraces the nucellus. A second round of mitosis produces a four-nucleate embryo sac (FG4; Figure 4E,M; Additional file 1: Figures S3J, S4E). This is followed by migration of the two chalazal nuclei from an orthogonal orientation to a chalazal-micropylar orientation. After nuclear migration, a third round of mitosis gives rise to eight nuclei in a 4n + 4n configuration (FG5; Additional file 1: Figure S3K). The two polar nuclei, one from each side, meet at the embryo sac’s micropylar half and fuse to form the central cell, while the antipodal nuclei start to degenerate (Additional file 1: Figure S4H). The central cell has formed and the antipodal nuclei are completed degenerated by FG7 (Additional file 1: Figures S3K, S4I). Prior to fertilization, one synergid nucleus degenerates, such that the embryo sac consists of one egg cell, one central cell and one synergid nucleus (FG8; Additional file 1: Figures S3L, S4J).Figure 4

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