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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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Early ovule development is disrupted in 35S:NTAP:AtCTF7∆B plants. Wild type (A-D) and 35S:NTAP:AtCTF7∆B (E-I ) ovules were analyzed by differential interference contrast microscopy. (A) Pre-meiotic ovule containing a single megaspore mother cell (MMC; stage 1-II). (B) Pre-meiotic ovule at stage 2-III. Inner and outer integuments start to initiate. (C) Meiotic ovule containing a dyad after meiosis I (stage 2-IV). (D) Meiotic ovule containing a tetrad after meiosis II. (E-F) 35S:NTAP:AtCTF7∆B ovules at pre-meiotic stages. (E) Right ovule containing an abnormal, enlarged cell (white arrow) adjacent to a MMC-like cell (black arrow). Adjacent ovules are different in size. (F-F’) Left ovule containing two abnormal, enlarged cells (white arrows) adjacent to a MMC-like cell (black arrow). Ovules are different in size and stage (left: stage 1-II, right: stage 2-III) and point in the same direction. (G-I) 35S:NTAP:AtCTF7∆B ovules at meiosis. (G) Ovule containing a large cell with prominent nucleus (arrow) resembling a MMC. An extra cell is present at the position of the degenerated megaspores, between the MMC and L1 cells. (H) Ovule containing a MMC-like cell with a prominent nucleus in the central region of the ovule. Ovule is enlarged and extra cells are between the MMC and L1 cells. (H’) Magnified view of MMC from H. (I) Ovule containing two cells with prominent nuclei (arrows) in the central region of the ovule. The two cells are separated and resemble a dyad. Extra cells surround the dyad. (I’) Magnified view of I. Size bar = 10 μm. Developmental stages are defined according to Schneitz et al. [32].
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Fig3: Early ovule development is disrupted in 35S:NTAP:AtCTF7∆B plants. Wild type (A-D) and 35S:NTAP:AtCTF7∆B (E-I ) ovules were analyzed by differential interference contrast microscopy. (A) Pre-meiotic ovule containing a single megaspore mother cell (MMC; stage 1-II). (B) Pre-meiotic ovule at stage 2-III. Inner and outer integuments start to initiate. (C) Meiotic ovule containing a dyad after meiosis I (stage 2-IV). (D) Meiotic ovule containing a tetrad after meiosis II. (E-F) 35S:NTAP:AtCTF7∆B ovules at pre-meiotic stages. (E) Right ovule containing an abnormal, enlarged cell (white arrow) adjacent to a MMC-like cell (black arrow). Adjacent ovules are different in size. (F-F’) Left ovule containing two abnormal, enlarged cells (white arrows) adjacent to a MMC-like cell (black arrow). Ovules are different in size and stage (left: stage 1-II, right: stage 2-III) and point in the same direction. (G-I) 35S:NTAP:AtCTF7∆B ovules at meiosis. (G) Ovule containing a large cell with prominent nucleus (arrow) resembling a MMC. An extra cell is present at the position of the degenerated megaspores, between the MMC and L1 cells. (H) Ovule containing a MMC-like cell with a prominent nucleus in the central region of the ovule. Ovule is enlarged and extra cells are between the MMC and L1 cells. (H’) Magnified view of MMC from H. (I) Ovule containing two cells with prominent nuclei (arrows) in the central region of the ovule. The two cells are separated and resemble a dyad. Extra cells surround the dyad. (I’) Magnified view of I. Size bar = 10 μm. Developmental stages are defined according to Schneitz et al. [32].

Mentions: To elucidate the function(s) of 35S:NTAP:AtCTF7∆B in ovule development, ovules from wild type and Line 11 plants were analyzed by differential interference contrast (DIC) microscopy. In wild type siliques, archesporial cells are specified from the subepidermal cell layers and differentiate into megaspore mother cells, which are initially unpolarized and become polarized prior to the start of meiosis (Additional file 1: Figure S3A). Two rounds of meiosis produce a tetrad of four haploid megaspores (Figure 3C,D; Additional file 1: Figure S3B-E) [32]. Prior to FG1, the megaspore mother cell, dyad and tetrad are all adjacent to L1 cells (Figures 3A-D) [33]. When the ovule reaches FG1, the megaspore at the chalazal-end differentiates into the functional megaspore while the other three megaspores undergo programmed cell death (Additional file 1: Figures S3F, G; Figures S4B, C) [34,35]. In wild type siliques, adjacent ovules are similar in size and point in opposite directions (Figures 3A,B).Figure 3


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)

Early ovule development is disrupted in 35S:NTAP:AtCTF7∆B plants. Wild type (A-D) and 35S:NTAP:AtCTF7∆B (E-I ) ovules were analyzed by differential interference contrast microscopy. (A) Pre-meiotic ovule containing a single megaspore mother cell (MMC; stage 1-II). (B) Pre-meiotic ovule at stage 2-III. Inner and outer integuments start to initiate. (C) Meiotic ovule containing a dyad after meiosis I (stage 2-IV). (D) Meiotic ovule containing a tetrad after meiosis II. (E-F) 35S:NTAP:AtCTF7∆B ovules at pre-meiotic stages. (E) Right ovule containing an abnormal, enlarged cell (white arrow) adjacent to a MMC-like cell (black arrow). Adjacent ovules are different in size. (F-F’) Left ovule containing two abnormal, enlarged cells (white arrows) adjacent to a MMC-like cell (black arrow). Ovules are different in size and stage (left: stage 1-II, right: stage 2-III) and point in the same direction. (G-I) 35S:NTAP:AtCTF7∆B ovules at meiosis. (G) Ovule containing a large cell with prominent nucleus (arrow) resembling a MMC. An extra cell is present at the position of the degenerated megaspores, between the MMC and L1 cells. (H) Ovule containing a MMC-like cell with a prominent nucleus in the central region of the ovule. Ovule is enlarged and extra cells are between the MMC and L1 cells. (H’) Magnified view of MMC from H. (I) Ovule containing two cells with prominent nuclei (arrows) in the central region of the ovule. The two cells are separated and resemble a dyad. Extra cells surround the dyad. (I’) Magnified view of I. Size bar = 10 μm. Developmental stages are defined according to Schneitz et al. [32].
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: Early ovule development is disrupted in 35S:NTAP:AtCTF7∆B plants. Wild type (A-D) and 35S:NTAP:AtCTF7∆B (E-I ) ovules were analyzed by differential interference contrast microscopy. (A) Pre-meiotic ovule containing a single megaspore mother cell (MMC; stage 1-II). (B) Pre-meiotic ovule at stage 2-III. Inner and outer integuments start to initiate. (C) Meiotic ovule containing a dyad after meiosis I (stage 2-IV). (D) Meiotic ovule containing a tetrad after meiosis II. (E-F) 35S:NTAP:AtCTF7∆B ovules at pre-meiotic stages. (E) Right ovule containing an abnormal, enlarged cell (white arrow) adjacent to a MMC-like cell (black arrow). Adjacent ovules are different in size. (F-F’) Left ovule containing two abnormal, enlarged cells (white arrows) adjacent to a MMC-like cell (black arrow). Ovules are different in size and stage (left: stage 1-II, right: stage 2-III) and point in the same direction. (G-I) 35S:NTAP:AtCTF7∆B ovules at meiosis. (G) Ovule containing a large cell with prominent nucleus (arrow) resembling a MMC. An extra cell is present at the position of the degenerated megaspores, between the MMC and L1 cells. (H) Ovule containing a MMC-like cell with a prominent nucleus in the central region of the ovule. Ovule is enlarged and extra cells are between the MMC and L1 cells. (H’) Magnified view of MMC from H. (I) Ovule containing two cells with prominent nuclei (arrows) in the central region of the ovule. The two cells are separated and resemble a dyad. Extra cells surround the dyad. (I’) Magnified view of I. Size bar = 10 μm. Developmental stages are defined according to Schneitz et al. [32].
Mentions: To elucidate the function(s) of 35S:NTAP:AtCTF7∆B in ovule development, ovules from wild type and Line 11 plants were analyzed by differential interference contrast (DIC) microscopy. In wild type siliques, archesporial cells are specified from the subepidermal cell layers and differentiate into megaspore mother cells, which are initially unpolarized and become polarized prior to the start of meiosis (Additional file 1: Figure S3A). Two rounds of meiosis produce a tetrad of four haploid megaspores (Figure 3C,D; Additional file 1: Figure S3B-E) [32]. Prior to FG1, the megaspore mother cell, dyad and tetrad are all adjacent to L1 cells (Figures 3A-D) [33]. When the ovule reaches FG1, the megaspore at the chalazal-end differentiates into the functional megaspore while the other three megaspores undergo programmed cell death (Additional file 1: Figures S3F, G; Figures S4B, C) [34,35]. In wild type siliques, adjacent ovules are similar in size and point in opposite directions (Figures 3A,B).Figure 3

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