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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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35S:NTAP:AtCTF7∆B plants exhibit reduced fertility. (A) Open siliques from wild type (Ai) and 35S:NTAP:AtCTF7∆B (Aii,Aiii) plants. i, Wild type silique with full seed set. ii, Silique from a 35S:NTAP:AtCTF7∆B plant (Line 19) exhibiting a weak phenotype. iii, Silique from a 35S:NTAP:AtCTF7∆B plant (Line 11) exhibiting a strong phenotype. Arrows indicate shriveled, unfertilized ovules. White stars show white, plump seeds, which aborted after fertilization. Scale bar = 0.5 cm. (B) Alexander staining of mature anthers from wild type (i) and 35S:NTAP:AtCTF7∆B (ii, iii) plants. i, Wild type anther. ii, Anther from Line 19. The anther is smaller and contains less pollen; all the pollen is viable. iii, Anther from Line 11. The anther is smaller and contains low numbers of viable pollen. Scale bar = 50 μm. (C). Expression analysis of AtCTF7 in wild type and 35S:NTAP:AtCTF7∆B plants. Transcript levels of total AtCTF7 (35S:NTAP:AtCTF7∆B and native AtCTF7) and native AtCTF7 are increased in 35S:NTAP:AtCTF7∆B plants with Line 11 plants exhibiting the highest levels. Buds of wild type, non-dwarf, 4th generation Line 11 plants and 4th generation Line19 plants were used for this experiment. Data are shown as means ± SD (n = 3).
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Fig1: 35S:NTAP:AtCTF7∆B plants exhibit reduced fertility. (A) Open siliques from wild type (Ai) and 35S:NTAP:AtCTF7∆B (Aii,Aiii) plants. i, Wild type silique with full seed set. ii, Silique from a 35S:NTAP:AtCTF7∆B plant (Line 19) exhibiting a weak phenotype. iii, Silique from a 35S:NTAP:AtCTF7∆B plant (Line 11) exhibiting a strong phenotype. Arrows indicate shriveled, unfertilized ovules. White stars show white, plump seeds, which aborted after fertilization. Scale bar = 0.5 cm. (B) Alexander staining of mature anthers from wild type (i) and 35S:NTAP:AtCTF7∆B (ii, iii) plants. i, Wild type anther. ii, Anther from Line 19. The anther is smaller and contains less pollen; all the pollen is viable. iii, Anther from Line 11. The anther is smaller and contains low numbers of viable pollen. Scale bar = 50 μm. (C). Expression analysis of AtCTF7 in wild type and 35S:NTAP:AtCTF7∆B plants. Transcript levels of total AtCTF7 (35S:NTAP:AtCTF7∆B and native AtCTF7) and native AtCTF7 are increased in 35S:NTAP:AtCTF7∆B plants with Line 11 plants exhibiting the highest levels. Buds of wild type, non-dwarf, 4th generation Line 11 plants and 4th generation Line19 plants were used for this experiment. Data are shown as means ± SD (n = 3).

Mentions: An AtCTF7 construct missing the C-terminal 46 amino acids (∆299-345) was generated, fused with NTAP [30] and expressed from the CaMV 35S promoter in wild-type Columbia plants (35S:NTAP:AtCTF7∆B; Additional file 1: Figure S1). Twenty out of the 36 independent lines examined exhibited reduced fertility, with fertility levels varying significantly between the lines. Plants exhibiting a weak phenotype, which accounted for two of the 20 reduced fertility lines, produced shorter siliques with reduced numbers of seeds, but the seeds appeared normal (Figure 1Aii). For example Line 19 produced 39.2 ± 3.9 seeds per silique (n = 35) compared to wild type plants that produce 54.2 ± 4.1 seeds per silique (n = 35). Anthers from Line 19 plants were smaller and contained reduced numbers of pollen (574 vs 1175 in wild type), but the pollen appeared viable (Figure 1Bii). The other 18 reduced fertility lines exhibited more severe defects. These plants produced siliques containing large numbers of unfertilized ovules and aborted seeds (Figure 1Aiii). Unfertilized ovules appeared as white dots, resembling the situation in atctf7-1 plants [17]. Aborted seeds appeared white and plump, similar to seeds containing arrested embryos in Atctf7-1+/− plants [16]. Seed set varied considerably between the lines, ranging from 13.3 ± 5.6 seeds per silique to 34.7 ± 8.4 seeds per silique. Anthers from these plants typically contained reduced numbers of pollen, much of which was not viable. For example, anthers from Line 11 produced on average approximately 210 pollen, of which only 20% was viable (Figure 1Biii). Given that most lines exhibited severe fertility defects, one representative line (#11) was chosen and characterized in detail.Figure 1


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)

35S:NTAP:AtCTF7∆B plants exhibit reduced fertility. (A) Open siliques from wild type (Ai) and 35S:NTAP:AtCTF7∆B (Aii,Aiii) plants. i, Wild type silique with full seed set. ii, Silique from a 35S:NTAP:AtCTF7∆B plant (Line 19) exhibiting a weak phenotype. iii, Silique from a 35S:NTAP:AtCTF7∆B plant (Line 11) exhibiting a strong phenotype. Arrows indicate shriveled, unfertilized ovules. White stars show white, plump seeds, which aborted after fertilization. Scale bar = 0.5 cm. (B) Alexander staining of mature anthers from wild type (i) and 35S:NTAP:AtCTF7∆B (ii, iii) plants. i, Wild type anther. ii, Anther from Line 19. The anther is smaller and contains less pollen; all the pollen is viable. iii, Anther from Line 11. The anther is smaller and contains low numbers of viable pollen. Scale bar = 50 μm. (C). Expression analysis of AtCTF7 in wild type and 35S:NTAP:AtCTF7∆B plants. Transcript levels of total AtCTF7 (35S:NTAP:AtCTF7∆B and native AtCTF7) and native AtCTF7 are increased in 35S:NTAP:AtCTF7∆B plants with Line 11 plants exhibiting the highest levels. Buds of wild type, non-dwarf, 4th generation Line 11 plants and 4th generation Line19 plants were used for this experiment. 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

Fig1: 35S:NTAP:AtCTF7∆B plants exhibit reduced fertility. (A) Open siliques from wild type (Ai) and 35S:NTAP:AtCTF7∆B (Aii,Aiii) plants. i, Wild type silique with full seed set. ii, Silique from a 35S:NTAP:AtCTF7∆B plant (Line 19) exhibiting a weak phenotype. iii, Silique from a 35S:NTAP:AtCTF7∆B plant (Line 11) exhibiting a strong phenotype. Arrows indicate shriveled, unfertilized ovules. White stars show white, plump seeds, which aborted after fertilization. Scale bar = 0.5 cm. (B) Alexander staining of mature anthers from wild type (i) and 35S:NTAP:AtCTF7∆B (ii, iii) plants. i, Wild type anther. ii, Anther from Line 19. The anther is smaller and contains less pollen; all the pollen is viable. iii, Anther from Line 11. The anther is smaller and contains low numbers of viable pollen. Scale bar = 50 μm. (C). Expression analysis of AtCTF7 in wild type and 35S:NTAP:AtCTF7∆B plants. Transcript levels of total AtCTF7 (35S:NTAP:AtCTF7∆B and native AtCTF7) and native AtCTF7 are increased in 35S:NTAP:AtCTF7∆B plants with Line 11 plants exhibiting the highest levels. Buds of wild type, non-dwarf, 4th generation Line 11 plants and 4th generation Line19 plants were used for this experiment. Data are shown as means ± SD (n = 3).
Mentions: An AtCTF7 construct missing the C-terminal 46 amino acids (∆299-345) was generated, fused with NTAP [30] and expressed from the CaMV 35S promoter in wild-type Columbia plants (35S:NTAP:AtCTF7∆B; Additional file 1: Figure S1). Twenty out of the 36 independent lines examined exhibited reduced fertility, with fertility levels varying significantly between the lines. Plants exhibiting a weak phenotype, which accounted for two of the 20 reduced fertility lines, produced shorter siliques with reduced numbers of seeds, but the seeds appeared normal (Figure 1Aii). For example Line 19 produced 39.2 ± 3.9 seeds per silique (n = 35) compared to wild type plants that produce 54.2 ± 4.1 seeds per silique (n = 35). Anthers from Line 19 plants were smaller and contained reduced numbers of pollen (574 vs 1175 in wild type), but the pollen appeared viable (Figure 1Bii). The other 18 reduced fertility lines exhibited more severe defects. These plants produced siliques containing large numbers of unfertilized ovules and aborted seeds (Figure 1Aiii). Unfertilized ovules appeared as white dots, resembling the situation in atctf7-1 plants [17]. Aborted seeds appeared white and plump, similar to seeds containing arrested embryos in Atctf7-1+/− plants [16]. Seed set varied considerably between the lines, ranging from 13.3 ± 5.6 seeds per silique to 34.7 ± 8.4 seeds per silique. Anthers from these plants typically contained reduced numbers of pollen, much of which was not viable. For example, anthers from Line 11 produced on average approximately 210 pollen, of which only 20% was viable (Figure 1Biii). Given that most lines exhibited severe fertility defects, one representative line (#11) was chosen and characterized in detail.Figure 1

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