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Liu and Makaroff BMC Plant Biology (2015) 15:74 DOI 10.1186/s12870-015-0452-2 RESEARCH ARTICLE Open Access Overexpression of a truncated CTF7 construct leads to pleiotropic defects in reproduction and vegetative growth in Arabidopsis Desheng Liu and Christopher A Makaroff* 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: AtCTF7Δ299–345 (AtCTF7ΔB) 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:AtCTF7ΔB displayed similar vegetative defects, suggesting the defects in 35S:NTAP:AtCTF7ΔB plants are caused by high-level expression of AtCTF7ΔB. Conclusions: High level expression of AtCTF7ΔB 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. Keywords: Meiosis, Sister chromatid cohesion, Megasporogenesis, Megagametogenesis, Megaspore mother cell, Functional megaspore-like, Epigenetic Background The precise establishment and release of sister chromatid cohesion are vital for cell division during mitosis and meiosis [1-3]. The cohesin complex, which forms a ring to entrap sister chromatids, is composed of four subunits: a heterodimer of Structural Maintenance of Chromosome (SMC) proteins, SMC1 and SMC3, an α-kleisin, Sister Chromatid Cohesion1 (SCC1) in somatic cells or REC8 in meiotic cells and SCC3 [2,4,5]. The core cohesin complex associates with a number of factors during the cohesion cycle [1-3,6-8] and its association with the chromosomes is controlled by * Correspondence: makaroca@miamioh.edu Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA modifications, such as acetylation, phosphorylation, sumoylation and proteolysis [9-13]. Sister chromatid cohesion is established during S phase by the Establishment of Cohesion protein, ECO1/CTF7 [14-18]. In budding yeast, ECO1/CTF7 acetylates lysine residues K112, K113 of SMC3 and K84, K210 of SCC1 to stabilize the ring and keep it closed [10,19,20]. ECO1/CTF1 is an essential gene in many organisms with the complete inactivation of CTF7 resulting in lethality [1-3,14-16,21]. Inactivation or mutations in ECO1/CTF1 leads to a broad range of defects, including chromosome mis-segregation, defects in DNA double strand break repair, defects in homologous recombination and transcriptional alterations [14,15,17,21-25]. Humans contain two ECO1/CTF7 orthologs, ESCO1 and ESCO2 [24]. Mutations in ESCO2 lead to Roberts syndrome © 2015 Liu and Makaroff; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu and Makaroff BMC Plant Biology (2015) 15:74 (RBS), which is associated with a variety of defects including growth retardation, limb reduction/asymmetric limb growth, cleft lip/palate and missing fingers/toes [26,27]. Arabidopsis contains a single CTF7 ortholog (AtCTF7), which can complement the temperature-sensitive yeast ctf7203 mutant [16]. AtCTF7 encodes a 345 amino acid protein, containing a PIP box at residues 82 to 86, a C2H2 zinc finger motif at residues 92–130 and an acetyltransferase domain from residues 184 to 335. The acetyltransferase domain can be further separated into three motifs: D (amino acids 184– 204), A (amino acids 266–301) and B (amino acids 311– 335). Motif D provides the framework of the acetyltransferase domain. Motif A participates in acetyl-CoA binding and is critical for catalytic activity, while motif B is the most Cterminal region and participates in substrate recognition and catalytic activity regulation [28,29]. Variations in AtCTF7 expression have been shown to result in a wide range of defects. Plants heterozygous for a T-DNA insertion in AtCTF7 grow normally but their siliques contain approximately 25% arrested seeds, consistent with the belief that CTF7 is an essential protein [16]. However, Atctf7 homozygous plants can be detected at very low frequencies [17]. Atctf7 plants exhibit a wide range of developmental defects, including extreme dwarfism and sterility with Atctf7 meiocytes exhibiting abnormal chromosome segregation and defective sister chromatid cohesion. Knockdown of AtCTF7 mRNA levels using RNAi leads to growth retardation and defective sister chromatid cohesion [18]. Finally, overexpression of full length AtCTF7 with the CaMV 35S promoter leads to ovule abortion, but plant growth is not affected [16]. To further investigate the roles of CTF7 in Arabidopsis and to identify AtCTF7 interacting proteins, transgenic plants expressing AtCTF7 constructs that encode either TAP-tagged [30] or non-tagged versions of full length or truncated AtCTF7 were generated and analyzed. Transgenic plants that express a truncated version of AtCTF7, missing motif B, from the 35S promoter exhibited impaired reproduction and vegetative growth. Ovules in 35S:NTAP:AtCTF7ΔB plants displayed alterations in cell identity and the timing of megasporogenesis and megagametogenesis, while male meiocytes exhibited alterations during meiosis II. The transgenic plants also displayed alterations in vegetative growth that were not inherited in a Mendelian fashion. Results Arabidopsis plants expressing high levels of NTAP: AtCTF7ΔB display reduced fertility An AtCTF7 construct missing the C-terminal 46 amino acids (Δ299-345) was generated, fused with NTAP [30] and expressed from the CaMV 35S promoter in wildtype Columbia plants (35S:NTAP:AtCTF7ΔB; Additional file 1: Figure S1). Twenty out of the 36 independent lines examined exhibited reduced fertility, with fertility levels Page 2 of 16 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. The reduced fertility phenotype of 35S:NTAP:AtCTF7ΔB plants suggested that co-suppression may be lowering AtCTF7 transcript levels in the lines. Therefore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted to examine transcript levels of native AtCTF7 as well as the CTF7 transgene. Surprisingly, total AtCTF7 (35S:NTAP:AtCTF7ΔB and native AtCTF7) transcript levels were approximately seven-fold higher in 35S: NTAP:AtCTF7ΔB plants than CTF7 transcript levels in wild type plants, while native AtCTF7 transcript levels were almost three-fold higher in the transgenic plants (Figure 1C). Therefore, the transgenic lines contained high levels of AtCTF7ΔB transcripts along with elevated levels of native AtCTF7 mRNA, indicating that the observed phenotypes are not the result of reduced AtCTF7 expression. Instead these results suggest that the 35S: NTAP:AtCTF7ΔB construct may be exerting a dominant negative effect. Reciprocal crossing experiments were carried out to determine the effect of the 35S:NTAP:AtCTF7ΔB construct on male and female gametes. The 35S:NTAP: AtCTF7ΔB construct was transmitted at reduced levels through both male and female gametes (Additional file 1: Table S1), consistent with our preliminary results that both male and female fertility were affected in 35S:NTAP:AtCTF7ΔB plants. Nonviable seeds were obtained when 35S:NTAP:AtCTF7ΔB plants were used as either the male or female parent; however the Liu and Makaroff BMC Plant Biology (2015) 15:74 Page 3 of 16 Figure 1 35S:NTAP:AtCTF7ΔB plants exhibit reduced fertility. (A) Open siliques from wild type (Ai) and 35S:NTAP:AtCTF7ΔB (Aii,Aiii) plants. i, Wild type silique with full seed set. ii, Silique from a 35S:NTAP:AtCTF7ΔB plant (Line 19) exhibiting a weak phenotype. iii, Silique from a 35S: NTAP:AtCTF7ΔB 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:AtCTF7ΔB (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: AtCTF7ΔB plants. Transcript levels of total AtCTF7 (35S:NTAP:AtCTF7ΔB and native AtCTF7) and native AtCTF7 are increased in 35S:NTAP:AtCTF7ΔB 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). relative proportion of nonviable seed was three times greater (9.3% verses 31.3%) when 35S:NTAP:AtCTF7ΔB plants served as the female. The number of nonviable seeds was greatest (65%) in self-pollenated plants. Therefore, while both male and female gametes are affected, the 35S:NTAP:AtCTF7ΔB construct has a greater effect on female reproduction. Inheritance of 35S:NTAP:AtCTF7ΔBassociated defects from both parents has a compounding effect on seed production. Male meiotic chromosome segregation is altered in 35S: NTAP:AtCTF7ΔB plants Previous studies showed that inactivation of AtCTF7 via TDNA insertion or reduction in AtCTF7 mRNA levels via RNAi disrupts sister chromatid cohesion, resulting in uneven chromosome segregation during meiosis and ultimately reduced pollen viability [17,18]. The reduced fertility observed in 35S:NTAP:AtCTF7ΔB plants suggested that meiosis might also be affected in these lines. Therefore, Liu and Makaroff BMC Plant Biology (2015) 15:74 chromosome spreading experiments were carried out to examine the effect of 35S:NTAP:AtCTF7ΔB expression on male meiosis. Male meiocytes in 35S:NTAP:AtCTF7ΔB plants resembled wild type during early stages of meiosis, with normal chromosome morphology during pachytene (Figure 2A,E), diakinesis (Figure 2B,F) and metaphase I (Figure 2C,G). The first noticeable defect was observed at telophase I when lagging chromosomes were observed (Figure 2D,H), followed by mis-segregated chromosomes at prophase II (Figure 2M). More than twenty individual chromosomes were typically observed in meiocytes beginning at metaphase II (Figure 2N), indicating that sister chromatid cohesion was prematurely lost. Chromosomes did not segregate evenly at anaphase II (Figure 2O) resulting in the production of polyads with varying DNA contents at tetrad stage (Figure 2P). Similar to atctf7 plants, a small number of relatively normal meiocytes were also observed throughout meiosis with fewer normal-appearing meiocytes in later stages of meiosis. For example, the percentages of defective meiocytes observed at various stages of meiosis in Line 11 were: metaphase I: 0% (0/41), telophase Page 4 of 16 I: 6.8% (7/87), prophase II: 23.5% (24/102), metaphase II: 40.7% (24/59), anaphase II: 56.8% (25/44) and telophase II: 64.0% (114/178). Immunolocalization experiments were then carried out to examine the distribution of the meiotic cohesin protein SYN1 [31] in 35S:NTAP:AtCTF7ΔB male meiocytes. Consistent with meiotic chromosome spreading experiments the overall distribution of SYN1 was not dramatically affected in meiocytes of 35S:NTAP: AtCTF7ΔB plants (Additional file 1: Figure S2). SYN1 exhibited diffuse nuclear labeling during interphase with strong signal observed on the developing chromosomal axes from early leptotene into zygotene (Additional file 1: Figures S2A,D). During late zygotene and pachytene the protein lined the chromosomes (Additional file 1: Figures S2B,E,C and F). SYN1 was released normally from the condensing chromosomes during diplotene and diakinesis and similar to the situation in wild type, SYN1 was barely detectable on chromosomes by prometaphase I. Therefore, cohesin appears to load and be removed normally from meiotic chromosomes in 35S: NTAP:AtCTF7ΔB plants. Figure 2 35S:NTAP:AtCTF7ΔB male meiocytes exhibit defective meiotic chromosome segregation. (A-D) and (I-L) Wild type meiocytes. (E-H) and (M-P) 35S:NTAP:AtCTF7ΔB meiocytes. A, E pachytene; B, F diakinesis; C, G metaphase I; D, H telophase I; I, M prophase II; J, N metaphase II; K, O anaphase II; L, P telophase II. Lagging chromosomes and/or chromosome fragments are denoted with arrows. Meiotic chromosomes are stained by 4’, 6-diamidino-2-phenylindole (DAPI). Scale bar = 10 μm. Liu and Makaroff BMC Plant Biology (2015) 15:74 Female gametophyte development is altered in 35S: NTAP:AtCTF7ΔB plants Previous studies showed that ovule development is very sensitive to AtCTF7 levels [16-18]. Approximately 50% of the ovules from Atctf7-1+/–plants contain nondegenerated antipodal nuclei [16], while ovules from atctf7 plants degenerate early [17]. RNAi directed reduction of AtCTF7 mRNA levels, as well as 35S-mediated increases in AtCTF7 transcript levels result in ovule arrest at Female Gametophyte (FG) 1 stage [16,18]. To elucidate the function(s) of 35S:NTAP:AtCTF7ΔB 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 S3BE) [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 Page 5 of 16 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). In mutant plants, defects were observed in ovules very early in development. Approximately 30.4% (41/135) of the ovules observed were defective at the premeiosis stage. Abnormally enlarged sub-epidermal cells were observed adjacent to normal looking megaspore mother cells in some pre-meiotic ovules (Figure 3E,F’). Adjacent ovules were often different in size and sometimes pointed in the same direction (Figure 3 F,F’). A functional megaspore was not identified in some ovules even though their shape and size was beyond FG1. Instead, large cells with prominent nuclei and megaspore-like characteristics [36] were found in the position of a normal embryo sac (Figures 3G-I). Ovules containing either one megaspore-like cell (Figure 3G,H) or two megaspore-like cells with dyad characteristics (Figure 3I) were also observed. This suggested that meiosis is delayed, altered and arrested in most megaspores. Extra cells were also observed between the megaspores and the L1 layer of cells (Figure 3G-I). In WT Figure 3 Early ovule development is disrupted in 35S:NTAP:AtCTF7ΔB plants. Wild type (A-D) and 35S:NTAP:AtCTF7ΔB (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:AtCTF7ΔB 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:AtCTF7ΔB 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]. Liu and Makaroff BMC Plant Biology (2015) 15:74 plants, a small number (6.7%, 8/120) of ovules were found to contain twin megaspore mother cells; however no defects were observed during or after meiosis. In Line 11 plants, 14.1% (19/135) of the ovules contained multiple megaspore mother-like cells at pre-meiosis. Furthermore, 72.5% (50/69) of ovules with dyads contained extra cells between the dyad and L1 layer, and 27.5% (19/69) the dyads were in elongated shape. Ultimately 50.4% (64/127) of the ovules observed were defective at FG1 stage. Therefore, alterations in archesporial cell differentiation, the onset and progression of meiosis and possibly somatic cell identity are observed in ovules of 35S:NTAP:AtCTF7ΔB plants. The effects of 35S:NTAP:AtCTF7ΔB 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). Ovule development is typically synchronous in wild type sliques with predominately one or two developmental stages present in a given pistil (Additional file 1: Table S2). In contrast, 35S:NTAP:AtCTF7ΔB ovule development appeared slowed and asynchronous (Additional file 1: Table S3). Female gametophytes in the same pistil were often at several different stages, indicating that the synchrony of gametophyte development was disturbed and embryo sac maturation was delayed. Ovules containing a megaspore (Figures 3G,H’), FG1 embryo sacs with a functional megaspore-like cell [37] and degrading megaspores (Figures 4G,H,N), and abnormal FG2 and FG3 embryo Page 6 of 16 sacs were commonly observed in the same slique (Figure 4I,O-P). Ovules containing degraded/degrading megasporocytes, and degrading FG1 embryo sacs were also observed (Additional file 1: Figures S5A-D). Alterations in nuclear division appeared to precede arrest in some megaspores (Additional file 1: Figure S5E-H). Most embryo sacs arrest at FG2/FG3; although some terminal ovules appeared to progress beyond FG3 (Additional file 1: Table S3). Common phenotypes included degraded/degrading nuclei (Additional file 1: Figure S5J-K), degenerated embryo sacs (Additional file 1: Figure S5M), polar nuclei fusion defects (Additional file 1: Figure S5N) and vacuole development defects (Additional file 1: Figure S5N-P). To determine if the alterations observed in Line 11 are representative of 35S:NTAP:AtCTF7ΔB 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:AtCTF7ΔB include a delay and alterations in both megasporogenesis and megagametogenesis with embryo sacs arresting at various stages of development. Because the ovules of 35S:NTAP:AtCTF7ΔB plants appeared to exhibit a delay in the onset of meiosis, qRTPCR was carried out to examine transcript levels for several genes important for meiosis and ovule development (Additional file 1: Figure S7A,B). Transcripts for WUS1 [38], MMD1 [39], SPO11-1 [40] and ZYP1a [41] were elevated between two and three fold in 35S:NTAP: AtCTF7ΔB plants relative to wild type. Transcript levels of DMC1 [42], SYN3 [43,44] and OSD1 [45] showed modest increases, while the transcript levels of other genes were unchanged (Additional file 1: Figures S7A,B). Liu and Makaroff BMC Plant Biology (2015) 15:74 Page 7 of 16 Figure 4 Embryo sac development is delayed and arrests early in 35S:NTAP:AtCTF7ΔB ovules. (A-E) and (J-M) Wild type ovules. (F-I) and (N-P) 35S:NTAP:AtCTF7ΔB 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:AtCTF7ΔB 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:AtCTF7ΔB 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. Expression of 35S:NTAP:AtCTF7ΔB causes pleiotropic growth defects During the analysis of 35S:NTAP:AtCTF7ΔB reduced fertility lines, plants displaying vegetative defects began to appear in the T2 or T3 generations. Specifically, later generations grew progressively worse in 12 of the 18 independent severely reduced fertility lines examined. The remaining six lines continued to display reduced fertility, but did not exhibit vegetative defects through the seventh generation. A wide range of morphological defects was observed in the 12 lines (Figure 6). The defects varied between lines and between progeny of the same line. The observed vegetative abnormalities included dwarf plants, fused stems and disruption of phyllotaxis (Figures 6C-E). The proportion of plants exhibiting vegetative alterations increased in successive generations. Likewise, the severity of the vegetative alterations also became successively worse in subsequent generations. For example, the frequency of dwarf plants increased from approximately 18% in the third generation to 82% by generation six (Figure 6; Table 1). The dwarf phenotype also became more severe with later generations containing smaller, more defective plants. Dwarf plants varied in morphology and exhibited a range of alterations, including acaulescent plants, floral abnormalities, homeotic changes and irregular leaves (Figure 6F-I). While some of the most severe 35S:NTAP: AtCTF7ΔB dwarf plants resembled atctf7-1 plants (Figure 6J), in general the phenotype was less severe than atctf7-1 plants. Furthermore, while most atctf7-1 plants exhibit early senescence, 35S:NTAP:AtCTF7ΔB plants typically did not. In order to determine if the phenotypic alterations were due to increased 35S:NTAP:AtCTF7ΔB expression, native CTF7 and total CTF7 levels were measured in both dwarf and non-dwarf plants of three different generations of 35S:NTAP:AtCTF7ΔB plants, which showed progressively more severe phenotypes. Native Liu and Makaroff BMC Plant Biology (2015) 15:74 Page 8 of 16 Figure 5 Phenotypes of 35S:NTAP:AtCTF7ΔB ovules from Line 15. (A-C) Pre-meiotic 35S:NTAP:AtCTF7ΔB 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. CTF7 levels ranged from 2.6-3.7 fold above wild type in the different plants, while total CTF7 transcript levels were elevated 5.4-7.6 fold relative to wild type. However, no consistent difference was observed between dwarf and nondwarf plants or between one generation and the other. Therefore, the phenotypic differences observed are not due to dramatic changes in AtCTF7ΔB transcript levels. 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 Figure 6 35S:NTAP:AtCTF7ΔB plant morphology changes in later generations. (A) Wild type Columbia plant. (B-E) Morphological alterations get progressively worse in 35S:NTAP:AtCTF7ΔB plants through self-pollination. (B) Second generation plants are normal, but reduced fertile. (C) Both dwarf and non-dwarf, reduced fertile plants are observed in third generation plants. (D) Fourth generation plants. (E) Sixth generation plants, showing a higher frequency of dwarf plants. Defects such as reduced apical dominance (arrow) and phyllotaxis disturbances (asterisks) are observed. (F-I) Representative 35S:NTAP:AtCTF7ΔB dwarf plants. Inflorescence defects include acaulescent (G), multiple inflorescence branches at the first node (H) and no inflorescence (I). Leave defects include aberrant rosette size and shape (F, H and I). (J) atctf7-1 plant showing an early senescence phenotype (arrow). Plants B to I are from Line 11. All plants are grown under the same conditions. Plants in A-H and J are approximately 30 days old; plant in I is 40 days old. Scale bar = 5 cm in A-E and 2 cm in F-J. Liu and Makaroff BMC Plant Biology (2015) 15:74 Table 1 Non-Mendelian inheritance in 35S:NTAP: AtCTF7ΔB plants Generation Dwarf plants (%) 2nd 3rd Reduced fertile, non-dwarf plants (%) 125 (100%) 24 (18.5%) 106 (81.5%) Seeds from dwarf Parent 16 (42.1% ) 22 (57.9%) Seeds from non-dwarf Parent 25 (45.5%) 30 (54.5%) Seeds from dwarf plants 55 (78.6%) 15 (21.4%) Seeds from non-dwarf plants 81 (67.5%) 39 (32.5%) Seeds from dwarf plants 55 (81.4%) 13 (18.6%) Seeds from non-dwarf plants 66 (82.5%) 14 (17.5%) 4th 5th 6th Plants were from Line 11. Seeds from reduced fertile, non-dwarf plants and from dwarf plants were collected and sown separately. 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 nondwarf 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:AtCTF7ΔB 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). Transcript levels of several cell cycle (RBR, CYCB1.1 and CYCA1.1) and DNA repair (BRCA1 and BRCA2B) genes have previously been shown to be elevated in atctf7 plants [17]. These genes were tested and found to also be elevated in 35S:NTAP:AtCTF7ΔB plants (Figure 7C). The similarities in morphological defects and expression patterns observed between 35S:NTAP:AtCTF7ΔB and atctf7-1 plants [17] are consistent with the hypothesis that the 35S:NTAP:AtCTF7ΔB construct is exerting a dominant negative effect. Finally, experiments were carried out to determine if the 35S:NTAP:AtCTF7ΔB-associated defects are due to the presence of the N-terminal tag or the absence of the Page 9 of 16 acetyltransferase B motif. A 35S:AtCTF7ΔB construct was generated and transformed into wild type Columbia plants (Additional file 1: Figure S1). Eight out of the 13 35S: AtCTF7ΔB transgenic lines examined exhibited reduced fertility, with the 35S:AtCTF7ΔB defects typically appearing more severe than those in 35S:NTAP:AtCTF7ΔB plants (Additional file 1: Figure S8). Like 35S:NTAP:AtCTF7ΔB plants, dwarf plants were not observed until the third generation in 35S: AtCTF7ΔB plants. However, dwarf plants appeared at higher frequencies and their phenotypes were more varied than 35S:NTAP:AtCTF7ΔB plants (Additional file 1: Figure S8B). For example, the viability of Line 21 decreased with successive generations, such that seeds from this line were not viable by the 4th generation. Likewise, while abnormalities associated with 35S:AtCTF7ΔB plants were similar to those observed in 35S:NTAP:AtCTF7ΔB plants, additional alterations were also present. For example, 35S:AtCTF7ΔB Line 24 segregated for two types of plants, those without an inflorescence and “normal” reduced fertility plants (Additional file 1: Figure S8C). 35S: AtCTF7ΔB Line 29 plants produced siliques that pointed downward and contained fewer seeds (46.4 ± 2.5 versus 54.2 ± 4.1 per silique in WT; n = 35) (Additional file 1: Figure S8D). This phenotype is similar to bp/knat1 mutations [51]. Finally, in order to investigate which aspect(s) of the 35S: NTAP:AtCTF7ΔB construct was causing the fertility and growth defects, several additional constructs were generated and introduced into wild type or Atctf7-1+/− plants. Wild type plants transformed with a 35S:NTAP:AtCTF7 construct (Additional file 1: Figure S1) resembled 35S: AtCTF7 plants [16]. Specifically, the plants grew normally and produced pollen, but exhibited reduced female fertility. A CTF7:AtCTF7ΔB construct (Additional file 1: Figure S1) was also created and transformed into Atctf7-1+/–plants. The native CTF7 promoter is expressed at low levels throughout the plant [17]. Wild type plants containing the CTF7:AtCTF7ΔB construct exhibited normal growth and development and normal fertility levels (52.0 ± 2.2 seed/silique, n = 33 versus 54.2 ± 4.1 seed/silique in wild type, n = 35). No alterations were observed in six different transgenic lines over six generations. atctf7-1 plants containing the CTF7:AtCTF7ΔB construct were obtained in T2 populations, but at frequencies (7%, 6/86) much lower than expected (25%) if the construct complemented the atctf7-1 mutation. While these plants were dwarf, they grew better than atctf7-1 plants (Additional file 1: Figure S8E). Somewhat similar to 35S:AtCTF7ΔB plants, plant morphology varied between plants with some plants appearing acaulescence or producing fewer rosette leaves. The plants produced approximately 40 ovules per silique; however they failed to set seed; siliques contained aborted ovules resembling atctf7-1 plants (Additional file 1: Figure S8G). Therefore, the defects observed in 35S:NTAP:AtCTF7ΔB plants Liu and Makaroff BMC Plant Biology (2015) 15:74 Page 10 of 16 Figure 7 Transcript levels of epigenetically regulated transposable elements and other select genes in 35S:NTAP:AtCTF7ΔB plants. (A) Transcript levels of MU1, COPIA28 and soloLTR, are increased dramatically in 35S:NTAP:AtCTF7ΔB 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). are caused by high-level expression of AtCTF7ΔB and not the presence of the NTAP tag. Rather, the presence of the NTAP appears to reduce the severity of the alterations, either by reducing the stability or activity of the protein, possibly by affecting its interaction with other proteins. Further, expression of a truncated version of CTF7, missing the B motif, can restore some vegetative growth to atctf7-1 plants; however the plants are still completely sterile. Therefore, an intact acetyltransferase domain is required for full CTF7 activity. Discussion The acetylation of cohesin complexes at conserved lysine residues by CTF7/Eco1 plays an essential role in the establishment of cohesion during S phase and therefore nuclear division. Consistent with this, CTF7/Eco1 null mutations are typically lethal. Organisms expressing altered CTF7/Eco1 levels or point mutations in the protein typically display relatively normal levels of cohesin during nuclear division, but exhibit a range of developmental alterations. For example, mutations in the