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Cited by 172Plant tissue culture and regenerationEpigenetics and DNA MethylationPlant Molecular Biology Research

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New Insights into Somatic Embryogenesis: LEAFY COTYLEDON1, BABY BOOM1 and WUSCHEL-RELATED HOMEOBOX4 Are Epigenetically Regulated in Coffea canephora

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Nic-Can, Geovanny I., López-Torres, Adolfo, Barredo-Pool, Felipe, Wrobel, Kazimierz, Loyola-Vargas, Víctor M., Rojas-Herrera, Rafael, De-la-Peña, Clelia·2013·PLoS ONE
DOI 10.1371/journal.pone.0072160PMC PMC3748027
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Abstract

Plant cells have the capacity to generate a new plant without egg fertilization by a process known as somatic embryogenesis (SE), in which differentiated somatic cells can form somatic embryos able to generate a functional plant. Although there have been advances in understanding the genetic basis of SE, the epigenetic mechanism that regulates this process is still unknown. Here, we show that the embryogenic development of Coffea canephora proceeds through a crosstalk between DNA methylation and histone modifications during the earliest embryogenic stages of SE. We found that low levels of DNA methylation, histone H3 lysine 9 dimethylation (H3K9me2) and H3K27me3 change according to embryo development. Moreover, the expression of LEAFY cotyledon1 (LEC1) and BABY BOOM1 (BBM1) are only observed after SE induction, whereas WUSCHEL-related homeobox4 (WOX4) decreases its expression during embryo maturation. Using a pharmacological approach, it was found that 5-Azacytidine strongly inhibits the embryogenic response by decreasing both DNA methylation and gene expression of LEC1 and BBM1. Therefore, in order to know whether these genes were epigenetically regulated, we used Chromatin Immunoprecipitation (ChIP) assays. It was found that WOX4 is regulated by the repressive mark H3K9me2, while LEC1 and BBM1 are epigenetically regulated by H3K27me3. We conclude that epigenetic regulation plays an important role during somatic embryogenic development, and a molecular mechanism for SE is proposed.

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Figure 1 Development of the somatic embryogenesis process in Coffea canephora. Leaf explants were cultured in liquid Yasuda medium in the presence of 5 µM of benzyl adenine under dark conditions. The embryogenic process starts with the thickening of leaf edges at 14 days after induction (dai), cellular proliferation and the development of the proembryogenic mass (Pm), between 21 and 28 dai. Differentiation of the first somatic embryogenic structures from Pm starts to appear at 35 dai and the differentiation of the late embryogenic stages begins at 42 dai until 56 dai. A close-up of the embryos is shown in circles. Bars = 4 mm.
Figure 1 Development of the somatic embryogenesis process in Coffea canephora. Leaf explants were cultured in liquid Yasuda medium in the presence of 5 µM of benzyl adenine under dark conditions. The embryogenic process starts with the thickening of leaf edges at 14 days after induction (dai), cellular proliferation and the development of the proembryogenic mass (Pm), between 21 and 28 dai. Differentiation of the first somatic embryogenic structures from Pm starts to appear at 35 dai and the differentiation of the late embryogenic stages begins at 42 dai until 56 dai. A close-up of the embryos is shown in circles. Bars = 4 mm.CC BY · PubMed Central
Figure 2 Scanning electron microscopy of the somatic embryo development in Coffea canephora. A) Proembryogenic mass (Pm) from a leaf explant at 21 days after induction (dai). B) Globular stage; the white arrowhead indicates the protoderm establisment. C) Heart stage; the white arrows indicate the beginning of the cotyledonary primordia in the embryo. D) Torpedo stage; the white arrow indicates the enlargement of the embryo. E) Early cotyledonary stage, where the establishment of the future cotyledons can be observed. F) Late cotyledonary stage. At this stage the cotyledons are fully developed. Bars = 100 µm.
Figure 2 Scanning electron microscopy of the somatic embryo development in Coffea canephora. A) Proembryogenic mass (Pm) from a leaf explant at 21 days after induction (dai). B) Globular stage; the white arrowhead indicates the protoderm establisment. C) Heart stage; the white arrows indicate the beginning of the cotyledonary primordia in the embryo. D) Torpedo stage; the white arrow indicates the enlargement of the embryo. E) Early cotyledonary stage, where the establishment of the future cotyledons can be observed. F) Late cotyledonary stage. At this stage the cotyledons are fully developed. Bars = 100 µm.CC BY · PubMed Central
Figure 3 Histological dissection of the somatic embryo at different developmental stages in Coffea canephora. A) Transverse cut of the early globular stage from the proembryogenic mass at 14 dai. B) Transverse cut of the globular stage. The red arrowhead indicates the cellular organization and the presence of a well-defined procambium. C) Longitudinal cut of the heart stage. The red arrowhead indicates the initiation of the elongation of the procambium zone and the beginning of the cotyledonary primordium is indicated with white stars. D) Longitudinal cut of the torpedo stage. The red arrowhead indicates the elongation of the procambium zone. E) Longitudinal cut of the early cotyledonay stage. The red arrowhead indicates the procambium zone, while the white arrowhead indicates the development of the early cotyledonary primordium. F) Longitudinal cut of the late cotyledonary stage. The red arrowheads indicate the procambium zone, and the white arrowhead indicates the development of cotyledonary primordium. Bars = 200 µm.
Figure 3 Histological dissection of the somatic embryo at different developmental stages in Coffea canephora. A) Transverse cut of the early globular stage from the proembryogenic mass at 14 dai. B) Transverse cut of the globular stage. The red arrowhead indicates the cellular organization and the presence of a well-defined procambium. C) Longitudinal cut of the heart stage. The red arrowhead indicates the initiation of the elongation of the procambium zone and the beginning of the cotyledonary primordium is indicated with white stars. D) Longitudinal cut of the torpedo stage. The red arrowhead indicates the elongation of the procambium zone. E) Longitudinal cut of the early cotyledonay stage. The red arrowhead indicates the procambium zone, while the white arrowhead indicates the development of the early cotyledonary primordium. F) Longitudinal cut of the late cotyledonary stage. The red arrowheads indicate the procambium zone, and the white arrowhead indicates the development of cotyledonary primordium. Bars = 200 µm.CC BY · PubMed Central
Figure 4 Global DNA methylation levels during somatic embryogenic induction and the separate embryogenic stages and tissues of Coffea canephora. A) Percentage of global DNA methylation during the development of the somatic embryogenesis process in Coffea canephora shown in Figure 1. B) DNA methylation levels of different tissues and developmental somatic embryo stages. Pm: Proembryogenic mass, G: Globular stage, H: Heart stage, T: Torpedo stage, C: Cotyledonary stage, P: C. canephora in vitro plantlets, Z: C. canephora zygotic embryo in cotyledonary stage. Bars represent the mean ± SE (n = 3). An asterisk represents the statistical significance of mean differences at a given time by the Tukey test (P≤0.05). Each experiment was carried out three times.
Figure 4 Global DNA methylation levels during somatic embryogenic induction and the separate embryogenic stages and tissues of Coffea canephora. A) Percentage of global DNA methylation during the development of the somatic embryogenesis process in Coffea canephora shown in Figure 1. B) DNA methylation levels of different tissues and developmental somatic embryo stages. Pm: Proembryogenic mass, G: Globular stage, H: Heart stage, T: Torpedo stage, C: Cotyledonary stage, P: C. canephora in vitro plantlets, Z: C. canephora zygotic embryo in cotyledonary stage. Bars represent the mean ± SE (n = 3). An asterisk represents the statistical significance of mean differences at a given time by the Tukey test (P≤0.05). Each experiment was carried out three times.CC BY · PubMed Central
Figure 5 Effects of 5-Azacytidine (5-AzaC) in the somatic embryogenesis of Coffea canephora. A) Temporal course of explants from C. canephora leaves under embryogenic induction with and without (control) the presence of 5-AzaC at 10 µM and 20 µM. The 5-AzaC was added into the medium every seven days starting at different independent time points (day 7, 14, 21 and 35) of the SE culture. The shaded rectangles mean the presence of 5-AzaC in the medium, as indicated in Materials and Methods. B) The number of every somatic embryo stage was counted after 56 days after induction (dai), with and without (control) the treatments with 5-AzaC added at different time points after the induction (day 7, 14, 21 and 35). The bars represent the mean ± SE (n = 3). Different letters in columns represent the statistical significance of mean differences between each embryogenic stage at a given time by Tukey test (P≤0.05). The experiments were performed three independent times.
Figure 5 Effects of 5-Azacytidine (5-AzaC) in the somatic embryogenesis of Coffea canephora. A) Temporal course of explants from C. canephora leaves under embryogenic induction with and without (control) the presence of 5-AzaC at 10 µM and 20 µM. The 5-AzaC was added into the medium every seven days starting at different independent time points (day 7, 14, 21 and 35) of the SE culture. The shaded rectangles mean the presence of 5-AzaC in the medium, as indicated in Materials and Methods. B) The number of every somatic embryo stage was counted after 56 days after induction (dai), with and without (control) the treatments with 5-AzaC added at different time points after the induction (day 7, 14, 21 and 35). The bars represent the mean ± SE (n = 3). Different letters in columns represent the statistical significance of mean differences between each embryogenic stage at a given time by Tukey test (P≤0.05). The experiments were performed three independent times.CC BY · PubMed Central
Figure 6 Histone H3 methylation patterns during somatic embryogenesis induction and in different embryogenic stages of Coffea canephora. A) Immunoblot analyses during the development of the somatic embryogenesis process in Coffea canephora shown in Figure 1. B) Immunoblot analyses of different tissues and developmental somatic embryo stages. Total histone extracted from leaf explants, as shown in Figure 1, from 0 until 49 days after induction and from different somatic embryo developmental stages were probed with specific antibodies in Western blots. Subsequent to the hybridization, membranes were stripped off and re-probed with antibodies specific to non-modified histone H3. Pm: proembryogenic mass, G: globular stage, H: heart stage, T: torpedo stage, C: cotyledonary stage.
Figure 6 Histone H3 methylation patterns during somatic embryogenesis induction and in different embryogenic stages of Coffea canephora. A) Immunoblot analyses during the development of the somatic embryogenesis process in Coffea canephora shown in Figure 1. B) Immunoblot analyses of different tissues and developmental somatic embryo stages. Total histone extracted from leaf explants, as shown in Figure 1, from 0 until 49 days after induction and from different somatic embryo developmental stages were probed with specific antibodies in Western blots. Subsequent to the hybridization, membranes were stripped off and re-probed with antibodies specific to non-modified histone H3. Pm: proembryogenic mass, G: globular stage, H: heart stage, T: torpedo stage, C: cotyledonary stage.CC BY · PubMed Central
Figure 7 Expression profiles of genes during embryogenic induction and development of the somatic embryos in Coffea canephora. A) Expression of LEC1, BBM1 and WOX4 was performed from total RNA samples for RT-PCR analysis that were isolated from leaf explants under embryogenic induction at days 0, 7, 14 and 21, and the different embryogenic stages were isolated and comparatively classified in proembryogenic mass (Pm), globular (G), heart (H), torpedo (T), cotyledonary (C) and zygotic embryo (Z) in cotyledonary stage. UBQ11 was used as a reference gene. B) Densitometric analysis of the gene expression showed in A. Relative expression of LEC1, BBM1 and WOX4 were normalized to the constitutive gene UBQ11. Different letters in columns represent the statistical significance of mean differences at a given time according to the Tukey test (P≤0.05). Each RT-PCR was conducted twice with three independent biological replicates.
Figure 7 Expression profiles of genes during embryogenic induction and development of the somatic embryos in Coffea canephora. A) Expression of LEC1, BBM1 and WOX4 was performed from total RNA samples for RT-PCR analysis that were isolated from leaf explants under embryogenic induction at days 0, 7, 14 and 21, and the different embryogenic stages were isolated and comparatively classified in proembryogenic mass (Pm), globular (G), heart (H), torpedo (T), cotyledonary (C) and zygotic embryo (Z) in cotyledonary stage. UBQ11 was used as a reference gene. B) Densitometric analysis of the gene expression showed in A. Relative expression of LEC1, BBM1 and WOX4 were normalized to the constitutive gene UBQ11. Different letters in columns represent the statistical significance of mean differences at a given time according to the Tukey test (P≤0.05). Each RT-PCR was conducted twice with three independent biological replicates.CC BY · PubMed Central
Figure 8 Histone H3-methylation patterns during somatic embryogenesis development in Coffea canephora using Chromatin Immunoprecipitation (ChIP). Samples were collected at the beginning of embryogenic induction (0d) and 14 days after embryogenic induction (14d) and during the proembryogenic mass (Pm), torpedo stage (T) and cotyledonary stage (C). The samples were examined for the Histone H3-tail methylation patterns, and the LEC1, BBM1 and WOX4 genes. Input (Input DNA): 10-fold diluted samples were used as templates for the input lanes. As negative control (-Ab), no antibody samples were treated in the same way as immunoprecipitated chromatin with H3K4me3, H3K9me2, H3K27me3 and H3K36me2. Amplified UBQ11 with specific primers was used as the control for the quality of samples and the same amounts was used to amplify LEC1, BBM1 and WOX4.
Figure 8 Histone H3-methylation patterns during somatic embryogenesis development in Coffea canephora using Chromatin Immunoprecipitation (ChIP). Samples were collected at the beginning of embryogenic induction (0d) and 14 days after embryogenic induction (14d) and during the proembryogenic mass (Pm), torpedo stage (T) and cotyledonary stage (C). The samples were examined for the Histone H3-tail methylation patterns, and the LEC1, BBM1 and WOX4 genes. Input (Input DNA): 10-fold diluted samples were used as templates for the input lanes. As negative control (-Ab), no antibody samples were treated in the same way as immunoprecipitated chromatin with H3K4me3, H3K9me2, H3K27me3 and H3K36me2. Amplified UBQ11 with specific primers was used as the control for the quality of samples and the same amounts was used to amplify LEC1, BBM1 and WOX4.CC BY · PubMed Central
Figure 9 Effects of 5-Azacytidine on the LEC1, BBM1 and WOX4 expression during the beginning of somatic embryogenesis in Coffea canephora. A) Gene expression under embryogenic conditions in the absence of 5-Azacytidine. B) Gene expression under embryogenic conditions with 10 µM 5-Azacytidine added every seven days from 0 to 21 dai. Total RNA was used for RT-PCR analysis. UBQ11 was used as a reference gene.
Figure 9 Effects of 5-Azacytidine on the LEC1, BBM1 and WOX4 expression during the beginning of somatic embryogenesis in Coffea canephora. A) Gene expression under embryogenic conditions in the absence of 5-Azacytidine. B) Gene expression under embryogenic conditions with 10 µM 5-Azacytidine added every seven days from 0 to 21 dai. Total RNA was used for RT-PCR analysis. UBQ11 was used as a reference gene.CC BY · PubMed Central
Figure 10 Proposed epigenetic regulatory mechanism during the somatic embryogenesis of Coffea canephora. Differentiated somatic cells from leaf explants are treated with plant growth regulators to induce somatic embryogenesis (SE). The embryogenic response proceeds through dynamic changes in DNA methylation and histone modification, each in turn contributing to epigenetic regulation of LEC, BBM1 and WOX4 genes. Under ideal somatic embryogenic induction, differentiated somatic cells from leaf explants (Le) initiate the first molecular and epigenetic changes. These changes start with the repression of LEC1 and BBM1 genes during the induction process, mainly by the accumulation of H3K27me3. WOX4 is highly expressed in this embryogenic stage, probably by the accumulation of H3K36me2 and the absence of H3K9me2. Furthermore, high levels of DNA methylation are observed. During the Pm development, the H3K27me3 mark on LEC1 loci is removed and the gene is expressed, while the expression of BBM1 is mainly accompanied by the accumulation of H3K4me3 and H3K36me2. At this stage, DNA methylation levels start to rapidly decrease. Finally, during the late developmental embryo stage, T, an increase of H3K9me2 promotes the transcriptional repression of WOX4 and H3K27me3 again starts accumulating on LEC1 and the expression of BBM1 decreases. At this embryogenic stage, high levels of DNA methylation are established. These findings suggest that dynamic changes in chromatin could be a crucial step for switching genes on or off during the dedifferentiation and differentiation events to develop a somatic embryo. Le: leaf explant; mRNA: messenger RNA; SE: somatic embryogenesis; Pm: proembryogenic mass; G: globular stage; H: heart stage; T: torpedo stage; C: cotyledonary stage. The arrows mean gene expression while the truncate lines mean gene repression. The abundance of the dashed lines means the abundance of the transcripts.
Figure 10 Proposed epigenetic regulatory mechanism during the somatic embryogenesis of Coffea canephora. Differentiated somatic cells from leaf explants are treated with plant growth regulators to induce somatic embryogenesis (SE). The embryogenic response proceeds through dynamic changes in DNA methylation and histone modification, each in turn contributing to epigenetic regulation of LEC, BBM1 and WOX4 genes. Under ideal somatic embryogenic induction, differentiated somatic cells from leaf explants (Le) initiate the first molecular and epigenetic changes. These changes start with the repression of LEC1 and BBM1 genes during the induction process, mainly by the accumulation of H3K27me3. WOX4 is highly expressed in this embryogenic stage, probably by the accumulation of H3K36me2 and the absence of H3K9me2. Furthermore, high levels of DNA methylation are observed. During the Pm development, the H3K27me3 mark on LEC1 loci is removed and the gene is expressed, while the expression of BBM1 is mainly accompanied by the accumulation of H3K4me3 and H3K36me2. At this stage, DNA methylation levels start to rapidly decrease. Finally, during the late developmental embryo stage, T, an increase of H3K9me2 promotes the transcriptional repression of WOX4 and H3K27me3 again starts accumulating on LEC1 and the expression of BBM1 decreases. At this embryogenic stage, high levels of DNA methylation are established. These findings suggest that dynamic changes in chromatin could be a crucial step for switching genes on or off during the dedifferentiation and differentiation events to develop a somatic embryo. Le: leaf explant; mRNA: messenger RNA; SE: somatic embryogenesis; Pm: proembryogenic mass; G: globular stage; H: heart stage; T: torpedo stage; C: cotyledonary stage. The arrows mean gene expression while the truncate lines mean gene repression. The abundance of the dashed lines means the abundance of the transcripts.CC BY · PubMed Central

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Authors
Nic-Can, G. I., López-Torres, A., Barredo-Pool, F., Wrobel, K., Loyola-Vargas, V. M., Rojas-Herrera, R., & De-la-Peña, C.
Affiliations
  • Autonomous University of Yucatán (MX)
  • Universidad de Guanajuato (MX)
  • Centro de Investigación Científica de Yucatán, A.C (MX)
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2013-08-20
Volume
8
Issue
8
Pages
e72160
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0072160
PMC
PMC3748027
OpenAlex
W1983625874
Cited by
164
References
78
Citations by year
2026: 14 · 2025: 6 · 2024: 20 · 2023: 13 · 2022: 13 · 2021: 14 · 2020: 26 · 2019: 13 · 2018: 14 · 2017: 7 · 2016: 13 · 2015: 7 · 2014: 4
FWCI
3.59
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
Consejo Nacional de Ciencia y Tecnología
Topics
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant tissue culture and regeneration
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
Keywords
somatic embryogenesis, Coffea canephora, epigenetic regulation, DNA methylation, histone modifications, H3K9me2, H3K27me3, WOX4, azacitidine, embryonic development, gene expression regulation
MeSH terms
Azacitidine, Histones, Plant Proteins, Protein Processing, Post-Translational, Seeds, Homeodomain Proteins, Gene Expression Regulation, Plant, Gene Expression Regulation, Developmental, DNA Methylation, CCAAT-Enhancer-Binding Proteins, Coffea, Epigenesis, Genetic, Plant Somatic Embryogenesis Techniques

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