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Male fertility in Arabidopsis requires active DNA demethylation of genes that control pollen tube function
  1. 1.

    Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Ambrosi, C., Manzo, M. & Baubec, T. Dynamics and context-dependent roles of DNA methylation. J. Mol. Biol. 429, 1459–1475 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    Luo, C., Hajkova, P. & Ecker, J. R. Dynamic DNA methylation: in the right place at the right time. Science 361, 1336–1340 (2018).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Matzke, M. A. & Mosher, R. A. RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat. Rev. Genet. 15, 394–408 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  5. 5.

    Stroud, H. et al. Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis. Nat. Struct. Mol. Biol. 21, 64–72 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6.

    Heard, E. & Martienssen, R. A. Transgenerational epigenetic inheritance: myths and mechanisms. Cell 157, 95–109 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Kawashima, T. & Berger, F. Epigenetic reprogramming in plant sexual reproduction. Nat. Rev. Genet. 15, 613–624 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. 8.

    Ingouff, M. et al. Live-cell analysis of DNA methylation during sexual reproduction in Arabidopsis reveals context and sex-specific dynamics controlled by noncanonical RdDM. Genes Dev. 31, 72–83 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Ibarra, C. A. et al. Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337, 1360–1364 (2012).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Calarco, J. P. et al. Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA. Cell 151, 194–205 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Park, K. et al. DNA demethylation is initiated in the central cells of Arabidopsis and rice. Proc. Natl Acad. Sci. USA 113, 15138–15143 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. 12.

    Choi, Y. et al. DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis. Cell 110, 33–42 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  13. 13.

    Satyaki, P. R. V. & Gehring, M. DNA methylation and imprinting in plants: machinery and mechanisms. Crit. Rev. Biochem. Mol. Biol. 52, 163–175 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Schoft, V. K. et al. Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte. Proc. Natl Acad. Sci. USA 108, 8042–8047 (2011).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  15. 15.

    Slotkin, R. K. et al. Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell 136, 461–472 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Martinez, G., Panda, K., Köhler, C. & Slotkin, R. K. Silencing in sperm cells is directed by RNA movement from the surrounding nurse cell. Nat. Plants 2, 16030 (2016).

  17. 17.

    He, S., Vickers, M., Zhang, J. & Feng, X. Natural depletion of histone H1 in sex cells causes DNA demethylation, heterochromatin decondensation and transposon activation. Elife 8, e42530 (2019).

  18. 18.

    Penterman, J. et al. DNA demethylation in the Arabidopsis genome. Proc. Natl Acad. Sci. USA 104, 6752–6757 (2007).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  19. 19.

    Gong, Z. et al. ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell 111, 803–814 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  20. 20.

    Francis, K. E. et al. Pollen tetrad-based visual assay for meiotic recombination in Arabidopsis. Proc. Natl Acad. Sci. USA 104, 3913–3918 (2007).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. 21.

    Hollister, J. D. et al. Transposable elements and small RNAs contribute to gene expression divergence between Arabidopsis thaliana and Arabidopsis lyrata. Proc. Natl Acad. Sci. USA 108, 2322–2327 (2011).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. 22.

    Schmitz, R. J. et al. Patterns of population epigenomic diversity. Nature 495, 193–198 (2013).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Gehring, M., Bubb, K. L. & Henikoff, S. Extensive demethylation of repetitive elements during seed development underlies gene imprinting. Science 324, 1447–1451 (2009).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 24.

    Batista, R. A. et al. The MADS-box transcription factor PHERES1 controls imprinting in the endosperm by binding to domesticated transposons. Elife 8, e50541 (2019).

  25. 25.

    Walker, J. et al. Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis. Nat. Genet. 50, 130–137 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  26. 26.

    Bewick, A. J. et al. On the origin and evolutionary consequences of gene body DNA methylation. Proc. Natl Acad. Sci. USA 113, 9111–9116 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  27. 27.

    Chang, F., Gu, Y., Ma, H. & Yang, Z. AtPRK2 promotes ROP1 activation via RopGEFs in the control of polarized pollen tube growth. Mol. Plant 6, 1187–1201 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28.

    Duckney, P. et al. Actin-membrane interactions mediated by NETWORKED2 in Arabidopsis pollen tubes through associations with Pollen Receptor-Like Kinase 4 and 5. N. Phytol. 216, 1170–1180 (2017).

    CAS  Article  Google Scholar 

  29. 29.

    Ge, Z. et al. Arabidopsis pollen tube integrity and sperm release are regulated by RALF-mediated signaling. Science 358, 1596–1600 (2017).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Lu, Y. et al. Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23, 81–93 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Qin, Y. et al. Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil. PLoS Genet. 5, e1000621 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  32. 32.

    Wang, T. et al. A receptor heteromer mediates the male perception of female attractants in plants. Nature 531, 241–244 (2016).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. 33.

    Muschietti, J. P. & Wengier, D. L. How many receptor-like kinases are required to operate a pollen tube. Curr. Opin. Plant Biol. 41, 73–82 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34.

    Evans, A. R., Hall, D., Pritchard, J. & Newbury, H. J. The roles of the cation transporters CHX21 and CHX23 in the development of Arabidopsis thaliana. J. Exp. Bot. 63, 59–67 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  35. 35.

    Loraine, A. E., McCormick, S., Estrada, A., Patel, K. & Qin, P. RNA-seq of Arabidopsis pollen uncovers novel transcription and alternative splicing. Plant Physiol. 162, 1092–1109 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Jones, A. M. et al. Border control–a membrane-linked interactome of Arabidopsis. Science 344, 711–716 (2014).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  37. 37.

    Dong, S. et al. Proteome-wide, structure-based prediction of protein-protein interactions/new molecular interactions viewer. Plant Physiol. 179, 1893–1907 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Tang, K., Lang, Z., Zhang, H. & Zhu, J.-K. The DNA demethylase ROS1 targets genomic regions with distinct chromatin modifications. Nat. Plants 2, 16169 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 39.

    Borges, F. et al. Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis. Nat. Genet. 50, 186–192 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    You, W. et al. Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana. BMC Plant Biol. 12, 51 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41.

    Dapper, A. L. & Wade, M. J. Relaxed selection and the rapid evolution of reproductive genes. Trends Genet. 36, 640–649 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Ossowski, S. et al. The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana. Science 327, 92–94 (2010).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  43. 43.

    Hesiod. Theogony (Start Publishing, 2017).

  44. 44.

    Higashiyama, T. & Yang, W. C. Gametophytic pollen tube guidance: attractant peptides, gametic controls, and receptors. Plant Physiol. 173, 112–121 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  45. 45.

    Johnson, M. A., Harper, J. F. & Palanivelu, R. A fruitful journey: pollen ttube navigation from germination to fertilization. Annu. Rev. Plant Biol. 70, 809–837 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  46. 46.

    Zhong, S. & Qu, L. J. Peptide/receptor-like kinase-mediated signaling involved in male-female interactions. Curr. Opin. Plant Biol. 51, 7–14 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  47. 47.

    Shirzadi, R. et al. Genome-wide transcript profiling of endosperm without paternal contribution identifies parent-of-origin-dependent regulation of AGAMOUS-LIKE36. PLoS Genet. 7, e1001303 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. 48.

    Lu, Y. RNA isolation from Arabidopsis pollen grains. Bio-protocol e67–e67 (2011).

  49. 49.

    Schoft, V. K. et al. SYBR green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content. Plant Reprod. 28, 61–72 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  50. 50.

    Borges, F. et al. FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei. Plant Methods 8, 44 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51.

    Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Krueger, F. & Andrews, S. R. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27, 1571–1572 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Huang, D. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    CAS  Article  Google Scholar 

Original Text (This is the original text for your reference.)

  1. 1.

    Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Ambrosi, C., Manzo, M. & Baubec, T. Dynamics and context-dependent roles of DNA methylation. J. Mol. Biol. 429, 1459–1475 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    Luo, C., Hajkova, P. & Ecker, J. R. Dynamic DNA methylation: in the right place at the right time. Science 361, 1336–1340 (2018).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Matzke, M. A. & Mosher, R. A. RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat. Rev. Genet. 15, 394–408 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  5. 5.

    Stroud, H. et al. Non-CG methylation patterns shape the epigenetic landscape in Arabidopsis. Nat. Struct. Mol. Biol. 21, 64–72 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6.

    Heard, E. & Martienssen, R. A. Transgenerational epigenetic inheritance: myths and mechanisms. Cell 157, 95–109 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Kawashima, T. & Berger, F. Epigenetic reprogramming in plant sexual reproduction. Nat. Rev. Genet. 15, 613–624 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. 8.

    Ingouff, M. et al. Live-cell analysis of DNA methylation during sexual reproduction in Arabidopsis reveals context and sex-specific dynamics controlled by noncanonical RdDM. Genes Dev. 31, 72–83 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Ibarra, C. A. et al. Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337, 1360–1364 (2012).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Calarco, J. P. et al. Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA. Cell 151, 194–205 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Park, K. et al. DNA demethylation is initiated in the central cells of Arabidopsis and rice. Proc. Natl Acad. Sci. USA 113, 15138–15143 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. 12.

    Choi, Y. et al. DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis. Cell 110, 33–42 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  13. 13.

    Satyaki, P. R. V. & Gehring, M. DNA methylation and imprinting in plants: machinery and mechanisms. Crit. Rev. Biochem. Mol. Biol. 52, 163–175 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Schoft, V. K. et al. Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte. Proc. Natl Acad. Sci. USA 108, 8042–8047 (2011).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  15. 15.

    Slotkin, R. K. et al. Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell 136, 461–472 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Martinez, G., Panda, K., Köhler, C. & Slotkin, R. K. Silencing in sperm cells is directed by RNA movement from the surrounding nurse cell. Nat. Plants 2, 16030 (2016).

  17. 17.

    He, S., Vickers, M., Zhang, J. & Feng, X. Natural depletion of histone H1 in sex cells causes DNA demethylation, heterochromatin decondensation and transposon activation. Elife 8, e42530 (2019).

  18. 18.

    Penterman, J. et al. DNA demethylation in the Arabidopsis genome. Proc. Natl Acad. Sci. USA 104, 6752–6757 (2007).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  19. 19.

    Gong, Z. et al. ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell 111, 803–814 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  20. 20.

    Francis, K. E. et al. Pollen tetrad-based visual assay for meiotic recombination in Arabidopsis. Proc. Natl Acad. Sci. USA 104, 3913–3918 (2007).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. 21.

    Hollister, J. D. et al. Transposable elements and small RNAs contribute to gene expression divergence between Arabidopsis thaliana and Arabidopsis lyrata. Proc. Natl Acad. Sci. USA 108, 2322–2327 (2011).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. 22.

    Schmitz, R. J. et al. Patterns of population epigenomic diversity. Nature 495, 193–198 (2013).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Gehring, M., Bubb, K. L. & Henikoff, S. Extensive demethylation of repetitive elements during seed development underlies gene imprinting. Science 324, 1447–1451 (2009).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 24.

    Batista, R. A. et al. The MADS-box transcription factor PHERES1 controls imprinting in the endosperm by binding to domesticated transposons. Elife 8, e50541 (2019).

  25. 25.

    Walker, J. et al. Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis. Nat. Genet. 50, 130–137 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  26. 26.

    Bewick, A. J. et al. On the origin and evolutionary consequences of gene body DNA methylation. Proc. Natl Acad. Sci. USA 113, 9111–9116 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  27. 27.

    Chang, F., Gu, Y., Ma, H. & Yang, Z. AtPRK2 promotes ROP1 activation via RopGEFs in the control of polarized pollen tube growth. Mol. Plant 6, 1187–1201 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28.

    Duckney, P. et al. Actin-membrane interactions mediated by NETWORKED2 in Arabidopsis pollen tubes through associations with Pollen Receptor-Like Kinase 4 and 5. N. Phytol. 216, 1170–1180 (2017).

    CAS  Article  Google Scholar 

  29. 29.

    Ge, Z. et al. Arabidopsis pollen tube integrity and sperm release are regulated by RALF-mediated signaling. Science 358, 1596–1600 (2017).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Lu, Y. et al. Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23, 81–93 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Qin, Y. et al. Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil. PLoS Genet. 5, e1000621 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  32. 32.

    Wang, T. et al. A receptor heteromer mediates the male perception of female attractants in plants. Nature 531, 241–244 (2016).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. 33.

    Muschietti, J. P. & Wengier, D. L. How many receptor-like kinases are required to operate a pollen tube. Curr. Opin. Plant Biol. 41, 73–82 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34.

    Evans, A. R., Hall, D., Pritchard, J. & Newbury, H. J. The roles of the cation transporters CHX21 and CHX23 in the development of Arabidopsis thaliana. J. Exp. Bot. 63, 59–67 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  35. 35.

    Loraine, A. E., McCormick, S., Estrada, A., Patel, K. & Qin, P. RNA-seq of Arabidopsis pollen uncovers novel transcription and alternative splicing. Plant Physiol. 162, 1092–1109 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Jones, A. M. et al. Border control–a membrane-linked interactome of Arabidopsis. Science 344, 711–716 (2014).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  37. 37.

    Dong, S. et al. Proteome-wide, structure-based prediction of protein-protein interactions/new molecular interactions viewer. Plant Physiol. 179, 1893–1907 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Tang, K., Lang, Z., Zhang, H. & Zhu, J.-K. The DNA demethylase ROS1 targets genomic regions with distinct chromatin modifications. Nat. Plants 2, 16169 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 39.

    Borges, F. et al. Transposon-derived small RNAs triggered by miR845 mediate genome dosage response in Arabidopsis. Nat. Genet. 50, 186–192 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    You, W. et al. Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana. BMC Plant Biol. 12, 51 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41.

    Dapper, A. L. & Wade, M. J. Relaxed selection and the rapid evolution of reproductive genes. Trends Genet. 36, 640–649 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Ossowski, S. et al. The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana. Science 327, 92–94 (2010).

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  43. 43.

    Hesiod. Theogony (Start Publishing, 2017).

  44. 44.

    Higashiyama, T. & Yang, W. C. Gametophytic pollen tube guidance: attractant peptides, gametic controls, and receptors. Plant Physiol. 173, 112–121 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  45. 45.

    Johnson, M. A., Harper, J. F. & Palanivelu, R. A fruitful journey: pollen ttube navigation from germination to fertilization. Annu. Rev. Plant Biol. 70, 809–837 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  46. 46.

    Zhong, S. & Qu, L. J. Peptide/receptor-like kinase-mediated signaling involved in male-female interactions. Curr. Opin. Plant Biol. 51, 7–14 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  47. 47.

    Shirzadi, R. et al. Genome-wide transcript profiling of endosperm without paternal contribution identifies parent-of-origin-dependent regulation of AGAMOUS-LIKE36. PLoS Genet. 7, e1001303 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. 48.

    Lu, Y. RNA isolation from Arabidopsis pollen grains. Bio-protocol e67–e67 (2011).

  49. 49.

    Schoft, V. K. et al. SYBR green-activated sorting of Arabidopsis pollen nuclei based on different DNA/RNA content. Plant Reprod. 28, 61–72 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  50. 50.

    Borges, F. et al. FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei. Plant Methods 8, 44 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51.

    Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Krueger, F. & Andrews, S. R. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27, 1571–1572 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Huang, D. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    CAS  Article  Google Scholar 

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