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One tissue, two fates: different roles of megagametophyte cells during Scots pine embryogenesis.

Vuosku J, Sarjala T, Jokela A, Sutela S, Sääskilahti M, Suorsa M, Läärä E, Häggman H - J. Exp. Bot. (2009)

Bottom Line: In the Scots pine (Pinus sylvestris L.) seed, embryos grow and develop within the corrosion cavity of the megagametophyte, a maternally derived haploid tissue, which houses the majority of the storage reserves of the seed.It was found that the megagametophyte was viable from the early phases of embryo development until the early germination of mature seeds.Their cell wall, plasma membrane, and nuclear envelope broke down with the release of cell debris and nucleic acids into the corrosion cavity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Oulu, PO Box 3000, 90014 Oulu, Finland. jaana.vuosku@oulu.fi

ABSTRACT
In the Scots pine (Pinus sylvestris L.) seed, embryos grow and develop within the corrosion cavity of the megagametophyte, a maternally derived haploid tissue, which houses the majority of the storage reserves of the seed. In the present study, histochemical methods and quantification of the expression levels of the programmed cell death (PCD) and DNA repair processes related genes (MCA, TAT-D, RAD51, KU80, and LIG) were used to investigate the physiological events occurring in the megagametophyte tissue during embryo development. It was found that the megagametophyte was viable from the early phases of embryo development until the early germination of mature seeds. However, the megagametophyte cells in the narrow embryo surrounding region (ESR) were destroyed by cell death with morphologically necrotic features. Their cell wall, plasma membrane, and nuclear envelope broke down with the release of cell debris and nucleic acids into the corrosion cavity. The occurrence of necrotic-like cell death in gymnosperm embryogenesis provides a favourable model for the study of developmental cell death with necrotic-like morphology and suggests that the mechanism underlying necrotic cell death is evolutionary conserved.

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Nuclear DNA fragmentation detected by TUNEL assay in Scots pine seed sections. (A) TUNEL-positive signals in the megagametophyte in the vicinity of the corrosion cavity at the early embryogeny stage. (B) TUNEL-positive signals in the megagametophyte in the arrow-shaped region in front of the embryo at the late embryogeny stage. (C) Slightly TUNEL-positive nuclei appeared in the inner part of the megagametophyte during the late embryogeny stage (sampling date IV). (D) TUNEL-positive nuclei in the nucellar layer at the late embryogeny stage. (E) TUNEL-positive nuclei in the suspensor cells at the early embryogeny stage. (F) TUNEL-positive nuclei detected by excitation at 543 nm in the inner part of the megagametophyte at the late embryogeny stage. (G) Lack of DNA fragmentation detected by excitation at 543 nm in the inner part of the megagametophyte at the early embryogeny stage. (H) Positive control (DNase treatment). (I) Negative control (omission of TdT). Bars: (C, D, F) 20 μm, (B, E, G, H, I) 50 μm, and (A) 100 μm.
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fig2: Nuclear DNA fragmentation detected by TUNEL assay in Scots pine seed sections. (A) TUNEL-positive signals in the megagametophyte in the vicinity of the corrosion cavity at the early embryogeny stage. (B) TUNEL-positive signals in the megagametophyte in the arrow-shaped region in front of the embryo at the late embryogeny stage. (C) Slightly TUNEL-positive nuclei appeared in the inner part of the megagametophyte during the late embryogeny stage (sampling date IV). (D) TUNEL-positive nuclei in the nucellar layer at the late embryogeny stage. (E) TUNEL-positive nuclei in the suspensor cells at the early embryogeny stage. (F) TUNEL-positive nuclei detected by excitation at 543 nm in the inner part of the megagametophyte at the late embryogeny stage. (G) Lack of DNA fragmentation detected by excitation at 543 nm in the inner part of the megagametophyte at the early embryogeny stage. (H) Positive control (DNase treatment). (I) Negative control (omission of TdT). Bars: (C, D, F) 20 μm, (B, E, G, H, I) 50 μm, and (A) 100 μm.

Mentions: Nuclear DNA fragmentation, i.e. DNA strand breaks leaving free 3′-OH termini, was shown by the TUNEL assay. TUNEL-positive nuclei were detected in the cells that displayed morphological features of necrotic cell death in ESR and in the arrow-shaped megagametophyte region in front of the dominant embryo throughout the development of the embryo (Fig. 2A, B). By contrast, no TUNEL-positive cells were found in the inner parts of the megagametophyte before the developmental stage of late embryogeny, when a weak signal could be detected (Fig. 2C). Furthermore, TUNEL-positive nuclei were also found in the cells of the nucellar layer covering the megagametophyte (Fig. 2D) as well as in the degrading suspensor cells (Fig. 2E). The TUNEL signals were located identically when examined by fluorescence microscopy as well as excitation at 543 nm (Fig. 2F, G).


One tissue, two fates: different roles of megagametophyte cells during Scots pine embryogenesis.

Vuosku J, Sarjala T, Jokela A, Sutela S, Sääskilahti M, Suorsa M, Läärä E, Häggman H - J. Exp. Bot. (2009)

Nuclear DNA fragmentation detected by TUNEL assay in Scots pine seed sections. (A) TUNEL-positive signals in the megagametophyte in the vicinity of the corrosion cavity at the early embryogeny stage. (B) TUNEL-positive signals in the megagametophyte in the arrow-shaped region in front of the embryo at the late embryogeny stage. (C) Slightly TUNEL-positive nuclei appeared in the inner part of the megagametophyte during the late embryogeny stage (sampling date IV). (D) TUNEL-positive nuclei in the nucellar layer at the late embryogeny stage. (E) TUNEL-positive nuclei in the suspensor cells at the early embryogeny stage. (F) TUNEL-positive nuclei detected by excitation at 543 nm in the inner part of the megagametophyte at the late embryogeny stage. (G) Lack of DNA fragmentation detected by excitation at 543 nm in the inner part of the megagametophyte at the early embryogeny stage. (H) Positive control (DNase treatment). (I) Negative control (omission of TdT). Bars: (C, D, F) 20 μm, (B, E, G, H, I) 50 μm, and (A) 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2657542&req=5

fig2: Nuclear DNA fragmentation detected by TUNEL assay in Scots pine seed sections. (A) TUNEL-positive signals in the megagametophyte in the vicinity of the corrosion cavity at the early embryogeny stage. (B) TUNEL-positive signals in the megagametophyte in the arrow-shaped region in front of the embryo at the late embryogeny stage. (C) Slightly TUNEL-positive nuclei appeared in the inner part of the megagametophyte during the late embryogeny stage (sampling date IV). (D) TUNEL-positive nuclei in the nucellar layer at the late embryogeny stage. (E) TUNEL-positive nuclei in the suspensor cells at the early embryogeny stage. (F) TUNEL-positive nuclei detected by excitation at 543 nm in the inner part of the megagametophyte at the late embryogeny stage. (G) Lack of DNA fragmentation detected by excitation at 543 nm in the inner part of the megagametophyte at the early embryogeny stage. (H) Positive control (DNase treatment). (I) Negative control (omission of TdT). Bars: (C, D, F) 20 μm, (B, E, G, H, I) 50 μm, and (A) 100 μm.
Mentions: Nuclear DNA fragmentation, i.e. DNA strand breaks leaving free 3′-OH termini, was shown by the TUNEL assay. TUNEL-positive nuclei were detected in the cells that displayed morphological features of necrotic cell death in ESR and in the arrow-shaped megagametophyte region in front of the dominant embryo throughout the development of the embryo (Fig. 2A, B). By contrast, no TUNEL-positive cells were found in the inner parts of the megagametophyte before the developmental stage of late embryogeny, when a weak signal could be detected (Fig. 2C). Furthermore, TUNEL-positive nuclei were also found in the cells of the nucellar layer covering the megagametophyte (Fig. 2D) as well as in the degrading suspensor cells (Fig. 2E). The TUNEL signals were located identically when examined by fluorescence microscopy as well as excitation at 543 nm (Fig. 2F, G).

Bottom Line: In the Scots pine (Pinus sylvestris L.) seed, embryos grow and develop within the corrosion cavity of the megagametophyte, a maternally derived haploid tissue, which houses the majority of the storage reserves of the seed.It was found that the megagametophyte was viable from the early phases of embryo development until the early germination of mature seeds.Their cell wall, plasma membrane, and nuclear envelope broke down with the release of cell debris and nucleic acids into the corrosion cavity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Oulu, PO Box 3000, 90014 Oulu, Finland. jaana.vuosku@oulu.fi

ABSTRACT
In the Scots pine (Pinus sylvestris L.) seed, embryos grow and develop within the corrosion cavity of the megagametophyte, a maternally derived haploid tissue, which houses the majority of the storage reserves of the seed. In the present study, histochemical methods and quantification of the expression levels of the programmed cell death (PCD) and DNA repair processes related genes (MCA, TAT-D, RAD51, KU80, and LIG) were used to investigate the physiological events occurring in the megagametophyte tissue during embryo development. It was found that the megagametophyte was viable from the early phases of embryo development until the early germination of mature seeds. However, the megagametophyte cells in the narrow embryo surrounding region (ESR) were destroyed by cell death with morphologically necrotic features. Their cell wall, plasma membrane, and nuclear envelope broke down with the release of cell debris and nucleic acids into the corrosion cavity. The occurrence of necrotic-like cell death in gymnosperm embryogenesis provides a favourable model for the study of developmental cell death with necrotic-like morphology and suggests that the mechanism underlying necrotic cell death is evolutionary conserved.

Show MeSH
Related in: MedlinePlus