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

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.

Fig. 1.

Acridine orange stained Scots pine embryos and megagametophyte tissue during embryogenesis. The double-stranded nucleic acids (i.e. DNA) fluoresce green and the single-stranded (i.e. RNA) fluoresce red. (A) Dominant and subordinate early embryos in the corrosion cavity on sampling date I. (B) Dominant early embryo in the corrosion cavity on sampling date II. (C) Dominant late embryo in the corrosion cavity on sampling date III. (D) Dominant late embryo and the dying subordinate embryos in the corrosion cavity on sampling date III. (E) Late embryo filling the corrosion cavity on sampling date IV. (F) Dying cells of subordinate embryos broke down, resulting in a leakage of nucleic acids into the surrounding extracellular space. (G) During embryo development, the cell wall, plasma membrane, and nuclear envelope of the megagametophyte cells lining the corrosion cavity broke down with the release of cell debris and nucleic acids into the corrosion cavity (early embryogeny, sampling date II). (H) During early embryogeny, nuclei in megagametophyte cells stained green, except for rRNA containing nucleoli, which were red. In the cytoplasmic region, the red colour indicated the presence of mRNA and active gene expression. (I) During late embryogeny, nuclei in megagametophyte cells appeared normal with the presence of nucleoli and with no sign of DNA fragmentation. (J) Control sample with no acridine orange staining. Bars: (G, H, I) 10 μm, (F) 20 μm, (A, C, J) 50 μm, and (B, D, E) 100 μm.

Fig. 2.

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.

Fig. 3.

Intact DNA of immature as well as mature Scots pine seeds and viable, proliferating megagametophytes of mature seeds. (A) The agarose gel shows intact DNA extracted from immature Scots pine seeds on sampling date I (lane 1), sampling date II (lane 2), sampling date III (lane 3), and sampling date IV (lane 4) as well as from the embryos (lane 5) and megagametophytes (lane 6) of mature seeds after 2 d imbibition. (B) Megagametophyte tissue of mature Scots pine seed was tested for viability using tetrazolium. The megagametophyte showed deep red when the seed coat was removed after the tetrazolium test. (C) Both the embryo and the megagametophyte were deep red in the mature seed, split in half after the tetrazolium test. (D) Tetrazolium test performed on mature seed boiled for 30 min as a negative control. (E) At the beginning of the proliferation test, embryos were excised from mature seeds and the halves of the megagametophytes were set on DCR medium. (F) After 10-d culture megagametophytes showed proliferation.

Fig. 4.

The expression of the putative RAD51, KU80, LIG, MCA, and TAT-D genes in developing Scots pine seeds. The expressions were quantified by real-time PCR and normalized by the expression derived from the housekeeping genes ACT, UBI, and GAPDH. The observed relative gene expressions are presented in relation to the effective temperature sum (d.d.) in the clones K818 (open circles) and K884 (closed circles), and the geometric mean values of the replicates pertaining to the same clone and sampling date are connected by dashed lines for K881 and solid lines for K884. There are five replicates per clone and per sampling date, except for the seeds of clone K884 on sampling dates III and IV, from which there are two and four replicates, respectively.