Expression of catalase and retinoblastoma-related protein genes associates with cell death processes in Scots pine zygotic embryogenesis

Abstract

Background: The cell cycle and cellular oxidative stress responses are tightly controlled for proper growth and development of Scots pine (Pinus sylvestris L.) seed. Programmed cell death (PCD) is an integral part of the embryogenesis during which megagametophyte cells in the embryo surrounding region (ESR) and cells in the nucellar layers face death. In the present study, we show both the tissue and developmental stage specific expression of the genes encoding the autophagy related ATG5, catalase (CAT), and retinoblastoma related protein (RBR) as well as the connection between the gene expressions and cell death programs.

Results: We found strong CAT expression in the cells of the developing embryo throughout the embryogenesis as well as in the cells of the megagametophyte and the nucellar layers at the early embryogeny. The CAT expression was found to overlap with both the ATG5 expression and hydrogen peroxide localization. At the late embryogeny, CAT expression diminished in the dying cells of the nucellar layers as well as in megagametophyte cells, showing the first signs of incipient cell death. Accumulation of starch and minor RBR expression were characteristic of megagametophyte cells in the ESR, whereas strong RBR expression was found in the cells of the nucellar layers at the late embryogeny.

Conclusions: Our results suggest that ATG5, CAT, and RBR are involved in the Scots pine embryogenesis and cell death processes. CAT seems to protect cells against hydrogen peroxide accumulation and oxidative stress related cell death especially during active metabolism. The opposite expression of RBR in the ESR and nucellar layers alongside morphological characteristics emphasizes the different type of the cell death processes in these tissues. Furthermore, the changes in ATG5 and RBR expressions specifically in the megagametophyte cells dying by necrotic cell death suggest the genetic regulation of developmental necrosis in Scots pine embryogenesis.

Figure 1

Early and late developmental stages of Scots pine embryogenesis. (A) The dominant embryo and subordinate embryos in the corrosion cavity at the early embryogeny surrounded by the embryo surrounding region (esr) of the megagametophyte highlighted with red color. The arrow-shaped region (asr) of the megagametophyte outside the esr highlighted with blue color. (B) The dominant embryo in the corrosion cavity at the late embryogeny. The esr of the megagametophyte highlighted with red color and asr highlighted with blue color. asr = arrow-shaped region, cc = corrosion cavity, e = embryo, esr = embryo surrounding region, m = megagametophyte, mm = megaspore membranes, nc = nucellar cap, nl = nucellar layers, nt = cellular nucellus, sr = suspensor remnants. Bars: 100 μm.

Figure 2

CAT expression in developing and mature Scots pine seeds. (A) The relative expression of CAT in developing seeds at the early and late embryogeny and in the embryos (e) and megagametophytes (m) of mature seeds. The expression was based on mRNA copy numbers generated with the absolute Q-RT-PCR analysis and values presented were normalized using the expression at the early embryogeny. A star denotes significant (P <0.05) difference in the expression. (B) The localization of CAT mRNAs by in situ hybridization with DIG-labelled RNA-probes (blue signal) in a developing Scots pine seed at the early embryogeny. (C) The localization of CAT mRNAs at the late embryogeny. (D) Intense CAT expression in the developing embryos and in the megagametophyte at the early embryogeny. (E) Intense CAT expression in the nucellar layers at the early embryogeny. (F) Minor CAT expression in the nucellar layers at the late embryogeny. (G) Intense CAT expression in the cells of the leading embryo and minor CAT expression in the megagametophyte cells at the late embryogeny. cc = corrosion cavity, e = embryo, esr = embryo surrounding region, m = megagametophyte, nc = nucellar cap, nl = nucellar layers, nt = cellular nucellus, sr = suspensor remnants. Bars: 100 μm.

Figure 3

The localization of CAT mRNAs in a mature Scots pine seed. (A) Intense CAT expression (blue signal) in the embryo and in the megagametophyte cells surrounding the corrosion cavity. (B) Intense CAT expression in the embryonic cells. (C) Minor CAT expression in the cells in the inner part of the megagametophyte. cc = corrosion cavity, e = embryo, m = megagametophyte. Bars: (B, C) 20 μm and (A) 200 μm.

Figure 4

The localization of H ₂ O ₂ in developing Scots pine seeds. (A) Intense blue colour from TMB indicates the presence of H₂O₂ in the seed coat and flight wing at the early embryogeny. (B) The localization of H₂O₂ in the nucellar cap, cellular nucellus, and in the nucellar layers at the early embryogeny. (C) The localization of H₂O₂ in the megaspore membranes at the early embryogeny. (D) The minor amount of H₂O₂ in the seed coat and flight wing and the large amount of H₂O₂ indicated by the dark blue colour in the nucellar layers at the late embryogeny. (E) The seed treated with Tris-acetate buffer without TMB. cc = corrosion cavity, e = embryo, esr = embryo surrounding region, fw = flight wing, m = megagametophyte, mm = megaspore membranes, nc = nucellar cap, nl = nucellar layers, nt = cellular nucellus, sc = seed coat, sr = suspensor remnants.

Figure 5

The localization of peroxidase activity in developing Scots pine seeds. (A, B) Oxidized DAB (brown colour) indicated peroxidase activity in the seed coat, nucellar layers, megagametophyte cells surrounding the corrosion cavity, suspensor cells and subordinate embryos at the early (A) and late (B) embryogeny. (C) The seed treated with the reaction buffer without DAB. asr = arrow-shaped region, e = embryo, esr = embryo surrounding region, m = megagametophyte, mm = megaspore membranes, nc = nucellar cap, nl = nucellar layers, nt = cellular nucellus, sc = seed coat, se = subordinate embryo, sr = suspensor remnants.

Figure 6

RBR expression in developing Scots pine seeds. (A) The relative expression of RBR in developing seeds at the early and late embryogeny and in the embryos (e) and megagametophytes (m) of mature seeds. The expression was based on mRNA copy numbers generated with the absolute Q-RT-PCR analysis and values presented were normalized using the expression at the early embryogeny. (B) The localization of RBR mRNAs (blue signal) in a developing Scots pine seed at the early embryogeny. (C) The localization of RBR mRNAs at the late embryogeny. (D) Minor RBR expression in the ESR of the megagametophyte at the early embryogeny. (E) Weak RBR expression in the cells of the nucellar layers at the early embryogeny. (F) Intense RBR expression in the cells of the nucellar layers at the late embryogeny. asr = arrow-shaped region, cc = corrosion cavity, e = embryo, esr = embryo surrounding region, m = megagametophyte, nc = nucellar cap, nl = nucellar layers, nt = cellular nucellus, sr = suspensor remnants. Bars: (D) 20 μm, (F) 50 μm, and (B, C, E) 100 μm.

Figure 7

The localization of ATG5 expression in developing and mature Scots pine seeds. (A) ATG5 expression (blue signal) in the esr of the megagametophyte and in the nucellar layers at the early embryogeny. (B) ATG5 expression in the esr of the megagametophyte and in the maturating tracheids of the embryo in a mature seed. asr = arrow-shaped region, cc = corrosion cavity, e = embryo, esr = embryo surrounding region, m = megagametophyte, nc = nucellar cap, nl = nucellar layers, sr = suspensor remnants. Bars: 100 μm.

Figure 8

Histochemical localization of proteins by amido black and starch by potassium iodide-iodine in developing Scots pine seeds. (A) Megagametophyte cells with few protein storage vacuoles at the early embryogeny. (B) Megagametophyte cells stocked with protein storage vacuoles at the late embryogeny. (C) Megagametophyte cells with starch grains in the vicinity of the corrosion cavity at the early embryogeny. (D) Starch grains in the cotyledons and root meristem of a late embryo as well as in the megagametophyte. asr = arrow-shaped region, cc = corrosion cavity, e = embryo, esr = embryo surrounding region, m = megagametophyte. Bars: (A, C) 100 μm and (B, D) 200 μm.