Fig 1.
Summary of differentially-expressed genes in the Norway spruce EM versus suspensor.
(A) Number of differentially expressed and GO annotated genes. (B) Glimma plot of expression values of RNA-seq detected genes normalized by their sequencing depth. Highlighted red are groups of genes up-regulated in the suspensor or EM. (C) Classes of transcriptionally up-regulated enzymes in the EM and in the suspensor.
Fig 2.
Schematic overview of flavonoid biosynthesis pathway (modified after [30]).
Highlighted in red are enzymes showing transcriptional up-regulation in the EM and their corresponding ConGenIE ID numbers.
Table 1.
Examples of enzymes transcriptionally up-regulated in the EM of Norway spruce and their known functions.
Table 2.
Transcription factors up-regulated in the EM of Norway spruce and functions of their Arabidopsis homologues.
Table 3.
Cell wall modifying enzymes transcriptionally up-regulated in the Norway spruce embryo-suspensor and functions of their Arabidopsis homologues.
Table 4.
Transcription factors up-regulated in the Norway spruce embryo-suspensor and the functions of their Arabidopsis homologues.
Table 5.
Examples of potential anti- and pro-cell-death genes up-regulated in the Norway spruce embryo-suspensor and functions of their Arabidopsis homologues.
Fig 3.
PaBI-1 deficiency impairs embryogenesis.
(A) Morphology of control (transformed with pMDC32::GUS, [6]) and PaBI-1 RNAi lines grown for 5 days without growth regulators and stained with Evan’s blue to detect dying or dead cells. Scale bars, 500 μm. (B) Normalized expression of PaBI-1 in the control and RNAi lines. *, P<0.01; vs control, Student’s t-test. (C) Length of suspensor cells. Data represent mean ± SEM for more than 80 Evan’s blue-positive suspensor cells from at least 10 different embryos per line. *, P<0.0001; vs control, Student’s t-test. (D) Frequency of aberrant early embryos lacking elongated suspensor. Data represent mean ± SEM. The experiment included more than 40 embryos per line and was repeated two times. *, P<0.0001; vs control, Student’s t-test. (E) Number of cotyledonary embryos formed after 7 weeks on ABA-containing medium. Data represent mean ± SEM from three independent experiments, each including one plate per line. *, P<0.001; vs control, Student’s t-test. FW, fresh weight. (F) Morphology of maturing embryos in control and PaBI-1 RNAi lines grown for 9 weeks on ABA-containing medium. Arrowheads indicate under-developed embryos in PaBI-1 RNAi line, compared to fully developed embryos that have already started germinating in the control line. Scale bars, 2 mm.
Fig 4.
PaBI-1 deficiency induces necrotic cell death.
(A) FDA and FM4-64 staining of control and PaBI-1 RNAi lines to detect necrosis. Arrowheads indicate shrunken and undigested protoplast in necrotic cells. DIC, differential interference contrast. Scale bars, 100 μm. (B) Frequency of necrotic cells. Data represents mean ± SEM from three independent experiments, each including more than 50 cells per line. (*, P<0.0001; vs control, Student’s t-test).
Fig 5.
A hypothetical model of transcriptionally regulated processes and corresponding up-regulated genes involved in terminal differentiation and death of the Norway spruce embryo-suspensor, as suggested by RNA-seq analysis.
Cells in the upper layers of the suspensor (i.e. adjacent to the embryonal mass) begin to expand with simultaneous reorganization of cell wall and enlargement of vacuole. Thereafter cells initiate PCD by expressing transcription factors and stress-responsive genes. The latter might act to suppress rapid cell death and to allow gradual cell dismantling characteristic for vacuolar PCD. At the basal end of the suspensor, hydrolytic enzymes (proteases and nucleases) execute PCD by processing protein and nucleic acid substrates. Note that some gene names correspond to Arabidopsis homologues.