Transcriptomic profiling identifies differentially expressed genes associated with programmed cell death of nucellar cells in Ginkgo biloba L

Abstract

Background: Previously, we demonstrated that pollen chamber formation (PCF) in G. biloba ovules was a process of programmed cell death (PCD) within the nucellar cells at the micropylar end. However, the signal triggering the cascades of the programmed events in these nucellar cells remains unexplored.

Results: A transcriptomic strategy was employed to unravel the mechanism underlying the nucellar PCD via the comparative profiles of RNA-seq between pre-PCF and post-PCF ovules. A total of 5599 differentially expressed genes (DEGs) with significance was identified from G. biloba ovules and classified into three main categories of GO annotation, including 17 biological processes, 15 cellular components and 17 molecular functions. KEGG analysis showed that 72 DEGs were enriched in "Plant hormone signal transduction". Furthermore, 99 DEGs were found to be associated with the PCD process, including the genes involved in ethylene signaling pathway, PCD initiation, and PCD execution. Moreover, calcium-cytochemical localization indicated that calcium could play a role in regulating PCD events within the nucellar cells during pollen chamber formation in G. biloba ovules.

Conclusions: A putative working model, consisting of three overlapping processes, is proposed for the nucellar PCD: at the stage of PCD preparation, ethylene signaling pathway is activated for transcriptional regulation of the downstream targets; subsequently, at the stage of PCD initiation, the upregulated expression of several transcription factors, i.e., NAC, bHLH, MADS-box, and MYB, further promotes the corresponding transcript levels of CYTOCHROME C and CALMODULINs, thereby, leads to the PCD initiation via the calcium-dependent signaling cascade; finally, at the stage of PCD execution, some proteases like metacaspases and vacuolar processing enzyme for hydrolysis, together with the process of autophagy, play roles in the clearance of cellular components. Afterwards, a pollen chamber is generated from the removal of specific nucellar cells in the developing ovule.

Keywords: Ginkgo biloba L.; Nucellus; Ovule; Pollen chamber; Programmed cell death (PCD); Transcriptomics.

Fig. 1

Pollen chamber formation within G. biloba ovule observed under the microscope. (a) At the early stage of ovule development, nucellar cells are morphologically similar in their size and shape. (b) At the later developmental stage, nucellar cells at the micropylar end (indicated by arrows), are elongated longitudinally, and distinguishable from other nucellar cells. (c) With the developmental process, pollen chamber (indicated by an arrow) is formed after the death and clearance of nucellar cells at the micropylar end within an ovule. Bars = 250 μm. Abbreviations: I, integument; M, micropyle; Nu, nucellus

Fig. 2

The assessment of correlations among the tested G. biloba ovules by analysis of replicate scatter (a and b) and PCA (c), with each sample group (pre-PCF or post-PCF) including three biological replicates (rep_1, 2, and 3), respectively

Fig. 3

The expression profiles of the identified DEGs. Red and blue points represent the significant DEGs with FDR ≤ 0.05 and log₂(fold change) > 1, and green ones show those without significance, respectively. Fold change refers to the values of FPKM change of post-PCF vs. pre-PCF libraries

Fig. 4

The Gene Ontology (GO) classification of 5599 DEGs. GO terms are summarized in three main categories of cellular component, molecular function and biological process

Fig. 5

Top 20 pathways of KEGG functional enrichment among DEGs. Coloring indicates -log₁₀(Q value) with higher in red and lower in green. And the lower Q value indicates the more significantly enriched. Point size indicates DEG number

Fig. 6

Heatmaps of expression patterns for DEGs involved in phytohormone signaling pathways, including auxin (a), ethylene (b), cytokinin (c), GA (d), BR (e), SA (f), ABA (g), and JA (h). Left and right box-columns represent pre-PCF and post-PCF libraries, respectively. Arrowheads indicate the significantly differentially expressed EIN3 genes

Fig. 7

The protein-protein interaction network of the significantly DEGs involved in the nucellar PCD. The nodes represent target proteins shown by their gene names, respectively. The color band is illustrated from red to yellow in descending order of degree values. Edge size is mapped according to its corresponding parameter EdgeBetweenness with lower value to smaller size

Fig. 8

Expression level validation of DEG using qRT-PCR, in comparation to corresponding data detected in RNA-Seq. Relative expression ratio of each DEG is presented in a log₂ value of post-PCF vs. pre-PCF libraries. The values are mean ± SE (n = 3)

Fig. 9

Subcellular localization of calcium within the nucellar cells involved in pollen chamber formation. (a) At the early stage of ovule development, a relatively higher Ca²⁺ precipitation is found in both of vacuoles and nucleus, compared with that in the cytoplasm. (b) A negative control of (a) without Ca²⁺ particles. (c), (d), (f), and (g) The nucellar cells at the micropylar end become elongated in shape, and distribution of Ca²⁺ precipitation is found to be increased in these nucellar cells. (d) Magnified view of the circled area in (c) shows numerous Ca²⁺ particles within the vacuole and cytoplasm. (e) A negative control of (c) without Ca²⁺ particles. (g) Deformed endoplasmic reticula are enclosed within the vacuole. (h) Few of Ca²⁺ particles are distributed along the cytoplasm debris, with no detectable aggregation of Ca²⁺ in the nucleus of the dying nucellar cell. Arrows indicate Ca²⁺ particles. Bars in (a), (b), (c), (e), and (h) = 2 μm, and in (d), (f), and (g) = 0.2 μm. CW, cell wall; N, nucleus; V, vacuole

Fig. 10

Tentative model for the nucellar PCD during pollen chamber formation in G. biloba ovules. Ovules at the stage of pre-PCF (a) and post-PCF (b) are sectioned longitudinally. The black-boxed area in (a) contains nucellar cells at the micropylar end of ovule, which are to undergo PCD to generated a pollen chamber, bounded by the white-box in (b). Other nucellar cells, represented by a white-boxed area in (a), keep growth throughout the stages of PCF

Fig. 11

Ovule collection from G. biloba strobili at the stage of pre-PCF (a) or post-PCF (b). Magnified view of the boxed area shows ovules (arrowheads) collected from megasporophyll, and red-dash lines marking the junction region between ovules and megasporophyll. G. biloba leaves (asterisks) are positioned along with ovules. Bars = 1 cm