Morphological observation and protein expression of fertile and abortive ovules in Castanea mollissima

Plant material

The anthesis of C. mollissima cv. ‘Huaihuang’ begins at mid-June. One hundred burrs from 20 chestnut trees were monitored at 20 days after anthesis (DAA) from the Chestnut Experiment Station in Huairou District, Beijing, China. At this time, fertile and abortive ovules in the same ovary could just be distinguished by the naked eyes.

Paraffin sections

Morphological structure of ovaries and ovules were observed according to the method described by Du et al. (2020). The ovaries and ovules were dehydrated in a graded series of ethanol (50%, 70%, 80%, 95% and 100%) followed by a xylene/ethanol series (ethanol/xylene 1:1 and 100% xylene) and wax/xylene series (wax/xylene 1:1 and 100% wax), and according to the size of samples, the timing of dehydration and the wax-impregnated were adjusted appropriately; Samples and the wax solution were poured into a stacked carton, the position of the ovaries and ovules were quickly adjusted before the wax solution could solidify. Paraffin-embedded samples were cut into slices of 7-μm thickness using a rotary microtome (Leica RM 2235) and dried on a hot plate at 45 °C overnight; The next day, the tissue biopsies were deparaffinized by a xylene/ethanol series (100% xylene, and ethanol/xylene 1:1) followed by an ethanol series (100%, 90%, 80%, 70%, 60% and 50%), then were hydrated in reverse.

Fluorescent labeling of cell nuclei

Propidium iodide (PI; Sigma, Kawasaki, Kanagawa, Japan) powder was dissolved at 30 ng/ml in PBS buffer. Then the tissue sections were incubated for 10–15 min in the PI stain (triplicates). DAPI (4′,6-diamidino-2-phenylindole; Sigma, Kawasaki, Kanagawa, Japan) powder was dissolved at 1 μg/ml in PBS buffer, and the tissue sections were incubated for 5–10 min in the DAPI stain (triplicates). After staining, the tissue biopsies were observed under a fluorescence microscope (Olympus BX51, Japan), and the images were captured with a CCD color camera.

Transmission electron microscopy

(1) The samples were fixed in 2.5% glutaraldehyde and then placed under vacuum for 2.5 h until the ovules were completely immersed in the fixative, then the tubes filled with the sample material were placed at 4 °C for overnight fixation. (2) After fixation, the ovules were washed 10 times for 10 min each with phosphate buffer (10 mM, pH 7.2). (3) Osmium acid was mixed with PBS in equal volumes in a centrifuge tube, and the sample was fully immersed in the mixture for 1.5 h with vibration. After osmic acid fixation was complete, the fixed ovules were washed 10 times for 10 min each with phosphate buffer. (4) After rinsing, the ovules were removed to fresh centrifuge tubes using a toothpick, then the fixed samples were dehydrated in a graded series of ethanol (15 min per step) at room temperature, followed by an acetone wash. (5) Acetone and a resin embedding agent were mixed at ratios of 1:1, 1:2 and 1:3. The ovules were impregnated separately for 8 h in the mixtures. (6) The samples were embedded at both ends of the embedding plate by the resin. (7) The embedding material was polymerized at 60 °C for 15 days. (8) The embedding block was cut into 50 nm slices by an ultra-thin slicing technique; the slices were then placed on a copper mesh. (9) The sections of the copper mesh were double-stained with 2% uranium acetate and 0.5% lead acetate. (10) The copper mesh was observed under a transmission electron microscope.

Protein digestion and iTRAQ labeling of samples

iTRAQ was used to identify differences in protein expression between fertile ovules and abortive ovules in early Chinese chestnut ovaries. This experiment was conducted at the Beijing Protein Innovation Co., Ltd. Three replications were carried out. The experimental procedures included: (1) Protein extraction. Triplicates of fertile ovules and abortive ovules were homogenized in precooled extraction buffer composed of 50 mM Tris-HCl, 2 mM EDTA, 100 mM KCl, 700 mM sucrose solutions and Phenol Tris. Following this step, the mixture was fully oscillated and centrifuged at 15,000×g for 10 min, then the supernatant obtained was mixed with ammonium acetate and acetone. Subsequently, the mixture was centrifuged at 15,000×g for 10 min, then the sediment obtained was mixed with lysis buffer (8 M Urea, 30 mM HEPES, 1 mM PMSF, 2 mM EDTA and 10 mM DTT). Then supernatant was precipitated with acetone again for 3 hours, the addition of redissolve buffer composed with 50% TEAB and 0.1% SDS to the sediment by centrifugation at 20,000×g for 30 min to get supernatant. Finally, the peptides were quantified by Bradford method. (2) Protein digestion. The peptides were digested overnight with 1 μg/μl trypsin in 100 μl TEAB (1 M) at 37 °C, which were freeze-dried and then redissolved with 30 μl TEAB solution (TEAB/water 1:1) for iTRAQ labelling. (3) Peptide marking. Peptide samples were divided into three batches and labeled with labeling reagents. For one batch, fertile ovules were labeled with 113, 114 and 115, for another batch, abortive ovules were labeled with 116, 117 and 118. Samples in each batch were mixed after labeled and then subjected to MS/MS analysis. (4) LC-MS/MS analysis. After desalted, the labeled peptides were separated and determined for the MS analysis by HPLC (High Performance Liquid Chromatography). This step was performed via a “trap and elute” configuration and the mobile phase included A (25% ACN, 10 mM KH₂PO₄) and B (25% ACN, 2 M KCL, 10 mM KH₂PO₄). The peptides were separated over 45 min by a gradient of 5–80% of mobile phase B at a flow rate of 400 nl/min combined with a Q Exactive mass spectrometer and the chromatographic column of model C18 was choice with a particle size of 5 μm. Then to evaluate the performance of this mass spectrometry on the iTRAQ labeled samples, two MS/MS acquisition modes, higher collision energy dissociation (HCD) was employed. And to optimize the MS/MS acquisition efficiency of HCD, normalized collision energy (NCE) was systemically examined 28, stepped 20%. (5) Qualitative and quantitative analysis of the proteins. Mass spectrograms were filtered by using Proteome Discoverer software v.1.3 and researched with mascot, searching parameters were defined as following: fixed modification-carbamidomethyl; sample style-iTRAQ 8 plex; peptide tol-15 ppm; MS/MS tol-20 mmu; digestion-trypsin; database-uni_rosids_71275. Then the search results and the spectrograms after the first step were quantitatively analyzed, quantitative analysis parameters were defined as following: protein ratio type-median; minimum peptides-1; normalisation method-median. In addition, the significant differences and the SD value of protein concentrations, total protein and DEPs expression ratio from fertile and abortive ovules were analyzed and calculated by SPSS software.

Bioinformatics analysis of differential proteins

The differently expressed proteins (DEPs) were defined with fold-change ratios of log2 > 1.2 or < 0.8. Analysis of DEPs were carried as previously described (Geng et al., 2018). In a short, additional filters were initially conducted to screen out proteins with p-value < 0.05 (95% confidence) and ion score or expected cutoff < 0.05 (95% confidence). These screened DEPs were subsequently characterized by Blast2GO and the non-redundant protein database. The significance of the GO analysis was further confirmed in the KEGG database, in which each DEP was classified into a specific pathway. Meanwhile, the mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD021317.

Western blot

This experiment was carried out at the Beijing Protein Innovation Co., Ltd., three replicates were performed. In this experiment, one peptide sequence containing the specific amino acids corresponding to the mature glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequence, was obtained by chemical synthesis. Synthetic peptide was purified by high-performance liquid chromatography and coupled with keyhole limpet hemocyanin. Then GAPDH was detected by western blotting, with steps below. First, filter paper, a PVDF membrane and an acrylamide gel were placed in the film transfer slot with transfer solution, and 100 V was applied for 2 hours to transfer the proteins to the PVDF membrane. The PVDF membrane was then placed in a primary antibody solution, followed by washing to remove non-specific binding. The membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G at room temperature for 1–2 h and then incubated in ECL2 reagent for 5 min in the dark. The PVDF membrane was then placed in a development folder, air bubbles were rolled out. The final color was developed with a solution containing 3,3-diaminobenzidine tetrahydrochloride as HRP substrate.

Morphologies of Chinese chestnut burrs, ovaries and ovules

The chestnut anthesis begins in mid-June. Female burrs were collected approximately 20 DAA. During this period, the chestnut spines become markedly stiffened and the ovary becomes enlarged (Figs. 1A, 1B). Fertile and abortive ovules in the same ovary could be distinguished by eyes. Based on the anatomy of the ovary, there were 12–18 ovules per ovary, and ovules were localized at the apex of the ovary connected to the style. Other ovules were aborted except the fertile ovule that could develop normally (Figs. 1C, 1D).

Microstructure of fertile and abortive ovules

Cross sections of the ovaries revealed 6–9 ventricles per ovary, and each ventricle had two different sizes of anatropous ovules; it is possible that the smaller one might be aborted earlier (Figs. 2A, 2B). The longitudinal section of the fertile ovules showed that they contained completely formed tissues, such as the embryo sac, embryo and endosperm, and the structures could support the normal processes of pollination, fertilization and embryogenesis. At this time (20 DAA), we could also see that the embryo developed into a sphere near the end of the funicle, and the inner integument was closely connected to the outer integument (Figs. 2C, 2D). But in the abortive ovules, there were some key parts missing, including embryo sac, embryo and endosperm; and obvious gap was present between the inner and outer integument compared with fertile ovules; furthermore, remnants of nucellus could still be seen in the abortive ovules (Figs. 2E, 2F).

Nuclei of fertile and abortive ovules cells

Fluorescence microscopy revealed that in the outer integument of fertile ovules, cell nuclei were in the shape of regular sphere, and the nuclear membrane could be seen in a sharp outline. However, the fluorescence intensity of DAPI labelling was higher than that of PI (Figs. 3A, 3D). In the inner integument of fertile ovules, no nuclei were detected in the cells (Figs. 3B, 3E). In the abortive ovules, there was little difference in the DAPI and PI fluorescence intensity. Notably, nucleoli of the outer integument and inner integument all exhibited intense fluorescence, and it was worth noting that the cells of inner integument had obvious nucleus. In addition, the cells of the outer and inner integument were abnormally shaped and had a smaller size. Cell nucleoli were detected as bright fluorescent spots (Figs. 3C, 3F).

Ultrastructure of fertile and abortive ovules

In the outer integument of fertile ovules, the cells were regularly shaped and contained a complete cell wall and plasma membrane. The cell nuclei were structurally intact and contained a clear and complete nuclear membrane and nucleoli. Meanwhile, mitochondria, Golgi apparatus, endoplasmic reticulum and other organelles all had complete structures, and several mitochondria were distributed around the cell membrane and nucleus (Figs. 4A–4C). Surprisingly, the ultrastructure features of the inner integument showed characteristics of apoptotic cells. There were distinct dividing lines between the inner and outer integuments (Fig. 4D). In the inner integument of fertile ovules, cell organelles were degraded, the cell contents were diminished, a cavity had formed inside the cells, and only starch grains were present in the cells (Figs. 4E, 4F). In the abortive ovules, the ultrastructure features of the outer integument showed an abnormal cell shape, irregular thickening of the cell walls, nuclear membrane rupture, substantial cytoplasm aggregation, and nuclear membrane damage. Furthermore, the organelles in these cells were difficult to distinguish (Figs. 4G–4I).

Primary data analysis and protein identification in the fertile and abortive ovules

Proteins were extracted from fertile and abortive ovules. The total protein content extracted from fertile ovules was 536 µg at 1.79 µg/µL and that of abortive ovules was 220 µg at 0.73 µg/µL (Fig. S1A). The protein content of fertile ovules was 2.43-fold greater than that of abortive ovules. In addition, there were apparent differences between fertile ovules and abortive ovules in the patterns of whole-cell proteins. The experimental results showed that 25–66 KD proteins were in the greatest abundance, followed by 14–25 KD and 66–97 KD proteins. Most proteins of fertile ovules were concentrated in the 25–97 KD range, and most proteins of abortive ovules were concentrated in the 14–66 KD range (Fig. S1B). This showed that the molecular weights of proteins in the fertile ovules were higher than those in the abortive ovules. Proteomics technology was used to identify differentially expressed proteins (DEPs). A volcano plot of the DEPs is shown in Fig. S2A. We defined DEPs as those with a P value < 0.05 and with a fold-change >1.20 or <0.833 between fertile ovules and abortive ovules. The green and red areas represent significantly different DEPs, and the black areas represent non-significantly different DEPs. In total, 3,230 DEPs between fertile and abortive ovules were identified. Overall, 1,748 DEPs were upregulated and 1,482 DEPs were downregulated in the abortive ovules (Figs. S2A, S2B).

Gene Ontology (GO) classification diagram and pathway enrichment of DEPs

GO annotation of the fertile and abortive ovules was performed using Blast2GO to identify the significantly enriched GO functional groups of the DEPs. The DEPs were grouped into three categories: molecular functions, cellular components and biological processes. For the molecular functions, the DEPs were predominantly distributed to the following categories: oxidoreductase, cationic binding enzyme, haem binding enzyme, antioxidant and calcium binding enzyme (Fig. S3). For the cellular components, the DEPs were predominantly distributed to the mitochondrial membrane system, respiratory chain reaction system, thylakoid, cell wall, proton transport ATP synthase complex and protease complex categories (Fig. S3). For the biological processes, the DEPs were predominantly distributed to the embryogenesis, hormone response, reactive oxygen metabolites, light intensity response, protein folding and cell death categories (Fig. S3). Significantly enriched KEGG pathways were determined based on a comparison of fertile and abortive ovules. DEPs were classified according to 17 KEGG categories. Among them, the pathways with more enriched differential proteins were amino acid synthesis and metabolism, biosynthesis of secondary metabolites, carbon fixation and carbon metabolism, and protein synthesis and hydrolysis (Fig. S4). We found that many pathways were involved in energy synthesis, such as glycolysis, pyruvate metabolism, oxidative phosphorylation and the TCA cycle. This indicated that energy synthesis was hindered in abortive ovules (Fig. S4). In addition, a number of DEPs were related to starch and sucrose metabolism, fatty acid synthesis and metabolism, purine and pyrimidine metabolism, the proteasome, and ascorbate and aldarate metabolism (Fig. S4).

Functional categories of DEPs and detection of GAPDH expression in the fertile and abortive ovules

The abnormal biosynthesis of protein in the abortive ovules

Seven DEPs were identified as being involved in the synthesis and folding of proteins. Four translation initiation factors (Q56WR4, W9S9Y5, A0A0B2PBC5 and M5W7G3) were downregulated in abortive ovules (Table 1, Fig. 5A), which indicated that transcription and translation were likely to be suppressed, further decreasing protein synthesis. Protein disulfide isomerase (PDI) is an essential folding catalyst and chaperone of the endoplasmic reticulum, what’s more, it could catalyze disulfide bond formation, reduction, or isomerization, all of which are important for the maturation and proper folding of secreted or plasma membrane proteins (Urade, 2019; Xia et al., 2018; Wilkinson & Gilbert, 2004; Cho et al., 2011). Three PDIs (W9RKP0, A0A067KKM7 and R0ING6) were downregulated in abortive ovules (Table 1, Fig. 5A); therefore, the formation of disulfide bonds would be inhibited during protein synthesis, and misfolded proteins would not be identified and corrected.

Table 1:

Down-regulated expression of the translation initiation factor in the abortive ovules.

Protein ID	Annotation	Expression ratio (A/F) ± SD	P value
Q56WR4	Translation initiation factor IF-2	0.7968 ± 0.0215	0.0001
W9S9Y5	Eukaryotic translation initiation factor 3 subunit B	0.7902 ± 0.0732	0.0017
A0A0B2PBC5	Eukaryotic initiation factor 4A-15	0.7317 ± 0.0409	0.0083
M5W7G3	Eukaryotic translation initiation factor 3 subunit B	0.5327 ± 0.0815	0.0001
W9RKP0	Protein disulfide-isomerase	0.7478 ± 0.0304	0.0302
A0A067KKM7	Protein disulfide-isomerase	0.6962 ± 0.0261	0.0001
R0ING6	Protein disulfide-isomerase	0.6896 ± 0.0534	0.0176

DOI: 10.7717/peerj.11756/table-1

Note:

“A” represents abortive ovules and “F” represents fertile ovules; P ≤ 0. 05.