Specific functions of single pistil S-RNases in S-gene homozygous Pyrus germplasm

doi:10.21203/rs.3.rs-3239468/v1

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Research Article

Specific functions of single pistil S-RNases in S-gene homozygous Pyrus germplasm

https://doi.org/10.21203/rs.3.rs-3239468/v1

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published 20 Nov, 2023

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Gametophytic self-incompatibility (SI) is regulated by S-allele recognition; that is, pollen in a style with the same S-genotype will undergo programmed cell death and stop growing so that it is unable to complete double fertilization, ultimately resulting in the SI response. S-RNase is the female determinant of SI in pear (Pyrus bretschneideri). In the Pyrus genome, there are two different S-RNase genes at the same S-locus, which generate two different S-RNase products in the pistil. The extracted S-glycoprotein is actually a protein complex. In this study, artificial self-pollination was conducted at the bud stage to overcome SI in ‘Huanghua’ (S₁S₂) pear. Seven plants homozygous for S₁-RNase and four homozygous for S₂-RNase were selected from the selfed progeny of ‘Huanghua’ by S-gene molecular identification biotechnology. We investigated the function of single S-RNases isolated from the pistils of S-gene homozygous Pyrus germplasm. The pollen of ‘Huanghua’ could smoothly pass through the style of the S-gene homozygous germplasm and complete fertilization. S-RNases were extracted from flower styles of different genotypes and used to treat different types of pollen. The S-RNase from ‘Huanghua’ completely inhibited the growth of S₁S_2,S₁S₁, and S₂S₂ pollen, while the S-RNase from homozygous germplasm allowed some S₁S₂ pollen and different single genotypes of pollen to continue growing. These results further validate the core events of SI including cytoskeleton depolymerization and programmed cell death. By iTRAQ-based proteomic analysis of style proteins, a total of 13 S-RNase-related proteins were identified. In summary, we have created reliable S-RNase gene homozygous germplasm, which will play a crucial role in further research on SI in pear and in the development of the pear industry.

Pyrus

S-gene identification biotechnology

Homozygote S-RNase

Proteomic analysis

Molecular mechanism

Self-incompatibility (SI) in flowering plants is a genetic mechanism that prevents inbreeding and promotes outcrossing. In many species, SI is controlled by a single multi-allelic S locus [1]. In the gametophytic self-incompatible (GSI) species of Solanaceae [2], Rosaceae [3], and Scrophulariaceae [4], the S locus encodes an allele-specific ribonuclease (S-RNase) that is expressed in the style. This S-RNase is responsible for inhibiting the growth of pollen tubes with the same S allele [5].

Pear (Pyrus pyrifolia L.), in the family Rosaceae, has an S-RNase-based gametophytic SI system. Using an in vitro system, we identified the characteristics of the S-RNase that specifically inhibits self-pollen germination and tube elongation [6–9]. Further studies showed that S-RNase induces the depolymerization of the actin cytoskeleton and DNA degradation in self-generated pollen tubes [10–12]. To further identify whether the results obtained in vitro reflect what happens in vivo, we evaluated the nuclear DNA of pollen tubes after different pollination events.

Pomologists have focused on the S-RNase in the Pyrus style as the key protein controlling SI in pear. In the Pyrus genome, there are two different S-RNase genes at the same S-locus, which generate two different S-RNase products in the pistil. The extracted S-glycoprotein is actually a protein complex comprising these two products. However, because it is difficult to separate and purify each S-glycoprotein component individually, there are few reports on the functional characteristics of each single pistil S-RNase.

To overcome the above issues, we generated S-gene homozygous germplasm and then extracted and purified single S-RNase products. Through comparing the different characteristics of these single S-RNases expressed in the pistil, we determined how each single S-RNase regulates pollen recognition and pollen tube growth, and clarified the effects of homozygous pollen on the ability of each S-RNase to trigger programmed cell death (PCD) and alter the cytoskeleton. The methods used here provide new opportunities to explore the specific functions of single pistil S-RNases in Pyrus, which may be relevant for clarifying the molecular mechanisms that function in pear pistils and pollen. More significantly, this research demonstrates the use of a new system to study the mechanism of pear gametophytic SI by detailed analyses of S-RNase gene homozygous germplasm.

2.1 Plant materials

Adult trees of the pear varieties ‘Huanghua’ (H-H, S₁S₂), ‘Xizilv’ (XZL, S₁S₄), and ‘Nijisseiki’ (NI, S₂S₄) growing in the orchards of Nanjing Agricultural University, Jiangsu, China were used in this study. The leaves, pistils, and pollen were collected and stored at − 80°C for later analyses. S-RNase gene homozygotes were identified from the seedlings of the selfed progeny of ‘Huanghua’. The S₁-homozygote H-7 (S₁S₁) and the S₂-homozygote H-9 (S₂S₂) were used in this study.

2.2 Bud stage artificial self-pollination

Artificial self-pollination of ‘Huanghua’ at the bud stage (4, 6, and 8 days before full bloom) was carried out in 2008 and 2009. Before anthesis, two to three flowers per inflorescence were selected, emasculated, and enclosed in insect-proof bags to prevent contamination after artificial self-pollination. Fruit set was recorded 3 weeks later and fruits were collected when mature.

2.3 PCR amplification and sequence analysis of genomic S-RNases

Genomic DNA was extracted from young leaves of each cultivar using the CTAB (cetyl trimethyl ammonium bromide) method (Macklin, Shanghai, China). S-RNase alleles were identified by PCR analysis using the primer pair PF and PR [13], which targeted the C1 consensus conserved region and the non-consensus conserved region located downstream of the C2 region of S-RNases, respectively (Supplemental Table 1). Amplifications were conducted on a PTC-200 thermal cycler (Bio-Rad, Hercules, CA, USA) with the following cycle parameters: 3 min at 94 ℃ for pre-denaturation, followed by ten cycles of 30 s at 94 ℃, 30 s at 50 ℃, and 45 s at 70 ℃, then 25 cycles of 30 s at 94 ℃, 30 s at 48 ℃, and 45 s at 70 ℃, with a final extension for 7 min at 70 ℃. The products were separated by electrophoresis on a 1.5% (w/v) agarose gel. Band scoring was carried out using a standard DL2000 ladder (TaKaRa, Dalian, China). S₁-RNase and S₂-RNase alleles were discriminated on the basis of fragment length.

2.4 S-RNase gene quantitative real-time PCR assay

S-RNase gene transcript levels were analyzed by quantitative RT-PCR using the Rotor-Gene 6 fluorescence quantitative PCR kit (Beijing Heros Technology Company, Shanghai, China). The RT-PCR system was as follows: 5 µL 5×PCR buffer, 4 µL MgCl₂ (25 mmol l^− 1), 1 µL dNTP mixture (10 mmol l^− 1), 0.5 µL specific primers (10 µmol l^− 1), 1.25 µL 20× Eva green fluorescent dye, 1.5 U Taq enzyme (2.5 U·µL^− 1), 1 µL template, and ddH₂O to 25 µL. The sample was pre-degenerated at 95 ℃ for 5 min, 95 ℃ for 15 s, and 65 ℃ for 35 s (for fluorescence detection) over 40 cycles. The S₁-RNase allele-specific primers were S₁-F and S₁-R. The S₂-RNase allele-specific primers were S₂-F and S₂-R. A relative quantification method was used to calculate relative gene transcript levels [14]. The transcript level of each gene in each sample was compared with that of Actin (AF386514), which allowed for direct comparison of Actin gene-normalized RT-PCR ratios among samples. The primers Pactin-F and Pactin-R were used to amplify Actin (Supplemental Table 1).

2.5 Analysis of nuclear DNA content by flow cytometry

Nuclear protoplasm was isolated by chopping 100 mg young leaf tissue with a sharp scalpel in a glass Petri dish containing 500 µL LB01 lysis buffer with the following composition: 15 mM Tris, 2 mM EDTA-2Na, 80 mM KCl, 20 mM NaCl, 0.5 mM spermine, 15 mM β-mercaptoethanol, and 0.1% (v/v) Triton X-100, with a final pH of 7.4 [15]. To remove leaf debris, the suspension of released nuclei was filtered through a 21-µm nylon mesh. The nuclei were stained with propidium iodide (PI) at 100 mg·mL^− 1 and incubated with 100 mg·mL^− 1 RNase for 15–30 min prior to analysis. Unstained chicken red blood cell nuclei were used as an internal standard and were added to suspensions of isolated nuclei prior to staining. After isolation, nuclei were fixed in ice-cold 3:1 fixative (ethanol/glacial acetic acid) and stored in 70% (v/v) ethanol at − 20°C. The PI-stained nuclei suspensions were analyzed with an Epics XL flow cytometer (Beckman Coulter, Miami, FL, USA) and sorter equipped with an argon ion laser at λ = 514 nm adjusted to a 100-mW power output. Integral fluorescence and the height and width of fluorescence pulses emitted from the nuclei were collected through a 620 nm long-pass filter.

2.6 Preparation, concentration, and activity of S-RNase

Four grams of styles were prepared for isolating each S-RNase following our previously described method [6, 8]. The isolated S-RNase sample was stored in Eppendorf tubes at − 80 ℃. The S-RNase concentration and activity were determined by the methods of Bradford [16] and Brown and Ho [17], respectively.

2.7 Pollination test and segregation of S-alleles

To check cross-(in)compatibility in the three cultivars/lines, self-pollination and cross-pollination tests were conducted in ‘Huanghua’, ‘H-7’, and ‘H-9’. Field pollination experiments were carried out in 2014 and 2015. Before anthesis, 2–3 flowers per inflorescence were selected, emasculated, and enclosed in insect-proof bags to prevent contamination after pollination. Subsequent fruit set was recorded 3 weeks later, and fruit were collected when mature. Pistils were harvested and fixed in FAA solution (5% v/v formalin, 5% v/v acetic acid, 45% v/v ethanol) 48 h after self-pollination. Pistils were washed in distilled water three times to remove FAA, and then softened by autoclaving for 30 min at 100 kPa in 5% (w/v) sodium sulfite solution. The pistils were stained for 24 h with 0.1% (v/v) aniline blue (0.1 N potassium phosphate, pH 8.7). After the epidermis was removed, the pistils were examined under a microscope (BX60, Olympus, Tokyo, Japan) equipped with a BH2-RFL-T2 ultraviolet light source and an Osram HBO 100 W/2 high-pressure mercury lamp (Olympus, Tokyo, Japan) [18]. To study the segregation of the S-RNase gene, seedling genotypes were detected by PCR using the primer set PF/PR for S-RNase.

2.8 Pollen culture and SI challenge

H-7 pollen grains were pre-cultured for 2 h at 25 ℃ in a basal medium in the dark according to the method of [6]. The basal medium consisted of a 2-(N-morpholine)–ethanesulfonic acid (MES)-NaOH buffer supplemented with 10% (w/v) sucrose, 15% (v/v) polyethylene glycol 4000, 0.01% (w/v) H₃BO₃, 0.07% (w/v) Ca(NO₃)₂ 4H₂O, 0.02% (w/v) MgSO₄·7H₂O and 0.01% (w/v) KNO₃, pH 6.0–6.5. After pre-culture, H-7 stylar S-RNase was added to the medium as an SI challenge, and H-9 stylar S-RNase was added to the medium as a compatible treatment. Medium without S-RNase was used as the control. The final activity of the S-RNases in the basal medium was 0.15 U.

2.9 Depolymerization assays and quantification of filamentous actin in vivo

An F-actin depolymerization assay was conducted and the amount of F-actin present in the samples was quantified as previously described [19]. After each reaction, the pollen samples were stabilized in 200 mM MES-buffered saline and fixed in 4% (w/v) paraformaldehyde. The samples were then stained individually with 5 mM rhodamine-phalloidin for the depolymerization assay or simultaneously with 5 mM rhodamine-phalloidin and 5 mM ethidium bromide (EB) for the quantification assay. A confocal microscope (LSM700, Zeiss, Jena, Germany) was used to examine the levels of actin depolymerization. Phalloidin fluorescence divided by EB fluorescence was used as an index of actin filament levels. The bound phalloidin and EB were eluted with methanol and quantified by spectrofluorometry with excitation and emission wavelengths of 492 and 514 nm, respectively, for phalloidin, and 513 and 615 nm, respectively, for EB.

2.10 Evaluation of programmed cell death in pollen tubes

We evaluated PCD in pollen tubes by terminal-deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining as previously described [20]. After fixing the samples in 4% (w/v) paraformaldehyde for 2 h, the pollen tubes were transferred to 70% (v/v) ethanol and incubated overnight at − 20 ℃. The Dead End Fluorometric TUNEL system (Promega, Beijing, China) was used to examine PCD in the samples. After the samples were washed with citrate buffer (pH 4.1), the pollen tubes were stained with 50 µg·mL^− 1 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature and then analyzed using a Fluoview FV1200 confocal microscope (Olympus). The TUNEL signal was detected at excitation and emission wavelengths of 540 and 620 nm, respectively, and the DAPI signal was detected at excitation and emission wavelengths of 360 and 420 nm, respectively. Positive TUNEL staining in the pollen tubes was indicative of PCD.

2.11 Protein extraction

One gram of frozen style from H-H, S₁S₁, and S₂S₂ was ground to a powder in liquid nitrogen. Each sample included three biological replicates. The dry powder was dissolved in 0.6 mL lysis buffer (1 M sucrose, 0.5 M Tris 8.0, 0.1 M KCl, 50 mM ascorbic acid, 1% NP 40, 1% NaDOC, 10 mM EDTA, 10 mM DTT, 1% protease inhibitor mixture and 1% phosphatase inhibitor mixture) and placed on ice for 10 min [21]. Then, 0.6 mL Tris-phenol was added to the samples, which were kept on ice for 10 min after intensive mixing. The samples were then centrifuged at 5500×g for 10 min at 4 ℃. The supernatant was transferred into a new tube, and four volumes of cold ammonium acetate/methanol were added to the tube, followed by incubation at − 20 ℃ overnight. After incubation, the pellet was collected by centrifugation at 1600×g for 10 min at 4 ℃, and then resuspended in cold methanol and kept at − 20 ℃ for 1 h. After centrifugation at 1600×g for 10 min at 4 ℃, the pellet was collected, resuspended in cold acetone, and washed twice. After washing, the mixture was centrifuged at 1600×g for 10 min at 4 ℃, and then the pellet collected and air-dried. Then, 0.5 mL lysis buffer (8 M Urea, 50 mM Tris 8.0, 1% NP40, 1% NaDOC, 10 mM EDTA, 5 mM DTT, 1% protease inhibitor mixture, and 1% phosphatase inhibitor mixture) was added. The samples were sonicated at 200 W for 5 min, and then centrifuged at 2000×g for 10 min at 4 ℃. The supernatant was collected, and the protein concentration was quantified using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions.

2.12 iTRAQ labeling and strong cation exchange (SCX) fractionation

From every sample, 100 µg protein was digested with Trypsin Gold (Promega, Madison, WI, USA) at a protein: trypsin ratio of 30:1 for 16 h at 37°C. After trypsin digestion, the peptides were dried by vacuum centrifugation, and then dissolved in 0.5 M TEAB using 8-plex iTRAQ reagent [22]. Then, one unit of iTRAQ reagent was thawed and diluted with 24 µL isopropanol. The samples were marked with iTRAQ labels as follows: iT-S₁S₂, iT-S₁S₁, iT-S₂S₂. The peptides were labeled with isobaric tags at 25 ℃ with 2 h. Finally, the marked peptide mixtures were combined and dried under vacuum centrifugation.

SCX Chromatography was conducted using an LC-20AB HPLC pump system (Shimadzu, Japan). The iTRAQ labeled peptide mixture was reconstituted in 2 mL buffer A (25 mM NaH₂PO₄ in 25% ACN, pH 2.7) and then loaded onto an Ultremex SCX column (particle size, 5 µm). The peptides were eluted in a gradient formed by buffer A and buffer B (1 M KCl in 25% ACN, 25 mM NaH₂PO₄, pH 2.7), according to the following program: buffer A for 10 min at 1 mL/min, then 5–60% buffer B over 27 min, and 60–100% buffer B over 1 min. The column was washed with 100% buffer B for 1 min and then equilibrated with buffer A for 10 min before the next injection. Fractions were collected every 1 min. The elution of products was detected by measuring absorbance at 214 nm. Finally, the eluted peptides collected in 20 fractions were desalted on a Strata X C18 column and then vacuum-dried [23].

2.13 LC–ESI–MS/MS analysis based on Triple TOF 5600

Each dried peptide fraction was reconstituted in buffer A (0.1% FA, 5% ACN) and then centrifuged at 20,000×g for 10 min. The average concentration of final peptides was about 0.5 µg/µL. For the LC analysis, 10 µL of supernatant was injected into a LC-20AD nanoHPLC equipped with a 2-cm C18 trap column. The peptides were separated on an internally packed analytical column. All samples were loaded at a rate of 8 µL/min over 4 min, and separated by a gradient of buffer A and B (0.1% FA, 95% ACN) supplied at a flow rate of 300 µL/min. The gradient program was as follows: 2–35% B over 35 min, increased with a linear gradient to 60% over 5 min, then to 80% over 2 min, and held at 80% B for 4 min before returning to 5% buffer B within 1 min.

Data were acquired from the Triple TOF 5600 System equipped with a pulled quartz tip as transmitter and a Nanospray III source, with the following settings: 30 psi curtain gas, 150 ℃ interface heater temperature, 15 psi atomization gas, and 2.5 kV ion spray voltage. A RP (≥ 30,000 FWHM) was used for operating the MS for TOF-MS scanning [24]. For IDA, if the threshold was exceeded at 120 counts per s above 2 to 5+, survey scans were performed every 250 ms and 30 production scans were assembled. The total cycle time was fixed at 3.3 s and the Q2 transfer window at 100 Da was set to 100%. By monitoring the 40 GHz multi-channel TDC detector with a four-anode channel detector, four time bins were summarized at 11 kHz pulse frequency per scan. The 35 ± 5 eV sweep collision energy setting was used in conjunction with iTRAQ to adjust the rolling collision energy to perform collision-induced dissociation of all precursor ions. Finally, dynamic exclusion was set to 50% of the peak width (15 s), and precursors were refreshed from the exclusion list.

2.14 iTRAQ protein identification and quantification

The raw data files obtained from the Triple TOF 5600 system were converted into MGF files using 5600 MS converter and then the MGF files were searched. The triple TOF 5600 mass spectrometer used in this study had a high mass accuracy (less than 2 ppm), allowing for reliable peptide identification. The charge states of peptides were set to + 2 and + 3. For proteome analyses, the MS data were processed using Proteome Discoverer software with default parameters to produce a list of peaks. Data were collected using Analyst QS 2.0 software, and proteins were identified and quantified by Mascot 2.3.02. For iTRAQ quantification, peptides for quantification were automatically selected by an algorithm to calculate reporter peak area, error factor (EF), and p-value (using the default parameters in the Mascot software package). The resulting data sets were automatically corrected for deviations to eliminate any changes due to uneven mixing of samples with different markers. The sequences of Chinese white pear proteins (https://www.peargenome.njau.edu.cn/) were identified using the Mascot search engine (Matrix Science). The ratios between samples were obtained directly based on the protein abundance for any given protein. Proteins showing 1.5-fold or higher difference in abundance between the treated and control samples and a p-value of less than 0.05 were identified as differentially expressed proteins (DEPs). Quantification was performed at the peptide level according to the procedure described at http://www.matrixscience.com/help/quant_statistics_help.html. Each treatment was repeated once for the iTRAQ analysis. The mass spectrometry proteomics data were stored in the PRIDE partner repository with the data set identifier PXD0043543 in the ProteomeXchange [25].

3.1 Overcoming SI to obtain homozygous S-RNase gene germplasm

Pyrus is a typical gametophytic SI fruit tree. Analyses of the spatio-temporal expression patterns of S₁-RNase and S₂-RNase in the floral tissues of ‘Huanghua’ pear at different developmental stages [− 8 days after flowering (DAF), − 6 DAF, − 4 DAF, and 0 DAF] revealed an increasing trend in their transcript levels until flowering (Fig. 1). Bud pollination was conducted to overcome SI in this cultivar and promote self-pollination. Fruit set and the average number of seeds after self-pollination were investigated after bud pollination (Table 1). Under natural conditions, ‘Huanghua’ showed the highest fruit set (43%) when it was self-pollinated at the bud stage at − 6 DAF. During the flowering period, the fruit setting rate was the lowest, which was only 1%. In the spring of 2008, a total of 29 progenies were obtained from 74 seeds of ‘Huanghua’. To determine the S-genotypes of the selfed progeny of ‘Huanghua’, S-RNase alleles were amplified by PCR from the genomic DNA of all individual progeny. Genomic PCR using the primers PF and PR yielded a 367-bp fragment corresponding to the S₁-RNase allele and a 1347-bp fragment corresponding to the S₂-RNase allele from the selfed progeny of ‘Huanghua’ (Fig. 2).

Table 1

Comparison of fruit set and selfed progeny among different bud stage artificial self-pollination in ‘Huanghua’
Days before flowering	Number of pollinated flowers	Fruited flowers	Fruit set ratio (%)	Number of seeds	Number of self-pollinated progenies
8	100	16	16.0	26	6
6	100	43	43.0	45	23
4	100	9	9.0	3	0
0	100	1	1.0	0	0

‘Huanghua’ and its selfed progeny were analyzed to give an accurate estimation of nuclear DNA content. Controls containing 2content (2C) DNA showed peak 1 at the position (channel 100) that had been determined by analyzing standards prepared from known diploid ‘Huanghua’. The histograms of its selfed progeny with 2C DNA showed this same peak 1 at channel 100 (Supplemental Fig. 1). These results strongly indicated that all seedlings produced from the ‘Huanghua’ selfed progeny were diploids, thus, no spontaneous triploids were detected by flow cytometry analysis. It can be inferred that each individual ‘Huanghua’ selfed progeny contained two S-RNase alleles in its genomic DNA. Through the identification of the S-genotype and S-gene copy number, six ’Huanghua’ selfed progeny (no. 7, 10, 12, 19, 21, and 25) were identified as S₁S₁ homozygotes, three selfed progeny (no. 9, 20 and 28) were identified as S₂S₂ homozygotes, and the remaining offspring were identified as S₁S₂ heterozygotes (Fig. 2).

3.2 Specific recognition functions between pollen and pistil in different homozygotes

We investigated the fruit set rate after self-pollination and cross-pollination with ‘HH’ (Huanghua, S₁S₂), ‘H-7’ (S₁S₁ homozygote, no. 7 selfed progeny of Huanghua), and ‘H-9’ (S₂S₂ homozygote, no. 9 selfed progeny of Huanghua). ‘HH’, ‘H-7’, and ‘H-9’ exhibited SI after self-pollination. Pollinations between various pairs of these three varieties were then conducted. When pollinated with ‘S₁S₂’ pollen, 67% of ‘S₁S₁’ flowers and 74% of ‘S₂S₂’ flowers set fruit, displaying cross-compatibility; however, the reciprocal pollination resulted in only a few ‘S₁S₂’ flowers setting fruit (Table 2). Cross-pollination between ‘S₁S₁’ and ‘S₂S₂’ displayed typical cross-compatibility.

Table 2

Fruit set percentage after cross- and self-pollinations of different *Pyrus* S-gene homozygote germplasm materials
Crosses for pollination	Number of pollinated flowers	Fruited flowers	Fruit set ratio (%)	Number of seeds per fruit	(In) Compatible
H-H(S₁S₂) selfed	100	0	0.0	0	SI
H-7(S₁S₁) selfed	100	1	1.0	0	SI
H-9 (S₂S₂) selfed	100	2	2.0	0	SI
H-7(S₁S₁)×H-H(S₁S₂)	100	67	67.0	4.5	CC
H-9 (S₂S₂)×H-H(S₁S₂)	100	74	74.0	4.8	CC
H-H(S₁S₂)×H-7(S₁S₁)	100	3	3.0	0	CI
H-H(S₁S₂)×H-9 (S₂S₂)	100	6	6.0	0	CI
H-7(S₁S₁)×H-9 (S₂S₂)	100	52	52.0	4.2	CC
H-9 (S₂S₂)×H-7(S₁S₁)	100	65	65.0	4.6	CC

Table 3

S-genotype in progeny obtained by different cross-pollinations of ‘H-7’ and ‘H-9’
Crosses for pollination	Number of pollinated flowers	Number of pollinated seeds	Number of pollinated progeny	S-gene type
H-H(S₁S₂)×H-7(S₁S₁)	100	0	0	/
H-H(S₁S₂)×H-9 (S₂S₂)	100	0	0	/
H-7(S₁S₁)×H-9(S₂S₂)	100	218	169	S₁S₂
H-9(S₂S₂)×H-7(S₁S₁)	100	299	216	S₁S₂
H-7(S₁S₁)×H-H(S₁S₂)	100	301	246	S₁S₂
H-9(S₂S₂)×H-H(S₁S₂)	100	355	273	S₁S₂
H-7(S₁S₁)×XZL(S₁S₄)	100	167	129	S₁S₄
XZL(S₁S₄)×H-7(S₁S₁)	100	0	0	/
H-9(S₂S₂)×NI(S₂S₄)	100	182	136	S₂S₄
NI(S₂S₄)×H-9(S₂S₂)	100	0	0	/

Pollen tube growth was subsequently investigated approximately 48 h after self-pollination or cross-pollination between various pairs of ‘S₁S₂’, ‘S₁S₁’, and ‘S₂S₂’ For all three types, after self-pollination, the pollen tubes could not complete normal development in the pistil or reach the ovary for fertilization. The ‘S₁S₂’ pollen was able to penetrate ‘S₁S₁’ or ‘S₂S₂’ pistils. However, ‘S₁S₁’ or ‘S₂S₂’ pollen could not penetrate ‘S₁S₂’ pistils (Fig. 3A, B). Notably, after cross-pollination between ‘S₁S₁’ and ‘S₂S₂’, the pollen tubes were able to reach the ovary. These results were consistent with the fruit set rate after self-pollination or cross-pollination in ‘S₁S₂’, ‘S₁S₁’, and ‘S₂S₂’.

Using the primers PF and PR, we amplified the genomic DNA of progeny of different cross-pollinations with ‘S₁S₁’ and ‘S₂S₂’ (Table 3). The bands S₁ and S₂ could be detected in all progeny of reciprocal cross-pollinations between ‘S₁S₁’ and ‘S₂S₂’. When ‘S₁S₁’ or ‘S₂S₂’ was the female parent and ‘S₁S₂’ was the male parent, the S-genotype of all cross-progeny was S₁S₂. When ‘XZL (S₁S₄)’ pollen was used to pollinate ‘S₁S₁’, the S-genotype of all cross-progeny was S₁S₄; however, the backcross showed typical cross-incompatibility. Similar results were also obtained in the reciprocal cross-pollination between ‘NI(S₂S₄)’ and ‘S₂S₂’. These results indicate that there is a specific recognition function between the pollen of the S-gene homozygotes and the pistil with the same S-locus.

3.3 Effect of single S-RNase on homozygote pollen germination and pollen tube growth

The in vitro system of pear SI is relatively mature. Here, we used in vitro experiments to verify the SI responses of pear S-gene homozygotes. Three types of pollen, S₁S₂, S₁S₁, and S₂S₂, were cultured in liquid medium, to which S₁-RNase and S₂-RNase were then added as appropriate. Liquid medium containing ‘HH’ stylar S-RNase (S₁S₂) was used as the control. As expected, ‘HH’ stylar S-RNase inhibited the growth of all three types of pollen tubes (Fig. 3C). In S₁-RNase liquid medium, pollen-tube elongation of ‘S₁S₁’ was significantly inhibited with a mean pollen tube length of 115 ± 12.3 µm (P < 0.05, n = 210); while the ‘S₁S₂’ and ‘S₂S₂’ pollen both grow normally, with mean pollen tube lengths of 210 ± 15.6 µm (P < 0.05, n = 205) and 280 ± 20.7 µm (P < 0.05, n = 207), respectively. In S₂-RNase liquid medium, ‘S₂S₂’ pollen tubes were strongly inhibited, with a mean pollen tube length of 108 ± 10.1 µm (P < 0.05, n = 208); while the ‘S₁S₂’ and ‘S₁S₁’ pollen tubes were not inhibited, with mean pollen tube lengths of 213 ± 12.1 µm (P < 0.05, n = 201) and 275 ± 18.9 µm (P < 0.05, n = 206), respectively (Fig. 3D). These results show that each single S-RNase has a significant inhibitory effect on the germination and growth of pollen containing the same S-gene type.

3.4 Each single homozygous S-RNase induces alterations in the actin cytoskeleton and PCD in homologous pollen tubes in vitro

The actin cytoskeleton of pollen tubes forms a dynamic cell framework that supports numerous fundamental cellular processes and is essential for their highly specialized polarized tip growth. Thus, we examined the effects of each single homozygous S-RNase on depolymerization of the actin cytoskeleton in homologous pollen tubes. We observed massive actin depolymerization in pear pollen tubes within minutes of an incompatible S-RNase challenge (Fig. 4A). We quantified the degree of F-actin depolymerization in response to 30 min of various treatments (Fig. 4B). The ‘HH’ stylar S-RNase (S₁S₂) led to significant actin depolymerization in ‘S₁S₂’, ‘S₁S₁’, and ‘S₂S₂’ pollen tubes. In S₁-RNase liquid media, ‘S₂S₂’ pollen tubes grew normally, while F-action depolymerization was highly significant in ‘S₁S₁’ pollen tubes, 68.34% (P < 0.05; n = 10). Similar results were obtained in S₂-RNase liquid media, in which the actin cytoskeleton remained intact in ‘S₁S₁’ pollen tubes, but showed a significant level of depolymerization in ‘S₂S₂’ pollen tubes, 67.25% (P < 0.05; n = 10). In S₁-RNase and S₂-RNase liquid media, the actin depolymerization rates in ‘S₁S₂’ pollen tubes were 19.21% and 21.32%, respectively (P < 0.05; n = 10). These findings show that each single homozygous S-RNase significantly depolymerized the actin cytoskeleton in homologous pollen tubes.

Senescent cells are precisely eliminated via PCD. Therefore, we used the TUNEL staining technique to assess DNA-strand breaks, which are indicative of PCD, in pear pollen tubes (Fig. 4C). The PCD ratio was the same, 58.43%, in ‘S₁S₁’ pollen tubes treated with S₁-RNase (P < 0.05; n = 10) and ‘S₂S₂’ pollen tubes treated with S₂-RNase (P < 0.05; n = 10) (Fig. 4D). These results show that the S-gene homozygotes generated in this study conformed to the characteristics of SI.

3.5 iTRAQ-based proteomic analysis of style proteins and S-RNase - related proteins in Huanghua and its homozygous S-genotype offspring

We used iTRAQ technology to investigate the protein expression characteristics of S-RNases in S-gene homozygous germplasm. By using MaxQuant to search the raw mass spectrometry data files against the protein database, a total of 30,503 identified spectra, 13,568 peptides, and 4,472 proteins were identified in the styles of Huanghua (S₁S₂) and its homozygous S-genotype offspring (S₁S₁ and S₂S₂). These identified proteins were predicted to be located in extracellular regions, cytoplasm, nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum, peroxisomes, vacuoles, nucleoplasm, and other cellular compartments. The number of DEPs among the three varieties was calculated (Fig. 5). According to the overall distribution of these proteins, the highest number of DEPs was between S₂S₂ and S₁S₂, while the lowest number of DEPs was between S₂S₂ and S₁S₁. This indicated that the protein expression patterns of S₂S₂ and S₁S₁ were more similar to each other, while both offspring samples showed significant differences in protein expression when compared with the parent S₁S₂.

In this study, we conducted DEP analyses focusing on S-RNases and the proteins that interact with them in the styles of the three pear varieties. Figure 6 shows the heat-map of the expression levels of the 13 identified S-RNase-related proteins, which reflects their differences in expression levels across the three samples. In terms of the overall expression trend, using the offspring sample S₁S₁ as a normal expression standard, most of the S-RNase-related proteins were down-regulated in the parent S₁S₂ (Fig. 6). In contrast, in the offspring sample S₂S₂, most of the S-RNase-related proteins were up-regulated relative to their levels in the other offspring sample S₁S₁, with only a few exceptions. Compared with the parent S₁S₂, both offspring samples, S₁S₁ and S₂S₂, showed up-regulated expression of 12 out of the 13 S-RNase-related proteins, except for S₁-RNase. Comparing S₁S₁ and S₂S₂, the expression levels of F-box/WD-repeat_TBL1XR1, SKP1-like_1A_2, and S₂-RNase were higher in S₁S₁ than in S₂S₂, while the expression levels of the other 10 S-RNase-related proteins were higher in S₂S₂ than in S₁S₁.

Pollination for fertilization is the initial step in reproductive development, and it directly ensures the fruit setting rate. In flowering plants, SI serves as a ubiquitous mechanism to limit self-fertilization; it prevents pollen tubes that possess the same S-allele from growing normally in the pistil, so that they fail to reach the ovary for fertilization [26]. It is time-consuming and laborious to collocate trees of different pollen types and to conduct artificial pollination in production practice. Pollination failure in pear trees can also result from flowering periods that do not overlap and extreme weather, leading to serious economic losses. Therefore, research on the mechanism of SI in Rosaceae species is important from both theoretical and practical perspectives. In this study, we used a bud pollination method to create homozygous germplasm of S-alleles and verified their applicability in production. These are unique materials for further research on the SI mechanism and directed breeding.

4.1 Self-pollination during the bud stage to overcome SI

Artificial self-pollination at the bud stage can break down the obstacles of SI to achieve fruit setting. Selection of the optimum pollination period is crucial for the success of bud fertilization [27]. If pollination is carried out too early, the immature style contains insufficient nutrients for the heterotrophic pollen tubes to elongate, and the pollen tubes fail to weave through the style because of the compact tissue. If pollination is carried out too late, up-regulation of the S-gene protein results in recognition of self-pollen, triggering SI. The specific period for successful bud pollination has been explored in several species. Previous studies have shown that artificial self-pollination of tomato (Solanum pennellii) at 3–5 days before flowering can effectively avoid SI-related barriers. In tobacco (N. tabacum), pollination at late stage 7 (when the length of floral buds reaches 34–38 mm) resulted in 100% fruit set. Late stage 7 is accompanied by the development of anther primordia and the differentiation of specific anther tissue. In our study, we conducted self-pollination of ‘Huanghua’ pear at different bud periods (4, 6, and 8 days before flowering) and obtained the highest fruit set rate (43%) after pollination at 6 days before flowering.

The failure of the SI mechanism at the bud stage is associated with the absence (or only tiny amounts) of incompatible compounds in the immature pistil. These compounds include S-RNase in the Rosaceae and SRK in the Cruciferae. During style development in cabbage (Brassica rapa var. pekinensis), the abundance of SRK, a female determinant regulating SI, gradually increases from the budding to the maturity stage of the style, and the intensity of SI increases concurrently [28]. In Rosaceae species, silencing of the S-RNase gene created a self-fertile apple (Malus × domestica Borkh), with most of the unaffiliated pollen tubes reaching the base of the style to achieve fertilization. The average self-pollinated fruit setting rate increased from 4% in wild-type to 31% in the S-RNase-silenced line [29]. More specifically, the Chinese pear (Pyrus × bretschneideri) cultivar ‘Yanzhuang’ lacks SI because of a mutation in the conserved glycine of the S-RNase C2 region [30].

We analyzed the spatio-temporal expression of S-RNase in the floral tissues of ‘Huanghua’ pear at different developmental stages (Fig. 1). The results show that the expression of S-RNase initiates at − 4 DAF and gradually increases with style development, reaching the peak at 0 DAF. It is highly likely that it correlates with the formation of the SI mechanism, confirming once again that S-RNase serves as a female determinant of the SI mechanism in pear. Consequently, self-pollination at the bud stage is capable of breaking the limitations of SI to complete fertilization by circumventing the influence of the S-RNase.

4.2 Some proteins are co-expressed with S-RNase in the style

A total of 13 S-RNase-related proteins were identified in the mass spectrometry analysis. The proteins could be divided into two categories: S-RNase family proteins, including S₁-RNase and S₂-RNase, and interactors of S-RNases, including skp1 family, cullin family, and f-box family proteins. These three types of proteins combine to form the SCF (Skp1-Cullins-F-box) complex, a multi-functional E3 ubiquitin ligase complex that can bind to S-RNase, leading to its oligo- or polyubiquitination. The complex is recognized by proteasomes and subsequently degraded, thus regulating the function of S-RNases (Fig. 7). Supplemental Tables 2 and 3 provide a summary of the changes in the expression levels of the 13 identified S-RNase-related proteins in the two sets of offspring samples relative to their parents, as well as the significance of the changes. As shown in the tables, the expression level of S₁-RNase was significantly down-regulated in S₁S₁ compared with S₁S₂, while that of S₂-RNase was not significantly different among the three samples. The expression level of the S-RNase-related protein F-box/WD-repeat_TBL1XR1 was significantly up-regulated in the S₁S₁ offspring relative to the parent S₁S₂, but it was not significantly different between the S₂S₂ offspring and the parent S₁S₂. The expression levels of F-box_PP2-B12 and F-box/LRR-repeat_13 were significantly up-regulated in the offspring sample S₂S₂ compared with the parent S₁S₂, but there was no significant difference in their expression levels in the S₁S₁ offspring sample compared with the parent S₁S₂. This may indicate that the regulatory pathways involving S-RNase include different sets of associated proteins in the two offspring samples, S₁S₁ and S₂S₂. These results provide a reference for further research on S-RNases and their related interacting proteins in styles.

4.3 Homozygous S-gene germplasms are excellent materials for the pear industry

Different S-allele genotypes are classified according to significant differences in the location of the SFBB/F-box genes relative to the S-RNase gene. However, this complex diploid genotype is not conducive to investigating the differences between pollen-specific recognition and pistil S-glycoprotein-specific recognition. In previous studies, the isolation of pure single S-gene proteins has been achieved by means of heterologous expression. For example, expression of the PbrS-RNase recombinant vector in Escherichia coli provided sufficient protein to perform biochemical analyses in vitro [20], thereby facilitating exploration of the detailed SI mechanism. That study revealed a new pathway in which PbrS-RNase induces the death of incompatible pollen tubes by directly binding to, and cleaving, F-actin [20]. However, single S-gene purified proteins are restricted to in vitro experimental studies and are not suitable for industrial applications in fruit trees. In this study, we obtained two S-RNase homozygous germplasm materials (S₁S₁ and S₂S₂) using the bud self-pollination method. These are excellent materials for further research on the SI mechanism from both theoretical and practical perspectives.

During SI pollination, pollen grains germinate into the style and the S-RNase is internalized into the pollen tube for recognition responses [31]. The same type of S-RNase will not be eliminated by the ubiquitination system and will eventually be released to exercise its cytotoxic effects, thereby inducing the typical SI response. This response includes depolymerization of microfilament structures, increased levels of reactive oxygen species, and PCD in unaffiliated pollen tubes. In our study, self-pollination of ‘S₁S₂’, ‘S₁S₁’, and ‘S₂S₂’ resulted in typical SI responses. Notably, the homozygous germplasms ‘S₁S₁’ and ‘S₂S₂’ were cross-compatible when used as the female parent, but cross-incompatible when used as the male parent. This implies that it will be useful to develop homozygous germplasms as major cultivars for pear orchard production.

In practice, pear trees must be pollinated with pollen with different S-genotypes as compared to the main cultivar to ensure a good fruit setting rate. At present, the main cultivars in China include ‘Dangshansu’ (S₇S₃₄), ‘Cuiguan’ (S₃S₅), ‘Huangguan’ (S₄S₁₆), and ‘Zhongli No. 1’ (S₄S₃₅), none of which contain S₁ or S₂. It is reasonable to match homozygous germplasm (S₁S₁ and S₂S₂) to completely circumvent the limitations of SI, thereby further safeguarding the fruit setting rate. In addition, these homozygous germplasm materials can serve as stable donors for efficiently integrating S₁ and S₂ into the genome of the main cultivar. New varieties can be developed with multiple functions as both the main cultivar and as an effective pollinator.

This study describes a reliable method for creating homozygous single S-genotype germplasm, which will accelerate fruit tree breeding programs by allowing for the directional insertion of S-alleles. This will be useful for the continuous enrichment of pear pollinator resources with different genotypes to promote the development of the pear industry.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethics approval and consent to participate

The experimental research on plants performed in this study complies with institutional, national and international guidelines. All the plant materials of this study are available

from the corresponding authors, upon request. No voucher specimen of this material has been deposited in a publicly available herbarium.
Consent for publication

Not applicable.
Availability of data and materials

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD043543.
Funding

This work was financially supported by the earmarked fund for Young Talents Program of Anhui Academy of Agricultural Sciences (QNYC- 202112), National Natural Science Foundation of China (31201598), and CARS(CARS-28)and National key research and development plan project (2019YFD1001404-1-3).

Authors' contributions

Yongjie Qi performed the experiments and wrote the paper. Zhenghui Gao, Na Ma, Liqing Lu, Fanjun Ke analyzed the data. Zhenghui Gao , Na Ma, Liqing Lu, contributed reagents/materials/ analysis tools. Yongjie Qi, Shaoling Zhang and Yiliu Xu discussed and analyzed the results. Yongjie Qi conceived and designed the experiments. All authors read and approved the final manuscript. All authors contributed to the article and approved the submitted version.
Acknowledgements

Not applicable.
Authors' information

¹Key Laboratory of Horticultural Crop Germplasm Innovation and Utilization(Co-construction by Ministry and Province)，Institute of Horticulture，Anhui Academy of Agricultural Sciences，Hefei ²30031, China ²College of Horticulture, Nanjing Agricultural University, Nanjing 210095,China ³Anhui University of Chinese Medicine, Hefei 230012, China

de Nettancourt D. Incompatibility in Angiosperms. Springer, Berlin. 1977.
McClure B.A., Haring V., Ebert P.P., Anderson M.A., Simpson R.J., Sakiyama F., Clarke A.E. Style self-incompatibility gene products of Nicotiana alata are ribonucleases. Nature. 1989; 342: 955-957.
Sassa H., Nishio T., Kowyama Y., Hirano H., Koba T., Ikehashi H. Self- incompatibility (SI) alleles of the Rosaceae encode members of a distinct class of the T2/S-ribonuclease superfamily. Molecular and Genetal Genetics. 1996; 250:547- 557.
Xue Y.R., Carpenter H.G.D., Coen E.S. Origin of allelic diversity in Antirrhinum S locus RNases. Plant Cell. 1996; 8:805-814.
Anderson，M.A.，Cornish，E.C.，Mau，S.L.，Williams，E.G.，Hoggart，R., Atkinson，A.，Bonig, l.,Grego,B.，Simpson，R.，Roche,P.J.，Haley,J.D.，Penschow,J.D.，Niall，H.D.,，Tregear，G.w.，Coghlan，J.P.,rawford，R.J.,and Clarke，A.E. Cloning of cDNA for a stylarglycoprotein associated with expression of self-incompatibility inNicotiana alata. Nature. 1986; 321, 38-44.
Hiratsuka S, Zhang S. L., Nakagawa E., Kawai Y. Selective inhibition of the growth of incompatible pollen tubes by S-protein in the Japanese pear. Sex Plant Reprod. 2001; 13: 209-215.
Zhang S.L., Hiratsuka S. Cultivar and developmental differences in S-protein concentration and self-incompatibility in the Japanese pear. Horticultural Science. 2000a; 35: 917-920.
Zhang S.L., Hiratsuka S. Effects of the stylar S-glycoproteins on the pollen germination and the tube growth in pears (Pyrus serotina Rhed.) in vitro. Acta Horticultural Sinica. 2000b; 27: 251-256.
Zhang S.L., Hiratsuka S. Variations in S-protein levels in styles of Japanese pears and the expression of self-incompatibility. Journal of the Japanese Society for Horticultural Science. 1999; 68: 911-918.
Liu Z.Q., Xu G.H., Zhang S.L. Pyrus pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen and tubes in vitro. Protoplasma. 2007; 232: 61-67.
Wang, C.L., Xu, G.H., Jiang, X.T, Chen, G., Wu, J., Wu, H.Q., and Zhang.s.L.. S-RNase triggers mitochondrial alteration and DNAdegradation in the incompatible pollen tube of Pyrus pyrifolia in vitro.Plant J. 2009; 57,220-229.
Wang C.L., Wu J., Xu G.H., Gao Y.B., Chen G., Wu J.Y., Wu H.Q., Zhang S.L. S-RNase disrupts tip-localized reactive oxygen species and induces nuclear DNA degradation in incompatible pollen tubes of Pyrus pyrifolia. Journal of Cell Science. 2010; 123: 4301-4309.
Ishimizu T., Inoue K., Shinonaka M., Saito T., Terai O., Norioka S. PCR-based method for identifying the S-genotypes of Japanese pear cultivars. Theoretical and Applied Genetics. 1999; 98:961-967.
Livak K.J., Schmittgen T.D . Analysis of relative gene expression data using real time quantitative PCR and the 2−⊿⊿CT method. Methods. 2001; 25:402-408.
Dolezel J., Binarova P., Lucretti S. Analysis of nuclear DNA content in plant cells by flow cytometry. Biology Plant. 1989; 31:113-120.
Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248-254.
Brown P.H, Ho THD. Barley aleurone layers secrete a nuclease in response to gibberellic acid. Plant Physiology. 1986; 82: 801-806.
Vilanova S., Badenes M.L., Burgos L., Martinez-Calvo J., Llacer G., Romero C. Self-compatibility of two apricot selections is associated with two pollen-part mutations of different nature. Plant Physiology. 2006; 142:629-641.
Huang, S., Gao, L., Blanchoin, L., and Staiger, C.J. Heterodimeric capping protein from Arabidopsis is regulated by phosphatidic acid. Molecular Biology of the Cell. 2006; 17: 1946–1958.
Chen J. Q., Wang P., de Graaf B. H. J., Zhang H., Jiao H. J., Tang C., Wu J. Y. Phosphatidic acid counteracts S-RNase signaling in pollen by stabilizing the actin cytoskeleton. Plant Cell. 2018; 30(5), 1023-1039.
Li J.R., Chen X.Z., Zhong L.T., Wang X.B., Zhou X.X., Tang Y., Liu Y.T., Zheng H., Zhan R.T., Chen L.K. Comparative iTRAQ- based proteomic analysis provides insight into a complex regulatory network of Pogostemon cablin in response to exogenous MeJA and Ethrel. Industrial Crops & Products. 2019; 140:111661.
Noirel J., Evans C., Salim M., Mukherjee J., Yen O.S., Pandhal J., Khoa Pham T.A., Biggs C.C., Wright P. Methods in quantitative: setting iTRAQ on the right track. Current Proteomics. 2011; 8:17–30.
Wang Y., Yang Q., Xiao G., Zhang Z., Guan C., Liu Z., Guan M., Wu X., Chen H., Li Q. iTRAQ-based quantitative proteomics analysis of an immature high-oleic acid near-isogenic line of rapeseed. Molecular Breeding. 2018; 38:2.
Unwin R.D., Griffiths J.R., Whetton A.D. Simultaneous analysis of relative protein expression levels across multiple samples using iTRAQ isobaric tags with 2D nano LC–MS/MS. Nature Protocols. 2010; 5:1574-1582.
Perez-Riverol Y., Bai J., Bandla C., Hewapathirana S., García-Seisdedos D., Kamatchinathan S., Kundu D., Prakash A., Frericks-Zipper A., Eisenacher M., Walzer M., Wang S., Brazma A., Vizcaíno J.A. (2022). The PRIDE database resources in 2022: A Hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res 50(D1): D543-D552
Allen, A.M., & Hiscock, S.J. Evolution and phylogeny of self-incompatibility systems in angiosperms. Self-Incompatibility in Flowering Plants - Evolution, Diversity, and Mechanisms. 2008; Chapter 4, 73-100.
Haring V., Gray J. E., McClure B. A., Anderson M. A., Clarke, A. E. Self-incompatibility: A self-recognition system in plants. Science. 1990; 250(4983), 937-941.
Huang J., Yang L., Yang L., Wu X., Cui X., Zhang L., Duan Q. Stigma receptors control intraspecies and interspecies barriers in brassicaceae. Nature. 2023; 614(7947), 303-308.
Broothaerts W., Keulemans J., Van Nerum I. Self-fertile apple resulting from S-RNase gene silencing. Plant Cell Reports. 2004; 22(7), 497-501.
Li Y., Wu J., Wu C., Yu J., Liu C., Fan W., Li W. A mutation near the active site of S-RNase causes self-compatibility in S-RNase -based self-incompatible plants. Plant Molecular Biology. 2020; 103(1-2), 129-139.
Luu D. T., Qin X., Morse D., Cappadocia M. S-RNase uptake by compatible pollen tubes in gametophytic self-incompatibility. Nature. 2000; 407(6804), 649-651.

No competing interests reported.

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Specific functions of single pistil S-RNases in S-gene homozygous Pyrus germplasm

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Journal Publication

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Abstract

Figures

1 Introduction

2 Materials and methods

2.1 Plant materials

2.2 Bud stage artificial self-pollination

2.3 PCR amplification and sequence analysis of genomic S-RNases

2.4 S-RNase gene quantitative real-time PCR assay

2.5 Analysis of nuclear DNA content by flow cytometry

2.6 Preparation, concentration, and activity of S-RNase

2.7 Pollination test and segregation of S-alleles

2.8 Pollen culture and SI challenge

2.9 Depolymerization assays and quantification of filamentous actin in vivo

2.10 Evaluation of programmed cell death in pollen tubes

2.11 Protein extraction

2.12 iTRAQ labeling and strong cation exchange (SCX) fractionation

2.13 LC–ESI–MS/MS analysis based on Triple TOF 5600

2.14 iTRAQ protein identification and quantification

3 Results

3.1 Overcoming SI to obtain homozygous S-RNase gene germplasm

3.2 Specific recognition functions between pollen and pistil in different homozygotes

3.3 Effect of single S-RNase on homozygote pollen germination and pollen tube growth

4 Discussion

4.1 Self-pollination during the bud stage to overcome SI

4.2 Some proteins are co-expressed with S-RNase in the style

4.3 Homozygous S-gene germplasms are excellent materials for the pear industry

5 Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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