Pistil

ClearSee solutions were prepared by mixing xylitol powder [#04; final 10% (w/v)], sodium deoxycholate [#07; final 15% (w/v)] and urea [#19; final 25% (w/v)] in water. Seedlings, leaves and pistils of A. thaliana and gametophores of P. patens were fixed with 4% (w/v) PFA for 30-120 min (seedlings, 30 min; leaves, 120 min; pistil or gametophores, 60 min) in PBS under vacuum (∼690 mmHg) at room temperature. Fixed tissues were washed twice for 1 min each in PBS and cleared with ClearSee at room temperature for 4 days to 4 weeks or more, depending on tissue type. The minimum incubation times for clearing were 4 days for leaves, roots and moss, 7 days for seedlings, 2 weeks for pistils, and 4 weeks for mature stems. In the case of pistils, incubation for 4 weeks improved clarity. ClearSee-treated samples could be stored at room temperature for at least 5 months. For post-staining, cleared tissues were stained with Calcofluor White (final 100 µg/ml) in ClearSee solution for 1 h, and Hoechst 33342 (final 10 µg/ml) in ClearSee solution overnight. After staining, tissues were washed in ClearSee for 1 h.

Two flowers per day from anthesis, two and three days after pollination were fixed in 4% formaldehyde freshly prepared from paraformaldehyde in 1x phosphate saline buffer (PBS) pH7.3, left overnight at 4ºC, and conserved then at 0.1% formaldehyde solution [83 (link)]. Then the pistils were dehydrated in an acetone series (30%, 50%, 70%, 90%, 100%), and embedded in Technovit 8100 (Kulzer and Co, Germany) for two days. The resin was polymerized at 4ºC, and sectioned at 4 μm thickness. Sections were placed in a drop of water on a slide covered with 2% (3-Aminopropyl) triethoxysilane - APTEX (Sigma-Aldrich), and dried at room temperature. Callose was identified with the anticallose antibody (AntiCal) that recognises linear β-(1,3)-glucan segments (anti-β-(1,3)-glucan; immunoglobulin G1), Biosupplies, Australia [49 (link)]. As a secondary antibody, Alexa 488 fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG was used (F-1763; Sigma). Additionally, a monoclonal antibody (mAbs) JIM13 [84 (link)] against AGPs glycosyl epitopes, and one mAb JIM11 [85 (link)] against extensin epitopes were obtained from Carbosource Services (University of Georgia, USA). Secondary antibodies were anti-rat IgG conjugated with the same Alexa 488 used above. Sections were incubated for 5 min in PBS pH7.3 followed by 5% bovine serum albumin (BSA) in PBS for 5 min. Then, sections were incubated at room temperature for 1h with AntiCal primary mAb, JIM13, and JIM11. After that, three washes in PBS of 5 minutes each preceded the incubation for 45 min in the dark with a 1/25 diluted secondary fluorescein isothiocyanate (FITC) conjugated with the antibody in 1% BSA in PBS, followed by three washes in PBS [83 (link)]. Sections were counterstained with calcofluor white for cellulose [86 (link)], mounted in PBS or Mowiol, and examined under a LEICA DM2500 epifluorescence microscope connected to a LEICA DFC320 camera. Filters were 355/455 nm for calcofluor white and 470/525 nm for the Alexa 488 fluorescein label of the antibodies (White Level?=?255; Black Level = 0; ϒ?=?1). Exposur (Exp) times were adapted to the best compromise in overlapping photographs for each antibody: AntiCal, Exp.?=?15.30ms (Calcofluor Exp. = 1.20ms); JIM13 Exp.?=?2.52ms (Calcofluor?=?0.41ms); JIM11, Exp. = 31.59 ms (Calcofluor Exp. = 1.40ms). Brightness and contrasts were adjusted to obtain the sharpest images with the Leica Application Suite software.

Flowers for histochemical examination were selected according to the pollen tube kinetics results, at anthesis, two, and three days after pollination. Two flowers - 10 styles - per day were fixed in 2.5% glutaraldehyde in 0.03M saline phosphate buffer pH7.3 for 4 h [78 (link)]. Then the pistils were washed in 0.03M saline phosphate buffer and sequentially dehydrated in an ethanol series (30%, 50%, 70%, and 96%), leaving them one hour in each ethanol concentration. The gynoecia were left for five days in the embedding solution at 4ºC, and then embedded in JB4 plastic resin (Polysciences Inc., 0226A). Both longitudinal and transversal sections 2μm thick were cut on a LEICA EM UC6 ultramicrotome with a glass knife and then placed onto distilled water on a glass slide previously coated with 1% gelatine. Polysaccharides were stained with periodic acid shift reagent-PAS [79 (link)] counterstained with 0.02% Toluidine Blue for general structure, and proteins with 0.25% Naphtol Blue Black in 1% acetic acid [80 (link)]. Also 0.07% calcofluor white for cellulose [48 (link)] and other polysaccharides [52 (link)], 0.01% auramine in 0.05M phosphate buffer for cutin and lipids [81 (link)], and 0.01% acridine orange in 0.03% phosphate buffer, pH7.4 [82 ] were used to observe the stylar morphology.
Slides were observed under bright field LEICA DM2500 microscope carrying 100W light source, and photographs were obtained with a Leica DFC320 camera linked to the software Leica Application Suite. Fluorescence observations were done with the same microscope provided with an epifluorescence source and connected to a CANON Power Shot S50 camera linked to the CANON Remote Capture software. Filters used were 355/455 nm for calcofluor white, and 450/510 nm for auramine and acridine orange stained sections.

Tissue-specific expression of the sPLA₂ and PLA₂-like members have been searched in various vegetative (leaf, stem, root, and seeds) and reproductive (flower, anther, pollen, pollen tube, carpels, pistil, ovary, ovule, and egg cells) tissues of Arabidopsis, Amborella, tomato, grape, rice, and maize using the CoNekT database (https://conekt.sbs.ntu.edu.sg/) (Proost and Mutwil, 2018 (link)). Gene expression was represented in transcripts per kilobase million (TPM)-based normalization because it can be used for both gene count comparisons within a sample or between samples of the same sample group (Abrams et al., 2019 (link)). The expression values were analyzed in the CIMminer one matrix server (discover.nci.nih.gov/cimminer).
Total RNA was isolated from tobacco leaves, roots, buds, flowers, imbibed pollen, germinating pollen grains and growing pollen tubes using Qiagen RNAeasy Kit, and Turbo DNA-free Kit (Applied Biosystems, Waltham, MA, USA) was used for DNA removal. cDNA synthesis was carried out using Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Penzberg, Germany) with anchored-oligo (DT)₁₈ primer according to manufacturer’s instructions. Semi-quantitative RT-PCR was performed with NtPLA₂ gene-specific oligonucleotides 1-6 (Supplementary Table 2) designed to span an intron in the corresponding genomic DNA sequence. Actin7 (Bosch et al., 2005 (link)) was used as load control. Amplification conditions were 94°C for 30 sec, 55°C for 30 sec, 68°C for 30 sec and final extension 68°C for 10 min for 28 or 34 cycles.

Tree peony (Paeonia rockii) with the same genetic origin was grown in the wild tree peony germplasm repository at Yangling, Shaanxi Province, China. They were identified by Professor Li-xin Niu from Northwest A&F University. The voucher specimens of Paeonia rockii were deposited into the Herbarium of the National Oil Peony Engineering Technology Research Center, China. Different tissues including the root, stem, leaf, calyx, petal, stamen, pistil, and developing seeds (20, 40, 60, 80, and 100 days after flowering) were collected for transcript level analysis. All the samples were immediately frozen in liquid nitrogen and stored at -80 ℃ for further studies. Arabidopsis thaliana (ecotype Columbia-0) and Nicotiana benthamiana plants used for transformation were grown in growth chambers at 21/25 ℃ (day/night) with a 16 h light/8 h dark.

Ten flower samples were respectively collected from five male and five female E. ulmoides individual trees growing on the campus of Northwest A&F University, Yangling, China (34°16′56″N, 108°04′27″E) in April 2021 for quantitative Real-Time PCR (qRT-PCR) analysis. The collected male and female flowers was about to open. Because there are no sepal and petal in either male or female flowers (Wang and Zhang, 2017 (link); Wuyun et al., 2018 (link); Zhu, 2019 ), only stamens and pistils were isolated and immediately immersed into liquid nitrogen before stored at −80°C until use. Total RNA extraction and cDNA synthesization were performed using RNeasy Plant Mini Kit (74904, Qiagen, German) and SuperScript™ IV VILO™ Master Mix (Thermo Fisher, United States of) respectively following the manufacturer’s instructions.
qRT-PCR analysis was conducted according to the manuals of Hieff qRT-PCR SYBR Green Master Mix (YEASEN, Shanghai, China) on a LightCycler 480 II Real-Time PCR Platform (Roche, Germany). The reaction system (20 μL) consisted of 10.0 μL of qRT-PCR Mix, 1.0 μL cDNA, 0.4 μL forward primer, 0.4 μL reverse primer, and 8.2 μL of ddH₂O. EuGAPDH gene was used as internal control for data normalization (Liu et al., 2018 (link)). 2^−ΔΔCT method was applied to calculate the relative gene expression of 14 A/B/C/D/E-class EuMADS genes (Livak and Schmittgen, 2001 (link)). Primers used for EuMADS and EuGAPDH genes (Supplementary Table S6) were designed using the online program Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/). Five biological replicates and two technical replicates were carried on for each gene. Differences of gene expression level between male and female flowers were analyzed via ANOVA followed by Student’s t-test in SPSS software (v.24, IBM).
The identified 14 A/B/C/D/E-class EuMADS genes were also functionally annotated by the online BLAST in NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The most similar genes in model plants (Arabidopsis, snapdragon/petunia) of flower development studies (Theißen et al., 2016 (link); Irish, 2017 (link); Ruelens et al., 2017 (link)) were screened as orthologous genes to predict gene function.