Ovule

Images were obtained with Olympus FV1000, Leica SP8 and Zeiss LSM780NLO confocal microscopes. SR2200 was excited with a 405-nm laser line and emission recorded between 415 and 476 nm (405/415–476); similar settings were used to detect DAPI. For fluorescent proteins, the following excitation/emission wavelengths were used: CFP (458/473–552), GFP (488/505–540), YFP (514/517–597), dsRed variants (561/565–615). For spectral unmixing of SR2200 and DAPI, images were obtained with a Zeiss LSM780NLO confocal microscopes equipped with a 32-channel GaAsP array for spectral detection (405/410–695) and processed with Zeiss ZEN software. Individual staining of Arabidopsis ovules with only SR2200 or DAPI, respectively, was performed to obtain reference spectra. 3D reconstructions and orthogonal sections were produced with ImageJ software.

SR staining solution, containing 0.1 % (v/v) SR2200 (Renaissance Chemicals; stock solution of the supplier was considered as 100 %), 1 % (v/v) DMSO (Carl-Roth, Cat.#7029.2), 0.05 % (w/v) Triton-X100, 5 % (w/v) glycerol (SIGMA-ALDRICH, Cat.#G5516) and 4 % (w/v) para-formaldehyde (SIGMA-ALDRICH, Cat.#6148) in PBS buffer (pH 8.0) was prepared freshly prior to use.
For imaging of developing embryos, immature seeds were collected in staining solution on a microscope slide and embryos were gently squeezed out of the ovule by applying pressure on a cover slip. Images of embryos were taken within 30 min after release from the ovules. For images of whole ovules and PTs, ovules were manually dissected out of the silique and collected in a drop of staining solution on a microscope slide. For better tissue penetration, soft vacuum was applied for 5 min at RT. Afterward, the staining solution was replaced by water and again incubated under soft vacuum for 5 min. After replacing the water with 10 % glycerol, the sample was mounted under a coverslip. After squeezing out of the ovule, torpedo stage embryos were processed in a similar fashion as whole ovules.

Growth medium for in vitro manipulations of pollen tubes [10 (link)] was determined to be optimal for also growing pollen tubes through a cut pistil. For the in vitro assays described here, pollen growth medium (3 ml) was poured into a 35 mm petri dish (Fisher Scientific, Hampton, USA). This volume of medium was ideal both for pollen tube growth and for microscopically viewing the interactions between pollen tubes and ovules. Excised pistils were pollinated under a dissection microscope (Zeiss Stemi 2000), cut with surgical scissors at the junction between the style and ovary (World Precision Instruments, Sarasota, USA), and placed horizontally on pollen growth medium. Pollen tubes emerged from the pistil ~3 hours after pollination and dispersed along the agarose surface for up to ~3 mm from the pistil.
Unlike previous reports [12 (link),18 (link)], ovules were excised dry under a dissection microscope with a 27.5 gauge needle, from pistils that were held horizontally on double-sided tape (Scotch brand, 3M, St. Paul, USA). Excised ovules were immediately placed on the pollen growth medium, ~2 mm from the pistil, a distance that was typically accessible by the emerging pollen tubes. To maximize pollen tube-ovule interactions, 8–10 ovules were placed at the base of a pistil as shown in Fig. 1d. Because pollen tubes tend to disburse and grow randomly after leaving the style, not all ovules, particularly those placed near the cut pistil, are visited by a pollen tube (Fig. 1e).
For time-lapse imaging, ovules were placed with their micropylar end closest to the pistil excision site. Although not essential for targeting, ovules were oriented in this manner to reduce the time elapsed before targeting was achieved. In vitro assays were typically performed by completely coating stigmas of cut pistils with pollen (>100 grains per stigma); in contrast, for the repulsion assays only 20–30 pollen grains were deposited per stigma, making it possible to clearly observe individual tube behaviour. Based on experiments with limited amounts of pollen, we typically observed 50–80% of the pollen grains produced tubes that emerged from the style.

The raw data of 144 seed coats RNA-seq data of six Brassica species, B. rapa (Parkland-R), B. oleracea (Chinese Kale-O), B. nigra (CR2748-N), B. napus (DH12075-P), B. juncea (AC Vulcan-J), B. carinata (C901163-C) with eight developmental stages (Unfertilized ovule integuments (UO; no embryo), 1- to 2-cell zygote stage (S1), 4- to 8-cell stage (S2, 8-cell stage shown), 16- to 64-cell stage (S3, globular stage shown), heart stage(S4), torpedo stage(S5), bent stage(S6), and mature (S7) stage of seed formation) were collected from Gene Expression Omnibus under accession no. GSE153257. Low-quality reads were removed from the raw reads using Cutadapt and Trimmomatic software to get clean reads [39 , 2 ]. Clean reads were mapped to the corresponding reference genome using HISAT2 software [51 (link)]. Gene expression levels of each gene were calculated using StringTie and Ballgown software [51 (link)]. The read counts of each gene were calculated using the htseq-count function in htseq software [1 (link)]. The R package DEseq2 (v1.16.1) was used to identify the differentially expressed genes (DEGs) between leaves of different colors based on the following criteria: padj < 0.05 & log2FoldChange > 2 [5 (link)].

Maximum fiber lengths were estimated by placing ovules on a watch-glass and gently spraying fibers with a stream of distilled water as described by Schubert et al. [27 ]. Ten to thirty cotton bolls were randomly selected from each biological replicate samples of G. arboreum (A₂-100 and SXY1), G. raimondii (D₅-6 and D₅-31) and G. hirsutum (TM-1, SG-747, DP-5690, Li₁, and Li₂). Single cotton seeds were randomly selected from an individual cotton boll. The distance between the chalazal end of the selected seeds and the tip of the spread fibers were measured to the nearest 0.1 mm with a digital caliper. Mean maximum fiber length of each cotton variety was obtained by measuring the randomly selected seeds from two biological replicates.

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.