Plant materials and growth conditions. T-DNA insertion lines SALK_021086 (ecs1) and SALK_090795 (ecs2) were obtained from the Arabidopsis Biological Resource Center (ABRC). Arabidopsis thaliana Columbia-0 (Col-0) was used as wild type (WT) control. Plants were grown in soil in a greenhouse under long‐day conditions (16 h light/8 h dark) at 22 °C.
Vector construction and plant transformation. All fragments were amplified by polymerase chain reaction (PCR) using the Phanta Kit (Vazyme Biotech). Primer sequences (synthesized by Sangon Biotech) are listed in Supplementary Table 1. All constructs were generated using restriction enzymes (New England Biolabs) and One Step Cloning Kit (Vazyme Biotech). To label egg cell membrane and chromosomes, DD45::GFP-LTI6b and DD45::CENH3-GFP fusion constructs were generated17, 24. A DNA fragment containing the promoter and coding sequence was amplified from A. thaliana ecotype Col-0 genomic DNA and inserted into the P094 vector. All constructs were transformed into Agrobacterium tumefaciens strain GV3101 and then into Arabidopsis plants by the floral dipping method26.
Ovule clearance. To analyze seed and embryo phenotypes, siliques at different developmental stages after fertilization were dissected with a needle and quickly cleared in Herr's solution (lactic acid:chloral hydrate:phenol:clove oil:xylene = 2:2:2:2:1, wt/wt) as described previously27. Images were acquired using an Olympus IX71 inverted microscope with differential interference contrast optics.
Confocal microscopy and image analysis. Ovules and early seeds were observed using a confocal microscope (Leica TCS SP8) and excited with a 488-nm laser and emitted light measured at 498–550 nm for enhanced GFP as well as excited with a 552-nm laser and emitted light measured at 600–650 nm for RFP. 3D images were generated using the LAS-X software (Leica) and 3D reconstructions were produced by Leica Aivia AI Image Analysis Software.
Flow cytometry. For flow cytometry analysis, two leaves were each harvested and chopped using a razor blade in a petri dish with 500 μl of nuclei extraction buffer (Partec CyStain). 1 ml of staining reagent (Partec CyStain UV Precise-Kit) was added and specimens were incubated at room temperature for 1 min. Samples were passed through a 40 µM nylon mesh into a 5 mL round-bottom polystyrene test tube (FALCON, 352235) and filtrates were analyzed using a Beckmann CytoFlex ploidy analyzer.
Chromosome spreads. For chromosome spreads, flower buds fixed in Carnoy’s solution were washed twice with 10 mM citrate buffer (pH 4.5) and digested with 0.3% cellulose and 0.3% pectolyase in citrate buffer at 25℃ for 1 h. After being washed twice with the same buffer and by adding DAPI solution (10μg/ml DAPI in citrate buffer), digested buds were squashed with a cover slip to release PMCs and samples were analyzed using a Leica SP8 CLSM.