Arabidopsis EED1 encoding a plant-specific nuclear protein is essential for early embryogenesis

Mai Yang^{1, 1},
Chun Yan^{2, 1},
Megan Griffith³,
Jinping Zhao⁴,
Yongbiao Zhang⁵,
Daoxin Xie⁶,
Jianbin Yan^{8, 9
, ,}

1.
MOE Key Laboratory of Bioinformatics, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China
2.
Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, Beihang University, Beijing, 100191, China
3.
Institute of Molecular and Cell Biology, Singapore, 117609, Singapore
4.
MOE Key Laboratory of Bioinformatics, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China
5.
Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, Beihang University, Beijing, 100191, China
6.
MOE Key Laboratory of Bioinformatics, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China
8.
MOE Key Laboratory of Bioinformatics, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China
9.
Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120, China

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Corresponding author: E-mail address: jianbinlab@caas.cn (Jianbin Yan)

These authors contributed equally to this work.

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These authors contributed equally to this work.

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Fig. 1. EED1 encodes a plant-specific nuclear protein essential for early embryogenesis in Arabidopsis. A: Schematic diagram of the gene structure of EED1 and the position of T-DNA insertion in the eed1 mutant (SALK_109357). Arrow indicates the direction of T-DNA insertion in eed1. The start and stop codons are labeled. B: eed1/+ mutant has no vegetative developmental defects. WT, Col-0; eed1/+, heterozygous plant; eed1:gEED1-7 and eed1:gEED1-8, two independent complementation lines (homozygous eed1 mutant complemented with the full-length genomic fragment of EED1). All plants are 5 weeks old. Scale bars, 2 cm. C–E: eed1/+ mutant shows an embryo-lethal phenotype which can be rescued by the EED1 gene. C: Phenotype of representative ovules in siliques of WT, eed1/+, and eed1:gEED1 complementation lines. White arrows indicate the aborted ovules. Scale bars, 1 mm. D: Phenotype of representative seeds from WT and eed1/+ plants. Black arrows indicate the aborted seeds. Scale bars, 500 μm. E: Segregation analysis of aborted ovules in siliques of corresponding plants. The χ² test with 0.05 significance level has accepted the hypothesis that segregation of aborted ovules in eed1/+ siliques is consistent with the expected ratio of 25%. F–I: Homozygous eed1 embryos show defects in early developmental stages. F: Normal embryo develops to the heart stage at 5 days after pollination (DAP). G: Higher magnification of normal embryo shown in (F). H: Mutant embryo remains small and is arrested very early. I: Higher magnification of mutant embryo shown in (H). Dotted lines highlight the mutant embryos. Normal and mutant embryos from the same silique were pretreated by whole-mount clearing and examined under differential interference contrast (DIC) microscopy. Scale bars, 50 μm (F and H) or 20 μm (G and I). J–L: Homozygous eed1 embryos are arrested at the 1-cell or 2/4-cell stage. At 3 DAP, normal embryo develops to the global stage (J), while mutant embryo is arrested at the 1-cell (K) or 2/4-cell stage (L). Right panels show higher magnification of embryos in left panels, respectively. White arrows indicate the nuclei. Normal and mutant embryos from the same silique were pretreated by propidium iodide (PI) staining and examined under confocal microscopy. Scale bars, 20 μm. M: Relative transcription levels of EED1 in various Arabidopsis tissues evaluated by quantitative real-time PCR (qRT-PCR). SE, seedlings; RT, roots; ST, stems; RL, rosette leaves; CL, cauline leaves; INF, inflorescences; SILQ1, siliques at 3 DAP; SILQ2, siliques at 5 DAP. Arabidopsis ACTIN8 (At1g49240) served as an internal control. Data are shown as mean ± SEM (n = 5). ANOVA was performed for statistical analysis; different letters indicate significantly differences from each other (P < 0.05).N–W: Tissue-specific expression of EED1 monitored in EED1::EED1-GUS transgenic plants (expressing EED1-GUS fusion protein under the control of the EED1 native promoter). N: A 7-day-old seedling. O and P: Shoot apex and leaf primordia (O) and apical root meristem (P) of the seedling shown in (N). Q–U: Rosette leaves (Q), mature stem (R), inflorescences of lateral shoot (S), inflorescences of main shoot (T) and unfertilized flower (U) of an adult plant. V and W: Embryos at transition stage (V) and heart stage (W), respectively. White arrows indicate the embryos. Scale bars, 1 mm (N–P and R‒T), 1 cm (Q), 500 μm (U), or 100 μm (V and W).X: EED1 encodes a nuclear-localized protein. The YFP-EED1 fusion protein (EED1 protein fused to the C terminus of yellow fluorescent protein) was transiently expressed in N. benthamiana leaf epidermal cells and examined under confocal microscopy 48 h after infiltration. The nuclei were indicated by 4′,6-diamidino-2-phenylindole (DAPI) staining. Scale bars, 50 μm. Y: EED1 expression is down-regulated by IAA treatment. 7-day-old Col-0 seedlings treated with EtOH (Mock) or 1 μM IAA for 1.5 h were collected for RNA extraction and qRT-PCR analysis. Arabidopsis ACTIN8 served as an internal control. Data are shown as mean ± SEM (n = 3); *P < 0.05; Student'st-test.

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