Fig 1.
Early embryo development in the wild-type plants and zar mutants.
(A) A mature wild-type embryo sac showing the egg cell (ec), central cell (cc) and synergid cells (sc) under confocal microscopy. (B-F) Early embryo in wild type showing zygote (zy) (B), elongated zygote (C), the first zygotic division to produce a small apical cell (ac) and a large basal cell (bc) (D), early globular embryo (em) (E), and globular embryo (F). (G-I) Early embryogenesis in zar1-1+/- seeds showing normal globular embryo (G), elongated and arrested zygote (H), and symmetrically divided zygote (I). (J-M) early embryogenesis in zar1-2 seeds showing elongated zygote (J), symmetric division of zygote (K), asymmetrically divided zygote (L), and globular embryo (M). dp, division plane; su, suspensor; red arrowheads indicate nuclei. Bars = 10 μm.
Fig 2.
Zygotic phenotype of zar1 and agb1 mutations.
Microscopic images showing the size of apical cell and basal cell in the wild-type Ler (A), zar1-1 (B), zar1-2 (C), agb1-2 (D), and zar1-2 agb1-2 (E) mutants. Statistical results on apical cell (F) and basal cell (G) are shown (n > 30). ac, apical cell; bc, basal cell; dp, division plane. Stars indicate significant difference. Bars = 10 μm.
Fig 3.
Ectopic expression of cell-specific markers in mutant embryo.
(A-N) Basal cell-specific marker pWOX8gΔ:NLS-vYFP3 and apical cell-specific marker pWOX2:DsRed2 are expressed in the wild-type (A-F), zar1-1 (G-H), and zar1-2 embryos (I-N). YFP-NLS signal of pWOX8gΔ:NLS-vYFP3 is only detected in zygote (A) and the basal cell lineage (B-F) in the wild-type, while in arrested zar1-1 zygote (G), very weak YFP signal is present, but no YFP in apical cell, or basal cell (H). The WOX8 is falsely expressed in the apical lineage besides the basal cell lineage in zar1-2 embryo (blue arrowhead) (K-N). And apical cell-specific marker pWOX2:DsRed2 is mis-expressed in arrested zar1-1 zygote (G). YFP signal is falsely colored with green and DsRed signal with purple. (O-T) Expression of zygote asymmetric division marker pWRKY2:NLS-vYFP3 in wild type (O-P), arrested zar1-1 seeds (Q-R) and zar1-2 ovules (S-T). Blue arrows show the ectopic expression of pWRKY2: NLS- vYFP3 in endosperm of zar1-2 ovules. ac, apical cell; bc, basal cell; dp, division plane; em, embryo; su, suspensor; zy, zygote. Bars = 10 μm.
Fig 4.
ZAR1 localization and expression pattern in embryo sac and early embryogenesis.
(A) A catoon depicting the domains of ZAR1 protein and insertion sites in alleles zar1-1, zar1-2 and zar1-3. SP, signal peptide; LRR, leucine-rich repeat; TM, transmembrane domain; CaMBD, calmodulin-binding domain; GβBS, Gβ binding site. (B) Co-localization of ZAR1-GFP with FM4-64 membrane probe in root cells over-expressing ZAR1-GFP. Plasmodesma was shown with white arrowhead. The bottom chart shows the profiling data of ZAR1-GFP and FM4-64. Bar = 5 μm. (C) Plasmolysis of root cells over-expressing ZAR1-GFP. Plasma membrane and cell wall were shown with white and red arrowheads, respectively. Bar = 10 μm. (D-L) Expression pattern of ZAR1. YFP signal from pZAR1:NLS-YFP transgenic plants is detected in the micropylar nuclei of eight-nucleate embryo sac (D), and in syngerid and central cells in mature embryo sac (E), and also in zygote (F) and endosperm after pollination (F, G); YFP is also detected in sperm and vegetative cells of both pollen grain (H) and pollen tube (I). Serial sections of the wild-type ovules were hybridized to ZAR1 antisense probe (J-L) showing ZAR1 expression in egg cell and synergids (J), central cell (K), zygote and endosperm (L) and ZAR1 sense probe (M). cc, central cell; cn, central cell nucleus; ec, egg cell; ed, endosperm; en, endosperm nucleus; sc, synergid cell; sn, synergid nucleus; sp, sperm nucleus; vn, vegetative nucleus; zy, zygote. Bars = 10 μm in (D-G) and (J-M). Bars = 5 μm in (H-I).
Fig 5.
(A) Pull-down assay showing interaction between His-kinase with GST-tagged CaM1 or CaM8 in a CaMBD dependent manner. (B) BiFC assay in Arabidopsis leaf protoplasts. No YFP reconstitution in control combinations: pCaM1-nYFP/pcYFP, pZAR1-cYFP/pnYFP, pZAR1-cYFP/pPMS1-nYFP, or pZAR1ΔCaMBD-cYFP/pCaM1-nYFP. YFP is restored only with pZAR1-cYFP/pCaM1-nYFP, indicative of the interaction between ZAR1 and CaM1 in a CaMBD dependent manner. a, pCaM1-nYFP and pcYFP; b, pnYFP and pZAR1-cYFP; c, pPMS1-nYFP and pZAR1-cYFP; d, pCaM1-nYFP and pZAR1ΔCaMBD-cYFP; e, pCaM1-nYFP and pZAR1-cYFP. (C) Co-IP assays showing ZAR1 and CaM1 interaction. FLAG-tagged ZAR1 and its variants were co-expressed with HA-tagged CaM1 in Arabidopsis leaf cells and FLAG-ZAR1 was co-immunoprecipitated with CaM1-HA using anti-HA antibody. (D) Co-localization of ZAR1-GFP (green) with CaM1-RFP (red) in plasma membrane. Bars = 20 μm.
Fig 6.
Interaction between ZAR1 and AGB1.
(A) Pull-down assay showing interaction of His-kinase or its variants with MBP-tagged AGB1 in a GβBS dependent manner. (B) BiFC assays in Arabidopsis leaf cells. No YFP restoration in control combinations: pAGB1-nYFP/pcYFP, pZAR1-cYFP/pPMS1-nYFP, pZAR1ΔGβBS-cYFP/pAGB1-nYFP, and pZAR1-cYFP/pnYFP. YFP was restored in pZAR1-cYFP/pAGB1-nYFP combinations, indicative of ZAR1 and AGB1 interaction. a, pAGB1-nYFP and pcYFP; b, pnYFP and pZAR1-cYFP; c, pPMS1-nYFP and pZAR1-cYFP; d, pAGB1-nYFP and pZAR1ΔGBS-cYFP; e and f, pAGB1-nYFP and pZAR1-cYFP. (C) Co-IP assay showing interaction of ZAR1 and AGB1 in vivo. FLAG-tagged ZAR1 and its variants were co-expressed with HA-tagged AGB1 in Arabidopsis leaf cells. HA-AGB1 was co-immunoprecipitated by FLAG-ZAR1 using anti-FLAG antibody. (D) Co-localization of ZAR1-GFP with AGB1-mCherry in plasma membrane foci (arrowheads). Bars = 20 μm.
Fig 7.
Ectopic expression of cell lineage markers in agb1-2 mutants.
(A-J) Basal cell-specific marker pWOX8gΔ:NLS-vYFP3 and apical cell specific-marker pWOX2:DsRed2 were expressed in the wild-type (A-E) and agb1-2 proembryos (F-J). The basal marker is mis-expressed in the apical lineage besides the basal cell lineage in agb1-2 embryo (blue arrowhead) (H-J). YFP signal is falsely colored with green and DsRed signal with purple. (K-N) Expression of zygote asymmetric division marker pWRKY2:NLS-vYFP3 in the wild-type (K-L) and agb1-2 (M-N) proembryos. Blue arrows show the ectopic expression of pWRKY2: NLS- vYFP3 in endosperm of agb1-2 proembryos. (O-P) Expression of basal cell-specific marker pWOX9:NLS-vYFP3 in the wild-type (O) and agb1-2 embryos (P). Star shows ectopic expression of basal cell lineage marker pWOX9:NLS-vYFP3 in apical cell lineage of agb1-2. ac, apical cell; bc, basal cell; em, embryo; en, endosperm; su, suspensor; zy, zygote. Bars = 10 μm.
Fig 8.
ZAR1’s interaction with CaM1 and AGB1 is activated by the binding.
(A) Pull-down assay showing that ZAR1 forms complex with CaM1 and AGB1. His-tagged ZAR1 intracellular domain was co-incubated with GST-CaM1 and MBP-AGB1. Either CaM-GST or AGB1-MBP pulled down the other two proteins. (B) Co-localization of ZAR1 with CaM1 and AGB1 in leaf cells. YFP fluorescence restored by ZAR1-cYFP and AGB1-nYFP interaction (green) is co-localized with CaM1-RFP (red) in plasma membrane foci. a, pZAR1-cYFP and pAGB1-nYFP; b, chlorophyll; c, pCaM1-RFP; d, merge. Bars = 20 μm (C) Co-IP assay showing ZAR1 interaction with CaM1 and AGB1 in planta. Co-IP was performed using total protein extract from plants overexpressing FLAG-ZAR1. Both AGB1 and CaM1 proteins were co-immunoprecipitated with FLAG-ZAR1 using anti-FLAG antibody. EGFP-LTi6b transgenic plant was used as negative control. (D) Pull-down assay showing auto-phosphorylation of ZAR1 kinase domain upon binding of CaM1. ZAR1 kinase and its variants tagged with His, CaM1 tagged with GST, CIP treatment as control. (E) Pull-down assay showing auto-phosphorylation of ZAR1 kinase domain upon binding of AGB1. ZAR1 kinase and its variants were tagged with His, and AGB1was tagged with MBP. Stars and arrows in D and E indicate phosphorylated and non-phosphorylated kinases, respectively.