Figure 1.
MYB64 and MYB119 are expressed during female gametogenesis.
(A) A schematic of female gametogenesis in Arabidopsis thaliana. Following meiosis, a haploid spore (FG1) undergoes two rounds of synchronous nuclear divisions without cytokinesis to generate a four-nucleate coenocyte (FG4). A third division is immediately followed by cellularization and differentiation (FG5 Transition) to generate the seven cells of the female gametophyte. Two polar nuclei in the central cell then fuse to form the secondary nucleus of the central cell (FG6). ac, antipodal cell; cc, central cell; ec, egg cell; pn, polar nuclei of the central cell; sc, synergid cell; sn, secondary nucleus (fused polar nuclei of the central cell). (B–D and F–H) Epifluorescent micrographs of wild-type female gametophytes expressing ProMYB64:MYB64-GFP (B–D), or ProMYB119:MYB119-GFP (F–H). (B, F) Expression at stage FG4. GFP is observed in all four nuclei (arrowheads) for both constructs. (C, G) Expression at stage FG5. GFP is observed in the unfused polar nuclei for both constructs. MYB64-GFP is additionally observed in the egg cell nucleus. (D, H) Expression at stage FG6. GFP is observed in the secondary nucleus for both constructs. MYB64-GFP is additionally observed in the egg cell nucleus. (E and I) Epifluorescent micrographs of wild-type female gametophytes expressing ProMYB64:H2B-GFP (E) and ProMYB119:H2B-GFP (I). For ProMYB64:H2B-GFP, GFP is observed in all nuclei of the female gametophyte (E). For ProMYB119:H2B-GFP, GFP is observed in the nuclei of the antipodal cells and central cell (I). acn, antipodal cell nuclei; en, egg nucleus; pn, unfused polar nuclei of the central cell; scn, synergid cell nucleus; sn, secondary nucleus (fused polar nuclei of the central cell). Scale bars are 25 µm.
Figure 2.
qRT-PCR analysis of MYB64 and MYB119 expression.
Relative expression of MYB64 and MYB119 in pistils, stamens, siliques at 36 hours after pollination, and seedlings at 10 days after germination. Error bars indicate standard deviations.
Table 1.
Segregation of myb64 alleles.
Table 2.
Segregation of myb119 alleles.
Figure 3.
myb64 myb119 gametophytes fail to initiate the FG5 transition.
(A–D) CSLM micrographs of wild-type female gametophytes. (A) A coenocytic, eight-nucleate female gametophyte at stage FG5. Only seven nuclei are visible in this projection. (B) A cellularized eight-nucleate female gametophyte at stage FG5. (C) A wild-type female gametophyte at stage FG6. (D) A mature wild-type female gametophyte at stage FG7. (E–H) CSLM micrographs of myb64-1 myb119-3 female gametophytes. (E) A coenocytic, eight-nucleate myb64 myb119 female gametophyte protruding from the micropyle. (F) An enlarged coenocytic myb64 myb119 female gametophyte containing supernumerary nuclei. (G) A cellularized myb64 myb119 gametophyte containing supernumerary, atypical cells. (H) A myb64 myb119 gametophyte that has collapsed and degenerated. (I–N) A Z-stack series of the ovule depicted in F. Fourteen nuclei are indicated by arrowheads. Abbreviations: ac, antipodal cells; cc, central cell; ec, egg cell; sc, synergid cell; WT, wild type. Scale bars are 20 µm.
Figure 4.
Cell-specific markers are misexpressed in myb64 myb119 gametophytes.
Epifluorescent micrographs of GFP expression in wild-type (A–D) and myb64-1 myb119-3 (E–H) female gametophytes. (A,E) Synergid cell marker ProDD31:GFP. (B,F) Egg cell marker ProDD45:GFP. (C,G) Central cell marker ProDD65:GFP. (D,H) Antipodal cell marker ProDD1:GFP. (I–L) Quantification of cell-specific marker expression in gametophytes from myb64-1/myb64-1 myb119-3/MYB119 plants. Bars represent the percentage of gametophytes expressing GFP. Wild-type expression was calculated as (GFP positive wild type)/(1/2 Total). Mutant expression was calculated as (GFP positive mutant)/(1/2 Total). Error bars indicate standard deviations. WT, wild type. Scale bars are 25 µm.
Table 3.
Analysis of myb64 myb119 seed phenotypes.
Figure 5.
myb64 myb119 gametophytes initiate autonomous endosperm development.
(A) A wild-type seed at 3 days after pollination. Arrow points to a pre-globular stage embryo. (B) A myb64-4 myb119-1 seed at 3 days after pollination containing proliferating endosperm but no embryo. (C) A myb64-4 myb119-1 seed at 3 days after pollination containing proliferating endosperm and a wild-type embryo (arrow). (D) A myb64-4 myb119-1 seed at 3 days after pollination containing proliferating endosperm and an embryo-like structure (arrow). (E) Magnification of the embryo-like structure in D. (F) A wild-type female gametophyte at 3 days after emasculation. (G) A degenerated myb64-4 myb119-1 gametophyte at 3 days after emasculation. (H) A myb64-4 myb119-1 autonomous seed-like structure at 3 days after emasculation. Abbreviations: ec, egg cell; emb, embryo; els, embryo-like structure; cc, central cell; DAP, days after pollination; DAE, days after emasculation; sc, synergid cell; WT, wild type. Scale bars are 40 µm.
Figure 6.
FIS2 is downregulated in myb64 myb119 gametophytes.
(A and B) Epifluorescent micrographs of ProFIS2:GFP expression in wild-type (A) and myb64-1 myb119-3 (B) gametophytes. (C) Quantification of ProFIS2:GFP expression in wild-type and myb64-1 myb119-3 gametophytes. Bars represent the percentage of gametophytes expressing GFP. Wild-type expression was calculated as (GFP positive wild type)/(1/2 Total). Mutant expression was calculated as (GFP positive mutant)/(1/2 Total). (D) Relative expression of FIS2 in wild-type and myb64-4/myb64-4 myb119-1/myb119-1 ovaries determined by qRT-PCR. WT, wild type. Error bars indicate standard deviations. Scale bars are 25 µm.
Figure 7.
MYB119 is downregulated in cki1 gametophytes.
(A, C and E) Epifluorescent micrographs of ProMYB64:H2B-GFP expression in wild-type and mutant gametophytes. (A) Expression in a wild-type female gametophyte. GFP is observed in all nuclei of the female gametophyte. (C) Expression in a cki1-9 female gametophyte. The polar nuclei are unfused. Arrowheads indicate antipodal cell nuclei that occupy abnormal positions. (E) Expression in a multinucleate rbr1-2 female gametophyte. (B, D and F) Epifluorescent micrographs of ProMYB119:H2B-GFP expression in wild-type and mutant gametophytes. (B) Expression in a wild-type female gametophyte. GFP is observed in the antipodal cell nuclei and central cell nucleus. (D) Expression in a cki1-9 female gametophyte. GFP expression is not detectable in cki1 female gametophytes. (F) Expression in a multinucleate rbr1-2 female gametophyte. (G) Quantification of female gametophytes expressing ProMYB64:H2B-GFP and ProMYB119:H2B-GFP in wild-type (N = 540 and 472, respectively), cki1-9/CKI1 (N = 378 and 456, respectively), and rbr1-2/RBR (N = 360 and 348 respectively) female gametophytes. Bars represent the percentage of gametophytes expressing GFP. *, p-val = 5.4E-10 for two sample t-test as compared to wild type; error bars indicate standard deviation. Abbreviations: acn, antipodal cell nuclei; en, egg cell nucleus; pn, polar nuclei of the central cell; scn, synergid cell nucleus; sn, secondary nucleus (fused polar nuclei of the central cell); WT, wild type. Scale bars are 25 µm.
Figure 8.
cki1 myb64 double mutant gametophytes fail to initiate the FG5 transition.
(A) A mature wild-type female gametophyte at stage FG7. (B) A cki1-9 female gametophyte at maturity. The central cell and antipodal cells are degenerated, whereas positioning and cellularization of the egg and synergid cells are unaffected. (C) A cki1-9 myb119-3 double mutant female gametophyte. cki1 myb119 gametophytes resemble cki1 single mutant gametophytes. (D) A cki1-9 myb64-1 female gametophyte. cki1 myb64 gametophytes are enlarged, contain supernumerary nuclei and resemble myb64 myb119 gametophytes. (E–J) A Z-stack series of the ovule depicted in D. Arrowheads indicate fourteen nuclei. Abbreviations: cc, central cell; ec, egg cell; sc, synergid cell; WT, wild type. Scale bars are 20 µm.
Figure 9.
CKI1 expression is restricted to the chalazal region during the FG5 transition.
CSLM micrographs of ProCKI1:CKI1-GFP expression in wild-type gametophytes. CKI1-GFP is localized primarily to the plasma membrane within the female gametophyte (A–F). Weak cytoplasmic localization is also visible during stages FG1 (A) and FG3 (B). During coenocytic development (stages FG1–FG4), ProCKI1:CKI1-GFP is expressed throughout the female gametophyte (A–C). Post-cellularization (stages FG5–FG7), CKI1-GFP expression becomes restricted to the central cell and antipodal cells (D–F). Abbreviations: ac, antipodal cells; cc, central cell. Scale bars are 25 µm.
Figure 10.
Regulation of the FG5 transition during female gametogenesis.
MYB64 and MYB119 redundantly regulate the FG5 transition. MYB119 expression is regulated by CKI1 whereas the regulation of MYB64 has yet to be determined. During the FG5 transition the embryo sac cellularizes, nuclear division ceases, cell growth attenuates, and the resulting cells differentiate.