Fig 1.
Silique and seed development in WT, ick12567, ick123567 and septuple mutants.
(A) Silique length. The average and standard deviation are shown for the length of fully extended siliques (4 plants per line with 10 siliques from each plant measured). (B) Number of aborted ovules per silique. Fully extended siliques were opened and aborted ovules counted under a dissecting microscope (4 plants with 6 siliques from each plant counted). The averages and standard deviations are shown. (C) Opened siliques showing silique length and aborted ovules of the septuple mutant. (D) Number of seeds per silique (4 plants per line with 6 siliques from each plant). The averages and standard deviations are shown. (E) Seed weight. Three different seed lots were used. For each lot, seeds were harvested from four plants in one pot and one thousand seeds were counted. The averages and standard deviations are shown. Data in (A, B, D, E) were analyzed using one-way ANOVA and post-hoc Tukey test, and significant differences are indicated by different letters (upper case) at p<0.01 level.
Fig 2.
Embryo sac development in WT and ick septuple mutant.
(A—I) DIC observation of embryo sac. Ovules from flowers just before opening were prepared and observed under a microscope with DIC optics. (A) A typical WT embryo sac showing secondary (central, C), egg (E) and synergid (S) nuclei. (B—C) Two optical sections of a septuple mutant ovule showing the embryo sac area with two sets of secondary (C), egg (E) and synergid (S) nuclei. The gamete and synergid nuclei are arranged in a configuration similar to that in the WT and the boundary between the two sets of gametes is clearly visible (arrow in B), indicating the presence of two embryo sacs in the ovule. (D—E) Two optical sections of a mutant ovule with three sets of secondary (C), egg (E) and synergid (S) nuclei. (F—I) Four optical sections of a mutant ovule with four set of secondary (C), egg (E) and synergid (S) nuclei. The synergids for the 3rd and 4th sets could not be easily observed. Different sets of gametes in the mutant are indicated by numbers “1”, “2”, “3” and “4”, respectively. The two syngergid nuclei of the same pair are differentiated with or without an “′”, e.g. “S1, S1′, S2, and S2′” referring to two pairs of synergid nuclei. (J—M) Ovules from the septuple mutant carrying a pEC1.1::GUS marker for the egg cell were stained for GUS. The GUS staining shows the presence of one to four egg cells in these ovules. (N—P) Ovules from the septuple mutant carrying a pFIS2::GUS marker for the central cell were stained for GUS. The GUS staining shows the presence of one to three central cells in these ovules. Scale bars in (A), (J), and (N) are for (A—I), (J—M), and (N—P) respectively, and all equal 10 μm.
Fig 3.
Development of megaspore mother cell and expression of the pKNU-GUS reporter in WT and septuple ovules.
Developing ovules at stage 2-II were observed with DIC microscopy for development of megaspore mother cell (A—H) and ovules at around stage 2-III were analyzed for the expression of the pKNU-nlsGUS reporter, which is strongly expressed in MMC (I—L). (A—B) WT ovules. Most of WT ovules had one enlarged cell (A) and about 5% of ovules had two enlarged cells (B). (C—H) Septuple mutant ovules with one (C), two (D), three (E), four (F) and five (G, H) enlarged cells (G and H show two focal planes of the same ovule). (I–J) Strong pKNU-nlsGUS reporter expression relative to the surrounding cells indicates the presence of one (I), two (J), three (K) and four (L) MMCs in the mutant ovules. Scale bar in (A) is for all images and equals 10 μm.
Fig 4.
Identification of megaspore mother cells (MMCs) in meiosis by DMC1 immunostaining.
Developing ovules were prepared and immunostained with an antibody against DMC1, which is specifically expressed during meiosis. (A—C) WT ovules at MMC (A), meiosis (B) and FG1 (C) stages. (D—I) Mutant ovules at MMC (D), meiosis (E—H) and FG1 (I) stages. One to four cells in the mutant ovules could be stained with DMC1 (E—H). Scale bars equal 5 μm.
Fig 5.
Analysis of callose deposition patterns in WT and septuple mutant ovules.
Ovules were stained for callose with aniline blue. For each ovule, the callose fluorescence image is shown at the top and the DIC image with superimposed callose staining at the bottom. (A—D) WT ovules at different stages. (A) Before meiosis, there was weak and punctate callose deposition surrounding the MMC. (B) Callose deposition after the first division of meiosis showing a bright callose band (disc) at the place of newly formed cell plate. (C) Typical callose deposition pattern at the end of meiosis usually with two prominent calluse bands (discs) and also focused accumulation of callose at the micropylar end. One of the callose bands came from the first division and one from the second of the division by the nucleus close to the chalazal end. This pattern indicates the result of meiosis by one MMC. (D) At functional megaspore stage, the callose deposition was much reduced. (E—H) Septuple mutant ovules showing callose deposition from one (E), two (F), three (G) and likely four (H) MMCs. Scale bar in (A) is for all images and equals 10 μm.
Fig 6.
Functional megaspore (FM) development in WT and ick septuple mutant.
(A—F) Ovules at functional megaspore stage and observed under a DIC microscope. (A) WT ovule with a typical FM. (B–E) ick septuple ovules with 1–4 FMs. A typical FM was present in (B). In (C), two pairs of nuclei appear to be derived from the second meiotic divisions. The two megaspores at the micropylar end are degenerating while the two close to the chalazal end were surviving. In (D), three megaspores of similar size and morphology were surviving, while in (E), four megaspores with different sizes and appearances were surviving. (F) No typical FM was observed in the ovule. (G—L) Callose staining and DIC images were obtained for the same mutant ovules at functional megaspore stage to determine which megaspore survived. The positions of megaspores from each MMC are indicated with a number, with position 1 indicating the megaspore closest to the chalazal end. (G, I, K) show callose staining while (H, J, M) show the corresponding DIC images with superimposed callose staining. (G, H) show that one MMC finished meiosis and two megaspores were surviving. (I, J) also show the result from one MMC, but megaspores at positions 1 and 3 (counting from the chalazal end) were surviving. Note that there are three callose bands plus the pointed callus deposition at the micropylar end. (K, L) indicate that two MMCs completed meiosis (K). While four megaspores derived from the MMC at one side were all surviving, no megaspore from the other MMC appeared to be surviving. Arrows in (C–E) indicate FMs, numbers in (G–L) indicate the megaspore positions counted from the chalazal end, and scale bars equal 10 μm.
Table 1.
Frequency of surviving megaspores at each of the four positions in ick septuple mutant ovules with one MMC.
Fig 7.
ICK4-YFP expression during megasporogenesis.
Developing ovules of ICK4-YFP transgenic line (WT background) were observed under a confocal microscopy. (A, D, G, J, M) show confocal images, (B, E, H, K, N) show bright field images, and (C, F, I, L, O) show the overlays of the confocal and bright field images. (A—C) A stage 2-I ovule. (D—F) A stage 2-II ovule. (G—I) A stage 2-III ovule. (J—L) A stage 2-IV ovule at the end of meiosis I. (M—O) A stage 3-I ovule after meiosis. The numbers in (N, O) indicate the positions of megaspores counting from the chalazal to micropylar end. The arrow in (N, O) points to the nucleus of functional megaspore. The scale bars in (A), (D), (G), (J) and (M) are for (A—C), (D—F), (G—I), (J—L) and (M—O) respectively.
Fig 8.
Embryo and endosperm development in WT and ick septuple mutant.
Three days after flowers opened, the young developing seeds were prepared for DIC observations or stained for GUS first and then prepared for observations. (A) A typical WT embryo sac with a globular embryo and endosperm nuclei of uniform morphology distributed throughout the embryo sac. (B—F) Embryo and endosperm development in mutant ovules. (B) In addition to the typical embryo and endosperm, a separate compartment is seen to contain a secondary nucleus. (C) In the chalazal area, there are also a few nuclei like the secondary nucleus (yellow star signs) in the endosperm as well as a secondary nucleus in a separate embryo sac (red star signs). (D) There is one embryo, and two separate compartments of endosperm. (E) There are two embryos, and two separate compartments of endosperm. (F) There are two embryos, and three separate compartments of endosperm. (G—I) Embryo and egg cells identified by pEC1.1::GUS expression in the septuple mutant. (G) A young embryo showing GUS staining. (H) In addition to the developing embryo, strong GUS staining is seen at the chalazal end of the embryo sac where a separate sac is often present. (I) In addition to the developing embryo, GUS staining is present at two other loci indicating the presence of extra egg cells. White arrows indicate embryos and black arrows indicate the boundaries between two separate compartments of endosperm. Stars indicate extra egg cells or secondary nuclei. Scale bar in (A) is for (A—I) and equals 10 μm.
Fig 9.
Occurrence of twin seedlings in the ick septuple mutant.
A typical 4-day Arabidopsis seedling (left) and twin mutant seedlings (right). Scale bar equals to 1 mm.