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First published online 28 November 2007
doi: 10.1242/dev.010108

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Chromatin assembly factor 1 regulates the cell cycle but not cell fate during male gametogenesis in Arabidopsis thaliana

¹ Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Department of Biological Sciences, 117604, Singapore.
² Section of Plant Biology, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, USA.

View larger version (69K): [in this window] [in a new window]   Fig. 1. Expression of CAF1 components in pollen. (A) RT-PCR was performed from RNAs extracted from mature Arabidopsis thaliana pollen grains and shows expression of MSI1, FAS1 and FAS2. (B-D) Wild-type pollen development. Nuclei are stained with DAPI. The uni-nucleate microspore (B) undergoes an asymmetrical mitosis, leading to the bicellular stage (C). At that stage the pollen grain contains a large vegetative cell and a small generative cell with a nucleus showing relatively higher chromatin compaction. (D) The generative cell divides, giving rise to two sperm cells with highly condensed chromatin. (E-G) Expression pattern of promMSI1-MSI1-mRFP1 in pollen in microspore (E), and developing pollen at bicellular stage (F) and tricellular stage (G). Confocal sections. Scale bars: 10 µm. G, generative cell; S, sperm cell; V, vegetative cell.

View larger version (56K): [in this window] [in a new window]   Fig. 2. Defects in pollen development in msi1/+ mutants. Pollen development in meiotic tetrads from qrt1/qrt1 and qrt1/qrt1; msi1-1/+ Arabidopsis plants. At uni-nucleate stage (A) microspore tetrads of wild-type and mutant plants are indistinguishable. In contrast to wild-type tetrads (B), msi1/+ tetrads at the bicellular stage contain one arrested microspore (C, arrowhead). (D-F) At the tricellular stage, qrt1/qrt1; msi1-1/+ plants produce tetrads containing three wild-type pollen grains and an abnormal pollen grain (arrowhead). The abnormal pollen is either a dead microspore (D), an arrested microspore (E) or a bicellular pollen grain (F). Epifluorescence micrographs of DAPI-stained pollen. Scale bars: 10 µm.

View larger version (9K): [in this window] [in a new window]   Fig. 3. Synergistic effects of combination between mutations in members of the CAF1 complex. Bar chart showing percentage of the two types of abnormal pollen at the tricellular mature stage in msi1-1/+, fas1-1/+, fas2-4/+, msi1-1/+;fas1-1/+ and fas1-1/+;fas2-4/+ mutant Arabidopsis plants. Gray bars correspond to arrested bicellular pollen and black bars to arrested microspores. Error bars correspond to standard errors calculated on the basis of several samples of 100 pollen grains, and the size of the total population analyzed (n) is indicated above each column.

View larger version (7K): [in this window] [in a new window]   Fig. 4. Effect of msi1 on DNA content in sperm cell nuclei. The DNA content in Arabidopsis sperm cells was estimated relative to the fluorescence intensity after DAPI staining. The relative DAPI fluorescence intensity between control wild-type sperm cells and single sperm-like cells in msi1-1;fas1-1 pollen and in duo1-1 pollen is indicated under the graph. Error bars correspond to standard deviations, and the size of the sample (n) is indicated above each column.

View larger version (70K): [in this window] [in a new window]   Fig. 5. Cell identities in bicellular msi1 pollen. (A-F) Wild-type Arabidopsis pollen at the tricellular stage. (G-L) Bicellular and tricellular pollen segregating from msi1/+ plants. (A,G) Fluorescence images of pollen grains stained with DAPI. (B,H) The same pollen grains as in A,G, respectively, expressing the vegetative cell marker AC26-Histone2B-mRFP1. (C,I) Pollen grains expressing the generative lineage marker pDUO1-DUO1-mRFP1. (D,J) Pollen grains expressing the generative lineage marker E1-GFP. (E,K) Pollen grains expressing the sperm cell marker HTR10-mRFP1. (F,L) Pollen grains expressing the sperm cell marker ATGEX1-GFP. GFP was examined using the 488 nm excitation line of an argon laser and an emission filter long pass of 510 nm. Fluorescence of mRFP1 was examined using the 543 nm excitation line of a HeNe laser and an emission filter of 585-615 nm. Scale bars: 10 µm.

View larger version (151K): [in this window] [in a new window]   Fig. 6. Pollination of wild-type ovules with msi1 pollen leads to single fertilization events. (A) Wild-type Arabidopsis seed with an embryo and an endosperm, 2 days after pollination (2 DAP). (B) Seed with an endosperm but without an embryo, 3 DAP. At the embryo region, the egg cell remains unfertilized. (C) Seed with an embryo but without endosperm, 3 DAP. At the side of the embryo the nucleus of central cell that remains unfertilized is still observed. (D) Fully aborted ovule without embryo and endosperm, 3 DAP. The egg cell and the central cell remain unfertilized. (E) Expression of the fluorescent KS22 GFP endosperm marker in a wild-type seed at 3 DAP. (F) Expression of KS22 GFP in a seed containing only endosperm from a cross between wild-type ovules and msi1-2/+; KS22/KS22 pollen. (G) Confocal section of a seed containing only an embryo, produced by a cross between a wild-type ovule and msi1-2 pollen. The nucleus of the unfertilized central cell remains associated with the surface of the embryo. Feulgen staining marks brightly the DNA in nuclei as described previously (Garcia et al., 2003). The inset shows ten chromocentres at pre-prophase in the nucleus from the single embryo. The single embryo is diploid and thus originates from a fertilized egg cell. Scale bars: 100 µm in A-D; 50 µm in E,F; 10 µm in G. cc, central cell; ec, egg cell; em, embryo; en, endosperm.

View larger version (38K): [in this window] [in a new window]   Fig. 7. Fate of the single sperm cell during msi1 pollen tube growth. (A) Percentage of pollen tubes transporting a single sperm cell from a msi1-1/+ Arabidopsis plant at different time points after pollen germination in vitro. Errors bars correspond to standard errors calculated on the basis of three replicates of 100 pollen grains. (B-D) Transport of sperm cells marked with HTR10-mRFP in pollen tube germinated in vitro from wild-type (B) and msi1/+ (C,D) plants. (B) At 6 hours after germination, wild-type pollen tubes transport two sperm cells (two fluorescent red dots). (C) By contrast, at 6 hours a msi1 pollen tube transports a single sperm cell. (D) At 7 hours after germination the msi1 single gamete had divided into two sperm cells. (B,C,D) Superimposed DIC images of the pollen tube with the mRFP1 fluorescence signal identifying the sperm cells. Scale bars: 20 µm.

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