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An ARID domain-containing protein within nuclear bodies is required for sperm cell formation in Arabidopsis thaliana.

Zheng B, He H, Zheng Y, Wu W, McCormick S - PLoS Genet. (2014)

Bottom Line: In plants, each male meiotic product undergoes mitosis, and then one of the resulting cells divides again, yielding a three-celled pollen grain comprised of a vegetative cell and two sperm cells.An arid1 mutant and antisense arid1 plants had an increased incidence of pollen with only a single sperm-like cell and exhibited reduced fertility as well as reduced expression of DUO1.In vitro and in vivo evidence showed that ARID1 binds to the DUO1 promoter.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China; Plant Gene Expression Center, USDA/ARS and Dept. of Plant and Microbial Biology, UC-Berkeley, Albany, California, United States of America.

ABSTRACT
In plants, each male meiotic product undergoes mitosis, and then one of the resulting cells divides again, yielding a three-celled pollen grain comprised of a vegetative cell and two sperm cells. Several genes have been found to act in this process, and DUO1 (DUO POLLEN 1), a transcription factor, plays a key role in sperm cell formation by activating expression of several germline genes. But how DUO1 itself is activated and how sperm cell formation is initiated remain unknown. To expand our understanding of sperm cell formation, we characterized an ARID (AT-Rich Interacting Domain)-containing protein, ARID1, that is specifically required for sperm cell formation in Arabidopsis. ARID1 localizes within nuclear bodies that are transiently present in the generative cell from which sperm cells arise, coincident with the timing of DUO1 activation. An arid1 mutant and antisense arid1 plants had an increased incidence of pollen with only a single sperm-like cell and exhibited reduced fertility as well as reduced expression of DUO1. In vitro and in vivo evidence showed that ARID1 binds to the DUO1 promoter. Lastly, we found that ARID1 physically associates with histone deacetylase 8 and that histone acetylation, which in wild type is evident only in sperm, expanded to the vegetative cell nucleus in the arid1 mutant. This study identifies a novel component required for sperm cell formation in plants and uncovers a direct positive regulatory role of ARID1 on DUO1 through association with histone acetylation.

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Altered histone acetylation in arid1-1.(A) Expanded pattern of histone acetylation signal in arid1-1 by immunofluorescence analysis. Mature pollen from wild type and arid1-1 plants was incubated with anti-H3 (control) and anti-H3K9ac antibodies. Representative examples for arid1-1 show histone acetylation in the vegetative nucleus (indicated by white arrowheads). 50–100 pollen grains for each genotype were examined. (B) Reduced histone acetylation at the DUO1 promoter in the arid1-1 mutant by ChIP. Inflorescences from wild type and arid1-1 were used for a ChIP assay with anti-H3, anti-H3K9ac, and anti-H3K4me3 antibodies. ChIP-DNA was used for PCR by amplifying 35 cycles for both EIF4A1 (negative control) and the DUO1_3 fragment, which bound ARID1. Similar results were obtained from three independent biological replicates; the results shown are from one replicate.
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pgen-1004421-g006: Altered histone acetylation in arid1-1.(A) Expanded pattern of histone acetylation signal in arid1-1 by immunofluorescence analysis. Mature pollen from wild type and arid1-1 plants was incubated with anti-H3 (control) and anti-H3K9ac antibodies. Representative examples for arid1-1 show histone acetylation in the vegetative nucleus (indicated by white arrowheads). 50–100 pollen grains for each genotype were examined. (B) Reduced histone acetylation at the DUO1 promoter in the arid1-1 mutant by ChIP. Inflorescences from wild type and arid1-1 were used for a ChIP assay with anti-H3, anti-H3K9ac, and anti-H3K4me3 antibodies. ChIP-DNA was used for PCR by amplifying 35 cycles for both EIF4A1 (negative control) and the DUO1_3 fragment, which bound ARID1. Similar results were obtained from three independent biological replicates; the results shown are from one replicate.

Mentions: Since ARID1 interacts with HDA8, we predicted that the in vivo histone acetylation level might be altered in the arid1-1 mutant. We therefore performed immunofluorescence with antibodies specific to H3K9 acetylation. In wild type pollen, the signal was only detected in the two sperm nuclei (Figure 6A, upper panel), but in the arid1-1 mutant, the immunofluorescence signal was also detected in the vegetative nucleus (Figure 6A, lower panel). The immunofluorescence signal with the Histone 3 antibody, used as a control, showed no difference between wild type and arid1-1 (Figure 6A). These results indicate that ARID1 is required to restrict histone acetylation to sperm cells.


An ARID domain-containing protein within nuclear bodies is required for sperm cell formation in Arabidopsis thaliana.

Zheng B, He H, Zheng Y, Wu W, McCormick S - PLoS Genet. (2014)

Altered histone acetylation in arid1-1.(A) Expanded pattern of histone acetylation signal in arid1-1 by immunofluorescence analysis. Mature pollen from wild type and arid1-1 plants was incubated with anti-H3 (control) and anti-H3K9ac antibodies. Representative examples for arid1-1 show histone acetylation in the vegetative nucleus (indicated by white arrowheads). 50–100 pollen grains for each genotype were examined. (B) Reduced histone acetylation at the DUO1 promoter in the arid1-1 mutant by ChIP. Inflorescences from wild type and arid1-1 were used for a ChIP assay with anti-H3, anti-H3K9ac, and anti-H3K4me3 antibodies. ChIP-DNA was used for PCR by amplifying 35 cycles for both EIF4A1 (negative control) and the DUO1_3 fragment, which bound ARID1. Similar results were obtained from three independent biological replicates; the results shown are from one replicate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109846&req=5

pgen-1004421-g006: Altered histone acetylation in arid1-1.(A) Expanded pattern of histone acetylation signal in arid1-1 by immunofluorescence analysis. Mature pollen from wild type and arid1-1 plants was incubated with anti-H3 (control) and anti-H3K9ac antibodies. Representative examples for arid1-1 show histone acetylation in the vegetative nucleus (indicated by white arrowheads). 50–100 pollen grains for each genotype were examined. (B) Reduced histone acetylation at the DUO1 promoter in the arid1-1 mutant by ChIP. Inflorescences from wild type and arid1-1 were used for a ChIP assay with anti-H3, anti-H3K9ac, and anti-H3K4me3 antibodies. ChIP-DNA was used for PCR by amplifying 35 cycles for both EIF4A1 (negative control) and the DUO1_3 fragment, which bound ARID1. Similar results were obtained from three independent biological replicates; the results shown are from one replicate.
Mentions: Since ARID1 interacts with HDA8, we predicted that the in vivo histone acetylation level might be altered in the arid1-1 mutant. We therefore performed immunofluorescence with antibodies specific to H3K9 acetylation. In wild type pollen, the signal was only detected in the two sperm nuclei (Figure 6A, upper panel), but in the arid1-1 mutant, the immunofluorescence signal was also detected in the vegetative nucleus (Figure 6A, lower panel). The immunofluorescence signal with the Histone 3 antibody, used as a control, showed no difference between wild type and arid1-1 (Figure 6A). These results indicate that ARID1 is required to restrict histone acetylation to sperm cells.

Bottom Line: In plants, each male meiotic product undergoes mitosis, and then one of the resulting cells divides again, yielding a three-celled pollen grain comprised of a vegetative cell and two sperm cells.An arid1 mutant and antisense arid1 plants had an increased incidence of pollen with only a single sperm-like cell and exhibited reduced fertility as well as reduced expression of DUO1.In vitro and in vivo evidence showed that ARID1 binds to the DUO1 promoter.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China; Plant Gene Expression Center, USDA/ARS and Dept. of Plant and Microbial Biology, UC-Berkeley, Albany, California, United States of America.

ABSTRACT
In plants, each male meiotic product undergoes mitosis, and then one of the resulting cells divides again, yielding a three-celled pollen grain comprised of a vegetative cell and two sperm cells. Several genes have been found to act in this process, and DUO1 (DUO POLLEN 1), a transcription factor, plays a key role in sperm cell formation by activating expression of several germline genes. But how DUO1 itself is activated and how sperm cell formation is initiated remain unknown. To expand our understanding of sperm cell formation, we characterized an ARID (AT-Rich Interacting Domain)-containing protein, ARID1, that is specifically required for sperm cell formation in Arabidopsis. ARID1 localizes within nuclear bodies that are transiently present in the generative cell from which sperm cells arise, coincident with the timing of DUO1 activation. An arid1 mutant and antisense arid1 plants had an increased incidence of pollen with only a single sperm-like cell and exhibited reduced fertility as well as reduced expression of DUO1. In vitro and in vivo evidence showed that ARID1 binds to the DUO1 promoter. Lastly, we found that ARID1 physically associates with histone deacetylase 8 and that histone acetylation, which in wild type is evident only in sperm, expanded to the vegetative cell nucleus in the arid1 mutant. This study identifies a novel component required for sperm cell formation in plants and uncovers a direct positive regulatory role of ARID1 on DUO1 through association with histone acetylation.

Show MeSH