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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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ARID1 physically associates with Histone Deacetylase 8 (HDA8).(A) Yeast cells co-expressing the indicated plasmids were grown on control (without Tryptophan and Leucine) or high stringency selection (without Tryptophan, Leucine, Histidine, and with 10 mM 3-AT) medium. ARID1, full length cDNA of ARID1; ARID1-C, the C-terminus-containing ELM2 domain of ARID1; HDA8, full length cDNA of HDA8. AD is pGAD10. The cultures from each of the indicated strains were diluted 100-fold and spotted. Three colonies were streaked for each pair. (B) ARID1 interacts with HDA8 by GST pulldown assay. Whole cell extracts from wild type or ARID1-GFP plants were applied onto GST and GST-HDA8 (abbreviated GST-H8) beads. GFP and Hsc70 (loading control) antibodies were used in immunoblotting. (C) Stained protein gel showing proteins used for the GST pulldown assay. 1/30 of the amount of each protein used in Figure 5A was resolved by SDS-PAGE. GST-H8, full length HDA8 fused to GST; GST alone was used as a control. GST-H8 is marked with an arrowhead. (D) ARID1 interacts with HDA8 by Co-IP. Inflorescences from ARID1-Myc; HDA8-YFP (abbreviated H8-YFP) doubly transgenic plants or from wild type were immunoprecipitated with Myc antibody. Myc, GFP and Hsc70 (loading control) antibodies were used for immunoblotting.
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pgen-1004421-g005: ARID1 physically associates with Histone Deacetylase 8 (HDA8).(A) Yeast cells co-expressing the indicated plasmids were grown on control (without Tryptophan and Leucine) or high stringency selection (without Tryptophan, Leucine, Histidine, and with 10 mM 3-AT) medium. ARID1, full length cDNA of ARID1; ARID1-C, the C-terminus-containing ELM2 domain of ARID1; HDA8, full length cDNA of HDA8. AD is pGAD10. The cultures from each of the indicated strains were diluted 100-fold and spotted. Three colonies were streaked for each pair. (B) ARID1 interacts with HDA8 by GST pulldown assay. Whole cell extracts from wild type or ARID1-GFP plants were applied onto GST and GST-HDA8 (abbreviated GST-H8) beads. GFP and Hsc70 (loading control) antibodies were used in immunoblotting. (C) Stained protein gel showing proteins used for the GST pulldown assay. 1/30 of the amount of each protein used in Figure 5A was resolved by SDS-PAGE. GST-H8, full length HDA8 fused to GST; GST alone was used as a control. GST-H8 is marked with an arrowhead. (D) ARID1 interacts with HDA8 by Co-IP. Inflorescences from ARID1-Myc; HDA8-YFP (abbreviated H8-YFP) doubly transgenic plants or from wild type were immunoprecipitated with Myc antibody. Myc, GFP and Hsc70 (loading control) antibodies were used for immunoblotting.

Mentions: A human ELM2 domain-containing protein, MI-ER, directly interacts with Histone Deacetylase 1 (HDAC1) [27], [28]. To determine whether ARID1 might be associated with plant histone deacetylase complexes, we performed yeast two hybrid experiments. We detected an interaction (Figure 5A) between ARID1 and HDA8 (Histone Deacetylase 8), which is highly expressed in the vegetative nucleus but not in the generative nucleus or sperm cell nuclei (Figure S6A). Moreover, we found that the ELM2 domain of ARID1 was important for the interaction with HDA8 (Figure 5A), as was shown for an ELM2 protein with a histone deacetylase in human cells [28]. To confirm the yeast results, a recombinant HDA8-GST protein (Figure 5C) was used for pulldown assays, with GST as a negative control. ARID1-GFP was pulled down by HDA8-GST but not by GST (Figure 5B). To confirm this association in vivo, we performed Co-IP experiments and showed that HDA8-YFP was co-immunoprecipitated with antibodies that recognize the ARID1-Myc fusion protein (Figure 5D).


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)

ARID1 physically associates with Histone Deacetylase 8 (HDA8).(A) Yeast cells co-expressing the indicated plasmids were grown on control (without Tryptophan and Leucine) or high stringency selection (without Tryptophan, Leucine, Histidine, and with 10 mM 3-AT) medium. ARID1, full length cDNA of ARID1; ARID1-C, the C-terminus-containing ELM2 domain of ARID1; HDA8, full length cDNA of HDA8. AD is pGAD10. The cultures from each of the indicated strains were diluted 100-fold and spotted. Three colonies were streaked for each pair. (B) ARID1 interacts with HDA8 by GST pulldown assay. Whole cell extracts from wild type or ARID1-GFP plants were applied onto GST and GST-HDA8 (abbreviated GST-H8) beads. GFP and Hsc70 (loading control) antibodies were used in immunoblotting. (C) Stained protein gel showing proteins used for the GST pulldown assay. 1/30 of the amount of each protein used in Figure 5A was resolved by SDS-PAGE. GST-H8, full length HDA8 fused to GST; GST alone was used as a control. GST-H8 is marked with an arrowhead. (D) ARID1 interacts with HDA8 by Co-IP. Inflorescences from ARID1-Myc; HDA8-YFP (abbreviated H8-YFP) doubly transgenic plants or from wild type were immunoprecipitated with Myc antibody. Myc, GFP and Hsc70 (loading control) antibodies were used for immunoblotting.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4109846&req=5

pgen-1004421-g005: ARID1 physically associates with Histone Deacetylase 8 (HDA8).(A) Yeast cells co-expressing the indicated plasmids were grown on control (without Tryptophan and Leucine) or high stringency selection (without Tryptophan, Leucine, Histidine, and with 10 mM 3-AT) medium. ARID1, full length cDNA of ARID1; ARID1-C, the C-terminus-containing ELM2 domain of ARID1; HDA8, full length cDNA of HDA8. AD is pGAD10. The cultures from each of the indicated strains were diluted 100-fold and spotted. Three colonies were streaked for each pair. (B) ARID1 interacts with HDA8 by GST pulldown assay. Whole cell extracts from wild type or ARID1-GFP plants were applied onto GST and GST-HDA8 (abbreviated GST-H8) beads. GFP and Hsc70 (loading control) antibodies were used in immunoblotting. (C) Stained protein gel showing proteins used for the GST pulldown assay. 1/30 of the amount of each protein used in Figure 5A was resolved by SDS-PAGE. GST-H8, full length HDA8 fused to GST; GST alone was used as a control. GST-H8 is marked with an arrowhead. (D) ARID1 interacts with HDA8 by Co-IP. Inflorescences from ARID1-Myc; HDA8-YFP (abbreviated H8-YFP) doubly transgenic plants or from wild type were immunoprecipitated with Myc antibody. Myc, GFP and Hsc70 (loading control) antibodies were used for immunoblotting.
Mentions: A human ELM2 domain-containing protein, MI-ER, directly interacts with Histone Deacetylase 1 (HDAC1) [27], [28]. To determine whether ARID1 might be associated with plant histone deacetylase complexes, we performed yeast two hybrid experiments. We detected an interaction (Figure 5A) between ARID1 and HDA8 (Histone Deacetylase 8), which is highly expressed in the vegetative nucleus but not in the generative nucleus or sperm cell nuclei (Figure S6A). Moreover, we found that the ELM2 domain of ARID1 was important for the interaction with HDA8 (Figure 5A), as was shown for an ELM2 protein with a histone deacetylase in human cells [28]. To confirm the yeast results, a recombinant HDA8-GST protein (Figure 5C) was used for pulldown assays, with GST as a negative control. ARID1-GFP was pulled down by HDA8-GST but not by GST (Figure 5B). To confirm this association in vivo, we performed Co-IP experiments and showed that HDA8-YFP was co-immunoprecipitated with antibodies that recognize the ARID1-Myc fusion protein (Figure 5D).

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
Related in: MedlinePlus