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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 binds to DUO1.(A) Schematic of subfragments of DUO1 genomic DNA. The black rectangles represent exons. The black triangle indicates the region where ARID1 was most abundant. The position of the ATG was set to 1, and the fragments upstream or downstream were numbered; for example fragment “1” is −1321 to −1019 bp. (B) ChIP performed with wild type (gray bars) or ARID1-GFP (black bars) with GFP antibody (upper panel) and No antibody control (lower panel). EIF4A1 was used an internal negative control. The results were reproducible in two biological replicates. Error bars show SD calculated from three technical replicates. (C) DNA binding assay. Proteins were resolved by SDS-PAGE and then immunoblotted using anti-ARID1. The input lanes have 1/5 of the amount of ARID1 protein used in the DNA binding assay. The lanes numbered 3, 4, 5 and 10 (corresponding to regions mentioned in (A)) have 1/6 of the DNA-bound protein.
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pgen-1004421-g003: ARID1 binds to DUO1.(A) Schematic of subfragments of DUO1 genomic DNA. The black rectangles represent exons. The black triangle indicates the region where ARID1 was most abundant. The position of the ATG was set to 1, and the fragments upstream or downstream were numbered; for example fragment “1” is −1321 to −1019 bp. (B) ChIP performed with wild type (gray bars) or ARID1-GFP (black bars) with GFP antibody (upper panel) and No antibody control (lower panel). EIF4A1 was used an internal negative control. The results were reproducible in two biological replicates. Error bars show SD calculated from three technical replicates. (C) DNA binding assay. Proteins were resolved by SDS-PAGE and then immunoblotted using anti-ARID1. The input lanes have 1/5 of the amount of ARID1 protein used in the DNA binding assay. The lanes numbered 3, 4, 5 and 10 (corresponding to regions mentioned in (A)) have 1/6 of the DNA-bound protein.

Mentions: To test how ARID1 affects DUO1 expression, we performed a ChIP assay to examine whether ARID1, as a transcription factor, binds DUO1 directly. We tested ARID1 occupancy at the genomic region of DUO1, including the ∼1.4 kb promoter region upstream of the ATG and the ∼300 bp UTR region downstream of the stop codon in ARID1-GFP transgenic plants (wild type plants as the negative control), using an antibody against GFP. We sub-divided the genomic region of DUO1 into ten subfragments of around 300 bp each (Figure 3A) and used EIF4A1 as a negative control. An obvious ARID1 occupancy over most of the DUO1 genomic region was detected in ARID1-GFP transgenic plants, with the peak of enrichment located between the ∼600–300 bp promoter region upstream of the ATG (Figure 3B, upper panel). In contrast, there was not much difference between wild type and ARID1-GFP plants for EIF4A1 enrichment (Figure 3B, upper panel), and enrichment from the no antibody control was negligible (Figure 3C, lower panel). To confirm the results from the ChIP assay, we performed an in vitro DNA binding assay. We expressed ARID1 with an in vitro transcription/translation system (Figure 3C, the band shown in the “input” lanes), and validated that ARID1 directly binds the DUO1 promoter region (Figure 3C). Therefore, both in vitro and in vivo evidence showed that ARID1 directly binds the DUO1 promoter. Together with the observation of reduced DUO1 expression in the arid1 mutant, we therefore conclude that ARID1 acts as an activator of DUO1, which is important for the initiation of the second mitosis for sperm cell formation.


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 binds to DUO1.(A) Schematic of subfragments of DUO1 genomic DNA. The black rectangles represent exons. The black triangle indicates the region where ARID1 was most abundant. The position of the ATG was set to 1, and the fragments upstream or downstream were numbered; for example fragment “1” is −1321 to −1019 bp. (B) ChIP performed with wild type (gray bars) or ARID1-GFP (black bars) with GFP antibody (upper panel) and No antibody control (lower panel). EIF4A1 was used an internal negative control. The results were reproducible in two biological replicates. Error bars show SD calculated from three technical replicates. (C) DNA binding assay. Proteins were resolved by SDS-PAGE and then immunoblotted using anti-ARID1. The input lanes have 1/5 of the amount of ARID1 protein used in the DNA binding assay. The lanes numbered 3, 4, 5 and 10 (corresponding to regions mentioned in (A)) have 1/6 of the DNA-bound protein.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004421-g003: ARID1 binds to DUO1.(A) Schematic of subfragments of DUO1 genomic DNA. The black rectangles represent exons. The black triangle indicates the region where ARID1 was most abundant. The position of the ATG was set to 1, and the fragments upstream or downstream were numbered; for example fragment “1” is −1321 to −1019 bp. (B) ChIP performed with wild type (gray bars) or ARID1-GFP (black bars) with GFP antibody (upper panel) and No antibody control (lower panel). EIF4A1 was used an internal negative control. The results were reproducible in two biological replicates. Error bars show SD calculated from three technical replicates. (C) DNA binding assay. Proteins were resolved by SDS-PAGE and then immunoblotted using anti-ARID1. The input lanes have 1/5 of the amount of ARID1 protein used in the DNA binding assay. The lanes numbered 3, 4, 5 and 10 (corresponding to regions mentioned in (A)) have 1/6 of the DNA-bound protein.
Mentions: To test how ARID1 affects DUO1 expression, we performed a ChIP assay to examine whether ARID1, as a transcription factor, binds DUO1 directly. We tested ARID1 occupancy at the genomic region of DUO1, including the ∼1.4 kb promoter region upstream of the ATG and the ∼300 bp UTR region downstream of the stop codon in ARID1-GFP transgenic plants (wild type plants as the negative control), using an antibody against GFP. We sub-divided the genomic region of DUO1 into ten subfragments of around 300 bp each (Figure 3A) and used EIF4A1 as a negative control. An obvious ARID1 occupancy over most of the DUO1 genomic region was detected in ARID1-GFP transgenic plants, with the peak of enrichment located between the ∼600–300 bp promoter region upstream of the ATG (Figure 3B, upper panel). In contrast, there was not much difference between wild type and ARID1-GFP plants for EIF4A1 enrichment (Figure 3B, upper panel), and enrichment from the no antibody control was negligible (Figure 3C, lower panel). To confirm the results from the ChIP assay, we performed an in vitro DNA binding assay. We expressed ARID1 with an in vitro transcription/translation system (Figure 3C, the band shown in the “input” lanes), and validated that ARID1 directly binds the DUO1 promoter region (Figure 3C). Therefore, both in vitro and in vivo evidence showed that ARID1 directly binds the DUO1 promoter. Together with the observation of reduced DUO1 expression in the arid1 mutant, we therefore conclude that ARID1 acts as an activator of DUO1, which is important for the initiation of the second mitosis for sperm cell formation.

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