Limits...
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.

Show MeSH
Specific expression of ARID1 in pollen and disruption of ARID1 results in defective sperm cell formation.(A) Expression of ARID1 by RT-PCR. Ro, roots; Se, seedlings; Le, leaves; Cf, closed flower buds; Of, open flowers; Pi, unpollinated pistils; Po, mature pollen; Si, siliques. The RT (−) control PCR was performed with UBQ5 primers. (B) Representative siliques of WT and arid1-1 and complementation test. Undeveloped ovules are indicated with arrows. (C) Percentage of normal seeds (dark grey), aborted seeds (lighter grey), and undeveloped ovules (lightest grey) from self-pollinated plants are shown. Error bars represent standard deviation from the mean. (D) Seed set analysis in antisense ARID1 transgenic plants. Numbers in the bottom row represent individual T1 lines, and the corresponding numbers in the top row indicate the percentage of reduced seed set in each line. The gel shows the expression of ARID1 as assessed by RT-PCR analysis. (E) Distribution of unicellular microspores (UM, lightest grey), bicellular pollen (BP, lighter grey), and tricellular pollen (TP, dark grey) in mature anthers. At least 600 pollen grains were stained with DAPI and used for statistical analysis; Error bars represent standard deviation from the mean.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109846&req=5

pgen-1004421-g001: Specific expression of ARID1 in pollen and disruption of ARID1 results in defective sperm cell formation.(A) Expression of ARID1 by RT-PCR. Ro, roots; Se, seedlings; Le, leaves; Cf, closed flower buds; Of, open flowers; Pi, unpollinated pistils; Po, mature pollen; Si, siliques. The RT (−) control PCR was performed with UBQ5 primers. (B) Representative siliques of WT and arid1-1 and complementation test. Undeveloped ovules are indicated with arrows. (C) Percentage of normal seeds (dark grey), aborted seeds (lighter grey), and undeveloped ovules (lightest grey) from self-pollinated plants are shown. Error bars represent standard deviation from the mean. (D) Seed set analysis in antisense ARID1 transgenic plants. Numbers in the bottom row represent individual T1 lines, and the corresponding numbers in the top row indicate the percentage of reduced seed set in each line. The gel shows the expression of ARID1 as assessed by RT-PCR analysis. (E) Distribution of unicellular microspores (UM, lightest grey), bicellular pollen (BP, lighter grey), and tricellular pollen (TP, dark grey) in mature anthers. At least 600 pollen grains were stained with DAPI and used for statistical analysis; Error bars represent standard deviation from the mean.

Mentions: Microarray data [25], [26] and our RT-PCR results (Figure 1A) showed that one ARID, At2g46040, here named ARID1, was expressed in a pollen-specific manner. In addition to the ARID domain, ARID1 contains an ELM2 domain at the C-terminus. In animals, ELM2 domains mediate histone modifications by interacting with histone deacetylase [27], [28]. The combination of ARID and ELM2 domains in a single protein is plant-specific. Given the importance of cell cycle regulation by ARID proteins in animals [19], and that a mouse mutant in a gene encoding an ARID protein is male infertile [15], we suspected that disrupting ARID1 function might lead to disorganized cell divisions during pollen development. The arid1-1 mutant has a T-DNA insertion in the only intron of ARID1 (Figure S1A). The insertion did not abolish expression, as a truncated transcript upstream of the inserted location was detected (Figure S1A), suggesting that arid1-1 might be a weak allele. Plants homozygous for arid1-1 had short siliques and reduced seed set (Figure 1B and 1C). We identified homozygous plants by genotyping a F2 population of arid1-1 backcrossed with wild type plants. As only 10 of the 96 F2 plants were homozygous, we hypothesized that there might be a transmission problem. Reciprocal crosses with wild type plants showed that transmission through the female was normal, but was perturbed through the male (Table 1). To investigate whether the arid1-1 phenotype was caused by the T-DNA insertion, we constructed two ARID1 transgenes by engineering GFP or RFP tags at the C-terminus to a genomic fragment of ARID1, driven by its native promoter. ARID1-GFP or ARID1-RFP completely complemented the reduced seed set phenotype (Figure 1B and 1C). Because arid1-1 appeared to be a weak allele, we explored whether a more complete loss of arid1 function would have similar or more severe phenotypes. We therefore generated a binary construct expressing antisense ARID1 under the control of the native ARID1 promoter. The seed set of 48 independent transgenic lines was examined: 42 plants showed reduced seed set, ranging from 15% to 95% (Figure 1D). Transcript analysis of representative antisense lines confirmed that the phenotype of reduced seed set correlated with reduced transcript levels of ARID1 (Figure 1D). Because immature seeds from antisense lines with severely reduced seed set finally shriveled, we performed further analyses using arid1-1.


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)

Specific expression of ARID1 in pollen and disruption of ARID1 results in defective sperm cell formation.(A) Expression of ARID1 by RT-PCR. Ro, roots; Se, seedlings; Le, leaves; Cf, closed flower buds; Of, open flowers; Pi, unpollinated pistils; Po, mature pollen; Si, siliques. The RT (−) control PCR was performed with UBQ5 primers. (B) Representative siliques of WT and arid1-1 and complementation test. Undeveloped ovules are indicated with arrows. (C) Percentage of normal seeds (dark grey), aborted seeds (lighter grey), and undeveloped ovules (lightest grey) from self-pollinated plants are shown. Error bars represent standard deviation from the mean. (D) Seed set analysis in antisense ARID1 transgenic plants. Numbers in the bottom row represent individual T1 lines, and the corresponding numbers in the top row indicate the percentage of reduced seed set in each line. The gel shows the expression of ARID1 as assessed by RT-PCR analysis. (E) Distribution of unicellular microspores (UM, lightest grey), bicellular pollen (BP, lighter grey), and tricellular pollen (TP, dark grey) in mature anthers. At least 600 pollen grains were stained with DAPI and used for statistical analysis; Error bars represent standard deviation from the mean.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004421-g001: Specific expression of ARID1 in pollen and disruption of ARID1 results in defective sperm cell formation.(A) Expression of ARID1 by RT-PCR. Ro, roots; Se, seedlings; Le, leaves; Cf, closed flower buds; Of, open flowers; Pi, unpollinated pistils; Po, mature pollen; Si, siliques. The RT (−) control PCR was performed with UBQ5 primers. (B) Representative siliques of WT and arid1-1 and complementation test. Undeveloped ovules are indicated with arrows. (C) Percentage of normal seeds (dark grey), aborted seeds (lighter grey), and undeveloped ovules (lightest grey) from self-pollinated plants are shown. Error bars represent standard deviation from the mean. (D) Seed set analysis in antisense ARID1 transgenic plants. Numbers in the bottom row represent individual T1 lines, and the corresponding numbers in the top row indicate the percentage of reduced seed set in each line. The gel shows the expression of ARID1 as assessed by RT-PCR analysis. (E) Distribution of unicellular microspores (UM, lightest grey), bicellular pollen (BP, lighter grey), and tricellular pollen (TP, dark grey) in mature anthers. At least 600 pollen grains were stained with DAPI and used for statistical analysis; Error bars represent standard deviation from the mean.
Mentions: Microarray data [25], [26] and our RT-PCR results (Figure 1A) showed that one ARID, At2g46040, here named ARID1, was expressed in a pollen-specific manner. In addition to the ARID domain, ARID1 contains an ELM2 domain at the C-terminus. In animals, ELM2 domains mediate histone modifications by interacting with histone deacetylase [27], [28]. The combination of ARID and ELM2 domains in a single protein is plant-specific. Given the importance of cell cycle regulation by ARID proteins in animals [19], and that a mouse mutant in a gene encoding an ARID protein is male infertile [15], we suspected that disrupting ARID1 function might lead to disorganized cell divisions during pollen development. The arid1-1 mutant has a T-DNA insertion in the only intron of ARID1 (Figure S1A). The insertion did not abolish expression, as a truncated transcript upstream of the inserted location was detected (Figure S1A), suggesting that arid1-1 might be a weak allele. Plants homozygous for arid1-1 had short siliques and reduced seed set (Figure 1B and 1C). We identified homozygous plants by genotyping a F2 population of arid1-1 backcrossed with wild type plants. As only 10 of the 96 F2 plants were homozygous, we hypothesized that there might be a transmission problem. Reciprocal crosses with wild type plants showed that transmission through the female was normal, but was perturbed through the male (Table 1). To investigate whether the arid1-1 phenotype was caused by the T-DNA insertion, we constructed two ARID1 transgenes by engineering GFP or RFP tags at the C-terminus to a genomic fragment of ARID1, driven by its native promoter. ARID1-GFP or ARID1-RFP completely complemented the reduced seed set phenotype (Figure 1B and 1C). Because arid1-1 appeared to be a weak allele, we explored whether a more complete loss of arid1 function would have similar or more severe phenotypes. We therefore generated a binary construct expressing antisense ARID1 under the control of the native ARID1 promoter. The seed set of 48 independent transgenic lines was examined: 42 plants showed reduced seed set, ranging from 15% to 95% (Figure 1D). Transcript analysis of representative antisense lines confirmed that the phenotype of reduced seed set correlated with reduced transcript levels of ARID1 (Figure 1D). Because immature seeds from antisense lines with severely reduced seed set finally shriveled, we performed further analyses using arid1-1.

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