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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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Reduced DUO1 and CYCB1 expression in arid1-1.(A) Expression of sperm-specific genes in mature pollen of wild type and arid1-1. Error bars represent the SE from the mean of three biological replicates. (B) Expression of DUO1-RFP in wild type and arid1-1. Representative images for each genotype were acquired with the same exposure times. White and red arrows indicate reduced DUO1-RFP signal in bicellular pollen and mature pollen, respectively. Scale bar, 10 µm. (C) Expression of CYCB1-GFP in the bicellular pollen of wild type and arid1-1, respectively. The red arrows indicate visible GFP accumulation of CYCB1 in the generative nucleus of wild type pollen, and the white arrows indicate unchanged GFP signal in the vegetative nuclei of the arid1-1 pollen. Scale bar, 10 µm.
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pgen-1004421-g002: Reduced DUO1 and CYCB1 expression in arid1-1.(A) Expression of sperm-specific genes in mature pollen of wild type and arid1-1. Error bars represent the SE from the mean of three biological replicates. (B) Expression of DUO1-RFP in wild type and arid1-1. Representative images for each genotype were acquired with the same exposure times. White and red arrows indicate reduced DUO1-RFP signal in bicellular pollen and mature pollen, respectively. Scale bar, 10 µm. (C) Expression of CYCB1-GFP in the bicellular pollen of wild type and arid1-1, respectively. The red arrows indicate visible GFP accumulation of CYCB1 in the generative nucleus of wild type pollen, and the white arrows indicate unchanged GFP signal in the vegetative nuclei of the arid1-1 pollen. Scale bar, 10 µm.

Mentions: The single sperm-like phenotype of arid1-1 was similar to the phenotypes of mutants such as duo1[3], cdka1;1[4], fbl17[6], and duo3[7]. We therefore used qPCR to examine whether the expression of these genes was disturbed in arid1-1. Of these, only DUO1 mRNA levels were reduced in arid1-1 (Figure 2A), suggesting that ARID1 positively regulates DUO1 at the transcriptional level, either directly or indirectly. DUO1 expression was also reduced in the antisense ARID1 plants with reduced seed set (Figure S2A). We also crossed a DUO1-RFP reporter into arid1-1 and saw that the DUO1-RFP signal was slightly reduced, in both bicellular pollen (Figure 2B, upper panels, yellow arrows) and mature pollen (Figure 2B, lower panels, yellow arrows). Since DUO1 is one of the targets of miR159 [12], we examined MIR159 expression in the arid1-1 mutant by qPCR, but found no change in miR159 levels (Figure S2B). As we recently showed that Anaphase Promoting Complex 8 (APC8) is involved in CYCB1 regulation at both the transcriptional level and protein degradation level [8], we crossed APC8-YFP and CYCB1-GFP into arid1-1. CYCB1 is mainly present during early pollen developmental stages but not in mature pollen [8], [9]. Overall CYCB1 expression in arid1-1 was not altered, as assessed by qPCR (Figure 2B). However, the weak accumulation of CYCB1 in the generative cell of wild type bicellular pollen was undetectable in arid1-1 (Figure 2C, red arrows), although no apparent change of CYCB1 accumulation in the vegetative cell was seen in arid1-1 (Figure 2C, white arrows). However, the APC8-YFP level was not affected in arid1-1 (Figure S2C). Similarly, we detected no effect on the expression of other known genes implicated in sperm cell function (Figure S2D), including HTR10[2], GEX2[10] and GEX1[29]. Taken together, these results suggest that ARID1 might promote DUO1 expression directly, but independently, of miR159.


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)

Reduced DUO1 and CYCB1 expression in arid1-1.(A) Expression of sperm-specific genes in mature pollen of wild type and arid1-1. Error bars represent the SE from the mean of three biological replicates. (B) Expression of DUO1-RFP in wild type and arid1-1. Representative images for each genotype were acquired with the same exposure times. White and red arrows indicate reduced DUO1-RFP signal in bicellular pollen and mature pollen, respectively. Scale bar, 10 µm. (C) Expression of CYCB1-GFP in the bicellular pollen of wild type and arid1-1, respectively. The red arrows indicate visible GFP accumulation of CYCB1 in the generative nucleus of wild type pollen, and the white arrows indicate unchanged GFP signal in the vegetative nuclei of the arid1-1 pollen. Scale bar, 10 µm.
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Related In: Results  -  Collection

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pgen-1004421-g002: Reduced DUO1 and CYCB1 expression in arid1-1.(A) Expression of sperm-specific genes in mature pollen of wild type and arid1-1. Error bars represent the SE from the mean of three biological replicates. (B) Expression of DUO1-RFP in wild type and arid1-1. Representative images for each genotype were acquired with the same exposure times. White and red arrows indicate reduced DUO1-RFP signal in bicellular pollen and mature pollen, respectively. Scale bar, 10 µm. (C) Expression of CYCB1-GFP in the bicellular pollen of wild type and arid1-1, respectively. The red arrows indicate visible GFP accumulation of CYCB1 in the generative nucleus of wild type pollen, and the white arrows indicate unchanged GFP signal in the vegetative nuclei of the arid1-1 pollen. Scale bar, 10 µm.
Mentions: The single sperm-like phenotype of arid1-1 was similar to the phenotypes of mutants such as duo1[3], cdka1;1[4], fbl17[6], and duo3[7]. We therefore used qPCR to examine whether the expression of these genes was disturbed in arid1-1. Of these, only DUO1 mRNA levels were reduced in arid1-1 (Figure 2A), suggesting that ARID1 positively regulates DUO1 at the transcriptional level, either directly or indirectly. DUO1 expression was also reduced in the antisense ARID1 plants with reduced seed set (Figure S2A). We also crossed a DUO1-RFP reporter into arid1-1 and saw that the DUO1-RFP signal was slightly reduced, in both bicellular pollen (Figure 2B, upper panels, yellow arrows) and mature pollen (Figure 2B, lower panels, yellow arrows). Since DUO1 is one of the targets of miR159 [12], we examined MIR159 expression in the arid1-1 mutant by qPCR, but found no change in miR159 levels (Figure S2B). As we recently showed that Anaphase Promoting Complex 8 (APC8) is involved in CYCB1 regulation at both the transcriptional level and protein degradation level [8], we crossed APC8-YFP and CYCB1-GFP into arid1-1. CYCB1 is mainly present during early pollen developmental stages but not in mature pollen [8], [9]. Overall CYCB1 expression in arid1-1 was not altered, as assessed by qPCR (Figure 2B). However, the weak accumulation of CYCB1 in the generative cell of wild type bicellular pollen was undetectable in arid1-1 (Figure 2C, red arrows), although no apparent change of CYCB1 accumulation in the vegetative cell was seen in arid1-1 (Figure 2C, white arrows). However, the APC8-YFP level was not affected in arid1-1 (Figure S2C). Similarly, we detected no effect on the expression of other known genes implicated in sperm cell function (Figure S2D), including HTR10[2], GEX2[10] and GEX1[29]. Taken together, these results suggest that ARID1 might promote DUO1 expression directly, but independently, of miR159.

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