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
ARID1 localization is developmentally dynamic during sperm cell formation.Representative images of unicellular microspores (UM) (A), bicellular pollen (BP) (B), tricellular pollen (TP) (C), and mature pollen (MP) (D) from ARID1-RFP transgenic plants. Yellow arrows indicate RFP signals in the generative nuclei and sperm cells. The percentages indicate incidence of each pattern. 600–800 pollen grains were analyzed for each stage. Scale bar, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004421-g004: ARID1 localization is developmentally dynamic during sperm cell formation.Representative images of unicellular microspores (UM) (A), bicellular pollen (BP) (B), tricellular pollen (TP) (C), and mature pollen (MP) (D) from ARID1-RFP transgenic plants. Yellow arrows indicate RFP signals in the generative nuclei and sperm cells. The percentages indicate incidence of each pattern. 600–800 pollen grains were analyzed for each stage. Scale bar, 10 µm.

Mentions: Considering that ARID1 can bind to the DUO1 promoter directly to promote DUO1 expression in both bicellular pollen and mature pollen (Figure 2, Figure 3, and Figure S2A), and DUO1 is specifically expressed from the early bicellular stage to mature pollen [3], we hypothesized that ARID1 might overlap with DUO1, in a cell type-specific pattern. We therefore examined the localization of ARID1 nuclear bodies at different developmental stages in ARID1-GFP and ARID1-RFP transgenic plants. To avoid interference due to the partial overlap of excitation wavelengths for DAPI and GFP, we mainly used ARID1-RFP transgenic plants for these observations. As in mature pollen (Figure S3B and S3C), the ARID1-RFP body was a single nuclear body in unicellular microspores (UM) (Figure 4A). Unexpectedly, increased numbers of nuclear bodies were observed in bicellular pollen (Figure S4). We categorized the ARID1-RFP nuclear bodies in bicellular pollen (BP) into four distinct patterns with similar incidences (Figure 4B): all foci within the vegetative nucleus; multiple foci in both the vegetative nucleus and the generative nucleus; several foci in the vegetative nucleus but a single dot in the generative nucleus; and much larger foci in the vegetative nucleus but a less intense single dot in the generative nucleus (yellow arrows indicate signals in the generative cell). Additional representative examples are shown in Figure S4C. Although the overall signal intensity in vegetative nuclei was much stronger than that in the generative cell, these images show that ARID1-RFP was in both the vegetative cell and generative cell at the bicellular stage, which is obviously different from the distribution in UM and MP. In TP, three patterns of ARID1-RFP bodies were identified (Figure 4C): most pollen (>60%) had a single ARID1-RFP body, as in mature pollen, but the rest had multiple foci in the vegetative nucleus or a single weakly fluorescent ARID1-RFP body in sperm nuclei (yellow arrows). Only a single nuclear body in the vegetative nucleus was observed in mature pollen (Figure S3B, S3C, and MP, Figure 4D). To further substantiate the biological significance of the presence of ARID1 in the generative cell, we constructed transgenic plants with ARID1 driven by LAT52 (LAT52:ARID1), a vegetative cell-specific promoter, and HTR10 (HTR10:ARID1), a generative cell and sperm cell-specific promoter, respectively. We observed reduced seed set (Figure S5A) and increased DUO1 expression (Figure S5B) in >10 independent T1 HTR10:ARID1 plants but not in LAT52:ARID1 plants, indicating that accumulation of ARID1 in the generative cell is biologically relevant during pollen development. Taken together, these data indicate that the subcellular distribution of ARID1-RFP nuclear bodies is variable during pollen development. The transient localization of ARID1-RFP in the generative nucleus is consistent with the idea that ARID1 might be required for initiation or progression of the second mitosis, by promoting DUO1 activation.


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 localization is developmentally dynamic during sperm cell formation.Representative images of unicellular microspores (UM) (A), bicellular pollen (BP) (B), tricellular pollen (TP) (C), and mature pollen (MP) (D) from ARID1-RFP transgenic plants. Yellow arrows indicate RFP signals in the generative nuclei and sperm cells. The percentages indicate incidence of each pattern. 600–800 pollen grains were analyzed for each stage. Scale bar, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004421-g004: ARID1 localization is developmentally dynamic during sperm cell formation.Representative images of unicellular microspores (UM) (A), bicellular pollen (BP) (B), tricellular pollen (TP) (C), and mature pollen (MP) (D) from ARID1-RFP transgenic plants. Yellow arrows indicate RFP signals in the generative nuclei and sperm cells. The percentages indicate incidence of each pattern. 600–800 pollen grains were analyzed for each stage. Scale bar, 10 µm.
Mentions: Considering that ARID1 can bind to the DUO1 promoter directly to promote DUO1 expression in both bicellular pollen and mature pollen (Figure 2, Figure 3, and Figure S2A), and DUO1 is specifically expressed from the early bicellular stage to mature pollen [3], we hypothesized that ARID1 might overlap with DUO1, in a cell type-specific pattern. We therefore examined the localization of ARID1 nuclear bodies at different developmental stages in ARID1-GFP and ARID1-RFP transgenic plants. To avoid interference due to the partial overlap of excitation wavelengths for DAPI and GFP, we mainly used ARID1-RFP transgenic plants for these observations. As in mature pollen (Figure S3B and S3C), the ARID1-RFP body was a single nuclear body in unicellular microspores (UM) (Figure 4A). Unexpectedly, increased numbers of nuclear bodies were observed in bicellular pollen (Figure S4). We categorized the ARID1-RFP nuclear bodies in bicellular pollen (BP) into four distinct patterns with similar incidences (Figure 4B): all foci within the vegetative nucleus; multiple foci in both the vegetative nucleus and the generative nucleus; several foci in the vegetative nucleus but a single dot in the generative nucleus; and much larger foci in the vegetative nucleus but a less intense single dot in the generative nucleus (yellow arrows indicate signals in the generative cell). Additional representative examples are shown in Figure S4C. Although the overall signal intensity in vegetative nuclei was much stronger than that in the generative cell, these images show that ARID1-RFP was in both the vegetative cell and generative cell at the bicellular stage, which is obviously different from the distribution in UM and MP. In TP, three patterns of ARID1-RFP bodies were identified (Figure 4C): most pollen (>60%) had a single ARID1-RFP body, as in mature pollen, but the rest had multiple foci in the vegetative nucleus or a single weakly fluorescent ARID1-RFP body in sperm nuclei (yellow arrows). Only a single nuclear body in the vegetative nucleus was observed in mature pollen (Figure S3B, S3C, and MP, Figure 4D). To further substantiate the biological significance of the presence of ARID1 in the generative cell, we constructed transgenic plants with ARID1 driven by LAT52 (LAT52:ARID1), a vegetative cell-specific promoter, and HTR10 (HTR10:ARID1), a generative cell and sperm cell-specific promoter, respectively. We observed reduced seed set (Figure S5A) and increased DUO1 expression (Figure S5B) in >10 independent T1 HTR10:ARID1 plants but not in LAT52:ARID1 plants, indicating that accumulation of ARID1 in the generative cell is biologically relevant during pollen development. Taken together, these data indicate that the subcellular distribution of ARID1-RFP nuclear bodies is variable during pollen development. The transient localization of ARID1-RFP in the generative nucleus is consistent with the idea that ARID1 might be required for initiation or progression of the second mitosis, by promoting DUO1 activation.

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