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OsABCG15 encodes a membrane protein that plays an important role in anther cuticle and pollen exine formation in rice.

Wu L, Guan Y, Wu Z, Yang K, Lv J, Converse R, Huang Y, Mao J, Zhao Y, Wang Z, Min H, Kan D, Zhang Y - Plant Cell Rep. (2014)

Bottom Line: Using map-based cloning, we found a spontaneous A-to-C transition in the fourth exon of OsABCG15 that caused an amino acid substitution of Thr-to-Pro in the predicted ATP-binding domain of the protein sequence.Our results suggested that OsABCG15 played an essential role in the formation of the rice anther cuticle and pollen exine.This role may include the secretion of the lipid precursors from the tapetum to facilitate the transfer of precursors to the surface of the anther epidermis as well as to microspores.

View Article: PubMed Central - PubMed

Affiliation: College of Agronomy and Biotechnology, Southwest University, Chongqing, 400715, China.

ABSTRACT

Key message: An ABC transporter gene ( OsABCG15 ) was proven to be involved in pollen development in rice. The corresponding protein was localized on the plasma membrane using subcellular localization. Wax, cutin, and sporopollenin are important for normal development of the anther cuticle and pollen exine, respectively. Their lipid soluble precursors, which are produced in the tapetum, are then secreted and transferred to the anther and microspore surface for polymerization. However, little is known about the mechanisms underlying the transport of these precursors. Here, we identified and characterized a member of the G subfamily of ATP-binding cassette (ABC) transporters, OsABCG15, which is required for the secretion of these lipid-soluble precursors in rice. Using map-based cloning, we found a spontaneous A-to-C transition in the fourth exon of OsABCG15 that caused an amino acid substitution of Thr-to-Pro in the predicted ATP-binding domain of the protein sequence. This osabcg15 mutant failed to produce any viable pollen and was completely male sterile. Histological analysis indicated that osabcg15 exhibited an undeveloped anther cuticle, enlarged middle layer, abnormal Ubisch body development, tapetum degeneration with a falling apart style, and collapsed pollen grains without detectable exine. OsABCG15 was expressed preferentially in the tapetum, and the fused GFP-OsABCG15 protein was localized to the plasma membrane. Our results suggested that OsABCG15 played an essential role in the formation of the rice anther cuticle and pollen exine. This role may include the secretion of the lipid precursors from the tapetum to facilitate the transfer of precursors to the surface of the anther epidermis as well as to microspores.

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Subcellular localization of the GFP-OsABCG15 fusion protein in Nicotiana benthamiana epidermal cells. a Transient expression of the GFP-OsABCG15 fusion protein, showing localization of the GFP-OsABCG15 protein on the plasma membrane. b Transient expression of the 35S:GFP control protein, showing that the expression of the GFP protein was distributed throughout the cell, including the cytoplasm and nucleus. c GFP-OsABCG15 imaging. d Fluorescent signal image of a FM4-64 stain. e Merged image of c and d. Scale bars 100 μm (a, b) and 10 μm (c–e)
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Fig7: Subcellular localization of the GFP-OsABCG15 fusion protein in Nicotiana benthamiana epidermal cells. a Transient expression of the GFP-OsABCG15 fusion protein, showing localization of the GFP-OsABCG15 protein on the plasma membrane. b Transient expression of the 35S:GFP control protein, showing that the expression of the GFP protein was distributed throughout the cell, including the cytoplasm and nucleus. c GFP-OsABCG15 imaging. d Fluorescent signal image of a FM4-64 stain. e Merged image of c and d. Scale bars 100 μm (a, b) and 10 μm (c–e)

Mentions: AtABCG26/WBC27, an Arabidopsisthaliana homolog of OsABCG15, was located in a separate dicot sub-clade of the phylogeny. Recent reports indicate that AtABCG26/WBC27 plays an important role in exporting sporopollenin precursors from tapetal cells and is a plasma membrane protein (Quilichini et al. 2010; Dou et al. 2011; Choi et al. 2011). In light of the homology of these two proteins and the similar pollen development defects in atabcg26/wbc27 and osabcg15 mutants, we predicted that OsABCG15 is also localized to the plasma membrane. To confirm this, we fused the full-length OsABCG15 coding region with GFP using a GFP-OsABCG15 pattern to screen recombinants. The fused construct and the GFP-only control, both driven by the cauliflower mosaic virus 35S promoter, were introduced into Nicotiana benthamiana epidermal cells by Agrobacterium infiltration (Voinnet et al. 2003). As expected, the GFP-OsABCG15 fusion protein was observed exclusively on the plasma membrane (Fig. 7a, c). In contrast, the free GFP signal occurred throughout the cell (Fig. 7b). Moreover, the green fluorescence, observed on the periphery of the GFP-OsABCG15 transformed cells, was similar to a membrane-specific FM4-64 stain signal (Bolte et al. 2004), and the two signals overlapped on the plasma membrane after merging the photomicrographs from each gene product (Fig. 7c–e). These observations support the prediction that OsABCG15 is localized to the plasma membrane and are consistent with the presumed function of exporting lipid-soluble precursors across the tapetal plasma membrane, resulting in the subcellular location observed in AtABCG26/WBC27.Fig. 7


OsABCG15 encodes a membrane protein that plays an important role in anther cuticle and pollen exine formation in rice.

Wu L, Guan Y, Wu Z, Yang K, Lv J, Converse R, Huang Y, Mao J, Zhao Y, Wang Z, Min H, Kan D, Zhang Y - Plant Cell Rep. (2014)

Subcellular localization of the GFP-OsABCG15 fusion protein in Nicotiana benthamiana epidermal cells. a Transient expression of the GFP-OsABCG15 fusion protein, showing localization of the GFP-OsABCG15 protein on the plasma membrane. b Transient expression of the 35S:GFP control protein, showing that the expression of the GFP protein was distributed throughout the cell, including the cytoplasm and nucleus. c GFP-OsABCG15 imaging. d Fluorescent signal image of a FM4-64 stain. e Merged image of c and d. Scale bars 100 μm (a, b) and 10 μm (c–e)
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Fig7: Subcellular localization of the GFP-OsABCG15 fusion protein in Nicotiana benthamiana epidermal cells. a Transient expression of the GFP-OsABCG15 fusion protein, showing localization of the GFP-OsABCG15 protein on the plasma membrane. b Transient expression of the 35S:GFP control protein, showing that the expression of the GFP protein was distributed throughout the cell, including the cytoplasm and nucleus. c GFP-OsABCG15 imaging. d Fluorescent signal image of a FM4-64 stain. e Merged image of c and d. Scale bars 100 μm (a, b) and 10 μm (c–e)
Mentions: AtABCG26/WBC27, an Arabidopsisthaliana homolog of OsABCG15, was located in a separate dicot sub-clade of the phylogeny. Recent reports indicate that AtABCG26/WBC27 plays an important role in exporting sporopollenin precursors from tapetal cells and is a plasma membrane protein (Quilichini et al. 2010; Dou et al. 2011; Choi et al. 2011). In light of the homology of these two proteins and the similar pollen development defects in atabcg26/wbc27 and osabcg15 mutants, we predicted that OsABCG15 is also localized to the plasma membrane. To confirm this, we fused the full-length OsABCG15 coding region with GFP using a GFP-OsABCG15 pattern to screen recombinants. The fused construct and the GFP-only control, both driven by the cauliflower mosaic virus 35S promoter, were introduced into Nicotiana benthamiana epidermal cells by Agrobacterium infiltration (Voinnet et al. 2003). As expected, the GFP-OsABCG15 fusion protein was observed exclusively on the plasma membrane (Fig. 7a, c). In contrast, the free GFP signal occurred throughout the cell (Fig. 7b). Moreover, the green fluorescence, observed on the periphery of the GFP-OsABCG15 transformed cells, was similar to a membrane-specific FM4-64 stain signal (Bolte et al. 2004), and the two signals overlapped on the plasma membrane after merging the photomicrographs from each gene product (Fig. 7c–e). These observations support the prediction that OsABCG15 is localized to the plasma membrane and are consistent with the presumed function of exporting lipid-soluble precursors across the tapetal plasma membrane, resulting in the subcellular location observed in AtABCG26/WBC27.Fig. 7

Bottom Line: Using map-based cloning, we found a spontaneous A-to-C transition in the fourth exon of OsABCG15 that caused an amino acid substitution of Thr-to-Pro in the predicted ATP-binding domain of the protein sequence.Our results suggested that OsABCG15 played an essential role in the formation of the rice anther cuticle and pollen exine.This role may include the secretion of the lipid precursors from the tapetum to facilitate the transfer of precursors to the surface of the anther epidermis as well as to microspores.

View Article: PubMed Central - PubMed

Affiliation: College of Agronomy and Biotechnology, Southwest University, Chongqing, 400715, China.

ABSTRACT

Key message: An ABC transporter gene ( OsABCG15 ) was proven to be involved in pollen development in rice. The corresponding protein was localized on the plasma membrane using subcellular localization. Wax, cutin, and sporopollenin are important for normal development of the anther cuticle and pollen exine, respectively. Their lipid soluble precursors, which are produced in the tapetum, are then secreted and transferred to the anther and microspore surface for polymerization. However, little is known about the mechanisms underlying the transport of these precursors. Here, we identified and characterized a member of the G subfamily of ATP-binding cassette (ABC) transporters, OsABCG15, which is required for the secretion of these lipid-soluble precursors in rice. Using map-based cloning, we found a spontaneous A-to-C transition in the fourth exon of OsABCG15 that caused an amino acid substitution of Thr-to-Pro in the predicted ATP-binding domain of the protein sequence. This osabcg15 mutant failed to produce any viable pollen and was completely male sterile. Histological analysis indicated that osabcg15 exhibited an undeveloped anther cuticle, enlarged middle layer, abnormal Ubisch body development, tapetum degeneration with a falling apart style, and collapsed pollen grains without detectable exine. OsABCG15 was expressed preferentially in the tapetum, and the fused GFP-OsABCG15 protein was localized to the plasma membrane. Our results suggested that OsABCG15 played an essential role in the formation of the rice anther cuticle and pollen exine. This role may include the secretion of the lipid precursors from the tapetum to facilitate the transfer of precursors to the surface of the anther epidermis as well as to microspores.

Show MeSH
Related in: MedlinePlus