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Suppression of ASKβ (AtSK32), a Clade III Arabidopsis GSK3, Leads to the Pollen Defect during Late Pollen Development.

Dong X, Nou IS, Yi H, Hur Y - Mol. Cells (2015)

Bottom Line: To dissect the function of ASKβ (AtSK32), ASKβ antisense transgenic plants were generated, revealing the effects of ASKβ down-regulation in Arabidopsis.The in silico analysis of promoter and the expression characteristics implicate ASKβ is associated with the expression of genes known to be involved in sperm cell differentiation.We speculate that ASKβ indirectly affects the transcription of its co-expressed genes through the phosphorylation of its target proteins during late pollen development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, College of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Korea.

ABSTRACT
Arabidopsis Shaggy-like protein kinases (ASKs) are Arabidopsis thaliana homologs of glycogen synthase kinase 3/SHAGGY-like kinases (GSK3/SGG), which are comprised of 10 genes with diverse functions. To dissect the function of ASKβ (AtSK32), ASKβ antisense transgenic plants were generated, revealing the effects of ASKβ down-regulation in Arabidopsis. Suppression of ASKβ expression specifically interfered with pollen development and fertility without altering the plants' vegetative phenotypes, which differed from the phenotypes reported for Arabidopsis plants defective in other ASK members. The strength of these phenotypes showed an inverse correlation with the expression levels of ASKβ and its co-expressed genes. In the aborted pollen of ASKβ antisense plants, loss of nuclei and shrunken cytoplasm began to appear at the bicellular stage of microgametogenesis. The in silico analysis of promoter and the expression characteristics implicate ASKβ is associated with the expression of genes known to be involved in sperm cell differentiation. We speculate that ASKβ indirectly affects the transcription of its co-expressed genes through the phosphorylation of its target proteins during late pollen development.

No MeSH data available.


Related in: MedlinePlus

Male gametophyte development in wild-type and ASKβ antisense transgenic lines. Male gametophytes were stained with modified Alexander’s stain (Peterson et al., 2010). Tetrad, uninucleate, bicellular, tricellular and mature indicate the different stages of pollen development. Bar = 20 μm.
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f4-molce-38-6-506: Male gametophyte development in wild-type and ASKβ antisense transgenic lines. Male gametophytes were stained with modified Alexander’s stain (Peterson et al., 2010). Tetrad, uninucleate, bicellular, tricellular and mature indicate the different stages of pollen development. Bar = 20 μm.

Mentions: Because the predominant expression of ASKβ has been observed in pollen (Tichtinsky et al., 1998; Wellmer et al., 2004), we suspected disrupted pollen development in the antisense transgenic plants resulted in the reduced fertility. To test this, we first investigated the morphology of mature pollen grains with a modified Alexander’s staining (Peterson et al., 2010). We found that significant amounts of pollen grains in the transgenic plants were aborted, even though overall anther morphology seemed to be normal (Supplementary Fig. S3A). The severity of the abortion phenotype showed a clear correlation with the strength of antisense effects on ASKβ expression (Fig. 3 and Supplementary Fig. S3A). As a first step in identifying the function of ASKβ in pollen development, the morphologies of developing male gametophytes at various developmental stages were systematically compared between wild-type and ASKβ antisense plants under light microscopy after Alexander staining (Fig. 4). Before tetrad stage, no differences were observed between wild-type and ASKβ antisense plants (data not show). As shown in Figure 4, the microspores were also very similar in the wild-type and transgenic plants at the tetrad and uninucleate stages. However, clear differences were detected after these stages. From the bicellular stage to the mature stage, wild-type microspores exhibited typical round, gradually enlarging forms. By contrast, some of the ASKβ microspores from the antisense transgenic lines displayed signs of cytosol shrinkage, which appeared to be responsible for the failure to develop intact pollen grains. The defects in ASKβ antisense pollen became more obvious at the tricellular stage. The loss of the inner contents of some mature pollen grains resulted in the production of smaller or shrunken pollen grains. To examine whether ASKβ knock-down also affects tapetal tissue development and differentiation and thereby indirectly causes defects in pollen development, semi-thin transverse sections were produced from anthers at stage 8 to 13 (Supplementary Fig. S4) (Sanders et al., 1999). Following the degradation of the callose wall, microspores are released at anther stage 8. From anther stage 9 to 12 (floral stage 10 to 12), microspores develop into pollen grains. Tapetum degradation occurs during stage 10 and 11, while pollen mitotic divisions are observed during anther stage 12, resulting in the formation of tricellular pollen grains. At stage 13 and 14, anther dehiscence and shrinkage of anther cells occurs, respectively (Ma, 2005). In ASKβ antisense transgenic plants producing defective pollen grains, tapetum differentiation seemed to be identical to that of wild-type plants. The normal anther morphology and tapetum differentiation observed in these lines (Supplementary Figs. S3A and S4) indicate that suppression of ASKβ levels specifically interferes with microspore development without affecting sporophytic tissues in the anther.


Suppression of ASKβ (AtSK32), a Clade III Arabidopsis GSK3, Leads to the Pollen Defect during Late Pollen Development.

Dong X, Nou IS, Yi H, Hur Y - Mol. Cells (2015)

Male gametophyte development in wild-type and ASKβ antisense transgenic lines. Male gametophytes were stained with modified Alexander’s stain (Peterson et al., 2010). Tetrad, uninucleate, bicellular, tricellular and mature indicate the different stages of pollen development. Bar = 20 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4469908&req=5

f4-molce-38-6-506: Male gametophyte development in wild-type and ASKβ antisense transgenic lines. Male gametophytes were stained with modified Alexander’s stain (Peterson et al., 2010). Tetrad, uninucleate, bicellular, tricellular and mature indicate the different stages of pollen development. Bar = 20 μm.
Mentions: Because the predominant expression of ASKβ has been observed in pollen (Tichtinsky et al., 1998; Wellmer et al., 2004), we suspected disrupted pollen development in the antisense transgenic plants resulted in the reduced fertility. To test this, we first investigated the morphology of mature pollen grains with a modified Alexander’s staining (Peterson et al., 2010). We found that significant amounts of pollen grains in the transgenic plants were aborted, even though overall anther morphology seemed to be normal (Supplementary Fig. S3A). The severity of the abortion phenotype showed a clear correlation with the strength of antisense effects on ASKβ expression (Fig. 3 and Supplementary Fig. S3A). As a first step in identifying the function of ASKβ in pollen development, the morphologies of developing male gametophytes at various developmental stages were systematically compared between wild-type and ASKβ antisense plants under light microscopy after Alexander staining (Fig. 4). Before tetrad stage, no differences were observed between wild-type and ASKβ antisense plants (data not show). As shown in Figure 4, the microspores were also very similar in the wild-type and transgenic plants at the tetrad and uninucleate stages. However, clear differences were detected after these stages. From the bicellular stage to the mature stage, wild-type microspores exhibited typical round, gradually enlarging forms. By contrast, some of the ASKβ microspores from the antisense transgenic lines displayed signs of cytosol shrinkage, which appeared to be responsible for the failure to develop intact pollen grains. The defects in ASKβ antisense pollen became more obvious at the tricellular stage. The loss of the inner contents of some mature pollen grains resulted in the production of smaller or shrunken pollen grains. To examine whether ASKβ knock-down also affects tapetal tissue development and differentiation and thereby indirectly causes defects in pollen development, semi-thin transverse sections were produced from anthers at stage 8 to 13 (Supplementary Fig. S4) (Sanders et al., 1999). Following the degradation of the callose wall, microspores are released at anther stage 8. From anther stage 9 to 12 (floral stage 10 to 12), microspores develop into pollen grains. Tapetum degradation occurs during stage 10 and 11, while pollen mitotic divisions are observed during anther stage 12, resulting in the formation of tricellular pollen grains. At stage 13 and 14, anther dehiscence and shrinkage of anther cells occurs, respectively (Ma, 2005). In ASKβ antisense transgenic plants producing defective pollen grains, tapetum differentiation seemed to be identical to that of wild-type plants. The normal anther morphology and tapetum differentiation observed in these lines (Supplementary Figs. S3A and S4) indicate that suppression of ASKβ levels specifically interferes with microspore development without affecting sporophytic tissues in the anther.

Bottom Line: To dissect the function of ASKβ (AtSK32), ASKβ antisense transgenic plants were generated, revealing the effects of ASKβ down-regulation in Arabidopsis.The in silico analysis of promoter and the expression characteristics implicate ASKβ is associated with the expression of genes known to be involved in sperm cell differentiation.We speculate that ASKβ indirectly affects the transcription of its co-expressed genes through the phosphorylation of its target proteins during late pollen development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, College of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Korea.

ABSTRACT
Arabidopsis Shaggy-like protein kinases (ASKs) are Arabidopsis thaliana homologs of glycogen synthase kinase 3/SHAGGY-like kinases (GSK3/SGG), which are comprised of 10 genes with diverse functions. To dissect the function of ASKβ (AtSK32), ASKβ antisense transgenic plants were generated, revealing the effects of ASKβ down-regulation in Arabidopsis. Suppression of ASKβ expression specifically interfered with pollen development and fertility without altering the plants' vegetative phenotypes, which differed from the phenotypes reported for Arabidopsis plants defective in other ASK members. The strength of these phenotypes showed an inverse correlation with the expression levels of ASKβ and its co-expressed genes. In the aborted pollen of ASKβ antisense plants, loss of nuclei and shrunken cytoplasm began to appear at the bicellular stage of microgametogenesis. The in silico analysis of promoter and the expression characteristics implicate ASKβ is associated with the expression of genes known to be involved in sperm cell differentiation. We speculate that ASKβ indirectly affects the transcription of its co-expressed genes through the phosphorylation of its target proteins during late pollen development.

No MeSH data available.


Related in: MedlinePlus