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Overexpression of PwTUA1, a pollen-specific tubulin gene, increases pollen tube elongation by altering the distribution of alpha-tubulin and promoting vesicle transport.

Yu Y, Li Y, Li L, Lin J, Zheng C, Zhang L - J. Exp. Bot. (2009)

Bottom Line: Here, a TUA1 gene from Picea wilsonii, which is specifically expressed in pollen, was isolated.Transient expression analysis in P. wilsonii pollen indicated that PwTUA1 improved pollen germination and pollen tube growth.The possible role of PwTUA1 in microtubule dynamics and organization was discussed.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian, Shandong 271018, PR China.

ABSTRACT
Tubulin genes are intimately associated with cell division and cell elongation, which are central to plant secondary cell wall development. However, their roles in pollen tube polar growth remain elusive. Here, a TUA1 gene from Picea wilsonii, which is specifically expressed in pollen, was isolated. Semi-quantitative RT-PCR analysis showed that the amount of PwTUA1 transcript varied at each stage of growth of the pollen tube and was induced by calcium ions and boron. Transient expression analysis in P. wilsonii pollen indicated that PwTUA1 improved pollen germination and pollen tube growth. The pollen of transgenic Arabidopsis overexpressing PwTUA1 also showed a higher percentage of germination and faster growth than wild-type plants not only in optimal germination medium, but also in medium supplemented with elevated levels of exogenous calcium ions or boron. Immunofluorescence and electron microscopy showed alpha-tubulin to be enriched and more vesicles accumulated in the apex region in germinating transgenic Arabidopsis pollen compared with wild-type plants. These results demonstrate that PwTUA1 up-regulated by calcium ions and boron contributes to pollen tube elongation by altering the distribution of alpha-tubulin and regulating the deposition of pollen cell wall components during the process of tube growth. The possible role of PwTUA1 in microtubule dynamics and organization was discussed.

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Alignment of the deduced PwTUA1 protein sequence (EU268195) with other plant TUA1s. Amino acid sequences used in the analysis are from Pseudotsuga menziesii (PmTUA1, AAV92379), Gossypium hirsutum (GhTUA1, AY345603), Oryza sativa (OsTUA1, Os03g0726100), and Arabidopsis thaliana (AtTUA1, At1g64740). Identical amino acid residues in this alignment are shaded in black. Possible phosphorylation sites were obtained with a PROSITE motif search: boxed amino acids are N-glycosylation sites, underlined amino acids are protein kinase C phosphorylation sites, amino acids underlined with dotted lines are casein kinase II phosphorylation sites, and the arrow indicates tubulin subunits α, β, and γ signatures. (This figure is available in colour at JXB online.)
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fig1: Alignment of the deduced PwTUA1 protein sequence (EU268195) with other plant TUA1s. Amino acid sequences used in the analysis are from Pseudotsuga menziesii (PmTUA1, AAV92379), Gossypium hirsutum (GhTUA1, AY345603), Oryza sativa (OsTUA1, Os03g0726100), and Arabidopsis thaliana (AtTUA1, At1g64740). Identical amino acid residues in this alignment are shaded in black. Possible phosphorylation sites were obtained with a PROSITE motif search: boxed amino acids are N-glycosylation sites, underlined amino acids are protein kinase C phosphorylation sites, amino acids underlined with dotted lines are casein kinase II phosphorylation sites, and the arrow indicates tubulin subunits α, β, and γ signatures. (This figure is available in colour at JXB online.)

Mentions: The TUA1 cDNA fragment was obtained from P. wilsonii by RT-PCR using primers designed according to conserved regions of known plant TUAs. After extension by 5′- and 3′-RACE-PCR, the complete open reading frame (ORF) was obtained. This gene was designated as PwTUA1 with the GenBank accession number EU268195. The full-length cDNA sequence was 1724 bp and contained an ORF of 1356 bp that tentatively encoded a PwTUA1 protein of 451 amino acids with a calculated mol. wt of 49.6 kDa and an isoelectric point (pI) of 4.97. A 207 bp 3′-UTR followed the stop codon (TGA) and a 161 bp 5′-UTR was upstream from the start codon (ATG). A BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) and multialignment analysis revealed that PwTUA1 was highly related to other plant TUAs, sharing homology of 88.47% with AtTUA1, 97.78% with GhTUA1, 96.9% with OsTUA1, and 99.78% with PmTUA1 (Fig. 1). The predicted protein sequence contained a GTP nucleotide-binding site (GGGTGSG), which was conserved and necessary for the polymerization of α-/β-tubulins (Kirschner and Mitchison, 1986). An MRECI domain, implicated in autoregulated post-transcriptional mechanisms (Yen et al., 1988a, b; Cleveland, 1989), was found at the N-terminus of PwTUA1. Phosphorylated residues were found at R79 and K336, and an acetylated residue at K40. PwTUA1 contained a C-terminal glutamic acid residue (referred to as an E-type), which was different from the more typical α-tubulins that contain a tyrosine at the C-terminus.


Overexpression of PwTUA1, a pollen-specific tubulin gene, increases pollen tube elongation by altering the distribution of alpha-tubulin and promoting vesicle transport.

Yu Y, Li Y, Li L, Lin J, Zheng C, Zhang L - J. Exp. Bot. (2009)

Alignment of the deduced PwTUA1 protein sequence (EU268195) with other plant TUA1s. Amino acid sequences used in the analysis are from Pseudotsuga menziesii (PmTUA1, AAV92379), Gossypium hirsutum (GhTUA1, AY345603), Oryza sativa (OsTUA1, Os03g0726100), and Arabidopsis thaliana (AtTUA1, At1g64740). Identical amino acid residues in this alignment are shaded in black. Possible phosphorylation sites were obtained with a PROSITE motif search: boxed amino acids are N-glycosylation sites, underlined amino acids are protein kinase C phosphorylation sites, amino acids underlined with dotted lines are casein kinase II phosphorylation sites, and the arrow indicates tubulin subunits α, β, and γ signatures. (This figure is available in colour at JXB online.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2692020&req=5

fig1: Alignment of the deduced PwTUA1 protein sequence (EU268195) with other plant TUA1s. Amino acid sequences used in the analysis are from Pseudotsuga menziesii (PmTUA1, AAV92379), Gossypium hirsutum (GhTUA1, AY345603), Oryza sativa (OsTUA1, Os03g0726100), and Arabidopsis thaliana (AtTUA1, At1g64740). Identical amino acid residues in this alignment are shaded in black. Possible phosphorylation sites were obtained with a PROSITE motif search: boxed amino acids are N-glycosylation sites, underlined amino acids are protein kinase C phosphorylation sites, amino acids underlined with dotted lines are casein kinase II phosphorylation sites, and the arrow indicates tubulin subunits α, β, and γ signatures. (This figure is available in colour at JXB online.)
Mentions: The TUA1 cDNA fragment was obtained from P. wilsonii by RT-PCR using primers designed according to conserved regions of known plant TUAs. After extension by 5′- and 3′-RACE-PCR, the complete open reading frame (ORF) was obtained. This gene was designated as PwTUA1 with the GenBank accession number EU268195. The full-length cDNA sequence was 1724 bp and contained an ORF of 1356 bp that tentatively encoded a PwTUA1 protein of 451 amino acids with a calculated mol. wt of 49.6 kDa and an isoelectric point (pI) of 4.97. A 207 bp 3′-UTR followed the stop codon (TGA) and a 161 bp 5′-UTR was upstream from the start codon (ATG). A BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) and multialignment analysis revealed that PwTUA1 was highly related to other plant TUAs, sharing homology of 88.47% with AtTUA1, 97.78% with GhTUA1, 96.9% with OsTUA1, and 99.78% with PmTUA1 (Fig. 1). The predicted protein sequence contained a GTP nucleotide-binding site (GGGTGSG), which was conserved and necessary for the polymerization of α-/β-tubulins (Kirschner and Mitchison, 1986). An MRECI domain, implicated in autoregulated post-transcriptional mechanisms (Yen et al., 1988a, b; Cleveland, 1989), was found at the N-terminus of PwTUA1. Phosphorylated residues were found at R79 and K336, and an acetylated residue at K40. PwTUA1 contained a C-terminal glutamic acid residue (referred to as an E-type), which was different from the more typical α-tubulins that contain a tyrosine at the C-terminus.

Bottom Line: Here, a TUA1 gene from Picea wilsonii, which is specifically expressed in pollen, was isolated.Transient expression analysis in P. wilsonii pollen indicated that PwTUA1 improved pollen germination and pollen tube growth.The possible role of PwTUA1 in microtubule dynamics and organization was discussed.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian, Shandong 271018, PR China.

ABSTRACT
Tubulin genes are intimately associated with cell division and cell elongation, which are central to plant secondary cell wall development. However, their roles in pollen tube polar growth remain elusive. Here, a TUA1 gene from Picea wilsonii, which is specifically expressed in pollen, was isolated. Semi-quantitative RT-PCR analysis showed that the amount of PwTUA1 transcript varied at each stage of growth of the pollen tube and was induced by calcium ions and boron. Transient expression analysis in P. wilsonii pollen indicated that PwTUA1 improved pollen germination and pollen tube growth. The pollen of transgenic Arabidopsis overexpressing PwTUA1 also showed a higher percentage of germination and faster growth than wild-type plants not only in optimal germination medium, but also in medium supplemented with elevated levels of exogenous calcium ions or boron. Immunofluorescence and electron microscopy showed alpha-tubulin to be enriched and more vesicles accumulated in the apex region in germinating transgenic Arabidopsis pollen compared with wild-type plants. These results demonstrate that PwTUA1 up-regulated by calcium ions and boron contributes to pollen tube elongation by altering the distribution of alpha-tubulin and regulating the deposition of pollen cell wall components during the process of tube growth. The possible role of PwTUA1 in microtubule dynamics and organization was discussed.

Show MeSH
Related in: MedlinePlus