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

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.

Fig. 1.

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.)

Fig. 2.

Phylogenetic tree of Picea wilsonii α-tubulin 1 (PwTUA1) and previously characterized α-tubulins. A Neighbor–Joining tree based on the deduced amino acid sequences of the TUAs. CrTUA was used as an outgroup. This bootstrap consensus tree was based on 1000 replicates. Numbers on nodes are bootstrap values. The accession numbers in GenBank and sources of the proteins are as follows: AtTUA1(At1g64740), AtTUA2 (At1g50010), AtTUA3 (At5g19770), AtTUA4 (At1g04820), AtTUA5 (At5g19780), and AtTUA6 (At4g14960) from Arabidopsis thaliana; CrTUA (AY182002) from Chlamydomonas reinhardtii; GhTUA1 (AY345603), GhTUA2(AY345604), GhTUA3 (AF106569), GhTUA4 (AY345605), and GhTUA5 (AF106571) from Gossypium hirsutum; HvTUA1 (X99623), HvTUA2 (Y08490), and HvTUA3 (AJ132399) from Hordeum vulgare; OsTUA1 (Os03g0726100), OsTUA2 (Os11g0247300), OsTUA3 (Os07g0574800), and OsTUA4 (Os03g0219300) from Oryza sativa; PmTUA1 (AY832610) from Pseudotsuga menziesii; and PtTUA1(AY229881), PtTUA2(AY229882), PtTUA3(EF583813), PtTUA4(EF584828), PtTUA5(EF583814), PtTUA6(EF583816), and PtTUA7(EF584829) from Populus tremuloides.

Fig. 3.

Expression of PwTUA1 in different tissues of Picea wilsonii. (A) Tissue-specific expression of PwTUA1 in P. wilsonii. Total RNA was isolated from needles (N), stems (S), roots (R), and pollen (P) (incubated for 0, 6, 12, 18, and 24 h). (B) Expression of PwTUA1 in pollen at different growth stages. (C) PwTUA1 gene expression induced by 0.1% (w/v) Ca²⁺ and 0.1% (w/v) H₃BO₃ at different time points.

Fig. 4.

Transient expression of PwTUA1 improves Picea wilsonii pollen germination and pollen tube growth. (A) The effect of PwTUA1 on pollen germination using microprojectile bombardment. After 12 h and 24 h of incubation, the percentage germination of pollen bombarded with the GFP-only construct showed no significant difference compared with the untreated control pollen (P <0.05). Significant differences were observed in the percentage of germination of pollen bombarded with 2.5 μg and 5 μg of the PwTUA1 gene construct compared with untreated pollen or GFP-transformed pollen (P <0.05). Error bars indicate averages from the number of pollen tubes measured (n≥81). (B) The effect of PwTUA1 on pollen tube length using microprojectile bombardment. After 12 h and 24 h of incubation, no significant differences were detected in the length of pollen tubes bombarded with GFP compared with untreated pollen tubes (P=0.47). Significant differences were seen in the length of pollen tubes bombarded with 5 μg of PwTUA1 compared with untreated pollen or GFP-transformed pollen (P <0.05). Error bars indicate averages from the number of pollen tubes measured (n≥65). (C) Microscopic analysis of pollen tube co-expression 24 h after gene transfer of different doses of PwTUA1 with GUS and GFP. Upper panel: fluorescence images. Lower panel: transmitted light images. The data were obtained from three independent experiments, and every condition was tested three times. Bars=100 μm.

Fig. 5.

Pollen tubes of wild-type (WT) and transgenic Arabidopsis cultured in different media. Pollen from wild-type Arabidopsis and transformants was germinated on medium containing 0.1% H₃BO₃, different concentrations of Ca²⁺, and 0.1 mM EGTA for 6 h and examined under a light microscope. Bars=50 μm.

Fig. 6.

Effects of PwTUA1 overexpression on plant pollen germination and pollen tube growth exposed to different concentrations of boron and Ca²⁺. (A) Comparison of tube growth of wild-type (WT) and transgenic plant pollen cultured in medium with 0.1% H₃BO₃ for 6 h. Only the pollen tubes of germinated pollen grains were measured. (B) Arabidopsis pollen was incubated in the germination medium with the indicated amounts of Ca²⁺. After 6 h of incubation, the percentage of germinating pollen grains was determined. To establish the pollen germination percentage, the percentage germination of pollen grains in at least 10 aliquots per treatment was determined and experiments were repeated three times. (C) Statistical analysis of pollen tube length after 6 h of incubation. Only the pollen tubes of germinated pollen grains were measured. Average lengths of >100 tubes from each line are shown. The experiments were repeated three times, and consistent results were obtained in all repeat experiments.

Fig. 7.

Subcellular localization of the α-tubulin protein in Arabidopsis pollen tubes. In vitro cultured pollen tubes were fixed, incubated with anti-α-tubulin antiserum, and detected with goat anti-mouse secondary antibody as described in the Materials and methods. The fluorescence of stained pollen tubes was observed under a confocal microscope. (A) Fluorescence from immunolabelling of Arabidopsis pollen tubes harvested at 1, 4, and 6 h after germination. WT indicates pollen of wild-type Arabidopsis; T indicates pollen of transgenic Arabidopsis. The transgenic tubes tip (arrows) were strongly labelled with antibody for α-tubulin, whereas wild-type pollen tubes showed a much weaker signal in the apical region (asterisks). These data were obtained from three independent experiments, and every condition was tested three times. Bars=20 μm. (B) Western blot of extracts (equal loading of samples) from Arabidopsis pollen tubes (lane 2) and P. wilsonii pollen tubes (lane 3) probed with anti-α-tubulin antibody. Lane 1 is the blank control.

Fig. 8.

Effects of PwTUA1 on the ultrastructure of pollen tubes in transgenic Arabidopsis. Pollen from wild-type and transgenic plants was cultured in vitro for 6 h. Bars=0.2 μm (A, B, E, F) or 0.5 μm (C, D). (A) Tip region of wild-type pollen tube showing organelles at the tip. (B) Tip region of a pollen tube expressing PwTUA1; MTs (arrowhead) appeared and the number of vacuoles and secretory vesicles increased. (C and D) Magnified images of boxed sections in A and B. (E) Pollen tube wall of wild-type Arabidopsis. (F) Pollen tube wall of transgenic Arabidopsis. For Arabidopsis expressing PwTUA1, analyses of two independent transgenic lines showed a similar ultrastructure of pollen tubes. Data from one transgenic line were used. CW, cell wall; Mi, mitochondria; D, dictyosome; SV, secretory vesicle; V, vacuole; L, lipid body.