Molecular and cellular characterization of the tomato pollen profilin, LePro1

Abstract

Profilin is an actin-binding protein involved in the dynamic turnover and restructuring of the actin cytoskeleton in all eukaryotic cells. We previously cloned a profilin gene, designated as LePro1 from tomato pollen. To understand its biological role, in the present study, we investigated the temporal and spatial expression of LePro1 during pollen development and found that the transcript was only detected at late stages during microsporogenesis and pollen maturation. Using antisense RNA, we successfully knocked down the expression of LePro1 in tomato plants using stable transformation, and obtained two antisense lines, A2 and A3 showing significant down-regulation of LePro1 in pollen resulting in poor pollen germination and abnormal pollen tube growth. A disorganized F-actin distribution was observed in the antisense pollen. Down-regulation of LePro1 also appeared to affect hydration of pollen deposited on the stigma and arrested pollen tube elongation in the style, thereby affecting fertilization. Our results suggest that LePro1 in conjunction with perhaps other cytoskeletal proteins, plays a regulatory role in the proper organization of F-actin in tomato pollen tubes through promoting actin assembly. Down-regulation of LePro1 leads to interruption of actin assembly and disorganization of the actin cytoskeleton thus arresting pollen tube growth. Based on the present and previous studies, it is likely that a single transcript of profilin gives rise to multiple forms displaying multifunctionality in tomato pollen.

Figure 1. Genomic organization of LePro1.

Panel A, DNA gel blot, showing single hybridization signal in each digestion by BamH1 (B1), EcoR1 (E1) and Hind III (H3). Panel B. genomic DNA fragment was amplified by polymerase chain reaction using 5′- and 3′- primers derived from the LePro1 coding sequence. A ∼650 bp PCR product (P) was obtained. Left column in panel b shows DNA size markers (M). Panel C, top row shows chromosome location of LePro1 based on the genome sequencing database of the Sol Genomics Network (http://solgenomics.net). Bottom row shows the structure of LePro1 containing 3 exons and 2 introns with 648 bp in length from the start codon (ATG) to the stop codon (TAA). A TATA box was found at 200 bp upstream to the start codon.

Figure 2. RNA gel blot and in situ hybridization of LePro1 in developing tomato flowers.

The panel on the tope left shows RNA gel blot analysis with hybridization signals on the top and loading control on the bottom. A, B, C, D and E represent anther sections from 3, 6, 9, 12 and 15 mm length flower buds, respectively. F, G, H, I and J are images showing in situ hybridization of DIG- labeled antisense LePro1 to tomato anther sections from flower buds at the same developmental stages as shown in A, B, C, D and E, respectively. K shows germinated pollen hybridized with antisense LePro1 probe, and L represents germinated pollen hybridized with sense LePro1 probe as control (Scale bar = 40 µm in F–J and 20 µm in K and L).

Figure 3. RNA gel blot analysis of the Lepro1 transcript in pollen grains of wildtype (C) and antisense plants (A1–A3), probed with ³²P-labeled LePro1 cDNA.

The bottom row shows ribosome RNA (rRNA) stained with ethidium bromide as loading control.

Figure 4. Immunoblot analysis of total soluble proteins extracted from pollen grains of wildtype (C), antisense (A1–A3) and sense (S1–5) plants.

Ten micrograms of proteins per sample were loaded in each well. A mixture of two antibodies, monoclonal anti-actin antibody 3H11 and polyclonal anti-profilin antibody Tp1 were used. A, actin (43 kD) and profilin (14 kD) were detected within the same blot. The upper bands represent actin signals and the lower bands represent profilin signals. The left column shows molecular weight standards (M) in kD. B, protein signal intensities obtained with a Bio-Rad Fluor-S MultiImager densitometer.

Figure 5. Ambient temperature SEM and low temperature SEM images of pollen in wildtype (panel C) and antisense (panels A2 & A3) plants.

A–C, ambient temperature low magnification of images of dehydrated pollen grains showing no significant difference in size or morphology (Scale bar = 9 µm). D–F, ambient temperature higher magnification images of the dehydrated pollen grains from all three lines showing no significant difference in the exine microstructure (Scale bar = 3 µm). G–I, low temperature SEM of selfed pollen, 2 hours after pollination. G, most wildtype pollen grains deposited on stigma are fully or partially hydrated; H and I, most pollen grains from antisense plants are not hydrated. G, arrow indicates pollen in hydrated condition or H and I, in non-hydrated condition. G, asterisk indicates stigmatic cell, and+indicates emerging pollen tube (Scale bar = 23 µm in G, and 30 µm in H and I).

Figure 6. Images of in vitro germinated wildtype and antisense pollen.

A, a high rate of pollen germination occurs in wildtype plants. B, a very low rate of pollen germination occurs in A3 line A few germinated pollen have short or abnormal pollen tubes (Scale bar = 100 µm). C, in vitro germination rate of different pollen type from antisense (A1–A3), sense (S1–S5) and wildtype plants (C). Error bars represent standard errors of 3 replications (p<0.001).

Figure 7. Comparison of in vivo germination of wildtype and antisense pollen.

A, wildtype plants pollen showing normal pollen growth down through the style (Scale bar = 300 µm). B, low pollen germination and growth are seen when A3 pollen is deposited on the A3 stigma (selfing) (Scale bar = 80 µm). C, low pollen growth seen when A3 pollen is deposited on the wildtype plants stigma (crossing) (Scale bar = 160 µm).

Figure 8. Comparison of seed-setting among LePro1 sense, antisense and wild type plants Seeds were collected from mature fruits and counted.

The number of seeds per cm fruit diameter was calculated (A). Error bars represent standard errors of 3 replications (p<0.001). B, shows examples of fruit sections from wild type (C), antisense 3 (A3) and sense 5 (S5) plants.

Figure 9. Confocal microscopy images of F-actin in germinated pollen in the wildtype and antisense line.

Pollen and pollen tubes were stained with rhodamine phalloidin. A normal F-actin strand distribution was seen in wildtype pollen tubes (A, B and C). F-actin appears disorganized and pollen tube growth arrested in antisense pollen (D, E and F). Scale bars are: 10 µm in A, B, C, D, F, and 5 µm in E.

Figure 10. Quantification of total actin and F-actin in antisense (A2) and wildtype (WT) pollen.

A, total actin quantified using anti-actin antibody followed by ELISA at the time course of 0, 2 and 4 hour germination. B, F-actin quantified using rhodamine phalloidin according to Gibbon and Staiger (2000). Error bars represent standard errors of 4 replications (p<0.005).

Figure 11. Comparison of protein sequences and 3-D structures of three tomato profilins and a tobacco pollen profilin.

A, amino acid sequence alignment of three tomato profilins with tobacco pollen profilin, ntPro3. The consensus sequence is indicated by star “*”, non-consensus sequence by colon “:” or dot “.” and missing sequence is indicated by “-”. The sequence data were obtained from Genbank with accession numbers of U50195 for tomato pollen profilin LePro1, AJ417553 for tomato fruit profilin Lyc e1, AY061819 for another tomato fruit profilin, and X93466 for tobacco pollen profilin ntPro3. B, 3-D protein structures were analyzed using Swiss Model (www.swissmodel.expasy.org). Ribbon structures of helices and β sheets are shown on the left panels. Additional loop structures are present in two pollen profilins, LePro1 and ntPro1 (Red arrow). Additional strand for β sheet are present in the tomato profilins (Blue arrow). The plots of the predicted B-factor for each residue are present on the right panel.