Molecular and Cellular Characterization of the Tomato Pollen Profilin, <i>LePro1</i>

<div><p>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 <i>LePro1</i> from tomato pollen. To understand its biological role, in the present study, we investigated the temporal and spatial expression of <i>LePro1</i> 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 <i>LePro1</i> in tomato plants using stable transformation, and obtained two antisense lines, A2 and A3 showing significant down-regulation of <i>LePro1</i> 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 <i>LePro1</i> 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 <i>LePro1</i> 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 <i>LePro1</i> 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.</p></div

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

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

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

<p><b>A</b>, 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 “<b>-</b>”. The sequence data were obtained from Genbank with accession numbers of U50195 for tomato pollen profilin <i>LePro1</i>, AJ417553 for tomato fruit profilin Lyc e1, AY061819 for another tomato fruit profilin, and X93466 for tobacco pollen profilin ntPro3. <b>B</b>, 3-D protein structures were analyzed using Swiss Model (<a href="http://www.swissmodel.expasy.org" target="_blank">www.swissmodel.expasy.org</a>). Ribbon structures of helices and β sheets are shown on the left panels. Additional loop structures are present in two pollen profilins, <i>LePro1</i> 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.</p

Genomic organization of <i>LePro1</i>.

<p>Panel <b>A</b>, DNA gel blot, showing single hybridization signal in each digestion by BamH1 (B1), EcoR1 (E1) and Hind III (H3). Panel <b>B</b>. genomic DNA fragment was amplified by polymerase chain reaction using 5′- and 3′- primers derived from the <i>LePro1</i> coding sequence. A ∼650 bp PCR product (P) was obtained. Left column in panel b shows DNA size markers (M). Panel <b>C</b>, top row shows chromosome location of <i>LePro1</i> based on the genome sequencing database of the Sol Genomics Network (<a href="http://solgenomics.net" target="_blank">http://solgenomics.net</a>). Bottom row shows the structure of <i>LePro1</i> 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.</p

Comparison of <i>in vivo</i> germination of wildtype and antisense pollen.

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

Comparison of seed-setting among <i>LePro1</i> sense, antisense and wild type plants Seeds were collected from mature fruits and counted.

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

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

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

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

<p>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. <b>A</b>, 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>B</b>, protein signal intensities obtained with a Bio-Rad Fluor-S MultiImager densitometer.</p

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

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

Identification and validation of single nucleotide polymorphic markers linked to Ug99 stem rust resistance in spring wheat

<div><p>Wheat stem rust (<i>Puccinia graminis</i> f. sp. <i>tritici</i> Eriks. and E. Henn.) is one of the most destructive diseases world-wide. Races belonging to Ug99 (or TTKSK) continue to cause crop losses in East Africa and threaten global wheat production. Developing and deploying wheat varieties with multiple race-specific genes or complex adult plant resistance is necessary to achieve durability. In the present study, we applied genome-wide association studies (GWAS) for identifying loci associated with the Ug99 stem rust resistance (SR) in a panel of wheat lines developed at the International Maize and Wheat Improvement Center (CIMMYT). Genotyping was carried out using the wheat 9K iSelect single nucleotide polymorphism (SNP) chip. Phenotyping was done in the field in Kenya by infection of <i>Puccinia graminis</i> f. sp. <i>tritici</i> race TTKST, the <i>Sr24</i>-virulent variant of Ug99. Marker-trait association identified 12 SNP markers significantly associated with resistance. Among them, 7 were mapped on five chromosomes. Markers located on chromosomes 4A and 4B overlapped with the location of the Ug99 resistance genes <i>SrND643</i> and <i>Sr37</i>, respectively. Markers identified on 7DL were collocated with <i>Sr25</i>. Additional significant markers were located in the regions where no <i>Sr</i> gene has been reported. The chromosome location for five of the SNP markers was unknown. A BLASTN search of the NCBI database using the flanking sequences of the SNPs associated with Ug99 resistance revealed that several markers were linked to plant disease resistance analogues, while others were linked to regulatory factors or metabolic enzymes. A KASP (Kompetitive Allele Specific PCR) assay was used for validating six marker loci linked to genes with resistance to Ug99. Of those, four co-segregated with the <i>Sr25</i>-pathotypes while the rest identified unknown resistance genes. With further investigation, these markers can be used for marker-assisted selection in breeding for Ug99 stem rust resistance in wheat.</p></div