A pollen-specific RALF from tomato that regulates pollen tube elongation

Abstract

Rapid Alkalinization Factors (RALFs) are plant peptides that rapidly increase the pH of plant suspension cell culture medium and inhibit root growth. A pollen-specific tomato (Solanum lycopersicum) RALF (SlPRALF) has been identified. The SlPRALF gene encodes a preproprotein that appears to be processed and released from the pollen tube as an active peptide. A synthetic SlPRALF peptide based on the putative active peptide did not affect pollen hydration or viability but inhibited the elongation of normal pollen tubes in an in vitro growth system. Inhibitory effects of SlPRALF were detectable at concentrations as low as 10 nm, and complete inhibition was observed at 1 mum peptide. At least 10-fold higher levels of alkSlPRALF, which lacks disulfide bonds, were required to see similar effects. A greater effect of peptide was observed in low-pH-buffered medium. Inhibition of pollen tube elongation was reversible if peptide was removed within 15 min of exposure. Addition of 100 nm SlPRALF to actively growing pollen tubes inhibited further elongation until tubes were 40 to 60 mum in length, after which pollen tubes became resistant to the peptide. The onset of resistance correlated with the timing of the exit of the male germ unit from the pollen grain into the tube. Thus, exogenous SlPRALF acts as a negative regulator of pollen tube elongation within a specific developmental window.

Figure 1.

Amino acid alignment of SlRALF with the pollen-expressed SlPRALF, PhanthRALF, and AtRALFL4. The underlined section of the alignment corresponds to the predicted signal peptide sequences, the dibasic site within the proregion is identified with an arrow, and the conserved Cys residues are indicated with asterisks. Identical residues are boxed in black, and similar residues are shaded gray. The predicted amino acid sequence encoded by the Pol2 cDNA differs from SlPRALF at a single Arg residue in the mature peptide (underlined and indicated by the caret), which in Pol2 is a His. Vegetatively expressed SlRALF corresponds to the translated tomato unigene SGN-U316452, SlPRALF corresponds to the translated pollen-expressed tomato unigene SGN-U324197, PhanthRALF corresponds to the translated P. hybrida anther-expressed unigene SGN-U207478, and AtRALFL4 corresponds to the translated Arabidopsis pollen-expressed At1g28270 gene.

Figure 2.

RNA gel-blot analysis of SlPRALF expression. The top panel shows the hybridization pattern of the Pol2 probe to RNA from designated tissues, and the bottom panel shows ethidium bromide-stained ribosomal RNA bands. DAP, Days after pollination.

Figure 3.

Detection of SlPRALF in pollen and medium. A, Immunolocalization of SlPRALF in tomato pollen tubes. Tomato pollen was germinated for 1 h, fixed and incubated with polyclonal antibodies raised against recombinant SlPRALF, and detected using TRITC-conjugated secondary antibodies (top) or preimmune serum detected using FITC-conjugated secondary antibodies (bottom). Pollen tubes were imaged both in bright-field and fluorescent modes with a confocal microscope. Fluorescent images of representative pollen tubes are presented on the left, bright-field images in the middle, and an overlay of both on the right. Intense signal in the exine is due to autofluorescence. B, Processed SlPRALF is secreted into the medium. The immunoblot was probed by anti-SlPRALF antibody. Lane 1, 20 pmol of synthetic active SlPRALF peptide; lane 2, 70 μg of total protein extracted from germinated pollen; lane 3, 25 μg of proteins secreted into PGM; lane 4, 25 pmol of recombinant preproSlPRALF expressed in Escherichia coli.

Figure 4.

Suspension cell culture alkalinization assay. Change in pH was measured in cell suspension culture 15 min after exposure to unaltered or alkalinized vegetative (SlRALF and alkSlRALF) and pollen (SlPRALF and alkSlPRALF) RALF peptides at 0.25 nm (white bars), 2.5 nm (light gray bars), 25 nm (dark gray bars), and 250 nm (black bars). A, S. peruvianum suspension cell culture assay. B, Arabidopsis suspension cell culture assay.

Figure 5.

Pollen viability assessed by FDA staining. A, Histogram of pollen fluorescence, with white bars representing positive FDA-stained fluorescent (live) pollen and gray bars representing nonfluorescent (dead) pollen from nontreated (no SlPRALF), treated (1 μm SlPRALF), and heat-killed pollen. FDA staining was done 1 h after the start of pollen to germination, ±sd, n ≥ 100 pollen grains. B, Micrograph of stained untreated pollen. C, Micrograph of stained SlPRALF-treated pollen.

Figure 6.

Effects of SlPRALF and alkSlPRALF on pollen tube germination and growth. Pollen was exposed to exogenous concentrations of SlPRALF peptide ranging from 0.005 to 1 μm. Peptide effects were classified according to pollen morphology. A, Micrograph of TGR class. B, Micrograph of TLR class. The arrow indicates the glebula. C, Micrograph of NGP class. D, Micrograph of BP class. Bars = 25 μm. E, The average percentage of hydrated pollen in each classification group, ±se, for pollen treated with each concentration of SlPRALF peptide for 1 h. F, The average percentage of hydrated pollen in each classification group, ±se, for pollen treated with SlPRALF peptide for 3 h. G, The average percentage of hydrated pollen in each classification group, ±se, for pollen treated with alkSlPRALF for 1 h. H, The average percentage of hydrated pollen in each classification group, ±se, for pollen treated with alkSlPRALF peptide for 3 h. n ≥ 100 for each treatment.

Figure 7.

Effects of peptide on pollen tube growth at different pH environments. Tomato pollen was germinated for 1 h in PGM-MES medium buffered to pH 5.5, 6, 6.5, or 7 containing either no peptide (white bars) or 0.05 μm SlPRALF (gray bars). Average number of normal pollen grains with tubes greater than the radius of the grain is shown as the percentage of normal pollen tubes ± se. n ≥ 100 for each pH.

Figure 8.

Reversibility of SlPRALF inhibition. A, Histogram of percentage of hydrated pollen with TGR ± se 1 h after time-limited treatment with 0.1 μm SlPRALF. Pollen was treated with peptide for designated exposure times, pelleted by centrifugation, and resuspended in medium containing 0.1 μm SlPRALF (constant SlPRALF, spin = black bars) or no peptide (SlPRALF removed, spin = dark gray bars). Samples not treated with peptide were analyzed without centrifugation (no SlPRALF, no spin = white bars) or with centrifugation (no SlPRALF, spin = light gray bars). n ≥ 100. B, Micrograph of pollen in peptide-removed PGM 1 h after 15 min of SlPRALF treatment. C, Micrograph of pollen in peptide-removed PGM 1 h after 20 min of SlPRALF treatment.

Figure 9.

SlPRALF effects on elongating pollen tubes. Histograms show pollen tube lengths of pollen tubes exposed to 0.1 μm SlPRALF at 40 (A), 50 (B), 60 (C), or 90 (D) min after addition of pollen to PGM. Pollen tube lengths were measured at the time of SlPRALF addition (green bars) and again 2 h later for nontreated (magenta bars) and 0.1 μm SlPRALF-treated (black bars) samples. Pollen tube lengths are grouped into 10-μm bin increments and represented as frequency of pollen tube lengths. The average pollen tube length for each treatment ± se is shown above the corresponding peak. n ≥ 100. The average pollen tube length for populations showing bimodal distributions (B and C) were calculated separately (P1 and P2).

Figure 10.

Migration of the MGU into pollen tubes analyzed by DAPI staining. A, Histogram of MGU components (vegetative nucleus and/or generative cell) in pollen tubes of different lengths. Bars are designated as follows: neither out (black bars) indicates the absence of either the vegetative nucleus or the generative cell in pollen tubes; one out (striped bars) indicates the presence of the leading edge of either (single) component in pollen tubes; and both out (white bars) indicates the presence of the leading edge of both the vegetative nucleus and the generative cell in pollen tubes. Total pollen number (n) analyzed is shown for each pollen tube bin length. B, Micrograph showing the diffusely stained vegetative nucleus (arrows) and intensely stained generative cell (arrowheads) entering tubes in DAPI-stained pollen. The top grain would be scored as neither out, and the bottom grain would be scored as both out.

Figure 11.

Summary of exogenous SlPRALF effects on pollen tube growth. Pollen tubes are sensitive to inhibition by SlPRALF peptide after hydration and early germination events, including glebula formation. This inhibition is reversible up to 15 min of exposure to peptide. Growing pollen tubes become resistant to exogenously added SlPRALF once they have reached lengths of 40 to 60 μm, when MGU migration is nearing completion.