A gain-of-function mutation of Arabidopsis lipid transfer protein 5 disturbs pollen tube tip growth and fertilization

Abstract

During compatible pollination of the angiosperms, pollen tubes grow in the pistil transmitting tract (TT) and are guided to the ovule for fertilization. Lily (Lilium longiflorum) stigma/style Cys-rich adhesin (SCA), a plant lipid transfer protein (LTP), is a small, secreted peptide involved in pollen tube adhesion-mediated guidance. Here, we used a reverse genetic approach to study biological roles of Arabidopsis thaliana LTP5, a SCA-like LTP. The T-DNA insertional gain-of-function mutant plant for LTP5 (ltp5-1) exhibited ballooned pollen tubes, delayed pollen tube growth, and decreased numbers of fertilized eggs. Our reciprocal cross-pollination study revealed that ltp5-1 results in both male and female partial sterility. RT-PCR and beta-glucuronidase analyses showed that LTP5 is present in pollen and the pistil TT in low levels. Pollen-targeted overexpression of either ltp5-1 or wild-type LTP5 resulted in defects in polar tip growth of pollen tubes and thereby decreased seed set, suggesting that mutant ltp5-1 acts as a dominant-active form of wild-type LTP5 in pollen tube growth. The ltp5-1 protein has additional hydrophobic C-terminal sequences, compared with LTP5. In our structural homology/molecular dynamics modeling, Tyr-91 in ltp5-1, replacing Val-91 in LTP5, was predicted to interact with Arg-45 and Tyr-81, which are known to interact with a lipid ligand in maize (Zea mays) LTP. Thus, Arabidopsis LTP5 plays a significant role in reproduction.

Figure 1.

The ltp5-1 Plant Has Defects in Pollen Tube Growth and Fertilization. (A) A portion of the unrooted neighbor-joining tree presented in Supplemental Figure 1 online showing phylogenetic relationships of SCA and SCA-like LTPs in Arabidopsis. Asterisk indicates lily SCA, maize LTP, and seven closely related Arabidopsis SCA-like LTPs. The values on the branches indicate the number of bootstrap replicates supporting the branch. Only bootstrap replication values >50 are shown. (B) Structures of two T-DNA insertion alleles for Arabidopsis LTP5. The white and gray triangles indicate T-DNA insertion sites in SALK_104674 (ltp5-1) and SALK_020545 plants, respectively. 674-5, 674-3, and LBa1 are PCR primers for genotyping analysis in (C). LTP5-5 and LTP5-3 are LTP5 gene-specific PCR primers for evaluating gene transcript levels in (D). LTP5-5B and LTP5C are forward and reverse primers for the first exon of LTP5 shown in (E). LTP5m-3K is the reverse primer for ltp5-1 whose putative translation termination codon (taa, underlined, bold, lowercase letters) was found in frame in the intron. Translation start (ATG) and termination codons (TGA) for LTP5 are shown in bold uppercase letters. Bold lowercase letters indicate both 5′- and 3′-splicing recognition sites. (C) PCR-based genotyping analysis for ltp5-1 plants. The LTP5 gene locus was not amplified in ltp5-1 when 674-5 and 674-3 primers were used for 35 cycles of PCR (LTP5-3′UTR) (top panel). This is due to a homozygous T-DNA insertion, identified by use of LBa1 and 674-3 primers (T-DNA-3′UTR) (bottom panel). (D) RT-PCR analysis for ltp5-1 plants from three replicates. LTP5 gene expression was not found in 35 cycles of PCR using the LTP5 gene-specific primer set (top panel). ACT2 is the PCR control (bottom panel). (E) Identification of aberrant LTP5 transcript in the ltp5-1 plant. Three replicates of RT-PCR using LTP5-5B and LTP5C primers showed the presence of the transcript of LTP5 exon1 in the wild type, ltp5-1, and SALK_020545. LTP5-5B and LTP5m-3K primers were used to amplify the transcript of aberrant LTP5. Thirty-five PCR cycles were performed. (F) Wild-type and ltp5-1 flowers at stage 14 (Smyth et al., 1990) were stained with aniline blue to visualize in vivo pollen tube growth in the pistil (n = 20). Arrow, pollen tube front; st, stigma; sty, style; ov, ovary; an, anther. Bars = 200 μm. (G) Mature siliques of wild-type and ltp5-1 plants were dissected to examine ovules (n = 10). Asterisks indicate unfertilized ovules in the ltp5-1 silique. Bars = 1 mm.

Figure 2.

In Vivo Reciprocal Cross-Pollination of ltp5-1 to Wild-Type Plants. (A) Flowers at stage 12 (Smyth et al., 1990) were emasculated a day before each cross-pollination (n = 15 per cross). At 12 h after pollination, six to seven pistils were fixed, and pollen tube growth was examined by aniline blue staining. The remaining pollinated pistils ripened into mature siliques in 8 d. Siliques were then dissected for examination of fertilized ovules. Bars = 200 μm. Arrows indicate the pollen tube front in the pistil. Asterisks designate unfertilized ovules in the silique. Bars = 1 mm. (B) and (C) In another set of reciprocal cross-pollinations, sizes of mature siliques (B) and numbers of seeds per silique (C) were examined 8 d after pollination. Control flowers were allowed to self-pollinate (asterisks). Data are shown as mean ± sd.

Figure 3.

The ltp5-1 Plants Showed Abnormal Pollen Tube Tip Growth in Vitro and Disturbed Pistil Function in Seed Set Formation. (A) and (B) In vitro pollen tube growth assay. (A) Pollen from mature flowers was grown on solid germination medium in vitro for 6 h at room temperature. Arrows indicate pollen tube tips. Bars = 100 μm. (B) Relative pollen tube lengths were measured at 6 h in vitro germination (n = 100). Data are shown as mean ± sd. Student's t test showed a significant difference in the comparison (P = 0.0001, 95% confidence interval for mean: 206 to 229 for the wild type and 101 to 124 for ltp5-1). (C) In vivo reciprocal cross-pollination of ltp5-1 heterozygote to the wild type. Pollination was allowed to grow for 12 h on the emasculated (previous day) pistils (n = 10). Aniline blue staining shows in vivo pollen tube growth. Arrows indicate the pollen tube front. Bars = 200 μm. Mature siliques at 8 d after pollination were decolorized with 100% ethanol to visualize seeds. Bars = 1 mm.

Figure 4.

Arabidopsis LTP5 Is Present in Pollen, Pollen Tubes, and the Pistil TT. (A) Gene expression levels of some SCA-like Arabidopsis LTPs were evaluated by two replicates of RT-PCR using the gene-specific primer sets. Thirty cycles of PCRs were performed for gene expression in the inflorescence apex and pistil tissues and 35 cycles for pollen. Among the LTP genes examined, LTP5 gene transcripts were shown to be present in both pollen and pistil at a low level. Only the second PCR using the first PCR products as templates was able to show a significant level of LTP5 transcript in pollen. (B) to (D) GUS assay of LTP5_pro:GUS flower. GUS signals were developed for 5 d to make the weak LTP5 gene level more visible. (B) Gene expression was shown in the style (sty), anthers (an), anther filaments (af), petals (pe), and the receptacle (re). Bar = 400 μm. (C) Weak GUS signals were shown in pollen grains. Bar = 100 μm. (D) A dissected pistil showed a low level of LTP5 gene expression in the pistil TT (arrow) Bar = 400 μm. (E) to (G) GUS assay of LTP5_pro:GUS pollen. Pollen tubes were grown on the solid medium in vitro for 6 h. GUS signals were developed for 3 d. Arrowhead indicates pollen grain right before germination; arrows indicate GUS signals in pollen grain and pollen tubes. Bars = 10 μm.

Figure 5.

Pollen-Targeted Overexpression of ltp5-1 or LTP5 Gene in Arabidopsis Plants. (A) RT-PCR analysis for LAT52_pro:ltp5-1 or LAT52_pro:LTP5 plants from two replicates. LTP5-5 and LTP5-3 primers shown in Figure 1B were used to examine LTP5 levels. LTP5-5B and LTP5m-3K primers were used to examine aberrant LTP5 levels. PCR was performed in 30 cycles. The ACT2 primer set was used as the PCR control. (B) In vivo pollen tube growth and silique examination for LAT52_pro:ltp5-1 and LAT52_pro:LTP5. Mature flowers were stained with aniline blue to visualize pollen tube growth in the pistil (n = 10). Arrows indicate the pollen tube front. Bars = 200 μm. Mature siliques were destained with 100% ethanol to examine seed sets (n = 20). Bars = 1 mm. (C) In vivo reciprocal cross-pollination of LAT52_pro:ltp5-1 or LAT52_pro:LTP5 pollen to wild-type pistils. Pollination was allowed for 12 h on the emasculated (previous day) wild-type pistils (n = 10). Aniline blue staining shows in vivo pollen tube growth. Arrows indicate the pollen tube front. Bars = 200 μm. (D) In vitro pollen tube growth assay. Pollen tubes were grown on the solid medium for 16 h. Bars = 50 μm.

Figure 6.

The ltp5-1 Protein Has an Additional Hydrophobic C-Terminal Tail. (A) Protein sequence alignment. Gray boxes indicate the eight Cys residues conserved in the plant LTP family. White boxes indicate Arg-45 and Tyr-81 in the conserved consensus pentapeptide motifs (Thr/Ser-X1-X2-Asp-Arg/Lys and Pro-Tyr-X-Ile-Ser) (Douliez et al., 2000b). Bold letters are additional C-terminal tail sequences in ltp5-1. (B) Superposition of ribbon representations of the structures of LTP5 and ltp5-1. The structures were generated using homology modeling and 1-ns molecular dynamics simulations. The additional, predominantly hydrophobic, C-terminal tail of ltp5-1 is shown to cap one side of the protein, which is known to be an entrance for a putative ligand to the internal hydrophobic cavity in maize LTP (Han et al., 2001). Red, LTP5; blue, ltp5-1; H1 to 4, helix 1 to 4. (C) Tyr-91 in ltp5-1, replacing Val-91 of LTP5, is shown to interact with Arg-45 and Tyr-81. A focused view of the superposition of (B) is shown, with residues of interest (Arg-45, Tyr-81, Val-91, and Tyr-91) depicted in ball and stick representations. Replacement of Val-91 with Tyr-91 results in stabilizing π-cation interactions with Arg-45 and π-stacking interactions with Tyr-81 (also see Supplemental Figure 7 online). The coloring scheme is the same as in (B).

Figure 7.

Model for the Role of LTP5 in Arabidopsis Sexual Reproduction. LTP5, present in pollen, may be secreted from the pollen tube tip and function in establishing or maintaining cell polarity at the tip of pollen tubes. Mutant pollen tubes showed a phenotype similar to that of mutants for Rop signaling (Fu et al., 2001; Gu et al., 2006; Zhang and McCormick, 2007), suggestive of a role of LTP5 in signaling for polar pollen tube tip growth. LTP5 was also found in the pistil TT, where pollen tubes grow. Our study showed a subtle effect of ltp5-1 on normal pistil function for seed formation. In the pistil, it may be both endocytosed into the pollen tube and interact with pectin in an adhesion-mediated guidance mechanism, as SCA does in lily (Kim et al., 2006). Other LTPs may also be involved in pollen tube guidance to the ovule for fertilization since several LTPs are known to be present in the Arabidopsis pistil TT (Thoma et al., 1994; Arondel et al., 2000; Tung et al., 2005). [See online article for color version of this figure.]