Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil

Abstract

Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell-cell interaction.

Figure 1. Pollen samples used in microarray analysis.

(A) A scanning electron micrograph of dry pollen grains collected by vacuum. (B) Bright field micrograph of the 0.5 h PT sample. Hydrated pollen grains (a representative grain shown by arrowhead) and grains that are beginning to germinate (arrow) are shown. (C) Pollen tube growth in a 4 h PT sample. Many grains exhibit elongated tubes (arrow); however, some pollen grains have not germinated (arrowhead). (D) Light micrograph of a petri dish containing semi in vivo grown pollen tubes from cut pistil explants. Approximately 800 pollen tube bundles were used for each microarray experiment. (E) Diagram of semi in vivo pollen tube growth. Bundles of pollen tubes that have emerged from the style (within the dotted box) were excised for RNA isolation. (F) A higher magnification of one cut pistil explant is shown. The dotted box indicates the SIV PT material collected for microarray analysis. Scale bars (A–C), 25 µm.

Figure 2. Differential gene expression in pollen and pollen tubes.

Genes were considered expressed in a given cell type or tissue if their mean, normalized expression value was greater than 100; the total number of genes expressed in each category is provided. (A) A two-way comparison of dry pollen (dry) with 0.5 h PT. (B) The set of 6,677 genes expressed in dry and 0.5 h PT in a 3-way comparison with 4 h PT and SIV PT. (C) Relative percentage of genes with selected GO terms that were significantly overrepresented in sectors 1–4 in Figure 2B. The number above each column denotes the cumulative number of genes detected for a particular GO term in the four sectors (also see Materials and Methods). (D) The set of 7,025 genes expressed in dry, 0.5 h PT, and 4 h PT in a 3-way comparison with the set of genes expressed in any of the seven sporophytic tissues analyzed (sporophyte) and SIV PT. (E) A 4-way comparison among the pollen samples of 2,040 pollen-enriched genes that were not expressed in any of the seven sporophytic tissues analyzed. (F) Number of genes with selected GO terms that were significantly overrepresented in the genes that are up or down regulated significantly in SIV PT compared to dry pollen or 4 h PT (Table S14 and Table S15).

Figure 3. qRT-PCR analysis of 21 pollen tube–enriched and pollen tube–expressed genes.

Total RNA from indicated tissues—dry pollen (pollen), 0.5 h PT, 4 h PT, 8- and 21-day-old seedlings (DS)—was analyzed by qRT-PCR. A heat map of the relative expression levels of the indicated genes by qRT-PCR is provided. Expression levels used for the heat map represent an average of gene expression values from four independent qRT-PCR reactions (two technical replicates each for two biological replicates). Relative expression is represented by a color scale that ranges from black (100%) to light yellow (0.01–0.1%). White represents relative expression levels that were below 0.01%. For each gene, the tissue which showed maximum expression was considered 100% (black), and the relative expression in the other four tissues was calculated based on this maximum level.

Figure 4. In vivo confirmation of pistil-dependent changes in pollen tube gene expression.

(A–D) Aniline blue staining of pollen tube growth in pistils. (A and C) are bright field images; (B and D) are epifluorescent images. (A and B) Pollinated pistils stained with aniline blue soon after pollen deposition. Tubes have not emerged from the grains but adhesion to stigmatic cells can be seen (black arrow). (C and D) Pollinated pistils stained with aniline blue 2 hours after pollen deposition; the pollen tube front in the style is indicated by white arrows. (E) A heat map of the relative expression levels (as described in Figure 3) measured by qRT-PCR of the indicated genes in dry pollen, emasculated ms1 pistils, pollinated pistils 1 minute (1 m pollinated pistil) or 2 hours (2 h pollinated pistil) after pollen deposition. Pistils were cut at the junction of the style and ovary (white dotted line); the portion with the stigma and style (B), or stigma, style, and pollen tubes (D), were used in qRT-PCR experiments (E). Scale bars, 100 µm.

Figure 5. At1g60420-1 and At3g18000-1 cause defective pollen tube growth in the pistil.

(A–I) ms1 pistils were hand-pollinated with pollen heterozygous for At1g60420-1, At3g18000-1, or a control insertion (does not affect pollen function), and were stained for GUS activity 24 hours after pollination. GUS is released into synergids from pollen tubes that successfully enter the micropyle and burst (arrows). (A–C) Ovules that have received GUS+ control pollen tubes. (D,F,G,I) Ovules that have received GUS activity from pollen tubes carrying the indicated insertion. (E,H) Some ovules attracted pollen tubes that failed to target the micropyle and burst. (F,I) Some ovules received GUS activity and have multiple additional tubes targeting the micropyle. Scale bars = 50 µm. (J) Quantitative analysis of ovule targeting. The number of ovules that received GUS activity following pollen tube burst was quantified for the control and indicated insertion lines and is plotted as a percentage (±s.d.) of the total number of ovules. *P value>0.01 (χ², expected = 50%).