Genome-wide identification of genes expressed in Arabidopsis pistils specifically along the path of pollen tube growth

Abstract

Plant reproductive development is dependent on successful pollen-pistil interactions. In crucifers, the pollen tube must breach the stigma surface and burrow through the extracellular matrix of the stigma epidermal cells and transmitting tract cells before reaching its ovule targets. The high degree of specificity in pollen-pistil interactions and the precision of directional pollen tube growth suggest that signals are continually being exchanged between pollen/pollen tubes and cells of the pistil that line their path. However, with few exceptions, little is known about the genes that control these interactions. The specialized functions of stigma epidermal cells and transmitting tract cells are likely to depend on the activity of genes expressed specifically in these cells. In order to identify these genes, we used the Arabidopsis (Arabidopsis thaliana) ATH1 microarray to compare the whole-genome transcriptional profiles of stigmas and ovaries isolated from wild-type Arabidopsis and from transgenic plants in which cells of the stigma epidermis and transmitting tract were specifically ablated by expression of a cellular toxin. Among the 23,000 genes represented on the array, we identified 115 and 34 genes predicted to be expressed specifically in the stigma epidermis and transmitting tract, respectively. Both gene sets were significantly enriched in predicted secreted proteins, including potential signaling components and proteins that might contribute to reinforcing, modifying, or remodeling the structure of the extracellular matrix during pollination. The possible role of these genes in compatible and incompatible pollen-pistil interactions is discussed.

Figure 1.

Effects of genetic ablation and cell type-specific expression in Arabidopsis pistils. Images of stigmas at stage 13 of floral bud development in wild-type (A) and ablated (B) pistils. Images of stigmas at stage 14 of floral bud development in wild-type (C) and ablated (D) pistils. Fluorescent micrographs of pollinated stigmas from stage 13 wild-type (E) and ablated (F) floral buds, and from stage 14 wild-type (G) and ablated (H) floral buds. Stigmas were pollinated with wild-type pollen grains, and pollen tubes were allowed to develop overnight. Nonradioactive whole-mount in situ hybridizations of wild-type stage 13 pistils using antisense riboprobes specific for selected genes in the stigma dataset: At5g59810 (I), At2g02850 (J), and At5g19880 (K). A sense riboprobe negative control is shown in L.

Figure 2.

Comparison of hybridization signals obtained on the ATH1 array and by reverse northern-blot analysis for 45 genes from the stigma dataset. cDNA clones were amplified and arrayed as described in “Materials and Methods.” Each data point represents the hybridization signal produced by one cDNA clone. RNA abundance is shown in arbitrary numbers. The β-tubulin4 (Tub4) signal was used to normalize the signals. Accession numbers are shown for cDNAs that produced higher signals on the reverse northern-blot analysis than on the ATH1 array.

Figure 3.

RNA gel-blot analysis of selected genes from the stigma dataset. RNA was extracted from anthers (A), whole pistils (P), ovaries (O), and stigmas (S) of wild-type and ablated plants. Ten micrograms of total RNA were loaded in each lane and blotted RNA was hybridized with probes specific for each of the listed genes. The intensity of ethidium bromide staining of ribosomal RNA served as a loading control. To the right of each image are the array hybridization signals (averages of two replicates) obtained with wild-type stigma (signal S) and ovary (signal O), and the SLR values (averages of two replicates) calculated for the stigma [SLR(S)] and ovary [SLR(O)] wild-type versus ablated comparisons. NC, No change. The SLR(O) of −0.9 scored for At3g03670 is not reflected in differential hybridization signals on gel blots of wild-type and ablated ovary RNA because the low transcript abundance (signal of 41.0 in wild-type ovary) is below the limits of detection of the RNA gel blot.

Figure 4.

Comparison of functional gene categories in the stigma and transmitting tract datasets. The bars indicate the number of genes assigned to each functional category as a percentage of the total number of genes in each dataset. The transmitting tract dataset contained no genes in the “cellular biogenesis of cell wall” category.