Genome-wide gene expression profiling reveals conserved and novel molecular functions of the stigma in rice

Abstract

In angiosperms, the stigma provides initial nutrients and guidance cues for pollen grain germination and tube growth. However, little is known about the genes that regulate these processes in rice (Oryza sativa). Here, we generate rice stigma-specific or -preferential gene expression profiles through comparing genome-wide expression patterns of hand-dissected, unpollinated stigma at anthesis with seven tissues, including seedling shoot, seedling root, mature anther, ovary at anthesis, seeds 5 d after pollination, 10-d-old embryo, 10-d-old endosperm, and suspension-cultured cells by using both 57 K Affymetrix rice whole-genome array and 10 K rice cDNA microarray. A high reproducibility of the microarray results was detected between the two different technology platforms. In total, we identified 548 genes to be expressed specifically or predominantly in the stigma papillar cells of rice. Real-time quantitative reverse transcription-polymerase chain reaction analysis of 34 selected genes all confirmed their stigma-specific expression. The expression of five selected genes was further validated by RNA in situ hybridization. Gene Ontology analysis shows that several auxin-signaling components, transcription, and stress-related genes are significantly overrepresented in the rice stigma gene set. Interestingly, most of them also share several cis-regulatory elements with known stress-responsive genes, supporting the notion of an overlap of genetic programs regulating pollination and stress/defense responses. We also found that genes involved in cell wall metabolism and cellular communication appear to be conserved in the stigma between rice and Arabidopsis (Arabidopsis thaliana). Our results indicate that the stigmas appear to have conserved and novel molecular functions between rice and Arabidopsis.

Figure 1.

Expression of five stigma preferential genes by RNA in situ hybridization. A, A rice pistil before pollination. B, D, E, F, G, and H, Hybridization by the antisense probes of LOC_Os02g46260, LOC_Os09g36700, LOC_Os10g33250, LOC_Os01g62070, AK071040, and LOC_Os01g09620, respectively. C, I, J, K, L, and M, Hybridization by their respective sense probes. Among them, the gene LOC_Os01g09620 (H and M) was used as a reference, because the microarray result showed that it was expressed in the entire mature pistil. The longitudinal section through the pistil before pollination was used for all the hybridizations. All of the slices were stained with Fluorescent Brightener 28 after hybridization. The hybridization signals are shown in dark purple. The structures of the pistil are illustrated in A and B. Bar = 100 μm.

Figure 2.

Putative cis-acting regulatory elements enriched in the stigma-specific or preferential genes. A to C, The top three candidate motifs of cluster I, cluster II, and cluster III, respectively. The overall height of each stack indicates the sequence conservation at that position (measured in bits), whereas the heights of symbols within the stack reflect the relative frequencies of the corresponding nucleic acids at that position.