The large-scale investigation of gene expression in Leymus chinensis stigmas provides a valuable resource for understanding the mechanisms of poaceae self-incompatibility

Abstract

Background: Many Poaceae species show a gametophytic self-incompatibility (GSI) system, which is controlled by at least two independent and multiallelic loci, S and Z. Until currently, the gene products for S and Z were unknown. Grass SI plant stigmas discriminate between pollen grains that land on its surface and support compatible pollen tube growth and penetration into the stigma, whereas recognizing incompatible pollen and thus inhibiting pollination behaviors. Leymus chinensis (Trin.) Tzvel. (sheepgrass) is a Poaceae SI species. A comprehensive analysis of sheepgrass stigma transcriptome may provide valuable information for understanding the mechanism of pollen-stigma interactions and grass SI.

Results: The transcript abundance profiles of mature stigmas, mature ovaries and leaves were examined using high-throughput next generation sequencing technology. A comparative transcriptomic analysis of these tissues identified 1,025 specifically or preferentially expressed genes in sheepgrass stigmas. These genes contained a significant proportion of genes predicted to function in cell-cell communication and signal transduction. We identified 111 putative transcription factors (TFs) genes and the most abundant groups were MYB, C2H2, C3H, FAR1, MADS. Comparative analysis of the sheepgrass, rice and Arabidopsis stigma-specific or preferential datasets showed broad similarities and some differences in the proportion of genes in the Gene Ontology (GO) functional categories. Potential SI candidate genes identified in other grasses were also detected in the sheepgrass stigma-specific or preferential dataset. Quantitative real-time PCR experiments validated the expression pattern of stigma preferential genes including homologous grass SI candidate genes.

Conclusions: This study represents the first large-scale investigation of gene expression in the stigmas of an SI grass species. We uncovered many notable genes that are potentially involved in pollen-stigma interactions and SI mechanisms, including genes encoding receptor-like protein kinases (RLK), CBL (calcineurin B-like proteins) interacting protein kinases, calcium-dependent protein kinase, expansins, pectinesterase, peroxidases and various transcription factors. The availability of a pool of stigma-specific or preferential genes for L. chinensis offers an opportunity to elucidate the mechanisms of SI in Poaceae.

Figure 1

The mature pistil that was collected just before anther dehiscence. A long red line showing the position where a stigma was excised and a short red line showing the cutting position between ovary and rachis.

Figure 2

A Venn diagram showing relationships between the three transcriptome datasets (stigmas, ovaries, and leaves). Numbers in parentheses are total number of expressed genes in each tissue type.

Figure 3

Validation of RNA-seq results by quantitative real time RT-PCR. Correlation plots indicating the relationship between qPCR results (fold change; Y-axis) of 18 selected genes expressed in the stigmas and leaves and the corresponding data from RNA-seq analysis (X-axis).

Figure 4

KOG function classification. The unigenes were aligned to the KOG database to predict and categorize possible functions. A total of 522 unigenes were assigned to 23 categories.

Figure 5

GO assignment of all identified genes expressed specifically or preferentially in the stigmas of sheepgrass, rice and Arabidopsis. The genes were mapped to three main categories: biological processes, cellular components and molecular functions. The right hand y-axis indicates the number of annotated genes.

Figure 6

Histogram presentation of the KEGG pathway categories. The Y-axis indicates the number of unigenes assigned to a specific pathway. The X-axis indicates the KEGG pathway.

Figure 7

Top 19 transcription factor families of the Leymus chinensis stigma dataset. The Y-axis indicates the number of unigenes assigned to a specific TF family. The X-axis indicates the top 19 TF families.

Figure 8

Expression pattern of four unigenes that best hit SI candidate genes of Lolium perrene. The relative expression levels are represented in arbitrary units (A.U.) normalized to the expression level of the actin gene, used as a reference, in each sample.