Profiling microRNA expression in Arabidopsis pollen using microRNA array and real-time PCR

Abstract

Background: MicroRNAs (miRNAs) are approximately 22-nt small non-coding RNAs that regulate the expression of specific target genes in many eukaryotes. In higher plants, miRNAs are involved in developmental processes and stress responses. Sexual reproduction in flowering plants relies on pollen, the male gametophyte, to deliver sperm cells to fertilize the egg cell hidden in the embryo sac. Studies indicated that post-transcriptional processes are important for regulating gene expression during pollen function. However, we still have very limited knowledge on the involved gene regulatory mechanisms. Especially, the function of miRNAs in pollen remains unknown.

Results: Using miRCURY LNA array technology, we have profiled the expression of 70 known miRNAs (representing 121 miRBase IDs) in Arabidopsis mature pollen, and compared the expression of these miRNAs in pollen and young inflorescence. Thirty-seven probes on the array were identified using RNAs isolated from mature pollen, 26 of which showed significant differences in expression between mature pollen and inflorescence. Real-time PCR based on TaqMan miRNA assays confirmed the expression of 22 miRNAs in mature pollen, and identified 8 additional miRNAs that were expressed at low level in mature pollen. However, the expression of 11 miRNA that were identified on the array could not be confirmed by the Taqman miRNA assays. Analyses of transcriptome data for some miRNA target genes indicated that miRNAs are functional in pollen.

Conclusion: In summary, our results showed that some known miRNAs were expressed in Arabidopsis mature pollen, with most of them being low abundant. The results can be utilized in future research to study post-transcriptional gene regulation in pollen function.

Figure 1

Expression of RNA silencing pathway genes in Arabidopsis mature pollen. ACT1(ACTIN1) was used as a positive control to ensure the quality of RNA and cDNA. PCR products amplified from cDNA are indicated by asterisks. Sequences for the cDNAs were confirmed by cloning and sequencing. PCR products at higher molecular weight in each sample were amplified from genomic DNA. DCL: Dicer-like; AGO, Argonaut; RDR, RNA-dependent RNA polymerases; DRB, Double-stranded RNA binding protein. AGO3, RDR1 and DRB2 were not detected.

Figure 2

Heat map and unsupervised hierarchical clustering of 26 differentially expressed miRNAs. The heat map diagram shows the result of the two-way hierarchical clustering of genes and samples. Each row represents a miRNA and each column represents a sample. The miRNA clustering tree is shown on the left, and the sample clustering tree appears at the top. The colour scale shown at the bottom illustrates the relative expression level of a miRNA across all samples: red represents an expression level above mean, blue represents expression lower than the mean. The clustering is performed on log₂(Hy3/Hy5) ratio which passed the filtering criteria on variation across samples; standard deviation > 0.50. miR172b* was not followed up in other experiments.

Figure 3

Expression of 27 miRNAs in inflorescence and mature pollen by RT-PCR. The adaptor sequence is 46 bp long. Depending on the length of the miRNA and the number of nucleotides added during the polyadenylation step, the PCR products range from 70 to 90 bp. I, inflorescence; P, mature pollen.