Sequencing-based approaches reveal low ambient temperature-responsive and tissue-specific microRNAs in phalaenopsis orchid

Abstract

Plant small RNAs (smRNAs) are short, non-coding RNA molecules that mediate RNA silencing and regulate a group of genes involved in plant development and responses to environmental stimuli. Low temperature is necessary to initiate stalk development in the orchid Phalaenopsis aphrodite subsp. formosana. To identify smRNAs in Phalaenopsis responding to low temperatures, a smRNA profiling analysis using high-throughput sequencing technology was performed. Subsequent bioinformatic analysis was applied to categorize the miRNAs identified. A total of 37,533,509 smRNA reads yielded 11,129 independent orchid miRNA sequences, representing 329 known miRNA families identified in other plant species. Because the genomic resources available for Phalaenopsis are limited, a transcriptomic database was established using deep sequencing data sets to identify miRNAs precursors and their target transcripts. Comparing small RNAs and the transcriptomic database, 14 putative miRNA precursors of 10 miRNA families were identified, as were hundreds of putative targets. Comparing sequencing data and smRNA northern hybridization results identified miR156, miR162, miR528 and miR535 as low temperature-induced miRNAs. In addition, tissue-specific expression of these miRNAs was investigated. It was concluded that miR156 and miR172 may be components of a regulatory pathway mediating transition from the vegetative to the reproductive phase in Phalaenopsis. The smRNA and transcriptomic databases could be the foundations for further research aimed at elucidating the control of the flowering time in orchids.

Figure 1. Size distribution of small RNA sequences in the separated tissues of native Phalaenopsis.

NL: leaves from untreated plants, CL: leaves after low ambient temperature treatment, S: stalks, FB: flower buds.

Figure 2. Characterization of conserved miRNAs in various tissues of Phalaenopsis orchid.

Size distribution of mature miRNAs in each tissue as a function of the sum of reads (A) and the number of unique sequences (B) NL: leaves from untreated plants, CL: leaves from low ambient temperature treatment, S: stalks, FB: flower buds.

Figure 3. Characterization of conserved miRNA families.

miRNA families pertaining to the number of unique sequences (A) and to the sum of reads in an individual miRNA family (B). NL: leaves from untreated plants, CL: leaves from low ambient temperature treatment, S: stalks, FB: flower buds.

Figure 4. Summary of low temperature-responsive miRNA selection.

Abundant smRNAs from untreated and low temperature-treated leaves (A); Characterization of abundant smRNAs (B); Fold change of small RNA unique sequences by comparing the dataset of the low temperature treated group to the untreated group (C). NL: leaves from untreated plants, CL: leaves from low ambient temperature treatment.

Figure 5. Secondary structure simulation of predicted miRNA precursors.

pha: abbreviation for Phalaenopsis aphrodite subsp. formosana.

Figure 6. Analysis of miRNA expression levels in Phalaenopsis aphrodite subsp. formosana using northern hybridization.

NL: leaves from untreated condition, CL: leaves from low ambient temperature treatment, S: stalks, FB: flower buds.

Figure 7. Identification of cleavage sites and expression patterns of PaSPL and PaAP2 in Phalaenopsis aphrodite subsp. formosana.

The arrows indicate the 5′ cleavage sites (A) that were sequenced from 5′-end cleavage products (B). The expressions of PaSPL and PaAP2 were analyzed using Q-PCR. The 18S rRNA expression level was used as a quantitative control. NL: leaves from untreated condition, CL: leaves from low ambient temperature treatment, S: stalks, FB: flower buds.