Identification of conserved and novel microRNAs that are responsive to heat stress in Brassica rapa

The species Brassica rapa includes various vegetable crops. Production of these vegetable crops is usually impaired by heat stress. Some microRNAs (miRNAs) in Arabidopsis have been considered to mediate gene silencing in plant response to abiotic stress. However, it remains unknown whether or what miRNAs play a role in heat resistance of B. rapa. To identify genomewide conserved and novel miRNAs that are responsive to heat stress in B. rapa, we defined temperature thresholds of non-heading Chinese cabbage (B. rapa ssp. chinensis) and constructed small RNA libraries from the seedlings that had been exposed to high temperature (46 degrees C) for 1 h. By deep sequencing and data analysis, we selected a series of conserved and novel miRNAs that responded to heat stress. In total, Chinese cabbage shares at least 35 conserved miRNA families with Arabidopsis thaliana. Among them, five miRNA families were responsive to heat stress. Northern hybridization and real-time PCR showed that the conserved miRNAs bra-miR398a and bra-miR398b were heat-inhibitive and guided heat response of their target gene, BracCSD1; and bra-miR156h and bra-miR156g were heat-induced and its putative target BracSPL2 was down-regulated. According to the criteria of miRNA and miRNA* that form a duplex, 21 novel miRNAs belonging to 19 miRNA families were predicted. Of these, four were identified to be heat-responsive by Northern blotting and/or expression analysis of the putative targets. The two novel miRNAs bra-miR1885b.3 and bra-miR5718 negatively regulated their putative target genes. 5'-Rapid amplification of cDNA ends PCR indicated that three novel miRNAs cleaved the transcripts of their target genes where their precursors may have evolved from. These results broaden our perspective on the important role of miRNA in plant responses to heat.