Conservation and divergence of microRNAs and their functions in Euphorbiaceous plants

Abstract

MicroRNAs (miRNAs) are approximately 21 nt non-coding RNAs which regulate post-transcriptional gene expression. miRNAs are key regulators of nearly all essential biological processes. Aiming at understanding miRNA's functions in Euphorbiaceae, a large flowering plant family, we performed a genome-scale systematic study of miRNAs in Euphorbiaceae, by combining computational prediction and experimental analysis to overcome the difficulty of lack of genomes for most Euphorbiaceous species. Specifically, we predicted 85 conserved miRNAs in 23 families in the Castor bean (Ricinus communis), and experimentally verified and characterized 58 (68.2%) of the 85 miRNAs in at least one of four Euphorbiaceous species, the Castor bean, the Cassava (Manihot esculenta), the Rubber tree (Hevea brasiliensis) and the Jatropha (Jatropha curcas) during normal seedling development. To elucidate their function in stress response, we verified and profiled 48 (56.5%) of the 85 miRNAs under cold and drought stresses as well as during the processes of stress recovery. The results revealed some species- and condition-specific miRNA expression patterns. Finally, we predicted 258 miRNA:target partners, and identified the cleavage sites of six out of ten miRNA targets by a modified 5' RACE. This study produced the first collection of miRNAs and their targets in Euphorbiaceae. Our results revealed wide conservation of many miRNAs and diverse functions in Euphorbiaceous plants during seedling growth and in response to abiotic stresses.

Figure 1.

The miRNA expression levels of 58 miRNAs, with respect to their 39 corresponding primers, measured by qRT–PCR in seedlings of the four Euphorbiaceous species. The relative quantity of expression is normalized to the expression of the internal control U6 gene in units of Log Fold Change to Cal. The ‘(a)’ and ‘(b)’ refer to the fold changes of expression that are, respectively, higher and lower than the expression of the internal control U6, and the first five primers of each were uniformly higher-expressed and lower-expressed than U6 in all four species.

Figure 2.

The identification of miRNA-guided cleavage products of target genes in Euphorbiaceous plants. The cleavage sites of six selected targets in four miRNA as identified by 5′-RACE analysis. For each miRNA, the target sequence is shown on the top and the miRNA sequence on the bottom. The numbers indicate the fraction of cloned PCR products when PCR were terminated at different positions. (a) The cleavage site of 30174.m009082 by miR156e. (b) The cleavage site of 29269.m000250 by miR156e. (c) The cleavage site of 30147.m014478 by miR156e. (d) The cleavage site of 27538.m000327 by miR160ab. (e) The cleavage site of 30138.m004055 by miR164. (f) The cleavage site of 29647.m002022 by miR393b.