Identification of miRNA precursors in the phloem of Cucurbita maxima

Plant development and responses to environmental cues largely depend on mobile signals including microRNAs (miRNAs) required for post-transcriptional silencing of specific genes. Short-range cell-to-cell transport of miRNA in developing tissues and organs is involved in transferring positional information essential for determining cell fate. Among other RNA species, miRNAs are found in the phloem sap. Long-distance transport of miRNA via the phloem takes a part in regulation of physiological responses to changing environmental conditions. As shown for regulation of inorganic phosphorus and sulfate homeostasis, mature miRNAs rather than miRNAs precursors are transported in the phloem as signaling molecules. Here, a bioinformatics analysis of transcriptomic data for Cucurbita maxima phloem exudate RNAs was carried out to elucidate whether miRNA precursors could also be present in the phloem. We demonstrated that the phloem transcriptome contained a subset of C. maxima pri-miRNAs that differed from a subset of pri-miRNA sequences abundant in a leaf transcriptome. Differential accumulation of pri-miRNA was confirmed by PCR analysis of C. maxima phloem exudate and leaf RNA samples. Therefore, the presented data indicate that a number of C. maxima pri-miRNAs are selectively recruited to the phloem translocation pathway. This conclusion was validated by inter-species grafting experiments, in which C. maxima pri-miR319a was found to be transported across the graft union via the phloem, confirming the presence of pri-miR319a in sieve elements and showing that phloem miRNA precursors could play a role in long-distance signaling in plants.