Cloning and characterization of small non-coding RNAs from grape

Small noncoding RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are effectors of regulatory pathways underlying plant development, metabolism and responses to biotic and abiotic stresses. To address the nature and functions of these regulators in grapevine (Vitis vinifera L.), we have produced a small RNA library from mixed-stage grape berries. Beside 13 conserved miRNAs belonging to 9 miRNA families, 6 non-conserved miRNAs were isolated and their expression and targets were described. Experimentally validated targets of non-conserved miRNAs included three genes encoding NB-LRR proteins putatively involved in pathogen resistance and a gene encoding a heavy metal ion transport/detoxification protein. Several endogenous and pathogen-derived siRNAs were also isolated. Among 8 endogenous siRNAs, 4 mapped to genes encoding RD22-like proteins and 2 to a gene encoding a cytokinin synthase. siRNA id65 targeted in antisense complementarity the cytokinin synthase gene transcript and was specifically expressed in mature berries where, accordingly, the expression of the cytokinin synthase gene was strongly repressed. Using 5' RACE, the transcript of this gene was shown to be processed in 21-nucleotide (nt) increments starting from siRNA id65 cleavage site. One of the resulting 21-nt siRNAs was also present in the library and mediated further cleavage of the transcript in cis. These results indicate that miRNA- and siRNA-mediated regulatory circuits have extended in grapevine to comprise processes associated with fruit ripening and broaden the range of small RNA-mediated regulation, which was previously associated with auxin, ABA and gibberellins, to encompass cytokinin metabolism.