Graft-induced Changes in MicroRNA Expression Patterns in Citrus Leaf Petioles

Grafting is an important, widely used plant propagation technique but its physiological effects are as yet insufficiently understood. Recent studies indicate that movement of proteins and small RNAs through the graft union might be involved. MicroRNAs are known to play a significant role in regulation of higher plants' developmental and metabolic traits. Extending this logic, we hypothesize that changes in activity of specific microRNAs are one of the mechanisms involved in physiological effects of grafting. The objective of the present study was to test this hypothesis. We determined the expression of a broad range of microRNAs in Citrus leaf petioles, as affected by grafting. Four stock/scion combinations ('Merav' mandarin and 'Star Ruby' grapefruit scions X 'Troyer' citrange and 'Volkamer' lemon rootstocks), rootstock auto-grafts and plants of the variety used as rootstock (= non-grafted) were examined. Grafting caused a dramatic reduction in the expression of the major microRNAs, miR156 (and miR157), which appear to be associated with reduction of juvenility in perennial woody plants. This effect was strongest in hetero-grafts but evident also in auto-grafts. Expression of miR894 also declined upon grafting. Differences in the expression of miR397 were found among grafted scion cultivars, while in non-grafted rootstocks expression of miR397 was barely detectable. Bioinformatic analysis confirmed the presence of miR397 in the citrus genome, validated its sequence and demonstrated its ability to form a stem loop. The differences in miR397 expression might be related to specific copper and other micronutrient requirements of citrus stock-scion combinations.Thus, our results support the hypothesis, indicating the involvement of specific microRNAs in engendering physiological effects of grafting in Citrus. The precise, underlying mechanism needs to be elucidated.