Involvement of Micro RNA Expression in Physiological Effects of Grafting: Experiments with Citrus

Grafting is an important, widely used plant propagation technique in horticultural crops, 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. Micro RNAs are known to play a significant role in regulation of higher plants’ developmental and metabolic traits. A distinction can be made, however, between major, ubiquitous, strongly expressed micro RNAs and minor, species-specific micro RNAs. The objective of the present study was to examine the possibility that micro RNAs play a role in the modulation of grafting effects. We adopted a screening approach, determining the expression of a broad range of micro RNAs in citrus leaf petioles, as affected by grafting. Four stock/scion combinations (‘Merav’ mandarin and ‘Star Ruby‘grapefruit scions x ‘Troyer’ citrange and Citrus ‘Volkameriana’ rootstocks), rootstock auto-grafts and the non-grafted rootstocks were examined. Microarray comparisons of miRNA expression revealed significant differences in major as well as minor micro RNAs. All grafting combinations caused a dramatic reduction in the expression of the major miR156 (and miR157) tha appear to be associated with reduction of juvenility in perennial woody plants. Expression of miR894 also declined upon grafting. Differential, rootstock and scion-dependent expression of the minor miR397 was found in petioles of grafted scions, while in non-grafted rootstocks the 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 copper and other micronutrient requirements of citrus stock-scion combinations. Differences in other micro RNAs’ expression will also be presented. These results suggest an involvement of specific micro RNAs in engendering physiological effects of grafting, although the precise mechanism remains to be elucidated. Supported by funds of the Israeli Citrus Marketing Board.