INTRODUCTION. Plant microRNAs (miRNAs) are key regulators of transcripts involved in stress response. In this study, we investigated the effects of drought stress on miRNA abundance, focusing on expression changes occurring in leaves and roots of Vitis vinifera cv. Cabernet Sauvignon (CS) and M4 rootstock (Vitis vinifera X Vitis berlandieri), and of their auto- and reciprocal grafts. MATERIALS AND METHODS. Potted grapevines were subjected to a water stress treatment, during which leaf water potential and gas exchanges were daily monitored on both treated (WS) and irrigated (IRR) plants, in order to assess the moment of maximum stress optimal for sampling (gs < 0.05 mmol H2O m-2 s-1 and Ψleaf ~ -1.4 MPa) . RNA samples were extracted in triplicate from WS and IRR leaves and roots of the two genotypes (CS, M4) and of different graft combinations (CS/CS, M4/M4, CS/M4, M4/CS) and were used i) to prepare cDNA libraries, processed by the high-throughput SOLiD sequencing system; ii) to carry out expression assays by RT-qPCR. RESULTS AND DISCUSSION. Most of the putative novel miRNAs identified by sequencing were differently expressed among target tissues: n_191 and n_327 were more abundant in leaf than in root, whereas n_47 (targeting NBS-LRR resistance proteins) was exclusively overexpressed in M4 root. Drought stress significantly reduced the expression of n_22 (targeting an ABC transporter), while it strongly increased the expression of n_312, in both CS and M4 leaves, and n_231 in CS tissues. The concentrations of n_191 and n_312 were overall lower in M4 than CS auto-grafted plants and, interestingly, their expression levels were differently modulated in the reciprocal grafts (CS/M4 and M4/CS) upon stress. Moreover, n_55 (targeting a serine/threonine kinase) and n_327 (targeting two SPL-like proteins) showed no expression differences between auto-grafted plants, but in hetero-grafts they were more expressed in M4 and CS leaves, respectively. CONCLUSIONS. These results show that grapevine miRNAs are regulated by drought stress and that the concentrations of some of the analyzed miRNAs are affected by genotype and graft combination, suggesting the possibility of transport events between rootstock and scion. Acknowledgments: AGER-SERRES N° 2010-2105 and CRC INTEFLAVI.
Characterization of novel putative MIRNAs differentially modulated by abiotic stress in two grapevine genotypes.
PAGLIARANI, CHIARA;FERRERO, MANUELA;VITALI, MARCO;LOVISOLO, Claudio;SCHUBERT, Andrea
2014-01-01
Abstract
INTRODUCTION. Plant microRNAs (miRNAs) are key regulators of transcripts involved in stress response. In this study, we investigated the effects of drought stress on miRNA abundance, focusing on expression changes occurring in leaves and roots of Vitis vinifera cv. Cabernet Sauvignon (CS) and M4 rootstock (Vitis vinifera X Vitis berlandieri), and of their auto- and reciprocal grafts. MATERIALS AND METHODS. Potted grapevines were subjected to a water stress treatment, during which leaf water potential and gas exchanges were daily monitored on both treated (WS) and irrigated (IRR) plants, in order to assess the moment of maximum stress optimal for sampling (gs < 0.05 mmol H2O m-2 s-1 and Ψleaf ~ -1.4 MPa) . RNA samples were extracted in triplicate from WS and IRR leaves and roots of the two genotypes (CS, M4) and of different graft combinations (CS/CS, M4/M4, CS/M4, M4/CS) and were used i) to prepare cDNA libraries, processed by the high-throughput SOLiD sequencing system; ii) to carry out expression assays by RT-qPCR. RESULTS AND DISCUSSION. Most of the putative novel miRNAs identified by sequencing were differently expressed among target tissues: n_191 and n_327 were more abundant in leaf than in root, whereas n_47 (targeting NBS-LRR resistance proteins) was exclusively overexpressed in M4 root. Drought stress significantly reduced the expression of n_22 (targeting an ABC transporter), while it strongly increased the expression of n_312, in both CS and M4 leaves, and n_231 in CS tissues. The concentrations of n_191 and n_312 were overall lower in M4 than CS auto-grafted plants and, interestingly, their expression levels were differently modulated in the reciprocal grafts (CS/M4 and M4/CS) upon stress. Moreover, n_55 (targeting a serine/threonine kinase) and n_327 (targeting two SPL-like proteins) showed no expression differences between auto-grafted plants, but in hetero-grafts they were more expressed in M4 and CS leaves, respectively. CONCLUSIONS. These results show that grapevine miRNAs are regulated by drought stress and that the concentrations of some of the analyzed miRNAs are affected by genotype and graft combination, suggesting the possibility of transport events between rootstock and scion. Acknowledgments: AGER-SERRES N° 2010-2105 and CRC INTEFLAVI.
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