Multi-omics analysis identifies Rch-miR156 as a regulator of salt tolerance in heterografted Rosa plants

Rosa chinensis is commonly grafted onto salt-tolerant rootstocks to mitigate salinity stress, yet the molecular mechanisms underlying heterografting-induced adaptations in both the scion and rootstock remain unclear. Through systematic rootstock screening, we identified R. multiflora ‘Inermis’ as an optimal salt-tolerant rootstock for grafting purposes. Integrated analysis combining small RNA and transcriptome sequencing of heterografted plants after 24 days revealed 142 and 273 differentially expressed miRNAs (DEMs) in the scion (R. chinensis) and rootstock (R. multiflora ‘Inermis’), respectively. Target prediction analyses indicated that these DEMs primarily modulate transcription factors (TFs) of the AUX/IAA, AP2/ERF, ARF, HD-ZIP, MYB, NAC, and bZIP families. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis demonstrated significant enrichment of DEM targets in histidine metabolism and lysine biosynthesis pathways, which are essential for osmotic adjustment and stress signaling. Notably, the Rch-miR156 isoform was markedly downregulated in heterografted scions, with RcNAC31 identified as its putative direct target TF. Functional validation demonstrated that Rch-miR156 overexpressing compromised salt tolerance in both R. chinensis and R. multiflora ‘Inermis’, as evidenced by increased reactive oxygen species (ROS) accumulation, elevated malondialdehyde (MDA) levels, and reduced chlorophyll content. Conversely, overexpression of RcNAC31 mitigated ROS accumulation, decreased MDA content, and consequently enhanced salt stress tolerance in R. chinensis. Collectively, these findings elucidate that heterografting in Rosa plants reshapes miRNA-mRNA regulatory networks and validates the Rch-miR156-RcNAC31 module for salt adaptation.