Investigating frost response, rootstock-dependent cold tolerance, and floral bud mortality in apple cultivars through transcriptomic insights

Late spring frosts threaten the productivity of deciduous fruit trees. In this study, we investigated frost tolerance in two apple (Malus domestica Borkh.) cultivars, ‘Fuji’ and ‘Gala’, grafted onto ten different rootstocks, over the springs of 2021–2023, to elucidate cold-responsive genes and regulatory mechanisms. Trees on the ‘B.9’ rootstock exhibited superior frost tolerance, with lower floral bud mortality compared to the susceptible ‘M.26’. Using RNA-sequencing, we analyzed floral buds (‘Gala’), scion leaves (‘Gala’), and rootstock sucker leaves (B9, M26). Samples were collected 12 h before and 6 h after a natural frost event in April 2021. Transcriptome analysis revealed extensive transcriptional changes, with 4549 genes upregulated and 5469 downregulated. Weighted gene co-expression network analysis (WGCNA) identified three significant modules based on module eigengenes (ME 6, ME 7, and ME 9) associated with the frost response. The ME 6 and ME 7 modules comprised 1210 and 1011 genes, respectively, while the ME 9 module included 163 genes, of which 6 were differentially expressed post-frost. Applying a 90 % module eigengene connectivity threshold, we identified key hub genes, including MdAFP (ABI five binding protein 3), MdCBF4 (C-repeat-binding factor 4), MdEXP8 (Expansin A8), MdHSFC1 (Heat shock transcription factor C1), MdHXXXD (HXXXD/BAHD-type acyl-transferase family protein), MdLRR-RK (Leucine-rich repeat receptor-like protein kinase), MdRPK2 (Receptor-like protein kinase 2), and MdWBC11 (White-Brown Complex homolog/ABC transporter protein). These findings elucidate the genetic basis of frost resilience in apple rootstocks and pinpoint potential targets for genetic enhancement of frost tolerance.