Systems-Level Analysis of Rootstock–Scion Interactions in Apple Reveals Mechanisms of Cold Tolerance Under Field Frost Events

Late spring frosts threaten apple (Malus × domestica Borkh.) productivity by damaging developing floral buds, yet the mechanisms underlying rootstock-dependent cold tolerance in orchard conditions remain poorly understood. 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 sensitive 'M.26' rootstock. To uncover the mechanisms underlying this tolerance, we integrated RNA sequencing, untargeted metabolomics, and soluble sugar profiling across floral buds ('Gala'), scion leaves ('Gala'), and rootstock sucker leaves (B.9, M.26), sampled 12 hours before and 6 hours after a naturally occurring frost in April 2021. Transcriptomic analysis identified cold-responsive gene networks involving transcription factors (MdCBF4, MdHSFC1), ABA signaling, ROS detoxification, and membrane remodeling. Co-expression network analysis revealed frost-associated hub genes and regulatory modules. Carbohydrate profiling showed that B.9 maintained more stable soluble sugars—such as sucrose, glucose, and sorbitol—during frost-sensitive stages, suggesting improved osmoprotection and energy balance. Metabolomic profiling revealed tissue-specific shifts in B.9, including increased ascorbate metabolism, arginine biosynthesis, and protective sugars like trehalose and melibiose. Lipid remodeling and signaling metabolites, such as colnelenic acid and LysoPA, were also enriched, pointing to dynamic membrane adaptation. Interestingly, despite exhibiting higher levels of reactive oxygen species (ROS), particularly superoxide and hydrogen peroxide, B.9 appeared to sustain redox homeostasis through coordinated antioxidant pathways, suggesting that ROS may function as protective signals rather than causing damage. In contrast, M.26 displayed higher bud mortality, weaker activation of cold-responsive genes, and metabolite profiles consistent with stress susceptibility, including elevated glutathione metabolism. Together, these results provide the first systems-level insight into tissue-specific natural frost responses in apple under orchard conditions. By identifying key candidate genes, metabolites, and regulatory pathways associated with frost resilience, this work lays the groundwork for future efforts in rootstock improvement and molecular breeding. These findings offer promising targets for developing cold-tolerant apple germplasm and improving orchard management, while more broadly providing a framework to understand how rootstock–scion interactions modulate complex abiotic stress responses.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Horticulture

Department dc:contributor.department

Horticulture

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2025

Author dc:creator

• Saini, Amolpreet Kaur

Chair dc:contributor.committeechair

• Sherif, Sherif Mohamed

Committee members dc:contributor.committeemember

• Artlip, Timothy S.
• Aćimović, Srđan G.
• Wright, Robert Clay

• Malus × domestica
• spring frost
• transcriptome
• metabolome
• soluble sugars
• rootstock
• apple
• grafting

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Dc Identifier Other

vt_gsexam:44447

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/136971