INTEGRATING GENOMIC, TRANSCRIPTOMIC AND PHYSIOLOGICAL INSIGHTS TO UNTANGLE COLD TOLERANCE, DORMANCY AND FLOWERING TIME IN SWEET CHERRY

Jonathan Thomas Magby

doi:10.7273/000007414

Low temperature stress is one of the most influential environmental factors limiting crop production worldwide. Phenotypic variation in sweet cherry (Prunus avium L.) cold tolerance provides key insights for cultivar selection and breeding. Using a multi-trait approach, along with transcriptomic and genomic analyses, this study explores the genetic regulation and physiological adaptations that underpin cold tolerance across paradormancy, endodormancy, and bud break. Lethal temperature (LT) was measured using Differential Thermal Analysis (DTA), cooler pull tests at ‘tight cluster’ and field damage (FD) assessments. In addition, changes in floral relative water content (RWC), number of primordia (florets) and flowering times were recorded. A new method, Low Temperature Exotherm Ratios (LTER), tracked the percentage of deep supercooling at the canopy level, linking deep supercooling to dormancy regulation. The onset and rates of dormancy acclimation and deacclimation, including the duration of endodormancy and ecodormancy, were linked to dynamic changes in cold tolerance and floral development. Specifically, cultivars with prolonged endodormancy periods exhibited enhanced cold tolerance during endodormancy. However, despite later flowering times, these cultivars became notably more susceptible to cold at ‘tight cluster’ compared to their endo-susceptible counterparts. This was linked to faster acclimation and deacclimation rates, resulting in a rapid cold hardiness loss. In parallel, transcriptomic analyses revealed seven transcripts and five genes with dual transcripts that were differentially expressed across all developmental stages. Abscisic acid (ABA) biosynthesis genes showed consistent differential expression across dormancy, emphasizing their roles in dormancy regulation, and cold adaptation. Ethylene and auxin showed stage-specific changes—increasing during paradormancy and decreasing at bud break in susceptible cultivars—supporting roles in acclimation and deacclimation. Additional factors such as plasmodesmata signaling (e.g., PRUPE_3G040900, RLK1) and copper-related genes (HMA5, RAN1) also contributed to dormancy and cold tolerance dynamics and flowering time variation. Dynamic changes in LT, LTER, and RWC were initially synchronized during acclimation but later diverged during late endodormancy. Decoupling of phenotypic traits revealed a conflict between ABA-driven drought/osmotic stress pathways and independent cold tolerance routes, especially noticeable in endo-susceptible cultivars. Downregulation of Dehydration Responsive Element Binding 1 (DREB1D, formerly annotated as DREB1B) from para-to-endodormancy, alongside an up-regulation of ABA biosynthesis, underscores its integral role in inducing and maintaining dormancy and managing dehydration, beyond merely preventing freezing. DREB1D had two transcripts, ONI28949.1 was downregulated in endo-susceptible cultivars from para-to-endodormancy, suggesting a potential dual role in heat- and cold-associated drought-osmotic stress and/or dormancy induction and maintenance. Thus heat- and cold-induced drought/osmotic stress over para-to-endodormancy decreased DREB1D function in endo-susceptible cultivars. DREB1D, ONI28950.1, was unique to endodormancy, coinciding with cold exposure during this phase. Manual protein sequence inspection revealed a partial ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif within the C-terminal region, aligning its function known transcriptional repressors such as CBF/DREB1D. The motif supports a model were upregulated DREB1D in endo-tolerant cultivars repressed drought/osmotic stress signaling— enhancing dormancy induction, maintenance and cold tolerance. These results underscore DREB1D’s roles in balancing stress signaling and dormancy regulation, particularly in dehydration-sensitive endo- susceptible cultivars. Taken together, DREB1D operates at the intersection of dehydration and osmotic stress to regulate dormancy, with deep supercooling potentially serving as the mechanistic overlap of these stresses. This coordination would explain how DREB1D supports cold acclimation and maintenance in high-chill cultivars during endodormancy, while being tightly linked to dormancy transitions. These findings form the basis for a testable model in which DREB1D integrates dehydration and osmotic stress signals to regulate dormancy transitions through modulation of deep supercooling capacity (i.e., the proportion of the buds acquiring deep supercooling), which in turn governs the overall cold tolerance (i.e., extent of deep supercooling) in high-chill cultivars. Alongside developing predictive markers to assist the sweet cherry industry, model forecasting applications for predicting LT, RWC, and LTER could serve practical uses for growers and researchers. This study provides insights into the genetic underpinnings of cold tolerance in sweet cherry and highlights the dynamic interplay between ABA-driven and independent cold tolerance pathways, particularly in endo-susceptible cultivars. These findings open new pathways to decode the molecular mechanisms involved in dormancy acclimation and deacclimation processes, ensuring that the plant’s growth and reproductive potential remain unaffected despite fluctuating temperature conditions.

INTEGRATING GENOMIC, TRANSCRIPTOMIC AND PHYSIOLOGICAL INSIGHTS TO UNTANGLE COLD TOLERANCE, DORMANCY AND FLOWERING TIME IN SWEET CHERRY

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