Omics-based characterization of physiological and transcriptomic resilience during cold de-acclimation and...

Cold acclimation, de-acclimation, and re-acclimation are critical adaptive phases that determine plant survival under fluctuating temperatures. Although the physiological and molecular basis of initial cold acclimation is well studied, the regulatory mechanisms governing the loss and re-acquisition of freezing tolerance remain poorly understood. Brassinosteroid (BR) signalling, mediated by glycogen synthase kinase-like (GSK-like) kinases, modulates both stress tolerance and plant growth; however, its role under fluctuating cold-related temperature conditions has not been explored in detail. This study investigated the physiological and transcriptomic responses of Arabidopsis thaliana wild-type (Col-4) and two BR-signalling mutants carrying T-DNA insertions in GSK-like kinase genes BIL1, (At2g30980); GSK3 (At1g06390) across cold acclimation, de-acclimation and re-acclimation phases.

All genotypes exhibited reduced leaf water content (~ 55%) and suppressed photosystem II efficiency during cold acclimation, with partial recovery during de-acclimation and re-acclimation. Notably, the genotypes showed divergent roles in re-acclimation responses: Col-4 showed the greatest freezing tolerance (LT₅₀ = -20 °C), whereas BIL1 and GSK3 mutants showed more moderate re-hardening (~-15 °C and - 13 °C, respectively). In contrast, both mutants showed higher post-freeze canopy regrowth (headroom recovery ~ 80%) compared with Col-4 (~ 20%) during de-acclimation, revealing a trade-off between maximal freezing tolerance and vegetative recovery. Chlorophyll fluorescence analysis showed that GSK3 achieved the strongest photosynthetic recovery, while BIL1 displayed persistently impaired electron transport efficiency. Transcriptomic profiling revealed a genotype-specific reprogramming switch: during de-acclimation, GSK3 produced the largest transcriptional response (1,433 differentially expressed genes), whereas during re-acclimation this pattern reversed, with BIL1 exhibiting the most extensive but poorly coordinated reprogramming (1,876 differentially expressed genes). The co-expression network analysis revealed a dense, highly connected re-acclimation module in GSK3, contrasting with a fragmented network architecture in BIL1, which indicates qualitatively different transcriptional coordination capacities between the two mutants.

Our findings suggest that GSK-like kinase signalling may contribute to cold resilience, potentially influencing the balance between freezing tolerance, photosynthetic recovery, and transcriptional coordination under fluctuating cold temperature conditions. The BIL1 mutant enhances post-freeze regrowth but compromises re-hardening and coherent transcriptional reprogramming, whereas GSK3 supports stronger photosynthetic and transcriptomic recovery. These findings identify GSK-like kinases as candidate regulators that warrant further functional validation as potential targets for engineering cold resilience in crops exposed to increasingly variable thermal environments.