Excess blue or violet-blue proportion in far-red supplemented white light induces temperature-dependent, contrasting effects on the metabolic adjustment and freezing tolerance of winter barley leaves

• Excess B 450 proportion reversed the low R:Fr ratio induced/enhanced pre-hardening. • WFr mainly enhanced amino acids at 15°C and sugars at 5°C, while cold increased both. • WFr+VB 410 increased amino acid concentrations at 5°C, augmenting the WFr effect. • Metabolic adjustment and related gene expression depended on spectral composition. • Effects of blue supplementations were wavelength- and temperature-dependent. Far-red supplemented white light (WFr) induces (at 15°C) or enhances (at 5°C) early cold acclimation (pre-hardening) in the leaves of young barley. However, its influence on metabolic remodelling and the role of blue light in these processes remain largely unexplored. We previously reported that supplementation of WFr with violet-blue LED (λ max = 410nm, WFr+VB 410 ) enhanced the WFr effect on leaf freezing tolerance at 5°C, although limited evidence suggests a negative interaction between WFr-induced changes and excess blue light in complex light environments. To address this, we compared the effects of WFr+VB 410 and WFr supplemented with another blue LED (λ max =450nm, WFr+B 450 ) at both 15°C and 5°C on young barley plants. Our results showed that WFr+B 450 negated the positive effects of WFr on leaf freezing tolerance, regardless of temperature. Metabolomic analysis distinguished far-red-dominant, far-red-blue-interfering, and blue-specific alterations, revealing key differences between far-red- and cold-induced pre-hardening at the metabolic level. At 15°C, WFr enhanced free amino acid accumulation more than it did soluble sugar content, whereas under cold exposure, both accumulated heavily. Although both blue light supplementation counteracted several WFr-induced changes at 15°C, WFr+VB 410 reversed the WFr-induced decrease in β-Alanine, L-Aspartic acid and 2-oxoglutaric acid levels. At 5°C, the effects of blue light supplementations diverged: WFr+B 450 negated the WFr-induced enhancement of soluble sugar accumulation, whereas WFr+VB 410 increased free amino acid levels. Light quality-dependent changes were also observed in the expression patterns of relevant genes.