Targeted and spatial metabolomics unveil how brassinolide enhances polyphenol and proline metabolism in cold-stressed jujube fruit

Brassinolide (BR) enhances cold tolerance in fruit and vegetables, yet the mechanisms through which BR prevents chilling injury in jujube fruit remain unclear. This study employed targeted and spatial metabolomic approaches to quantify the accumulation and distribution of key enzymes involved in the metabolism of lignin, phenolic compounds, and proline in jujube. Brassinolide treatment mitigated chilling injury in jujube fruit, preserving the ascorbic acid content, cell wall structure, firmness, and flavor of jujube fruit during cold storage. Brassinolide effectively inhibited increases in fruit malondialdehyde content and relative conductivity caused by chilling. In jujube fruit, BR downregulated genes involved in lignin synthesis, thereby slowing the rate of lignification. This finding was supported by a spatial metabolomics analysis which illustrated the diffusion of substances related to lignin metabolism from the outside to the inside of the fruit. Furthermore, BR promoted the accumulation of phenolics, leading to an even distribution of 4-methylumbelliferone, glutathione, and hesperetin throughout each fruit. By upregulating genes associated with proline synthesis, while inhibiting its degradation, BR treatment increased proline accumulation. Notably, glutathione was evenly distributed within BR-treated jujube fruit, whereas arginine was concentrated in the outer pulp. These findings lay the groundwork for the use of exogenous brassinolide to alleviate cold-induced fruit lignification and preserve jujube fruit quality.