Participation of phenylalanine ammonia-lyase (PAL) in increased phenolic compounds in fresh cold stressed walnut (Juglans regia L.) kernels

• Increased activity and indication of new synthesis of PAL were observed under cold.
• 2,4-Dihydroxybenzoic acid and PCE were identified in walnuts.
• PAL was closely associated with increases in 4 out of 7 phenolic compounds.
• Increased ellagic acid was largely independent on PAL activity.
• Protocatechuic acid and its ethyl ester (PCE) changes were independent of PAL.

Mature fresh walnut (Juglans regia L.) kernels showed increased phenolics under cold storage in a previous study. The aim of the present work was to investigate changes in individual soluble phenolic compounds under cold stress in relation to changes in total and specific activity of phenylalanine ammonia-lyase (PAL) as an approach to study the association of particular compounds with phenylpropanoid pathway (PhP). Treatments with inhibitors of phenylalanine ammonia-lyase (PAL), mRNA, RNA and protein synthesis, as well as, activities of the enzymes polyphenol oxidase (PPO) and peroxidase (POD) were included in the experiment. Treated samples were stored at 1 °C and 90% RH for 20 d. Results revealed increases in specific and total activity of PAL during cold storage. Among phenolics, 2,4-dihydroxybenzoic acid and protocatechuic acid ethyl ester were identified by HPLC–DAD–ESI-MS. Increases in acids 4-hydroxybenzoic, 2,4-dihydroxybenzoic, syringic and vanillic agreed with PAL activity increases, whereas in ellagic were largely independent on PAL. Increases in protocatechuic acid and decreases in protocatechuic acid ethyl ester exhibited no direct relation to PAL.