Husk and pellicle as the agri-food waste in the walnut-product industry are in soaring demand because of their rich polyphenol content. This study investigated the differential compounds related to walnut polyphenol between husk and pellicle during fruit development stage. By using ultra-high performance liquid chromatography-quadrupole-orbitrap (UHPLC-Q-Orbitrap), a total of 110 bioactive components, including hydrolysable tannins, flavonoids, phenolic acids and quinones, were tentatively identified, 33 of which were different between husk and pellicle. The trend of dynamic content of 16 polyphenols was clarified during walnut development stage by high-performance liquid chromatography (HPLC). This is the first time to comprehensive identification of phenolic compounds in walnut husk and pellicle, and our results indicated that the pellicle is a rich resource of polyphenols. The dynamic trend of some polyphenols was consistent with total phenols. The comprehensive characterization of walnut polyphenol and quantification of main phenolic compounds will be beneficial for understanding the potential application value of walnut and for exploiting its metabolism pathway.

Walnut (Juglans regia L.) is a major nut crop of the Juglandaceae family and is well-known for its high nutritional value, which is achieved by a rich array of polyphenolic compounds. Phenolics are considered beneficial to human health because of their antioxidant, antimutagenic, and free radical scavenging properties. However, the phenolic biosynthetic pathway in walnut remains poorly studied. In this study, we cloned a 5-enolpyruvylshikimate 3-phosphate synthase (JrEPSPS) gene from walnut, a key gene involved in the shikimate pathway that catalyzes the penultimate step of the shikimate pathway toward the biosynthesis of aromatic amino acids. Subsequent sequence analysis revealed that the JrEPSPS protein harbors an N-terminal helixturn-helix-like motif, which is known to mediate EPSPS function by acting as a transcription factor and regulating the expression of genes in the phenylpropanoid pathway in poplar. Subcellar localization analysis suggested JrEPSPS was localized in chloroplasts. The transient overexpression of JrEPSPS in persimmon (Diospyros kaki Thunb.) leaves and fruit discs showed significantly increased phenolic accumulation by elevating the expression of phenolic biosynthetic pathway genes. These results provide novel insights into the roles of EPSPS involved in phenolic biosynthesis in plants.