Walnuts (Juglans regia L.) are a valuable dietary source of polyphenols and lipids, with increasing worldwide consumption. California is a major producer, with ’Chandler’ and ’Tulare’ among the cultivars more widely grown. ’Chandler’ produces kernels with extra light color at a higher frequency than other cultivars, gaining preference by growers and consumers. Here we performed a deep comparative proteome analysis of kernel pellicle tissue from these two valued genotypes at three harvest maturities, detecting a total of 4937 J. regia proteins. Late and early maturity stages were compared for each cultivar, revealing many developmental responses common or specific for each cultivar. Top protein biomarkers for each developmental stage were also selected based on larger fold-change differences and lower variance among replicates, including proteins for biosynthesis of lipids and phenols, defense-related proteins and desiccation stress-related proteins. Comparison between the genotypes also revealed the common and specific protein repertoires, totaling 321 pellicle proteins with differential abundance at harvest stage. The proteomics data provides clues on antioxidant, secondary, and hormonal metabolism that could be involved in the loss of quality in the pellicles during processing for commercialization.

The biosynthesis of melanin in plants remains an enduring biochemical enigma. Unlike the well-characterized pathways of animals and fungi that produce the canonical “true melanins”, the enzymatic origins and physiological functions of melanin-like pigments in plants are poorly described. Recent advances in Juglans regia (walnut) have begun to illuminate this “dark metabolism,” revealing a dual polyphenol oxidase (PPO) system, constitutive JrPPO1 and stress-inducible JrPPO2, that orchestrates the oxidation of phenolics into amorphous, heterogeneous polymeric pigments. Functional studies demonstrate that JrPPO1 maintains tyrosine and redox homeostasis, while silencing triggers a lesion-mimic phenotype, highlighting the enzyme’s role in detoxifying reactive intermediates. In contrast, JrPPO2 responds to redox and pathogen stress, driving pigment formation as part of the defense response. The integration of biological evidence, encompassing genomics, genetics, and phenotyping, reveals that walnut pigmentation represents a genetically encoded, developmentally regulated adaptation balancing metabolic cost and oxidative protection. Decoding this system reframes melanin biosynthesis in plants as a strategic redox resilience mechanism, one that transforms potentially toxic phenolic oxidation into protective polymerization, bridging primary metabolism, defense, and evolution.

Biomanufacturing enables novel sources of compounds with constant demand, such as food coloring and preservatives, as well as new compounds with peak demand, such as diagnostics and vaccines. The COVID-19 pandemic has highlighted the need for alternative sources of research materials, thrusting research on diversification of biomanufacturing platforms. Here, we show initial results exploring the walnut somatic embryogenic system expressing the recombinant receptor binding domain (RBD) and ectodomain of the spike protein (Spike) from the SARS-CoV-2 virus. Stably transformed walnut embryo lines were selected and propagated in vitro. Both recombinant proteins were detected at 3–14 µg/g dry weight of tissue culture material. Although higher yields of recombinant protein have been obtained using more conventional biomanufacturing platforms, we also report on the production of the red pigment betanin in somatic embryos, reaching yields of 650 mg/g, even higher than red beet Beta vulgaris. This first iteration shows the potential of biomanufacturing using somatic walnut embryos that can now be further optimized for different applications sourcing specialized proteins and metabolites.