Protoplast transient transformation facilitates subcellular localization and functional analysis of walnut proteins

Abstract Walnut (Juglans regia L.), an important contributor to oil production among woody plants, encounters research constraints due to difficulties in the subcellular localization and functional analysis of its proteins. These limitations arise from the protracted fruiting cycle and the absence of a reliable transient gene transformation system and organelle markers. In this study, we established a transient expression system using walnut protoplasts and generated fluorescent-tagged organelle markers, whose localization was validated against Arabidopsis (Arabidopsis thaliana) organelle markers. The versatility of this system was demonstrated through pharmaceutical treatments, confirming its ability to determine the subcellular localization of endogenous proteins. We determined the subcellular localization of walnut oleosin proteins and explored protein–protein interactions through bimolecular fluorescence complementation analysis. We also explored the effects of abscisic acid signaling on oil body morphology and the regulation of walnut WRINKLED1 (JrWRI1) in lipid biosynthesis. Overall, this stable and versatile protoplast-based transient expression system, integrated with walnut organelle markers, enhances the subcellular localization and functional studies of uncharacterized walnut proteins. This advancement accelerates research into walnut gene function and streamlines molecular breeding processes with high-throughput efficiency.