Three-dimensional imaging of plant cuticle architecture using confocal scanning laser microscopy

Full appreciation of the roles of the plant cuticle in numerous aspects of physiology and development requires a comprehensive understanding of its biosynthesis and deposition; however, much is still not known about cuticle structure, trafficking and assembly. To date, assessment of cuticle organization has been dominated by 2D imaging, using histochemical stains in conjunction with light and fluorescence microscopy. This strategy, while providing valuable information, has limitations because it attempts to describe a complex 3D structure in 2D. An imaging technique that could accurately resolve 3D architecture would provide valuable additions to the growing body of information on cuticle molecular biology and biochemistry. We present a novel application of 3D confocal scanning laser microscopy for visualizing the architecture, deposition patterns and micro-structure of plant cuticles, using the fluorescent stain auramine O. We demonstrate the utility of this technique by contrasting the fruit cuticle of wild-type tomato (Solanum lycopersicum cv. M82) with those of cutin-deficient mutants. We also introduce 3D cuticle modeling based on reconstruction of serial optical sections, and describe its use in identification of several previously unreported features of the tomato fruit cuticle.