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

Abstract

SUMMARYFull 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.

Analyses of Tomato Fruit Brightness Mutants Uncover Both Cutin-Deficient and Cutin-Abundant Mutants and a New Hypomorphic Allele of GDSL Lipase

“…fruit growth, visual aspect, cracking, water loss, resistance to pathogens, and postharvest shelf-life) are highly dependent on fruit cuticle (Bargel and Neinhuis, 2005;Saladié et al, 2007;Matas et al, 2009;Domínguez et al, 2011;Parsons et al, 2012). An increasing number of studies highlight the possibilities offered by tomato for analyzing cuticle architecture, mechanical properties, and permeability (López-Casado et al, 2007;Saladié et al, 2007;MintzOron et al, 2008;Buda et al, 2009;Isaacson et al, 2009;Wang et al, 2011) and for discovering genes contributing to cuticle synthesis and regulation (Hovav et al, 2007;Mintz-Oron et al, 2008;Girard et al, 2012;Nadakuduti et al, 2012;Yeats et al, 2012b;Shi et al, 2013). Nevertheless, to further our understanding of the relationships between cuticle composition and architecture and cuticle properties and performance in plants, new tomato cuticle mutants are highly needed (Domínguez et al, 2011).…”

The Tomato FRUITFULL Homologs TDR4/FUL1 and MBP7/FUL2 Regulate Ethylene-Independent Aspects of Fruit Ripening

“…A Sudan IV (Sigma-Aldrich) stock solution (0.1% [w/v] in isopropanol) was diluted 3:2 with distilled water, mixed well by rotation for 30 min, and filtered through a syringe filter to remove precipitates (Buda et al, 2009). Thin Br stage pericarp sections were incubated for 10 min in Sudan IV solution, rinsed first with 50% isopropanol and then with water.…”

Cuticle Biosynthesis in Tomato Leaves Is Developmentally Regulated by Abscisic Acid

“…Tissue fixation and embedding were performed as by Buda et al (2009). A saturated solution of Oil Red O (Alfa Aesar) in isopropyl alcohol was diluted 3:2 with distilled water, mixed well, left for 30 min at room temperature, and then filtered with a syringe filter of 0.8/0.2 mm pore size (Acrodisc syringe filters; Pall Corporation) to remove precipitates.…”