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An efficient method for medium throughput screening of cuticular wax composition in different plant species



Most aerial plant organs are covered by a cuticle, which largely consists of cutin and wax. Cuticular waxes are mixtures of dozens of compounds, mostly very-long-chain aliphatics that are easily extracted by solvents. Over the last four decades, diverse cuticular wax analysis protocols have been developed, most of which are complex and time-consuming, and need to be adapted for each plant species or organ. Plant genomics and breeding programs often require mid-throughput metabolic phenotyping approaches to screen large numbers of individuals and obtain relevant biological information.


To generate a fast, simple and user-friendly methodology able to capture most wax complexity independently of the plant, cultivar and organ.


Here we present a simple GC–MS method for screening relatively small wax amounts, sampled by short extraction with a versatile, uniform solvent. The method will be tested and validated in leaves and fruits from three different crop species: tomato (Solanum lycopersicum), apple (Malus domestica) and hybrid aspen (Populus tremula × tremuloides).


Consistent results were obtained in tomato cultivar M82 across three consecutive years (2010–2012), two organs (leaf and fruit), and also in two different tomato (M82 and MicroTom) and apple (Golden Delicious and Granny Smith) cultivars. Our results on tomato wax composition match those reported previously, while our apple and hybrid aspen analyses provide the first comprehensive cuticular wax profile of these species.


This protocol allows standardized identification and quantification of most cuticular wax components in a range of species.

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  1. Adato, A., Mandel, T., Mintz-Oron, S., et al. (2009). Fruit-surface flavonoid accumulation in tomato is controlled by a SlMYB12-regulated transcriptional network. PLoS Genetics, 5, e1000777.

  2. Albert, Z., Ivanics, B., Molnár, A., Miskó, A., Tóth, M., & Papp, I. (2013). Candidate genes of cuticle formation show characteristic expression in the fruit skin of apple. Plant Growth Regulation, 70, 71–78.

  3. Bauer, S., Schulte, E., & Their, H.-P. (2004a). Composition of the surface wax from tomatoes. I. Identification of the components by GC/MS. European Food Research and Technology, 219, 223–228.

  4. Bauer, S., Schulte, E., & Their, H.-P. (2004b). Composition of the surface wax from tomatoes. II. Quantification of the components at the ripe red stage and during ripening. European Food Research and Technology, 219, 487–491.

  5. Belding, R. D., Blankenship, S. M., Young, E., & Leidy, R. B. (1998). Composition and variability of epicuticular waxes in apple cultivars. Journal of the American Society for Horticultural Science, 123, 348–356.

  6. Belding, R. D., Sutton, T. B., Blankenship, S. M., & Young, E. (2000). Relationship between apple fruit epicuticular wax and growth of Peltaster fructicola and Leptodontidi umelatius, two fungi that cause sooty blotch disease. Plant Disease, 8, 767–772.

  7. Buschhaus, C., & Jetter, R. (2011). Composition differences between epicuticular and intracuticular wax substructures: How do plants seal their epidermal surfaces? Journal of Experimental Botany, 62, 841–853.

  8. Caligiani, A., Malavasi, G., Palla, G., Marseglia, A., Tgnolini, M., & Bruni, R. (2013). A simple GC-MS method for the screening of betulinic, corosolic, maslinic, oleanolic and ursolic acid contents in commercial botanicals used as food supplement ingredients. Food Chemistry, 136, 735–741.

  9. Cameron, K. D., Teece, M. A., Bevilacqua, E., & Smart, B. (2002). Diversity of cuticular wax among Salix species and Populus species hybrids. Phytochemistry, 60, 715–725.

  10. Dobson, G., Vasukuttan, V., & Alexander, C. J. (2012). Evaluation of different protocols for the analysis of lipophilic plant metabolites by gas chromatography-mass spectrometry using potato as a model. Metabolomics, 8, 880–893.

  11. Domínguez, E., Cuartero, J., & Heredia, A. (2011). An overview on plant cuticle biomechanics. Plant Science, 181, 77–84.

  12. Eshed, Y., & Zamir, D. (1995). An introgression line population of Lycopersicon pennellii in the cultivated tomato enables the identification and fine mapping of yield-associated QTL. Genetics, 141, 1147–1162.

  13. Hen-Avivi, S., Lashbrooke, J., Costa, F., & Aharoni, A. (2014). Scratching the surface: Genetic regulation of cuticle assembly in fleshy fruit. Journal of Experimental Botany, 65, 4653–4664.

  14. Hovav, R., Chehanovsky, N., Moy, M., Jetter, R., & Schaffer, A. A. (2007). The identification of a gene (Cwp1), silenced during Solanum evolution, which causes cuticle microfisuring and dehydration when expressed in tomato fruit. Plant Journal, 52, 627–639.

  15. Isaacson, T., Kosma, D. K., Matas, A. J., et al. (2009). Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss. Plant Journal, 60, 363–377.

  16. Jones, H. G. (1992). Plants and microclimate: A quantitative approach to environmental plant physiology (2nd ed.). New York: Cambridge University Press.

  17. Kimbara, J., Yoshida, M., Ito, H., et al. (2012). A novel class of sticky peel and light green mutations causes cuticle deficiency in leaves and fruits of tomato (Solanum lycopersicum). Planta, 236, 1559–1570.

  18. Lara, I., Belge, B., & Goulao, L. F. (2014). The fruit cuticle as a modulator of postharvest quality. Postharvest Biology and Technology, 87, 103–112.

  19. Lashbrooke, J., Aharoni, A., & Costa, F. (2015). Genome investigation suggests MdSHN3, and APETALLA2-domain transcription factor gene, to be a positive regulator of apple fruit cuticle formation and a inhibitor of russet development. Journal of Experimental Botany. doi:10.1093/jxb/erv366.

  20. Leide, J., Hildebrandt, U., Reussing, K., Riederer, M., & Vogg, G. (2007). The developmental pattern of tomato fruit wax accumulation and its impact on cuticular transpiration barrier properties: Effects of a deficiency in a β-ketoacyl-Coenzyme A synthase (LeCER6). Plant Physiology, 144, 1667–1679.

  21. Leide, J., Hildebrandt, U., Vogg, G., & Riederer, M. (2011). The positional sterile (ps) mutation affects cuticular transpiration and wax biosynthesis of tomato fruit. Journal of Plant Physiology, 168, 871–877.

  22. Liu, J., Xu, X., & Deng, X. (2005). Intergeneric somatic hybridization and its application to crop genetic improvement. Plant Cell, Tissue and Organ Culture, 82, 19–44.

  23. Matsukura, C., Yamaguchi, I., Inamura, M., Ban, Y., Kobayashi, Y., Yin, Y., et al. (2007). Generation of gamma irradiation-induced mutant lines of the miniature tomato (Solanum lycopersicum L.) cultivar ‘Micro-Tom’. Plant Biotechnology, 24, 39–44.

  24. Riederer, M., & Müller, C. (2006). Biology of the plant cuticle. Oxford: Blackwell Pub.

  25. Samuels, L., Kunst, L., & Jetter, R. (2008). Sealing plant surface: Cuticular wax formation by epidermal cells. Annual Review of Plant Biology, 59, 683–707.

  26. Smith, R. M., Marshall, J. A., Davey, M. R., Lowe, K. C., & Power, B. (1996). Comparison of volatiles and waxes in leaves of genetically engineered tomatoes. Phytochemistry, 43, 753–758.

  27. Szakiel, A., Pąckowski, C., Pensec, F., & Bertsch, C. (2012). Fruit cuticular waxes as a source of biologically active triterpenoids. Phytochemistry Reviews, 11, 263–284.

  28. Veraverbeke, E. A., Lammertyn, J., Saevels, S., & Nicalï, B. M. (2001). Changes in chemical wax composition of three different apple (Malus domestica Borkh.) cultivars during storage. Postharvest Biology and Technology, 23, 197–208.

  29. Vogg, G., Fischer, S., Leide, J., et al. (2004). Tomato fruit cuticular waxes and their effects on transpiration barrier properties: Functional characterization of a mutant deficient in a very-long-chain fatty acid β-ketoacyl-CoA synthase. Journal of Experimental Botany, 55, 1401–1410.

  30. Wang, Z., Guhling, O., Yao, R., Li, F., Yeats, T. H., Rose, J. K. C., & Jetter, R. (2011). Two Oxidosqualene cyclases responsible for biosynthesis of tomato fruit cuticular triterpenoids. Plant Physiology, 155, 540–552.

  31. Watanabe, S., Mizoguchi, T., Aoki, K., et al. (2007). Ethylmethanesulfonate (EMS) mutagenesis of Solanum lycopersicum cv. Micro-Tom for large-scale mutant screens. Plant Biotechnology, 24, 33–38.

  32. Yeats, T. H., Buda, G. J., Wang, Z., et al. (2012). The fruit cuticles of wild tomato species exhibit architectural and chemical diversity, providing a new model for studying the evolution of cuticle function. Plant Journal, 69, 655–666.

  33. Yeats, T. H., & Rose, J. K. C. (2013). The formation and function of plant cuticles. Plant Physiology, 163, 5–20.

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We thank the Adelis Foundation, the Leona M. and Harry B. Helmsley Charitable Trust, the Jeanne and Joseph Nissim Foundation for Life Sciences, the Tom and Sondra Rykoff Family Foundation Research and the Raymond Burton Plant Genome Research Fund for supporting the A. A. lab activity. A. A. is the incumbent of the Peter J. Cohn Professorial Chair. A. A., A. G. and J. P. F. M. thank COST FA1106 Quality Fruit for STSM and networking activities.


Research at the IBMCP was supported by MINECO Grant BIO2013-42193-R and from EC H2020 TRADITOM SFS7a-2014- (contract 634561) to Antonio Granell and by FPU-MECD personal Grant to Josefina Patricia Fernandez Moreno (AP-2007-01905). Research at the Weizmann Institute of Sciences was supported by the Israel Science Foundation (ISF) personal Grant to Asaph Aharoni (ISF Grant No. 646/11). We also thank COST FA1106 Quality Fruit for funding networking activities.

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Correspondence to Asaph Aharoni or Antonio Granell.

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Fernandez-Moreno, J., Malitsky, S., Lashbrooke, J. et al. An efficient method for medium throughput screening of cuticular wax composition in different plant species. Metabolomics 12, 73 (2016).

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  • Metabolic profiling
  • Cuticular waxes
  • Fruit surface
  • Fleshy fruit