A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis

Plant J. 2004 Jan;37(1):139-46. doi: 10.1046/j.1365-313x.2003.01946.x.


The epidermis of higher plants generates the cuticle layer that covers the outer surface of each plant. The cuticle plays a crucial role in plant development, and some mutants with defective cuticle exhibit morphological abnormalities, such as the fusion of organs. The way in which the cuticle forms and its contribution to morphogenesis are poorly understood. Conventional detection of the cuticle by transmission electron microscopy (TEM) requires laborious procedures, which include fixation, staining with osmium, and preparation of ultra-thin sections. It is also difficult to survey entire surfaces of expanded leaves because of the limited size of specimens that can be examined. Thus, TEM is unsuitable for large-scale screening for mutants with defective cuticle. We describe here a rapid and inexpensive method, designated the toluidine-blue (TB) test, for detection of cuticular defects in whole leaves. We demonstrated the validity of the TB test using mutants of Arabidopsis thaliana, including abnormal leaf shape1 (ale1), fiddlehead (fdh), and five eceriferum (cer) mutants, in which the structure and/or function of the cuticle is abnormal. Genetic screening for mutants using the TB test allowed us to identify seven loci. The cuticle-defective regions of leaves of the mutants revealed five intrinsic patterns of surface defects (classes I through V), suggesting that formation of functional cuticle on leaves involves various spatially regulated factors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics
  • Arabidopsis / growth & development*
  • Arabidopsis Proteins / genetics
  • Cell Surface Extensions / drug effects
  • Cell Surface Extensions / genetics
  • Cell Surface Extensions / physiology
  • Chromosome Mapping
  • Coloring Agents / pharmacology
  • Mutation
  • Phenotype
  • Plant Epidermis / drug effects
  • Plant Epidermis / genetics
  • Plant Epidermis / growth & development*
  • Plant Leaves / genetics
  • Plant Leaves / growth & development*
  • Serine Endopeptidases / genetics
  • Tolonium Chloride / pharmacology


  • Arabidopsis Proteins
  • At2g26250 protein, Arabidopsis
  • CER protein, Arabidopsis
  • Coloring Agents
  • Tolonium Chloride
  • ALE1 protein, Arabidopsis
  • Serine Endopeptidases