, Volume 226, Issue 6, pp 1459–1473 | Cite as

Cloning and expression analysis of candidate genes involved in wax deposition along the growing barley (Hordeum vulgare) leaf

  • Andrew Richardson
  • Alexandre Boscari
  • Lukas Schreiber
  • Gerhard Kerstiens
  • Mike Jarvis
  • Pawel Herzyk
  • Wieland Fricke
Original Article


The aim of the present study was to isolate clones of genes which are likely to be involved in wax deposition on barley leaves. Of particular interest were those genes which encode proteins that take part in the synthesis and further modification of very long chain fatty acids (VLCFAs), the precursors of waxes. Previously, it had been shown that wax deposition commences within a spatially well-defined developmental zone along the growing barley leaf (Richardson et al. in Planta 222:472–483, 2005). In the present study, a barley microarray approach was used to screen for candidate contig-sequences ( that are expressed particularly in those leaf zones where wax deposition occurs and which are expressed specifically within the epidermis, the site of wax synthesis. Candidate contigs were used to screen an established in-house cDNA library of barley. Six full-length coding sequences clones were isolated. Based on sequence homologies, three clones were related to Arabidopsis CER6/CUT1, and these clones were termed HvCUT1;1, HvCUT1;2 and HvCUT1;3. A fourth clone, which was related to Arabidopsis Fiddlehead (FDH), was termed HvFDH1;1. These clones are likely to be involved in synthesis of VLCFAs. A fifth and sixth clone were related to Arabidopsis CER1, and were termed HvCER1;1 and HvCER1;2. These clones are likely to be involved in the decarbonylation pathway of VLCFAs. Semi-quantitative RT-PCR confirmed microarray expression data. In addition, expression analyses at 10-mm resolution along the blade suggest that HvCUT1;1 (and possibly HvCUT1;2) and HvCER1;1 are involved in commencement of wax deposition during barley leaf epidermal cell development.


Cuticle Epidermal cell development Hordeum Leaf growth Microarray Very long chain fatty acid elongation Wax 



Acyl-carrier protein


Fatty acid elongase


Fatty acid synthase


Glossy 1


Glossy 8




3(β)-Ketoacyl-CoA synthase


Long-chain acyl-CoA synthetase


Point of emergence


Point of leaf insertion


Very long chain fatty acid



This research was supported by the Biotechnology and Biological Sciences Research Council (BBSRC), UK, Grant 61/P18283, and benefited also from a research grant of The Leverhulme Trust, UK (to WF). We would like to thank Tobias Wojciechowski (who was funded through a studentship by University of Paisley) for his help with constructing the barley cDNA library and John Christie (Glasgow University) for use of the electroporator. We would also like to thank two anonymous referees for constructive comments on an earlier version of the manuscript.

Supplementary material

425_2007_585_MOESM1_ESM.xls (2.7 mb)
(XLS 2723 kb)


  1. Aarts MGM, Keijzer CJ, Stiekema WJ, Pereira A (1995) Molecular characterization of the CER1 gene of Arabidopsis involved in epicuticular wax biosynthesis and pollen fertility. Plant Cell 7:2115–2127PubMedCrossRefGoogle Scholar
  2. Barnes J, Percy K, Paul N, Jones P, McLauchlin C, Mullineaux P, Creissen G, Wellburn A (1996) The influence of UV-B radiation on the physiochemical nature of tobacco (Nicotiana tabacum L.) leaf surface. J Exp Bot 47:99–109CrossRefGoogle Scholar
  3. Barthlott W, Neinhuis C (1997) Purity of the sacred lotus, or escape from the contamination in biological science. Planta 202:1–8CrossRefGoogle Scholar
  4. Breitling R, Herzyk P (2005) Rank-based methods as a non-parametric alternative of the t-statistic for the analysis of biological microarray data. J Bioinform Comput Biol 3:1171–1189PubMedCrossRefGoogle Scholar
  5. Breitling R, Armengaud P, Amtmann A, Herzyk P (2004a) A simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments. FEBS Lett 573:83–92PubMedCrossRefGoogle Scholar
  6. Breitling R, Amtmann A, Herzyk P (2004b) Iterative group analysis (iGA): a simple tool to enhance sensitivity and facilitate interpretation of microarray experiments. BMC Bioinformatics 5:34–41PubMedCrossRefGoogle Scholar
  7. Broadwater JA, Haas JA, Fox BG (1998) The fundamental, versatile role of diiron enzymes in lipid metabolism. Fett/Lipid 100:103–111CrossRefGoogle Scholar
  8. Brown BA, Cloix C, Jiang GH, Kaiserli E, Herzyk P, Kliebenstein DJ, Jenkins GI (2005) A UV-B-specific signaling component orchestrates plant UV protection. Proc Natl Acad Sci USA 102:18225–18230PubMedCrossRefGoogle Scholar
  9. Chen X, Goodwin SM, Boroff VL, Liu X, Jenks MA (2003) Cloning and characterization of the WAX2 gene of Arabidopsis involved in cuticle membrane and wax production. Plant Cell 15:1170–1185PubMedCrossRefGoogle Scholar
  10. Close TJ, Wanamaker SI, Caldo RA, Turner SM, Ashlock DA, Dickerson JA, Wing RA, Muehlbauer GJ, Kleinhofs A, Wise R (2004) A new resource for cereal genomics:22K barley GeneChip comes of age. Plant Physiol 134:960–968PubMedCrossRefGoogle Scholar
  11. Costaglioli P, Joubès J, Garcia C, Stef M, Arveiler B, Lessire R, Garbay B (2005) Profiling candidate genes involved in wax biosynthesis in Arabidopsis thaliana by microarray analysis. Biochim Biophys Acta 1734:247–258PubMedGoogle Scholar
  12. Dietrich CR, Perera MADN, Yandeau-Nelson MD, Meeley RB, Nikolau BJ, Schnable PS (2005) Characterization of the GL8 paralogs reveals that the 3-ketoacyl reductase component of fatty acid elongase is essential for maize (Zea mays L.) development. Plant J 42:844–861PubMedCrossRefGoogle Scholar
  13. Eigenbrode SD (1996) Plant surface waxes and insect behaviour. In: Kerstiens G (ed) Plant cuticles—an integrated functional approach. BIOS Scientific Publishers Limited, Oxford, pp 201–222Google Scholar
  14. Fiebig A, Mayfield JA, Miley NL, Chau S, Fischer RL, Preuss D (2000) Alterations in CER6, a gene identical to CUT1, differentially affects long-chain lipid content on the surface of pollen and stems. Plant Cell 12:2001–2008PubMedCrossRefGoogle Scholar
  15. Focke M, Gieginger E, Schwan S, Jänsch L, Binder S, Braun H-P (2003) Fatty acid biosynthesis in mitochondria of grasses: malonyl-coenzyme A is generated by a mitochondrial-localized acetylcoenzyme A carboxylase. Plant Physiol 133:875–884PubMedCrossRefGoogle Scholar
  16. Fricke W, Flowers TJ (1998) Control of leaf cell elongation in barley. Generation rates of osmotic pressure and turgor, and growth-associated water potential gradients. Planta 206:53–65CrossRefGoogle Scholar
  17. Fricke W, Peters WS (2002) The biophysics of leaf growth in salt-stressed barley. A study at the cell level. Plant Physiol 129:374–388PubMedCrossRefGoogle Scholar
  18. Hansen JD, Pyee J, Xia Y, Wen T-J, Robertson DS, Kolattukudy PE, Nikolau BJ, Schnable PS (1997) The glossy1 locus of maize and an epidermis-specific cDNA from Kleinia odora define a class of receptor-like proteins required for the normal accumulation of cuticular waxes. Plant Physiol 113:1091–1100PubMedCrossRefGoogle Scholar
  19. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP (2003) Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res 31:e15PubMedCrossRefGoogle Scholar
  20. Jakobsen MK, Poulsen LR, Schulz A, Fleurat-Lessard P, Møller A, Husted S, Schiøtt M, Amtmann A, Palmgren MG (2005) Pollen development and fertilization in Arabidopsis is dependent on the MALE GAMETOGENESIS IMPAIRED ANTHERS gene encoding a type V P-type ATPase. Genes Develop 19:2757–2769PubMedCrossRefGoogle Scholar
  21. James DW Jr, Lim E, Keller J, Plooy I, Ralston E, Dooner HK (1995) Direct tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator. Plant Cell 7:309–319PubMedCrossRefGoogle Scholar
  22. Jeffery IB, Higgins DJ, Culhane AC (2006) Comparison and evaluation of methods for generating differentially expressed gene lists from microarray data. BMC Bioinformatics 7:359PubMedCrossRefGoogle Scholar
  23. Jenks MA, Ashworth EN (1999) Plant epicuticular waxes: function, production and genetics. Hortic Rev 23:1–68Google Scholar
  24. Jenks MA, Joly RJ, Peters PJ, Rich PJ, Axtell JD, Ashworth EN (1994) Chemically induced cuticle mutation affecting epidermal conductance to water vapour and disease susceptibility in Sorghum bicolor (L.) Moench. Plant Physiol 105:1239–1245PubMedGoogle Scholar
  25. Jenks MA, Tuttle HA, Eigenbrode SD, Feldman KA (1995) Leaf epicuticular waxes of the eceriferum mutants in Arabidopsis. Plant Physiol 108:369–377PubMedGoogle Scholar
  26. Jung KH, Han MJ, Lee DY, Lee YS, Schreiber L, Franke R, Faust A, Yephremov A, Saedler H, Kim YW, Hwang I, An G (2006) Wax-deficient anther1 is involved in cuticle and wax production in rice anther walls and is required for pollen development. Plant Cell 18:3015–3032PubMedCrossRefGoogle Scholar
  27. Kerstiens G (1996) Cuticular water permeability and its physiological significance. J Exp Bot 47:1813–1832CrossRefGoogle Scholar
  28. Kerstiens G (2006) Water transport in plant cuticles: an update. J Exp Bot 57:2493–2499PubMedCrossRefGoogle Scholar
  29. Kunst L, Samuels AL (2003) Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res 42:51–80PubMedCrossRefGoogle Scholar
  30. Kunst L, Jetter R, Samuels AL (2006) Biosynthesis and transport of plant cuticular waxes. In: Riederer M, Muller C (eds) Biology of the plant cuticle. Blackwell Publishing, Oxford, pp 182–215Google Scholar
  31. Kurata T, Kawabata-Awai C, Sakuradani S, Okada K, Wada T (2003) The YOREYORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis. Plant J 36:55–66PubMedCrossRefGoogle Scholar
  32. Lundqvist U, Lundqvist A (1988) Mutagen specificity in barley for 1580 eceriferum mutants localized to 79 loci. Hereditas 108:1–12Google Scholar
  33. Millar AA, Kunst L (1997) Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme. Plant J 12:121–131PubMedCrossRefGoogle Scholar
  34. Millar AA, Clemens S, Zachgo S, Giblin EM, Taylor DC, Kunst L (1999) CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme. Plant Cell 11:825–838PubMedCrossRefGoogle Scholar
  35. Richardson A, Franke R, Kerstiens G, Jarvis M, Schreiber L, Fricke W (2005) Cuticular wax deposition in growing barley (Hordeum vulgare) leaves commences in relation to the point of emergence of epidermal cells from the sheaths of older leaves. Planta 222:472–483PubMedCrossRefGoogle Scholar
  36. Richardson A, Wojciechowski T, Franke R, Schreiber L, Kerstiens G, Jarvis M, Fricke W (2007) Cuticular permeance in relation to wax and cutin development along the growing barley (Hordeum vulgare) leaf. Planta 225:1471–1481PubMedCrossRefGoogle Scholar
  37. Riederer M, Schreiber L (2001) Protecting against water loss: analysis of the barrier properties of plant cuticles. J Exp Bot 52:2023–2032PubMedCrossRefGoogle Scholar
  38. Rossak M, Smith M, Kunst L (2001) Expression of the FAE1 gene and FAE1 promoter activity in developing seeds of Arabidospsis thaliana. Plant Mol Biol 46:717–725PubMedCrossRefGoogle Scholar
  39. Rowland O, Zheng H, Hepworth SR, Lam P, Jetter R, Kunst L (2006) CER4 encodes an alcohol-forming fatty acyl-coenzyme A reductase involved in cuticular wax production in Arabidopsis. Plant Physiol 142:866–877PubMedCrossRefGoogle Scholar
  40. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbour Laboratory Press, New YorkGoogle Scholar
  41. Sayanova O, Beaudoin F, Libisch B, Castel A, Shewry PR, Napier JA (2001) Mutagenesis and heterologous expression in yeast of a plant Δ6-fatty acid desaturase. J Exp Bot 52:1581–1585PubMedCrossRefGoogle Scholar
  42. Schnurr J, Shockey J, Browse J (2004) The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis. Plant Cell 16:629–642PubMedCrossRefGoogle Scholar
  43. Schreiber L, Schönherr J (1993) Mobilities of organic compounds in reconstituted cuticular wax of barley leaves: determination of diffusion coefficients. Pestic Sci 38:353–361CrossRefGoogle Scholar
  44. Schreiber L, Skrabs M, Hartmann K, Becker D, Cassagne C, Lessire R (2000) Biochemical and molecular characterization of corn (Zea mays L.) root elongases. Biochem Soc Trans 28:647–649PubMedCrossRefGoogle Scholar
  45. Schreiber L, Franke R, Lessire R (2005) Biochemical characterization of elongase activity in corn (Zea mays L.) roots. Phytochemistry 66:131–138PubMedCrossRefGoogle Scholar
  46. Shanklin J, Whittle E, Fox BG (1994) Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase. Biochemistry 33:12787–12794PubMedCrossRefGoogle Scholar
  47. Shen L, Gong J, Caldo RA, Nettleton D, Cook D, Wise RP, Dickerson JA (2005) BarleyBase—an expression profiling database for plant genomics. Nucleic Acids Res 33:D614–D618PubMedCrossRefGoogle Scholar
  48. Sturaro M, Hartings H, Schmelzer E, Velasco R, Salamini F, Motto M (2005) Cloning and characterization of GLOSSY1, a maize gene involved in cuticle membrane and wax production. Plant Physiol 138:478–489PubMedCrossRefGoogle Scholar
  49. Suh MC, Samuels AL, Jetter R, Kunst L, Pollard M, Ohlrogge J, Beisson F (2005) Cuticular lipid composition, surface structure, and gene expression in Arabidospis stem epidermis. Plant Physiol 139:1649–1665PubMedCrossRefGoogle Scholar
  50. Tacke E, Korfhage C, Michel D, Maddaloni M, Motto M, Lanzani S, Salamini F, Doring H-P (1995) Transposon tagging of the maize Glossy2 locus with the transposable element En/Spm. Plant J 8:907–917PubMedGoogle Scholar
  51. Todd J, Post-Beittenmiller D, Jaworski JG (1999) KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase affecting wax biosynthesis in Arabodopsis thaliana. Plant J 17:119–130PubMedCrossRefGoogle Scholar
  52. Vogg G, Fischer S, Leide J, Emmanuel E, Jetter R, Levy AA, Riederer M (2004) Tomato fruit cuticular waxes and the effects on transpiration barrier properties: functional characterisation of a mutant deficient in a very-long-chain fatty acid β-ketoacyl-CoA synthase. J Exp Bot 55:1401–1410PubMedCrossRefGoogle Scholar
  53. von Wettstein-Knowles P (2007) Analyses of barley spike mutant waxes identify alkenes, cyclopropanes and internally branched alkanes with dominating isomers at carbon 9. Plant J 49:250–264CrossRefGoogle Scholar
  54. Xu X, Dietrich CR, Delledonne M, Xia Y, Wen T-J, Robertson DS, Nikolau BJ, Schnable PS (1997) Sequence analysis of the cloned glossy8 gene of maize suggests that it may encode for a β-ketoacyl reductase required for the biosynthesis of cuticular waxes. Plant Physiol 115:501–510PubMedCrossRefGoogle Scholar
  55. Xu X, Dietrich CR, Lessire R, Nikolau BJ, Schnable PS (2002) The endoplasmic reticulum-associated maize GL8 protein is a component of the acyl-coenzyme A elongase involved in the production of cuticular waxes. Plant Physiol 128:924–934PubMedCrossRefGoogle Scholar
  56. Yephremov A, Wisman E, Huijser P, Huijser C, Wellesen K, Saedler H (1999) Characterization of the FIDDLEHEAD gene of Arabidopsis reveals link between adhesion response and cell differentiation in the epidermis. Plant Cell 11:2187–2201PubMedCrossRefGoogle Scholar
  57. Zheng H, Rowland O, Kunst L (2005) Disruptions of the Arabidopsis enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis. Plant Cell 17:1467–1481PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Andrew Richardson
    • 1
    • 7
  • Alexandre Boscari
    • 1
    • 8
  • Lukas Schreiber
    • 2
  • Gerhard Kerstiens
    • 3
  • Mike Jarvis
    • 4
  • Pawel Herzyk
    • 5
  • Wieland Fricke
    • 1
    • 6
  1. 1.Division of Biological SciencesUniversity of PaisleyPaisleyScotland, UK
  2. 2.Department of Ecophysiology, IZMBUniversity of BonnBonnGermany
  3. 3.Department of Biological Sciences, Lancaster Environment CentreLancaster UniversityLancasterUK
  4. 4.Department of ChemistryUniversity of GlasgowGlasgowScotland, UK
  5. 5.Sir Henry Wellcome Functional Genomics Facility, Institute of Biomedical and Life SciencesUniversity of GlasgowGlasgowUK
  6. 6.UCD School of Biology and Environmental Science, UCD Science Centre WestUniversity College DublinDublin 4Ireland
  7. 7.MilliporeDundeeUK
  8. 8.Interactions Plantes Microorganismes et Santé VégétaleUMR INRA 1064/Université de Nice-Sophia Antipolis/CNRS 6192Sophia Antipolis CedexFrance

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