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Planta

, Volume 228, Issue 4, pp 675–685 | Cite as

Wax Crystal-Sparse Leaf1 encodes a β–ketoacyl CoA synthase involved in biosynthesis of cuticular waxes on rice leaf

  • Dongmei Yu
  • Kosala Ranathunge
  • Huasun Huang
  • Zhongyou Pei
  • Rochus Franke
  • Lukas Schreiber
  • Chaozu HeEmail author
Original Article

Abstract

Cuticular waxes, forming the plant/atmosphere interface of plants colonizing the terrestrial environment, are complex mixtures of very-long chain fatty acids (VLCFAs) and their derivatives. In VLCFAs biosynthesis, β-ketoacyl CoA synthase (E.C.2.3.1.119, KCS) is the key enzyme. Using T-DNA insertional mutagenesis, we identified a cuticle-deficient rice mutant, which displayed a pleiotropic phenotype including reduced growth, leaf fusion, sparse wax crystals, enhanced sensitivity to drought and low fertility. Further analysis indicated that T-DNA was inserted in the 5′-UTR intron of the affected gene, Wax Crystal-Sparse Leaf1 (WSL1), and abnormal transcript caused the loss-of-function of WSL1 gene. Genetic complementation experiment confirmed the function of the candidate gene. WSL1 was predicted to encode a polypeptide containing a conserved FAE1_CUT1_RppA domain typical of the KCS family proteins. Qualitative and quantitative wax composition analyses by gas chromatography–mass spectrometry (GC–MS) demonstrated a marked reduction of total cuticular wax load on wsl1 leaf blades and sheaths, and VLCFA precursors of C20–C24 decreased in both. Moreover, ubiquitous expression of the WSL1 gene gave a hint that WSL1-catalyzed elongation of VLCFAs might participate in a wide range of rice growth and development processes beyond biosynthesis of cuticular waxes.

Keywords

Cuticular waxes β-Ketoacyl CoA synthase Rice (Oryza sativa L.) T-DNA insertional mutagenesis Wax Crystal-Sparse Leaf1 

Abbreviations

VLCFA

Very-long chain fatty acid (>C18)

KCS

β-Ketoacyl CoA synthase

SEM

Scanning electron microscopy

TEM

Transmission electron microscopy

GC–MS

Gas chromatography–mass spectrometry

TAIL-PCR

Thermal asymmetric interlaced PCR

Notes

Acknowledgments

This work was supported by the Basic Research Program of Ministry of Science and Technology of China (Grant 2006CB101900) and by the Alexander-von-Humboldt-Stiftung (Postdoc grant awarded to K. R).

Supplementary material

425_2008_770_MOESM1_ESM.tif (1.9 mb)
Fig. S1 Morphology of the complemented line HC-8, wild-type (wt) and wsl1 plants. The plant size and vigor of HC-8 was similar to those of the wild-type (TIFF 1,907 kb)
425_2008_770_MOESM2_ESM.tif (1.2 mb)
Fig. S2 Multiple alignment of WSL1 and six well-characterized KCS family members in Arabidopsis. Arrowheads indicated the conserved Cys223, His391 and Asn424 in all KCS family members (TIFF 1,253 kb)
425_2008_770_MOESM3_ESM.tif (1.5 mb)
Fig. S3 Composition of the cutin on leaves of wild-type, wsl1 and the complemented line HC-8. No significant difference was found. Cutin was analyzed as described in Jung et al. (2006) (TIFF 1,498 kb)

References

  1. Blacklock BJ, Jaworski JG (2006) Substrate specificity of Arabidopsis 3-ketoacyl-CoA synthases. Biochem Biophys Res Commun 346:583–590PubMedCrossRefGoogle Scholar
  2. 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
  3. Chung BYW, Simons C, Firth AE, Brown CM, Hellens RP (2006) Effect of 5′-UTR introns on gene expression in Arabidopsis thaliana. BMC Genomics 7:120PubMedCrossRefGoogle Scholar
  4. Dietrich CR, Perera MA, Yandeau-Nelson MD, Meeley RB, Nikolau BJ, Schnable PS (2005) Characterization of two 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
  5. Dunn TM, Lynch DV, Michaelson LV, Napier JA (2004) A post-genomic approach to understanding sphingolipid metabolism in Arabidopsis thaliana. Ann Bot 93:483–497PubMedCrossRefGoogle Scholar
  6. Ecker R, Yaniv Z (1993) Genetic control of fatty acid composition in seed oil of Sinapis alba L. Euphytica 69:45–49CrossRefGoogle Scholar
  7. Fehling E, Mukherjee KD (1991) Acyl-CoA elongase from a higher plant (Lunaria annua): metabolic intermediates of very-long-chain acyl-CoA products and substrate specificity. BBA-Lipids Lipid Metab 1082:239–246CrossRefGoogle Scholar
  8. Fiebig A, Mayfield JA, Miley NL, Chau S, Fischer RL, Preuss D (2000) Alterations in CER6, a gene identical to CUT1, differentially affect long-chain lipid content on the surface of pollen and stems. Plant Cell 12:2001–2008PubMedCrossRefGoogle Scholar
  9. Franke R, Schreiber L (2007) Suberin—a biopolyester forming apoplastic plant interfaces. Curr Opin Plant Biol 10:252–259PubMedCrossRefGoogle Scholar
  10. Ghanevati M, Jaworski JG (2001) Active-site residues of a plant membrane-bound fatty acid elongase β-ketoacyl-CoA synthase, FAE1 KCS. BBA-Lipids Lipid Meta 1530:77–85Google Scholar
  11. Ghanevati M, Jaworski JG (2002) Engineering and mechanistic studies of the Arabidopsis FAE1 β-ketoacyl-CoA synthase, FAE1 KCS. Eur J Biochem 269:3531–3539PubMedCrossRefGoogle Scholar
  12. Graca J, Pereira H (2000) Methanolysis of bark suberins: analysis of glycerol and acid monomers. Phytochem Anal 11:45–51CrossRefGoogle Scholar
  13. Grant L (1987) Diffuse and specular characteristics of leaf reflectance. Remote Sens Environ 22:309–322CrossRefGoogle Scholar
  14. Gray JE, Holroyd GH, van der Lee FM, Bahrami AR, Sijmons PC, Woodward FI, Schuch W, Hetherington AM (2000) The HIC signalling pathway links CO2 perception to stomatal development. Nature 408:713–716PubMedCrossRefGoogle Scholar
  15. Gülz PG, Markstädter C, Riederer M (1994) Isomeric alkyl esters in Quercus robur leaf cuticular wax. Phytochemistry 35:79–81CrossRefGoogle Scholar
  16. Haas K, Brune T, Rücker E (2001) Epicuticular wax crystalloids in rice and sugar cane leaves are reinforced by polymeric aldehydes. J Appl Bot 75:178–187Google Scholar
  17. Han JX, Luhs W, Sonntag K, Zahringer U, Borchardt DS, Wolter FP, Heinz E, Frentzen M (2001) Functional characterization of β-ketoacyl-CoA synthase genes from Brassica napus L. Plant Mol Biol 46:229–239PubMedCrossRefGoogle Scholar
  18. Hansen JD, Pyee J, Xia Y, Wen TJ, 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. Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282PubMedCrossRefGoogle Scholar
  20. James DW, Lim E, Keller J, Plooy I, Ralston E, Dooner HK (1995) Directed tagging of the Arabidopsis FATTY-ACID ELONGATION1 (FAE1) gene with the maize transposon Activator. Plant Cell 7:309–319PubMedCrossRefGoogle Scholar
  21. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  22. Jenks MA, Rich PJ, Peters PJ, Axtell JD, Ashworth EN (1992) Epicuticular wax morphology of bloomless (bm) mutants in Sorghum bicolor. Int J Plant Sci 153:311–319CrossRefGoogle Scholar
  23. 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
  24. Kolattukudy PE (1966) Biosynthesis of wax in Brassica oleracea. Relation of fatty acids to wax. Biochemistry 5:2265–2275PubMedCrossRefGoogle Scholar
  25. Kumar S, Sridhar R (1987) Significance of epicuticular wax in the specificity of blast fungus to rice varieties. Int J Tropical Plant 5:131–139Google Scholar
  26. Kunst L, Clemens S (2001) Plant long chain fatty acid biosynthetic enzyme. Patent Cooperation Treaty Int Patent Appl WO 0107586Google Scholar
  27. Kunst L, Samuels AL (2003) Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res 42:51–80PubMedCrossRefGoogle Scholar
  28. Lassner MW, Lardizabal K, Metz JG (1996) A jojoba β-ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants. Plant Cell 8:281–292PubMedCrossRefGoogle Scholar
  29. Lessire R, Bessoule J-J, Cassagne C (1989) Involvement of a β-ketoacyl-CoA intermediate in acyl-CoA elongation by an acyl-CoA elongase purified from leek epidermal cells. BBA-Lipids Lipid Meta 1006:35–40Google Scholar
  30. Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8:457–463PubMedCrossRefGoogle Scholar
  31. Lolle SJ, Cheung AY, Sussex IM (1992) Fiddlehead: an Arabidopsis mutant constitutively expressing an organ fusion program that involves interactions between epidermal cells. Dev Biol 152:383–392PubMedCrossRefGoogle Scholar
  32. 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
  33. Miyamoto N, Steudle E, Hirasawa T, Lafitte R (2001) Hydraulic conductivity of rice roots. J Exp Bot 52:1835–1846PubMedCrossRefGoogle Scholar
  34. Moon H, Smith MA, Kunst L (2001) A condensing enzyme from the seeds of Lesquerella fendleri that specifically elongates hydroxy fatty acids. Plant Physiol 127:1635–1643PubMedCrossRefGoogle Scholar
  35. Moon H, Chowrira G, Rowland O, Blacklock BJ, Smith MA, Kunst L (2004) A root-specific condensing enzyme from Lesquerella fendleri that elongates very-long-chain saturated fatty acids. Plant Mol Biol 56:917–927PubMedCrossRefGoogle Scholar
  36. Moose SP, Sisco PH (1996) Glossy15, an APETALA2-like gene from maize that regulates leaf epidermal cell identity. Gene Dev 10:3018–3027PubMedCrossRefGoogle Scholar
  37. O’Toole JC, Cruz RT, Seiber JN (1979) Epicuticular wax and cuticular resistance in rice. Physiol Plant 47:239–244CrossRefGoogle Scholar
  38. Ohlrogge JB, Jaworski JG, Post-Beittenmiller D (1993) De novo fatty acid biosynthesis. In: Moore TS (ed) Lipid metabolism in plants. CRC Press, Boca Raton, pp 3–32Google Scholar
  39. Oxley D, Bacic A (1999) Structure of the glycosylphosphatidylinositol anchor of an arabinogalactan protein from Pyrus communis suspension-cultured cells. Proc Natl Acad Sci USA 96:14246–14251PubMedCrossRefGoogle Scholar
  40. Post-Beittenmiller D (1996) Biochemistry and molecular biology of wax production in plants. Annu Rev Plant Physiol Plant Mol Biol 47:405–430PubMedCrossRefGoogle Scholar
  41. Pruitt RE, Vielle-Calzada JP, Ploense SE, Grossniklaus U, Lolle SJ (2000) FIDDLEHEAD, a gene required to suppress epidermal cell interactions in Arabidopsis, encodes a putative lipid biosynthetic enzyme. Proc Natl Acad Sci USA 97:1311–1316PubMedCrossRefGoogle Scholar
  42. Qin YM, Pujol FM, Hu CY, Feng JX, Kastaniotis AJ, Hiltunen JK, Zhu YX (2007) Genetic and biochemical studies in yeast reveal that the cotton fibre-specific GhCER6 gene functions in fatty acid elongation. J Exp Bot 58:473–481PubMedCrossRefGoogle Scholar
  43. 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 T 28:647–649CrossRefGoogle Scholar
  44. Schreiber L, Franke R, Lessire R (2005) Biochemical characterization of elongase activity in corn (Zea mays L.) roots. Phytochemistry 66:131–138PubMedCrossRefGoogle Scholar
  45. Sha Y, Li S, Pei Z, Luo L, Tian Y, He C (2004) Generation and flanking sequence analysis of a rice T-DNA tagged population. Theor Appl Genet 108:306–314PubMedCrossRefGoogle Scholar
  46. Tacke E, Korfhage C, Michel D, Maddaloni M, Motto M, Lanzini 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
  47. Taleisnik E, Peyrano G, Cordoba A, Arias C (1999) Water retention capacity in root segments differing in the degree of exodermis development. Ann Bot 83:19–27CrossRefGoogle Scholar
  48. Todd J, Post-Beittenmiller D, Jaworski JG (1999) KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase affecting wax biosynthesis in Arabidopsis thaliana. Plant J 17:119–130PubMedCrossRefGoogle Scholar
  49. Vogg G, Fischer S, Leide J, Emmanuel E, Jetter R, Levy AA, Riederer M (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. J Exp Bot 55:1401–1410PubMedCrossRefGoogle Scholar
  50. von Wettstein-Knowles PM (1982) Elongase and epicuticular wax biosynthesis. Physiol Veg 20:797–809Google Scholar
  51. Woodhead S, Padgham DE (1988) The effect of plant surface characteristics on resistance of rice to the brown planthopper Nilaparvata lugens. Entomol Exp Appl 47:15–22CrossRefGoogle Scholar
  52. Xu XJ, Dietrich CR, Delledonne M, Xia YJ, Wen TJ, Robertson DS, Nikolau BJ, Schnable PS (1997) Sequence analysis of the cloned glossy8 gene of maize suggests that it may code for a beta-ketoacyl reductase required for the biosynthesis of cuticular waxes. Plant Physiol 115:501–510PubMedCrossRefGoogle Scholar
  53. Yephremov A, Wisman E, Huijser P, Huijser C, Wellesen K, Saedler H (1999) Characterization of the FIDDLEHEAD gene of Arabidopsis reveals a link between adhesion response and cell differentiation in the epidermis. Plant Cell 11:2187–2201PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Dongmei Yu
    • 1
    • 2
  • Kosala Ranathunge
    • 3
  • Huasun Huang
    • 4
  • Zhongyou Pei
    • 1
  • Rochus Franke
    • 3
  • Lukas Schreiber
    • 3
  • Chaozu He
    • 1
    Email author
  1. 1.State Key Laboratory of Plant Genomics, Institute of MicrobiologyChinese Academy of SciencesBeijingPeople’s Republic of China
  2. 2.Graduate School of the Chinese Academy of SciencesBeijingPeople’s Republic of China
  3. 3.Institute of Cellular and Molecular Botany, Department of EcophysiologyUniversity of BonnBonnGermany
  4. 4.Rubber Research InstituteChinese Academy of Tropical Agricultural SciencesHainanPeople’s Republic of China

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