Applied Microbiology and Biotechnology

, Volume 99, Issue 15, pp 6405–6415 | Cite as

Extracellular esterases of phylloplane yeast Pseudozyma antarctica induce defect on cuticle layer structure and water-holding ability of plant leaves

  • Hirokazu Ueda
  • Ichiro Mitsuhara
  • Jun Tabata
  • Soichi Kugimiya
  • Takashi Watanabe
  • Ken Suzuki
  • Shigenobu Yoshida
  • Hiroko Kitamoto
Applied microbial and cell physiology


Aerial plant surface (phylloplane) is a primary key habitat for many microorganisms but is generally recognized as limited in nutrient resources. Pseudozyma antarctica, a nonpathogenic yeast, is commonly isolated from plant surfaces and characterized as an esterase producer with fatty acid assimilation ability. In order to elucidate the biological functions of these esterases, culture filtrate with high esterase activity (crude enzyme) of P. antarctica was applied onto leaves of tomato and Arabidopsis. These leaves showed a wilty phenotype, which is typically associated with water deficiency. Furthermore, we confirmed that crude enzyme-treated detached leaves clearly lost their water-holding ability. In treated leaves of both plants, genes associated to abscisic acid (ABA; a plant stress hormone responding osmotic stress) were activated and accumulation of ABA was confirmed in tomato plants. Microscopic observation of treated leaf surfaces revealed that cuticle layer covering the aerial epidermis of leaves became thinner. A gas chromatography-mass spectrometry (GC-MS) analysis exhibited that fatty acids with 16 and 18 carbon chains were released in larger amounts from treated leaf surfaces, indicating that the crude enzyme has ability to degrade lipid components of cuticle layer. Among the three esterases detected in the crude enzyme, lipase A, lipase B, and P. antarctica esterase (PaE), an in vitro enzyme assay using para-nitrophenyl palmitate as substrate demonstrated that PaE was the most responsible for the degradation. These results suggest that PaE has a potential role in the extraction of fatty acids from plant surfaces, making them available for the growth of phylloplane yeasts.


Pseudozyma antarctica Plant surface Cuticle layer Biodegradable plastic-degrading enzyme Drought stress response 



We thank D. H. Kaku for technical advise, Dr. E. Suto for the valuable comments, and Ms. X. Cao for technical assistance in this research. This research was financially supported by the National Institute for Agro-Environmental Sciences, Japan, and Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry.


  1. Abo M, Fujita Y, Itami R, Matsukura M (1992) Method for avoiding pitch troubles by use of thermostable lipase. WO1992013130Google Scholar
  2. Abuqamar S, Luo H, Laluk K, Mickelbart MV, Mengiste T (2009) Crosstalk between biotic and abiotic stress responses in tomato is mediated by the AIM1 transcription factor. Plant J 58(2):347–360. doi: 10.1111/j.1365-313X.2008.03783.x PubMedCrossRefGoogle Scholar
  3. Baldwin IT, Zhang Z-P, Diab N, Ohnmeiss TE, McCloud ES, Lynds GY, Schmelz EA (1997) Quantification, correlations and manipulations of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris. Planta 201:397–404. doi: 10.1007/s004250050082 CrossRefGoogle Scholar
  4. Bashi E, Fokkema NJ (1977) Environmental factors limiting growth of Sporobolomyces roseus, an antagonist of Cochliobolus sativus, on wheat leaves. Trans Br Mycol Soc 68(1):17–25. doi: 10.1016/S0007-1536(77)80146-0 CrossRefGoogle Scholar
  5. Bessire M, Chassot C, Jacquat AC, Humphry M, Borel S, Petetot JM, Metraux JP, Nawrath C (2007) A permeable cuticle in Arabidopsis leads to a strong resistance to Botrytis cinerea. EMBO J 26(8):2158–2168. doi: 10.1038/sj.emboj.7601658 PubMedCentralPubMedCrossRefGoogle Scholar
  6. Buda GJ, Isaacson T, Matas AJ, Paolillo DJ, Rose JK (2009) Three-dimensional imaging of plant cuticle architecture using confocal scanning laser microscopy. Plant J 60(2):378–385. doi: 10.1111/j.1365-313X.2009.03960.x PubMedCrossRefGoogle Scholar
  7. Buschhaus C, Jetter R (2012) Composition and physiological function of the wax layers coating Arabidopsis leaves: β-amyrin negatively affects the intracuticular water barrier. Plant Physiol 160(2):1120–1129. doi: 10.1104/pp. 112.198473
  8. Chassot C, Metraux J (2005) The cuticle as source of signals for plant defense. Plant Biosyst 139(1):28–31. doi: 10.1080/112635000500056344 CrossRefGoogle Scholar
  9. Chen S, Su LQ, Chen J, Wu J (2013) Cutinase: characteristics, preparation, and application. Biotechnol Adv 31(8):1754–1767. doi: 10.1016/j.biotechadv.2013.09.005 PubMedCrossRefGoogle Scholar
  10. Cominelli E, Galbiati M, Tonelli C (2008) Integration of water stress response: cell expansion and cuticle deposition in Arabidopsis thaliana. Plant Signal Behav 3(8):556–557. doi: 10.1111/j.1365-313X.2007.03310.x PubMedCentralPubMedCrossRefGoogle Scholar
  11. Finkelstein R (2013) Abscisic acid synthesis and response. The Arabidopsis Book 11:e0166. doi: 10.1199/tab.0166
  12. Fonseca ÁI (2006) Phylloplane yeasts. In: Rosa CAPG (ed) Biodiversity and ecophysiology of yeasts Springer. London, Berlin, pp 264–301Google Scholar
  13. Franke R, Briesen I, Wojciechowski T, Faust A, Yephremov A, Nawrath C, Schreiber L (2005) Apoplastic polyesters in Arabidopsis surface tissues—a typical suberin and a particular cutin. Phytochemistry 66(22):2643–2658. doi: 10.1016/j.phytochem.2005.09.027 PubMedCrossRefGoogle Scholar
  14. Holm HC, Nielsen PM (2012) Esterification process. WO2012130961Google Scholar
  15. Ichihara K, Fukubayashi Y (2009) Preparation of fatty acid methyl esters for gas-liquid chromatography. J Lipid Res 51(3):635–640. doi: 10.1194/jlr.D001065 PubMedCrossRefGoogle Scholar
  16. Isaacson T, Kosma DK, Matas AJ, Buda GJ, He Y, Yu B, Pravitasari A, Batteas JD, Stark RE, Jenks MA, Rose JK (2009) Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss. Plant J 60(2):363–377. doi: 10.1111/j.1365-313X.2009.03969.x PubMedCrossRefGoogle Scholar
  17. Ishii M (1988) Positionally non-specific lipase from Candida sp., a method for producing it, its use and a recombinant DNA process for produce it. WO 88/02775Google Scholar
  18. Iuchi S, Kobayashi M, Taji T, Naramoto M, Seki M, Kato T, Tabata S, Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K (2001) Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J 27(4):325–333. doi: 10.1046/j.1365-313x.2001.01096.x PubMedCrossRefGoogle Scholar
  19. Karpushova A, Brummer F, Barth S, Lange S, Schmid RD (2005) Cloning, recombinant expression and biochemical characterisation of novel esterases from Bacillus sp. associated with the marine sponge Aplysina aerophoba. Appl Microbiol Biotechnol 67(1):59–69. doi: 10.1007/s00253-004-1780-6 PubMedCrossRefGoogle Scholar
  20. Kitamoto HK, Shinozaki Y, Cao XH, Morita T, Konishi M, Tago K, Kajiwara H, Koitabashi M, Yoshida S, Watanabe T, Sameshima-Yamashita Y, Nakajima-Kambe T, Tsushima S (2011) Phyllosphere yeasts rapidly break down biodegradable plastics. AMB Express 1:44. doi: 10.1186/2191-0855-1-44 PubMedCentralPubMedCrossRefGoogle Scholar
  21. Kmieciak M, Simpson CG, Lewandowska D, Brown JW, Jarmolowski A (2002) Cloning and characterization of two subunits of Arabidopsis thaliana nuclear cap-binding complex. Gene 283(1–2):171–183. doi: 10.1016/S0378-1119(01)00859-9 PubMedCrossRefGoogle Scholar
  22. Kolattukudy PE (1980) Biopolyester membranes of plants: cutin and suberin. Science 208(4447):990–1000. doi: 10.1126/science.208.4447.990 PubMedCrossRefGoogle Scholar
  23. Kurtzman C, Fell J, Boekhout T (2011) The yeasts: a taxonomic study, 5th ed./edited by Cletus P. Kurtzman, Jack W. Fell, and Teun Boekhout edn. Elsevier, New York: OxfordGoogle Scholar
  24. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685. doi: 10.1038/227680a0 PubMedCrossRefGoogle Scholar
  25. L'Haridon F, Besson-Bard A, Binda M, Serrano M, Abou-Mansour E, Balet F, Schoonbeek HJ, Hess S, Mir R, Leon J, Lamotte O, Metraux JP (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunity. PLoS Pathog 7(7):e1002148. doi: 10.1371/journal.ppat.1002148 PubMedCentralPubMedCrossRefGoogle Scholar
  26. Lindow SE, Brandl MT (2003) Microbiology of the phyllosphere. Appl Environ Microbiol 69(4):1875–1883. doi: 10.1128/AEM. 69.4.1875-1883.2003 PubMedCentralPubMedCrossRefGoogle Scholar
  27. Nadakuduti SS, Pollard M, Kosma DK, Allen C Jr, Ohlrogge JB, Barry CS (2012) Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato. Plant Physiol 159(3):945–960. doi: 10.1104/pp. 112.198374 PubMedCentralPubMedCrossRefGoogle Scholar
  28. Nielsen TB, Ishii M, Kirke O (1999) Lipases A and B from the yeast Candida antarctica. In: Margesin RaS F (ed) Biotechnological applications of cold-adapted organisms. Springer, Berlin Heidelberg, pp 49–61CrossRefGoogle Scholar
  29. Nitsch LM, Oplaat C, Feron R, Ma Q, Wolters-Arts M, Hedden P, Mariani C, Vriezen WH (2009) Abscisic acid levels in tomato ovaries are regulated by LeNCED1 and SlCYP707A1. Planta 229(6):1335–1346. doi: 10.1007/s00425-009-0913-7 PubMedCrossRefGoogle Scholar
  30. Park JJ, Jin P, Yoon J, Yang JI, Jeong HJ, Ranathunge K, Schreiber L, Franke R, Lee IJ, An G (2010) Mutation in Wilted Dwarf and Lethal 1 (WDL1) causes abnormal cuticle formation and rapid water loss in rice. Plant Mol Biol 74(1–2):91–103. doi: 10.1007/s11103-010-9656-x PubMedCrossRefGoogle Scholar
  31. Patkar S, Bjorkling F, Zundell M, Schulein M, Svendsen A, Hansen HH, Gormsen E (1993) Purification of two lipases from Candida antarctica and their inhibition by various inhibitors. Indian J Chem 32B:76–80Google Scholar
  32. Riederer M, Schonherr J (1986) Thermodynamic analysis of nonelectrolyte sorption in plant cuticles: the effects of concentration and temperature on sorption of 4-nitrophenol. Planta 169(1):69–80. doi: 10.1007/BF01369777 PubMedCrossRefGoogle Scholar
  33. Saito S, Hirai N, Matsumoto C, Ohigashi H, Ohta D, Sakata K, Mizutani M (2004) Arabidopsis CYP707As encode (+)-abscisic acid 8′-hydroxylase, a key enzyme in the oxidative catabolism of abscisic acid. Plant Physiol 134(4):1439–1449. doi: 10.1104/pp. 103.037614 PubMedCentralPubMedCrossRefGoogle Scholar
  34. Schonherr J (1976) Water permeability of isolated cuticular membranes: the effect of pH and cations on diffusion, hydrodynamic permeability and size of polar pores in the cutin matrix. Planta 128(2):113–126. doi: 10.1007/BF00390312 PubMedCrossRefGoogle Scholar
  35. Seo S, Okamoto M, Seto H, Ishizuka K, Sano H, Ohashi Y (1995) Tobacco MAP kinase: a possible mediator in wound signal transduction pathways. Science 270(5244):1988–1992. doi: 10.1126/science.270.5244.1988 PubMedCrossRefGoogle Scholar
  36. Shinozaki Y, Morita T, Cao XH, Yoshida S, Koitabashi M, Watanabe T, Suzuki K, Sameshima-Yamashita Y, Nakajima-Kambe T, Fujii T, Kitamoto HK (2013) Biodegradable plastic-degrading enzyme from Pseudozyma antarctica: cloning, sequencing, and characterization. Appl Microbiol Biotechnol 97(7):2951–2959. doi: 10.1007/s00253-012-4188-8 PubMedCrossRefGoogle Scholar
  37. Sieber P, Schorderet M, Ryser U, Buchala A, Kolattukudy P, Metraux JP, Nawrath C (2000) Transgenic Arabidopsis plants expressing a fungal cutinase show alterations in the structure and properties of the cuticle and postgenital organ fusions. Plant Cell 12(5):721–737. doi: 10.1105/Tpc.12.5.721 PubMedCentralPubMedCrossRefGoogle Scholar
  38. Tabata J, Narai Y, Sawamura N, Hiradate S, Sugie H (2012) A new class of mealybug pheromones: a hemiterpene ester in the sex pheromone of Crisicoccus matsumotoi. Naturwissenschaften 99(7):567–574. doi: 10.1007/s00114-012-0935-z PubMedCrossRefGoogle Scholar
  39. Watanabe T, Shinozaki Y, Yoshida S, Koitabashi M, Sameshima-Yamashita Y, Fujii T, Fukuoka T, Kitamoto HK (2014) Xylose induces the phyllosphere yeast Pseudozyma antarctica to produce a cutinase-like enzyme which efficiently degrades biodegradable plastics. J Biosci Bioeng 117(3):325–329. doi: 10.1016/j.jbiosc.2013.09.002 PubMedCrossRefGoogle Scholar
  40. Yeats TH, Rose JK (2013) The formation and function of plant cuticles. Plant Physiol 163(1):5–20. doi: 10.1104/pp. 113.222737 PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Hirokazu Ueda
    • 1
  • Ichiro Mitsuhara
    • 2
  • Jun Tabata
    • 1
  • Soichi Kugimiya
    • 1
  • Takashi Watanabe
    • 1
  • Ken Suzuki
    • 1
  • Shigenobu Yoshida
    • 1
  • Hiroko Kitamoto
    • 1
  1. 1.National Institute for Agro-Environmental Sciences (NIAES)TsukubaJapan
  2. 2.National Institute of Agrobiological Sciences (NIAS)TsukubaJapan

Personalised recommendations