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Changes in cuticular wax coverage and composition on developing Arabidopsis leaves are influenced by wax biosynthesis gene expression levels and trichome density

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Abstract

Main conclusion

Wax coverage on developing Arabidopsis leaf epidermis cells is constant and thus synchronized with cell expansion. Wax composition shifts from fatty acid to alkane dominance, mediated by CER6 expression.

Epidermal cells bear a wax-sealed cuticle to hinder transpirational water loss. The amount and composition of the cuticular wax mixture may change as organs develop, to optimize the cuticle for specific functions during growth. Here, morphometrics, wax chemical profiling, and gene expression measurements were integrated to study developing Arabidopsis thaliana leaves and, thus, further our understanding of cuticular wax ontogeny. Before 5 days of age, cells at the leaf tip ceased dividing and began to expand, while cells at the leaf base switched from cycling to expansion at day 13, generating a cell age gradient along the leaf. We used this spatial age distribution together with leaves of different ages to determine that, as leaves developed, their wax compositions shifted from C24/C26 to C30/C32 and from fatty acid to alkane constituents. These compositional changes paralleled an increase in the expression of the elongase enzyme CER6 but not of alkane pathway enzymes, suggesting that CER6 transcriptional regulation is responsible for both chemical shifts. Leaves bore constant numbers of trichomes between 5 and 21 days of age and, thus, trichome density was higher on young leaves. During this time span, leaves of the trichome-less gl1 mutant had constant wax coverage, while wild-type leaf coverage was initially high and then decreased, suggesting that high trichome density leads to greater apparent coverage on young leaves. Conversely, wax coverage on pavement cells remained constant over time, indicating that wax accumulation is synchronized with cell expansion throughout leaf development.

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Abbreviations

CER:

ECERIFERUM

FAE:

Fatty acid elongase

KCS:

3-Ketoacyl-CoA synthase

References

  • Andriankaja M, Dhondt S, De Bodt S, Vanhaeren H, Coppens F, De Milde L, Mühlenbock P, Skirycz A, Gonzalez N, Beemster GTS et al (2012) Exit from proliferation during leaf development in Arabidopsis thaliana: a not-so-gradual process. Dev Cell 22:64–78

    Article  CAS  PubMed  Google Scholar 

  • Atkin DSJ, Hamilton RJ (1950) The changes with age in the epicuticular wax of Sorghum bicolor. J Nat Prod 45:697–703

    Article  Google Scholar 

  • Bazzaz FA, Chiariello NR, Coley PD, Pitelka LF (2016) Allocating resources to reproduction and defense. Bioscience 37:58–67

    Article  Google Scholar 

  • Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B 57:289–300

    Google Scholar 

  • Bennett RN, Wallsgrove RM (1994) Secondary metabolites in plant defence mechanisms. New Phytol 127:617–633

    Article  CAS  Google Scholar 

  • Bernard A, Domergue F, Pascal S, Jetter R, Renne C, Faure J-D, Haslam RP, Napier JA, Lessire R, Joubès J (2012) Reconstitution of plant alkane biosynthesis in yeast demonstrates that Arabidopsis ECERIFERUM1 and ECERIFERUM3 are core components of a very-long-chain alkane synthesis complex. Plant Cell 24:3106–3118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bourdenx B, Bernard A, Domergue F, Pascal S, Léger A, Roby D, Pervent M, Vile D, Haslam RP, Napier JA et al (2011) Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses. Plant Physiol 156:29–45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bringe K, Schumacher CFA, Schmitz-Eiberger M, Steiner U, Oerke E-C (2006) Ontogenetic variation in chemical and physical characteristics of adaxial apple leaf surfaces. Phytochemistry 67:161–170

    Article  CAS  PubMed  Google Scholar 

  • 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 160:1120–1129

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Byrne ME (2005) Networks in leaf development. Curr Opin Plant Biol 8:59–66

    Article  PubMed  Google Scholar 

  • Chen X, Wang H, Li J, Huang H, Xu L (2013) Quantitative control of ASYMMETRIC LEAVES2 expression is critical for leaf axial patterning in Arabidopsis. J Exp Bot 64:4895–4905

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chu C-C, Freeman TP, Buckner JS, Henneberry TJ, Nelson DR, Natwick ET (2001) Susceptibility of upland cotton cultivars to Bemisia tabaci Biotype B (Homoptera: Aleyrodidae) in relation to leaf age and trichome density. Ann Entomol Soc Am 94:743–749

    Article  Google Scholar 

  • Coley PD, Bryant JP, Chapin SFI (1985) Resource availability and plant antiherbivore defense. Science 230:895–899

    Article  CAS  PubMed  Google Scholar 

  • Donnelly PM, Bonetta D, Tsukaya H, Dengler RE, Dengler NG (1999) Cell cycling and cell enlargement in developing leaves of Arabidopsis. Dev Biol 215:407–419

    Article  CAS  PubMed  Google Scholar 

  • Efroni I, Eshed Y, Lifschitz E (2010) Morphogenesis of simple and compound leaves: a critical review. Plant Cell 22:1019–1032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Esch JJ, Chen M, Sanders M, Hillestad M, Ndkium S, Idelkope B, Neizer J, Marks MD (2003) A contradictory GLABRA3 allele helps define gene interactions controlling trichome development in Arabidopsis. Development 130:5885–5894

    Article  CAS  PubMed  Google Scholar 

  • Fabre G, Mazurek S, Daraspe J, Mucciolo A, Sankar M, Humbel BM, Nawrath C (2016) The ABCG transporter PEC1/ABCG32 is required for the formation of the developing leaf cuticle in Arabidopsis. New Phytol 209:192–201

    Article  CAS  PubMed  Google Scholar 

  • Fehling E, Mukherjee K (1991) Acyl-CoA elongase from a higher plant (Lunaria annua): metabolic intermediates of very-long-chain acyl-CoA products and substrate specificity. Biochem Biophys Acta 1082:239–246

    Article  CAS  PubMed  Google Scholar 

  • 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 12:2001–2008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fraenkel G (1959) The raison d’etre of secondary plant substances. Science 129:1466–1470

    Article  CAS  PubMed  Google Scholar 

  • Glover BJ (2000) Differentiation in plant epidermal cells. J Exp Bot 51:497–505

    Article  CAS  PubMed  Google Scholar 

  • Granier C, Massonnet C, Turc O, Muller B, Chenu K, Tardieu F (2002) Individual leaf development in Arabidopsis thaliana: a stable thermal-time-based programme. Ann Bot 89:595–604

    Article  PubMed  PubMed Central  Google Scholar 

  • 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 408:713–716

    Article  CAS  PubMed  Google Scholar 

  • Guimil S, Dunand C (2007) Cell growth and differentiation in Arabidopsis epidermal cells. J Exp Bot 58:3829–3840

    Article  CAS  PubMed  Google Scholar 

  • Gülz P-G, Prasad RBN, Müller E (1992) Surface structures and chemical composition of epicuticular waxes during leaf development of Fagus sylvatica L. Z Naturforsch 47c:190–196

    Google Scholar 

  • Haas K, Schönherr J (1979) Composition of soluble cuticular lipids and water permeability of cuticular membranes from Citrus leaves. Planta 146:399–403

    Article  CAS  PubMed  Google Scholar 

  • Haslam TM, Mañas Fernández A, Zhao L, Kunst L (2012) Arabidopsis ECERIFERUM2 is a component of the fatty acid elongation machinery required for fatty acid extension to exceptional lengths. Plant Physiol 160:1164–1174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Haslam TM, Haslam R, Thoraval D, Pascal S, Delude C, Domergue F, Fernández AM, Beaudoin F, Napier JA, Kunst L et al (2015) CER2-LIKE proteins have unique biochemical and physiological functions in very-long-chain fatty acid elongation. Plant Physiol 167:682–692

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Herms DA, Mattson WJ (2016) The dilemma of plants: to grow or defend. Q Rev Biol 67:283–335

    Article  Google Scholar 

  • Hooker TS, Millar AA, Kunst L (2002) Significance of the expression of the CER6 condensing enzyme for cuticular wax production in Arabidopsis. Plant Physiol 129:1568–1580

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Isaacson T, Kosma DK, Matas AJ, Buda GJ, He Y, Yu B, Pravitasari A, Batteas JD, Stark RE, Jenks MA 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 J 60:363–377

    Article  CAS  PubMed  Google Scholar 

  • Iwakawa H, Iwasaki M, Kojima S, Ueno Y, Soma T, Tanaka H, Semiarti E, Machida Y, Machida C (2007) Expression of the ASYMMETRIC LEAVES2 gene in the adaxial domain of Arabidopsis leaves represses cell proliferation in this domain and is critical for the development of properly expanded leaves. Plant J 51:173–184

    Article  CAS  PubMed  Google Scholar 

  • Jakoby MJ, Falkenhan D, Mader MT, Brininstool G, Wischnitzki E, Platz N, Hudson A, Hülskamp M, Larkin J, Schnittger A (2008) Transcriptional profiling of mature Arabidopsis trichomes reveals that NOECK encodes the MIXT-like transcriptional regulator MYB106. Plant Physiol 148:1538–1602

    Article  Google Scholar 

  • Jenks MA, Tuttle HA, Eigenbrode SD, Feldmann KA (1995) Leaf epicuticular waxes of the eceriferum mutants in Arabidopsis. Plant Physiol 108:369–377

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jenks MA, Tuttle HA, Feldmann KA (1996) Changes in epicuticular waxes on wildtype and ECERIFERUM mutants in Arabidopsis during development. Phytochemistry 42:29–34

    Article  CAS  Google Scholar 

  • Jetter R, Schäffer S (2001) Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development. Plant Physiol 126:1725–1737

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Joubès J, Raffaele S, Bourdenx B, Garcia C, Laroche-Traineau J, Moreau P, Domergue F, Lessire R (2008) The VLCFA elongase gene family in Arabidopsis thaliana: phylogenetic analysis, 3D modelling and expression profiling. Plant Mol Biol 67:547–566

    Article  PubMed  Google Scholar 

  • Kearns EV, Assmann SM (1993) The guard cell-environment connection. Plant Physiol 102:711–715

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kim M-S, Shim K-B, Park S-H, Kim K-S (2009) Changes in cuticular waxes of developing leaves in sesame (Sesamum indicum L.). J Crop Sci Biotechnol 12:161–167

    Article  Google Scholar 

  • Kim J, Jung JH, Lee SB, Go YS, Kim HJ, Cahoon R, Cahoon EB, Markham JE, Suh MC (2013) Arabidopsis 3-ketoacyl-CoA synthase 9 is involved in the synthesis of tetracosanoic acids as precursors of cuticular waxes, suberins, sphingolipids, and phospholipids. Plant Physiol 162:567–580

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lai C, Kunst L, Jetter R (2007) Composition of alkyl esters in the cuticular wax on inflorescence stems of Arabidopsis thaliana cer mutants. Plant J 50:189–196

    Article  CAS  PubMed  Google Scholar 

  • Larkin JC, Brown ML, Schiefelbein J (2003) How do cells know what they want to be when they grow up? Lessons from epidermal patterning in Arabidopsis. Annu Rev Plant Biol 54:403–430

    Article  CAS  PubMed  Google Scholar 

  • 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 Physiol 144:1667–1679

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li F, Wu X, Lam P, Bird D, Zheng H, Samuels L, Jetter R, Kunst L (2008) Identification of the wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase WSD1 required for stem wax ester biosynthesis in Arabidopsis. Plant Physiol 148:97–107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lu S, Song T, Kosma DK, Parsons EP, Rowland O, Jenks MA (2009) Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis. Plant J 59:553–564

    Article  CAS  PubMed  Google Scholar 

  • Marks MD (1997) Molecular genetic analysis of trichome development in Arabidopsis. Annu Rev Plant Physiol Plant Mol Biol 48:137–163

    Article  CAS  PubMed  Google Scholar 

  • Marks MD, Wenger JP, Gilding E, Jilk R, Dixon RA (2009) Transcriptome analysis of Arabidopsis wild-type and gl3–sst sim trichomes identifies four additional genes required for trichome development. Mol Plant 2:803–822

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mauricio R (2005) Ontogenetics of QTL: The genetic architecture of trichome density over time in Arabidopsis thaliana. Genetica 123:75–85

    Article  PubMed  Google Scholar 

  • Millar A, Kunst L (1997) Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme. Plant J 12:121–131

    Article  CAS  PubMed  Google Scholar 

  • Nath U, Crawford BCW, Carpenter R, Coen E (2003) Genetic control of surface curvature. Science 299:1404–1407

    Article  CAS  PubMed  Google Scholar 

  • Ni Y, Guo Y-J, Wang J, Xia R-E, Wang X-Q, Ash G, Li J-N (2013) Responses of physiological indexes and leaf epicuticular waxes of Brassica napus to Sclerotinia sclerotiorum infection. Plant Pathol 63:174–184

    Article  Google Scholar 

  • Pascal S, Bernard A, Sorel M, Pervent M, Vile D, Haslam RP, Napier JA, Lessire R, Domergue F, Joubès J (2013) The Arabidopsis cer26 mutant, like the cer2 mutant, is specifically affected in the very-long-chain fatty acid elongation process. Plant J 73:733–746

    Article  CAS  PubMed  Google Scholar 

  • Peschel S, Franke R, Schreiber L, Knoche M (2007) Composition of the cuticle of developing sweet cherry fruit. Phytochemistry 68:1017–1025

    Article  CAS  PubMed  Google Scholar 

  • Pfaffl M (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Prasad RBN, Gülz P (1990) Developmental and seasonal variations in the epicuticular waxes of beech leaves (Fagus sylvatica L.). Zeitschrift für Naturforsch C 45:805–812

    CAS  Google Scholar 

  • R Core Team (2015) R: a language and environment for statistical computing

  • Ramsay NA, Glover BJ (2005) MYB-bHLH-WD40 protein complex and the evolution of cellular diversity. Trends Plant Sci 10:63–70

    Article  CAS  PubMed  Google Scholar 

  • Reisberg EE, Hildebrandt U, Riederer M, Hentschel U (2012) Phyllosphere bacterial communities of trichome-bearing and trichomeless Arabidopsis thaliana leaves. Antonie Van Leeuwenhoek 101:551–560

    Article  PubMed  Google Scholar 

  • 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 222:472–483

    Article  CAS  PubMed  Google Scholar 

  • 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 142:866–877

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Salasoo I (1983) Effect of leaf age on epicuticular wax alkanes in Rhododendron. Phytochemistry 22:461–463

    Article  CAS  Google Scholar 

  • Samuels L, Kunst L, Jetter R (2008) Sealing plant surfaces: cuticular wax formation by epidermal cells. Annu Rev Plant Biol 59:683–707

    Article  CAS  PubMed  Google Scholar 

  • Schönherr J (1976) Water permeability of isolated cuticular membranes: the effect of cuticular waxes on diffusion of water. Planta 131:159–164

    Article  PubMed  Google Scholar 

  • Stocker H, Ashton L (1975) Changes in the composition of coffee wax with development. Phytochemistry 14:1919–1920

    Article  CAS  Google Scholar 

  • Suh MC, Samuels AL, Jetter R, Kunst L, Pollard M, Ohlrogge J, Beisson F (2005) Cuticular lipid composition, surface structure, and gene expression in Arabidopsis stem epidermis. Plant Physiol 139:1649–1665

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sylvester AW, Cande WZ, Freeling M (1990) Division and differentiation during normal and liguleless-1 maize leaf development. Development 110:985–1000

    CAS  PubMed  Google Scholar 

  • Telfer A, Bollman KM, Poethig RS (1997) Phase change and the regulation of trichome distribution in Arabidopsis thaliana. Development 124:645–654

    CAS  PubMed  Google Scholar 

  • Tian D, Tooker J, Peiffer M, Chung SH, Felton GW (2012) Role of trichomes in defense against herbivores: comparison of herbivore response to woolly and hairless trichome mutants in tomato (Solanum lycopersicum). Planta 236:1053–1066

    Article  CAS  PubMed  Google Scholar 

  • 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 17:119–130

    Article  CAS  PubMed  Google Scholar 

  • Trenkamp S, Martin W, Tietjen K (2004) Specific and differential inhibition of very-long-chain fatty acid elongases from Arabidopsis thaliana by different herbicides. Proc Natl Acad Sci USA 101:11903–11908

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsubaki S, Sugimura K, Teramoto Y, Yonemori K, Azuma J (2013) Cuticular membrane of Fuyu persimmon fruit is strengthened by triterpenoid nano-fillers. PLoS One 8:1–13

    Article  Google Scholar 

  • Tulloch AP (1973) Composition of leaf surface waxes of Triticum species: variation with age and tissue. Phytochemistry 12:2225–2232

    Article  CAS  Google Scholar 

  • van Maarseveen C, Han H, Jetter R (2009) Development of the cuticular wax during growth of Kalanchoe daigremontiana (Hamet et Perr. de la Bathie) leaves. Plant Cell Environ 32:73–81

    Article  PubMed  Google Scholar 

  • Viougeas MA, Rohr R, Chamel A (1995) Structural changes and permeability of ivy (Hedera helix L.) leaf cuticles in relation to leaf development and after selective chemical treatments. New Phytol 130:337–348

    Article  CAS  Google Scholar 

  • Wagner GJ, Wang E, Shepherd RW (2004) New approaches for studying and exploiting an old protuberance, the plant trichome. Ann Bot 93:3–11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yeats TH, Rose JKC (2013) The formation and function of plant cuticles. Plant Physiol 163:5–20

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zust T, Joseph B, Shimizu KK, Kliebenstein DJ, Turnbull LA (2011) Using knockout mutants to reveal the growth costs of defensive traits. Proc R Soc B Biol Sci 278:2598–2603

    Article  Google Scholar 

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Acknowledgments

The authors thank Mrs. Yan Cao for skillful technical assistance. Seeds of the trichome-less gl1 mutant were obtained from the Arabidopsis Biological Resource Center (ABRC). This work has been supported by the Natural Sciences and Engineering Research Council (Canada), the Canada Foundation for Innovation, the British Columbia Knowledge Development Fund, and the Canada Research Chairs Program.

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    Authors and Affiliations

    1. Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1, Canada

      Lucas Busta & Reinhard Jetter

    2. Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, BC, V6T 1Z4, Canada

      Daniela Hegebarth & Reinhard Jetter

    3. Department of Statistics, University of British Columbia, 3182 Earth Sciences Building, 2207 Main Mall, Vancouver, BC, V6T 1Z4, Canada

      Edward Kroc

    4. Department of Educational and Counselling Psychology, and Special Education, University of British Columbia, 2125 Main Mall, Vancouver, BC, V6T 1Z4, Canada

      Edward Kroc

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    1. Lucas Busta
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    Correspondence to Reinhard Jetter.

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    L. Busta and D. Hegebarth contributed equally to this work.

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    Busta, L., Hegebarth, D., Kroc, E. et al. Changes in cuticular wax coverage and composition on developing Arabidopsis leaves are influenced by wax biosynthesis gene expression levels and trichome density. Planta 245, 297–311 (2017). https://doi.org/10.1007/s00425-016-2603-6

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