Skip to main content

BnA1.CER4 and BnC1.CER4 are redundantly involved in branched primary alcohols in the cuticle wax of Brassica napus

Abstract

Key message

The mutations BnA1.CER4 and BnC1.CER4 produce disordered wax crystals types and alter the composition of epidermal wax, causing increased cuticular permeability and sclerotium resistance.

Abstract

The aerial surfaces of land plants are coated with a cuticle, comprised of cutin and wax, which is a hydrophobic barrier for preventing uncontrolled water loss and environmental damage. However, the mechanisms by which cuticle components are formed are still unknown in Brassica napus L. and were therefore assessed here. BnA1.CER4 and BnC1.CER4, encoding fatty acyl-coenzyme A reductases localizing to the endoplasmic reticulum and highly expressed in leaves, were identified and functionally characterized. Expression of BnA1.CER4 and BnC1.CER4 cDNA in yeast (Saccharomyces cerevisiae) induced the accumulation of primary alcohols with chain lengths of 26 carbons. The mutant line Nilla glossy2 exhibited reduced wax crystal types, and wax composition analysis showed that the levels of branched primary alcohols were decreased, whereas those of the other branched components were increased. Further analysis showed that the mutant had reduced water retention but enhanced resistance to Sclerotinia sclerotiorum. Collectively, our study reports that BnA1.CER4 and BnC1.CER4 are fatty acyl-coenzyme A reductase genes in B. napus with a preference for branched substrates that participate in the biosynthesis of anteiso-primary alcohols.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

References

  1. Bach L, Michaelson LV, Haslam R, Bellec Y, Gissot L, Marion J (2008) The very-long-chain hydroxy fatty acyl-coa dehydratase pasticcino2 is essential and limiting for plant development. Proceed Nat Acad Sci United States 105:14727–14731. https://doi.org/10.1073/pnas.0805089105

    Article  Google Scholar 

  2. Beaudoin F et al (2009) Functional characterization of the Arabidopsis beta-ketoacyl-coenzyme A reductase candidates of the fatty acid elongase. Plant Physiol 150:1174–1191. https://doi.org/10.1104/pp.109.137497

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Bernard A et al (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. https://doi.org/10.1105/tpc.112.099796

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Bodnaryk RP (1992) Leaf epicuticular wax, an antixenotic factor in brassicaceae that affects the rate and pattern of feeding of flea beetles, phyllotreta cruciferae (Goeze). Can J Plant Sci 72:1295–1303. https://doi.org/10.4141/cjps92-163

    Article  Google Scholar 

  5. Bonaventure G, Salas JJ, Pollard MR, Ohlrogge JB (2003) Disruption of the FATB gene in arabidopsis demonstrates an essential role of saturated fatty acids in plant growth. Plant Cell 15:1020–1033. https://doi.org/10.1105/tpc.008946

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Busta L, Jetter R (2017) Structure and biosynthesis of branched wax compounds on wild type and wax biosynthesis mutants of arabidopsis thaliana. Plant Cell Physiol 58:1059–1074. https://doi.org/10.1093/pcp/pcx051

    CAS  Article  PubMed  Google Scholar 

  7. Chalhoub B et al (2014) Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome. Science 345:950–953. https://doi.org/10.1126/science.1253435

    CAS  Article  Google Scholar 

  8. Cheesbrough TM, Kolattukudy PE (1984) Alkane biosynthesis by decarbonylation of aldehydes catalyzed by a particulate preparation from pisum sativum. Proceed Nat Acad Sci United States of Am Biol Sci 81:6613–6617. https://doi.org/10.1073/pnas.81.21.6613

    CAS  Article  Google Scholar 

  9. Chen W et al (2011) Male Sterile2 encodes a plastid-localized fatty acyl carrier protein reductase required for pollen exine development in Arabidopsis. Plant Physiol 157:842–853. https://doi.org/10.1104/pp.111.181693

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Domergue F et al (2010) Three arabidopsis fatty acyl-coenzyme A reductases, FAR1, FAR4, and FAR5, generate primary fatty alcohols associated with suberin deposition. Plant Physio 153:1539–1554. https://doi.org/10.1104/pp.110.158238

    CAS  Article  Google Scholar 

  11. Dong X et al (2019) Fine-mapping and transcriptome analysis of BoGL-3, a wax-less gene in cabbage (Brassica oleracea L. var. capitata). Mol Genet Genom 294:1231–1239. https://doi.org/10.1007/s00438-019-01577-5

    CAS  Article  Google Scholar 

  12. Dun X et al (2011) BnaC.Tic40, a plastid inner membrane translocon originating from Brassica oleracea, is essential for tapetal function and microspore development in Brassica napus. Plant J 68:532–545. https://doi.org/10.1111/j.1365-313X.2011.04708.x

    CAS  Article  PubMed  Google Scholar 

  13. 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. https://doi.org/10.1105/tpc.12.10.2001

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Gniwotta F, Vogg G, Gartmann V, Carver TL, Riederer M, Jetter R (2005) What do microbes encounter at the plant surface? Chemical composition of pea leaf cuticular waxes. Plant Physio 139:519–530. https://doi.org/10.1104/pp.104.053579

    CAS  Article  Google Scholar 

  15. Greer S, Wen M, Bird D, Wu X, Samuels L, Kunst L, Jetter R (2007) The cytochrome p450 enzyme CYP96A15 is the midchain alkane hydroxylase responsible for formation of secondary alcohols and ketones in stem cuticular wax of arabidopsis. Plant Physiol 145:653–667. https://doi.org/10.1104/pp.107.107300

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Han L, Lobo S, Reynolds KA (1998) Characterization of beta-ketoacyl-acyl carrier protein synthase III from Streptomyces glaucescens and its role in initiation of fatty acid biosynthesis. J Bacteriol 180:4481–4486

    CAS  Article  Google Scholar 

  17. Haslam TM, Manas-Fernandez 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. https://doi.org/10.1104/pp.112.201640

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Heredia A (2003) Biophysical and biochemical characteristics of cutin, a plant barrier biopolymer. Biochimica Et Biophysica Acta-General Subjects 1620:1–7. https://doi.org/10.1016/s0304-4165(02)00510-x

    CAS  Article  Google Scholar 

  19. Holloway PJ, Brown GA, Baker EA, Macey MJK (1977) Chemical composition and ultrastructure of the epicuticular wax in three lines of Brassica napus (L). Chem Phy Lipids 19:114–127. https://doi.org/10.1016/0009-3084(77)90092-5

    CAS  Article  Google Scholar 

  20. Jenks MA, Eigenbrode SD, Lemieux B (2002) Cuticular waxes of Arabidopsis. Arabidopsis Book. https://doi.org/10.1199/tab.0016

    Article  PubMed  PubMed Central  Google Scholar 

  21. Joubes J et al (2008) The VLCFA elongase gene family in Arabidopsis thaliana: phylogenetic analysis, 3D modelling and expression profiling. Plant Mol Biol 67:547–566. https://doi.org/10.1007/s11103-008-9339-z

    CAS  Article  PubMed  Google Scholar 

  22. Kerstiens G (1996) Signalling across the divide: a wider perspective of cuticular structure-function relationships. Trends Plant Sci 1:125–129. https://doi.org/10.1016/S1360-1385(96)90007-2

    Article  Google Scholar 

  23. Kim DH et al (2001) Trafficking of phosphatidylinositol 3-phosphate from the trans-Golgi network to the lumen of the central vacuole in plant cells. Plant Cell 13:287–301. https://doi.org/10.1105/tpc.13.2.287

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Kim KS, Park SH, Kim DK, Jenks MA (2007) Influence of water deficit on leaf cuticular waxes of soybean (Glycine max L. Merr.). Int J Plant Sci 168:307–316. https://doi.org/10.1086/510496

    CAS  Article  Google Scholar 

  25. Kunst L, Samuels AL (2003) Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res 42:51–80. https://doi.org/10.1016/S0163-7827(02)00045-0

    CAS  Article  PubMed  Google Scholar 

  26. Li F et al (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. https://doi.org/10.1104/pp.108.123471

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Liu F et al (2015) Overexpression of barley oxalate oxidase gene induces partial leaf resistance to Sclerotinia sclerotiorum in transgenic oilseed rape. Plant Pathol 64:1407–1416. https://doi.org/10.1111/ppa.12374

    CAS  Article  Google Scholar 

  28. Liu D et al (2017) Cgl2 plays an essential role in cuticular wax biosynthesis in cabbage (Brassica oleracea L. var. capitata). Bmc Plant Biol 17. https://doi.org/10.1186/s12870-017-1162-8

  29. Liu D et al (2018) Fine mapping and candidate gene identification for wax biosynthesis locus, boWax1 in Brassica oleracea L. var. capitata. Front Plant Sci 9. https://doi.org/10.3389/fpls.2018.00309

  30. Lu S et al (2012) Arabidopsis ECERIFERUM9 involvement in cuticle formation and maintenance of plant water status. Plant Physiol 159:930–944. https://doi.org/10.1104/pp.112.198697

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Lue 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. https://doi.org/10.1111/j.1365-313X.2009.03892.x

    CAS  Article  Google Scholar 

  32. McFarlane HE, Shin JJ, Bird DA, Samuels AL (2010) Arabidopsis ABCG transporters, which are required for export of diverse cuticular lipids, dimerize in different combinations. Plant Cell 22:3066–3075. https://doi.org/10.1105/tpc.110.077974

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Mentzen WI, Peng J, Ransom N, Nikolau BJ, Wurtele ES (2008) Articulation of three core metabolic processes in Arabidopsis: fatty acid biosynthesis, leucine catabolism and starch metabolism. BMC Plant Biol 8:76. https://doi.org/10.1186/1471-2229-8-76

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. Michelmore RW, Paran I, Kesseli RV (1991) Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Natl Acad Sci USA 88:9828–9832. https://doi.org/10.1073/pnas.88.21.9828

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Mo JG, Li WQ, Yu Q, Bodnaryk RP (1995) Inheritance of the waxless character of Brassica napus Nilla glossy. Can J Plant Sci 75:893–894. https://doi.org/10.4141/cjps95-148

    Article  Google Scholar 

  36. Ni Y, Guo YJ, Wang J, Xia RE, Wang XQ, Ash G, Li JN (2014) Responses of physiological indexes and leaf epicuticular waxes ofBrassica napustoSclerotinia sclerotioruminfection. Plant Pathol 63:174–184. https://doi.org/10.1111/ppa.12060

    CAS  Article  Google Scholar 

  37. Pascal S et al (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. https://doi.org/10.1111/tpj.12060

    CAS  Article  PubMed  Google Scholar 

  38. Piquemal J et al (2005) Construction of an oilseed rape (Brassica napus L.) genetic map with SSR markers. TAG Theoretical and applied genetics Theoretische und angewandte Genetik 111:1514–1523. https://doi.org/10.1007/s00122-005-0080-6

    CAS  Article  PubMed  Google Scholar 

  39. Pollard M, Beisson F, Li Y, Ohlrogge JB (2008) Building lipid barriers: biosynthesis of cutin and suberin. Trends in Plant Sci 13:236–246. https://doi.org/10.1016/j.tplants.2008.03.003

    CAS  Article  Google Scholar 

  40. Pu Y et al (2013) A novel dominant glossy mutation causes suppression of wax biosynthesis pathway and deficiency of cuticular wax in Brassica napus. Bmc Plant Biol. https://doi.org/10.1186/1471-2229-13-215

    Article  PubMed  PubMed Central  Google Scholar 

  41. Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66. https://doi.org/10.1016/s0304-3940(02)01423-4

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Riederer M, Schreiber L (2001) Protecting against water loss: analysis of the barrier properties of plant cuticles. J Exp Bot 52:2023–2032. https://doi.org/10.1093/jexbot/52.363.2023

    CAS  Article  PubMed  Google Scholar 

  43. 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. https://doi.org/10.1104/pp.106.086785

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. Samuels L, Kunst L, Jetter R (2008) Sealing plant surfaces: Cuticular wax formation by epidermal cells. Annual Rev Plant Biol 59:683–707. https://doi.org/10.1146/annurev.arplant.59.103006.093219

    CAS  Article  Google Scholar 

  45. Schneider-Belhaddad F, Kolattukudy P (2000) Solubilization, partial purification, and characterization of a fatty aldehyde decarbonylase from a higher plant, Pisum sativum. Archiv Biochem Biophy 377:341–349. https://doi.org/10.1006/abbi.2000.1798

    CAS  Article  Google Scholar 

  46. 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 12:721–737. https://doi.org/10.1105/tpc.12.5.721

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025. https://doi.org/10.1038/nprot.2006.286

    CAS  Article  PubMed  Google Scholar 

  48. Tafolla-Arellano JC, Báez-Sañudo R, Tiznado-Hernández ME (2018) The cuticle as a key factor in the quality of horticultural crops. Scientia Horticulturae 232:145–152. https://doi.org/10.1016/j.scienta.2018.01.005

    Article  Google Scholar 

  49. Tassone EE et al (2016) Chemical variation for leaf cuticular waxes and their levels revealed in a diverse panel of Brassica napus L. Industrial Crops Prod 79:77–83. https://doi.org/10.1016/j.indcrop.2015.10.047

    CAS  Article  Google Scholar 

  50. Uppalapati SR et al (2012) Loss of abaxial leaf epicuticular wax in Medicago truncatula irg1/palm1 mutants results in reduced spore differentiation of anthracnose and nonhost rust pathogens. Plant Cell 24:353–370. https://doi.org/10.1105/tpc.111.093104

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. Vos P et al (1995) AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 23:4407–4414. https://doi.org/10.1093/nar/23.21.4407

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. Wang ZY, Xiong L, Li W, Zhu JK, Zhu J (2011) The plant cuticle is required for osmotic stress regulation of abscisic acid biosynthesis and osmotic stress tolerance in Arabidopsis. Plant Cell 23:1971–1984. https://doi.org/10.1105/tpc.110.081943

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. Wang W et al (2015) Cucumber ECERIFERUM1 (CsCER1), which influences the cuticle properties and drought tolerance of cucumber, plays a key role in VLC alkanes biosynthesis. Plant Mol Biol 87:219–233. https://doi.org/10.1007/s11103-014-0271-0

    CAS  Article  PubMed  Google Scholar 

  54. Wang M et al (2016) Three TaFAR genes function in the biosynthesis of primary alcohols and the response to abiotic stresses in Triticum aestivum. Sci Rep 6:25008. https://doi.org/10.1038/srep25008

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. Wang C, Li H, Li Y, Meng Q, Xie F, Xu Y, Wan Z (2019) Genetic characterization and fine mapping BrCER4 in involved cuticular wax formation in purple cai-tai (Brassica rapa L. var. purpurea). Mol Breed 39. https://doi.org/10.1007/s11032-018-0919-6

  56. Willemsen V, Wolkenfelt H, de Vrieze G, Weisbeek P, Scheres B (1998) The HOBBIT gene is required for formation of the root meristem in the Arabidopsis embryo. Development 125:521–531. https://doi.org/10.1242/dev.125.3.521

  57. Wu R et al (2011) CFL1, a WW domain protein, regulates cuticle development by modulating the function of HDG1, a class IV homeodomain transcription factor, in rice and Arabidopsis. Plant Cell 23:3392–3411. https://doi.org/10.1105/tpc.111.088625

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. Xu L et al (2017) Overexpression of the novel arabidopsis gene At5g02890 alters inflorescence stem wax composition and affects phytohormone homeostasis. Front Plant Sci 8:68. https://doi.org/10.3389/fpls.2017.00068

    Article  PubMed  PubMed Central  Google Scholar 

  59. Yang X et al (2017) The Acyl Desaturase CER17 Is Involved in Producing Wax Unsaturated Primary Alcohols and Cutin Monomers. Plant Physiol 173:1109–1124. https://doi.org/10.1104/pp.16.01956

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  60. Yeats TH, Rose JK (2013) The formation and function of plant cuticles. Plant Physiol 163:5–20. https://doi.org/10.1104/pp.113.222737

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  61. Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protoc 2:1565–1572. https://doi.org/10.1038/nprot.2007.199

    CAS  Article  Google Scholar 

  62. Zeisler-Diehl V, Muller Y, Schreiber L (2018) Epicuticular wax on leaf cuticles does not establish the transpiration barrier, which is essentially formed by intracuticular wax. J Plant Physiol 227:66–74. https://doi.org/10.1016/j.jplph.2018.03.018

    CAS  Article  PubMed  Google Scholar 

  63. 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 17:1467–1481. https://doi.org/10.1105/tpc.104.030155

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We would like to thank Xianpeng Yang (Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden) for providing the yeast expression vector. We also thank Jinhua Nan (College of Plant Science and Technology, Huazhong Agricultural University) for providing the wax analysis method.

Funding

This work was supported by the National Key Research and Development of China (2017YFD0102004) and the Fundamental Research Funds for the Central Universities (2662016PY063)

Author information

Affiliations

Authors

Contributions

JL acquired, analyzed, and interpreted most of the data and was a major contributor in writing the manuscript. BY designed the work. LX Z participated in the development of molecular markers and contributed to language modification. BQ W and HD W participated in the planting and collection of plant materials. SQ Z and IM contributed to language modification. JW, CZ M, CD, JX T, JX S, and TD F provided instruments, equipment, and technical support. All authors approved the final manuscript.

Corresponding author

Correspondence to Bin Yi.

Ethics declarations

Conflicts of interest

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Communicated by Maria Laura Federico.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 680 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, J., Zhu, L., Wang, B. et al. BnA1.CER4 and BnC1.CER4 are redundantly involved in branched primary alcohols in the cuticle wax of Brassica napus. Theor Appl Genet 134, 3051–3067 (2021). https://doi.org/10.1007/s00122-021-03879-y

Download citation

Keywords

  • Brassica napus
  • Branched alcohols
  • BnA1.CER4
  • BnC1.CER4
  • Cuticular wax
  • Fatty acyl-CoA reductase