Advertisement

Plant Molecular Biology

, Volume 80, Issue 3, pp 289–297 | Cite as

Transposon insertion in a cinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize

  • Wei Chen
  • Nathan VanOpdorp
  • Dennis Fitzl
  • Jagdish Tewari
  • Peter Friedemann
  • Tom Greene
  • Steve Thompson
  • Siva Kumpatla
  • Peizhong ZhengEmail author
Article

Abstract

Maize brown midrib1 (bm1) mutant plants have reduced lignin content and offer significant advantages when used in silage and biofuel applications. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the conversion of hydroxycinnamyl aldehydes to monolignols, a key step in lignin biosynthesis. Maize CAD2 has been implicated as the underlying gene for bm1phenotypes since bm1 plants have reduced CAD activity and lower CAD2 transcript level. Here, we describe a Dow AgroSciences maize bm1 mutant (bm1-das1) that contains a 3,444-bp transposon insertion in the first intron of CAD2 gene. As a result of chimeric RNA splicing, cad2 mRNA from bm1-das1 contains a 409-bp insert between its 1st and 2nd exons. This insertion creates a premature stop codon and is predicted to result in a truncated protein of 48 amino acids (AA), compared to 367 AA for the wild type (WT) CAD2. We have also sequenced cad2 from the reference allele bm1-ref in 515D bm1 stock and showed that it contains a two-nucleotide (AC) insertion in the 3rd exon, which is predicted to result in a truncated protein of 147 AA. The levels of cad2 mRNA in the midribs of bm1-das1 and bm1-ref are reduced by 91 and 86 % respectively, leading to reductions in total lignin contents by 24 and 30 %. Taken together, our data show that mutations in maize CAD2 are responsible for maize bm1 phenotypes. Based on specific changes in bm1-das1 and bm1-ref, high throughput TaqMan and KBioscience’s allele specific PCR assays capable of differentiating mutant and WT alleles have been developed to accelerate bm1 molecular breeding.

Keywords

Brown midrib bm1 Cinnamyl alcohol dehydrogenase Lignin Transposon Mutant 

Notes

Acknowledgments

The authors would like to thank Ryan Gibson and Seshasai Parthasarathy for assistance in plant material generation, TaqMan and KASPar genotyping; Jafar Mammadov for critical review of the manuscript; David Meyer for supporting this project.

Supplementary material

11103_2012_9948_MOESM1_ESM.pdf (214 kb)
Supplementary material 1 (PDF 213 kb)

References

  1. Barrière Y, Ralph J, Méchin V, Guillaumie S, Grabber JH, Argillier O, Chabbert B, Lapierre C (2004) Genetic and molecular basis of grass cell wall biosynthesis and degradability. II. Lessons from brown-midrib mutants. Comptes Rendus Biol 327:847–860CrossRefGoogle Scholar
  2. Cao J (2007) Genetic dissection of the rf2a-mediated fertility restoration pathway in maize—appendix: reverse genetic analysis of maize cinnamyl alcohol dehydrogenase 2 gene. PhD Dissertation, Iowa State UniversityGoogle Scholar
  3. Chen W, VanOpdorp N, Channabasavaradhya C, Kumpatla SP (2011) Use of brown midrib-3 gene specific markers in maize for trait introgression. US Patent application 20110283427Google Scholar
  4. Cherney JH, Cherney DJ, Akin DE, Axtell JD (1991) Potential of brown-midrib low lignin mutants for improving forage quality. Adv Agron 46:157–198CrossRefGoogle Scholar
  5. Conti E, Izaurralde E (2005) Nonsense-mediated mRNA decay: molecular insights and mechanistic variations across species. Curr Opin Cell Biol 17:316–325PubMedCrossRefGoogle Scholar
  6. Cui X, Wise RP, Schnable PS (1996) The rf2 nuclear restorer of male-sterile T-cytoplasm maize. Science 272:1334–1336PubMedCrossRefGoogle Scholar
  7. Dien BS, Sarath G, Pedersen JF, Sattler SE, Chen H, Funnell-Harris DL, Nichols NN, Cotta MA (2009) Improved sugar conversion and ethanol yield for forage sorghum (Sorghum bicolor L. Moench) lines with reduced lignin content. Bio Energy Res 2:153–164Google Scholar
  8. Eyster WH (1926) Chromosome VIII in maize. Science 64:22PubMedCrossRefGoogle Scholar
  9. Guillaumie S, Pichon M, Martinant J-P, Bosio M, Goffner D, Barrière Y (2007) Differential expression of phenylpropanoid and related genes in brown-midrib bm1, bm2, bm3, and bm4 young near-isogenic maize plants. Planta 226:235–250PubMedCrossRefGoogle Scholar
  10. Halpin C, Holt K, Chojecki J, Oliver D, Chabbert B, Monties B, Edwards K, Barakate A, Foxon GA (1998) Brown-midrib maize (bm1)—a mutation affecting the cinnamyl alcohol dehydrogenase gene. Plant J 14:545–553PubMedCrossRefGoogle Scholar
  11. Haney LJ, Hake S, Scott MP (2008) Allelism testing of Maize Coop Stock Center lines containing unknown brown midrib alleles. Maize Genet Coop Newslett 82:4–5Google Scholar
  12. Hernandez M, Duplan MN, Berthier G, Vaietilingom M, Hauser W, Freyer R, Pla M, Bertheau Y (2004) Development and comparison of four real-time Polymerase Chain Reaction systems for specific detection and quantification of Zea mays. J Agric Food Chem 52:4632–4637PubMedCrossRefGoogle Scholar
  13. Jorgensen LR (1931) Brown midrib in maize and its linkage relations. J Am Soc Agron 23:549–557CrossRefGoogle Scholar
  14. Kim S-J, Kim M-R, Bedgar DL, Moinuddin SGA, Cardenas GL, Davin LB, Kang C, Lewis NG (2004) Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proc Natl Acad Sci USA 101:1455–1460PubMedCrossRefGoogle Scholar
  15. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  16. Milligan BG (1998) Total DNA isolation. In: Hoelzel AR (ed) Molecular genetic analysis of population: a practical approach, 2nd edn. Oxford University Press, Oxford, pp 29–64Google Scholar
  17. Morrow SL, Mascia P, Self KA, Altschuler M (1997) Molecular characterization of a brown midrib3 deletion mutation in maize. Mol Breed 3:351–357CrossRefGoogle Scholar
  18. Neuffer MG, Jones L, Zuber MS (1968) The mutants of maize: a pictorial survey in color of the usable mutant genes in maize with gene symbols and linkage map positions. In: Matthias S, Hamilton H (eds) Crop Science Society of America. Madison, WIGoogle Scholar
  19. Pillonel C, Mulder MM, Boon JJ, Forster B, Binder A (1991) Involvement of cinnamyl-alcohol dehydrogenase in the control of lignin formation in Sorghum bicolor L. Moench. Planta 185:538–544Google Scholar
  20. Pohlman RF, Fedoroff NV, Messing J (1984) The nucleotide sequence of the maize controlling element Activator. Cell 37:635–643PubMedCrossRefGoogle Scholar
  21. Provan GJ, Scobbie L, Chesson A (1997) Characterization of lignin from CAD and OMT deficient bm mutants of maize. J Sci Food Agric 73:133–142CrossRefGoogle Scholar
  22. Rubin E, Lithwick G, Levy AA (2001) Structure and evolution of the hAT transposon superfamily. Genetics 158:949–957PubMedGoogle Scholar
  23. Saballos A, Ejeta G, Sanchez E, Kang CH, Vermerris W (2009) A genomewide analysis of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L.) Moench] identifies SbCAD2 as the brown midrid6 gene. Genetics 181:783–795PubMedCrossRefGoogle Scholar
  24. Sattler SE, Saathoff AJ, Haas EJ, Palmer NA, Funnell-Harris DL, Sarath G, Pedersen JF (2009) A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is responsible for the sorghum brown midrid6 phenotype. Plant Physiol 150:584–595PubMedCrossRefGoogle Scholar
  25. Sattler SE, Funnell-Harris DL, Pedersen JF (2010a) Brown midrib mutations and their importance to the utilization of maize, sorghum and pearl millet lignocellulosic tissues. Plant Sci 178:229–238CrossRefGoogle Scholar
  26. Sattler SE, Funnell-Harris DL, Pedersen JF (2010b) Efficacy of singular and stacked brown midrib 6 and 12 in the modification of lignocellulose and grain chemistry. J Agric Food Chem 58:3611–3616PubMedCrossRefGoogle Scholar
  27. Shi C, Koch G, Ouzunova M, Wenzel G, Zein I, Lübberstedt T (2006) Comparison of maize brown-midrib isogenic lines by cellular UV-microspectrophotometry and comparative transcript profiling. Plant Mol Biol 62:269–714CrossRefGoogle Scholar
  28. Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, Rock JM, Wu YY, Katibah GE, Zhifang G, McCaskill D, Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD (2009) Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459(7245):437–441PubMedCrossRefGoogle Scholar
  29. Tobias CM, Chow EK (2005) Structure of the cinnamyl-alcohol dehydrogenase gene family in rice and promoter activity of a member associated with lignification. Planta 220:678–688PubMedCrossRefGoogle Scholar
  30. Vermerris W (2009) Cell wall biosynthetic genes of maize and their potential for bioenergy production. In: Bennetzen JL, Hake S (eds) Handbook of maize: genetics and genomics. Springer, New York, pp 741–767Google Scholar
  31. Vermerris W (2011) Survey of genomics approaches to improve bioenergy traits in maize, sorghum and sugarcane. J Integr Plant Biol 53:105–119PubMedCrossRefGoogle Scholar
  32. Vermerris W, Thompson KJ, McIntyre LM (2002) The maize Brown midrib1 locus affects cell wall composition and plant development in a dose-dependent manner. Heredity 88:450–457PubMedCrossRefGoogle Scholar
  33. Vermerris W, Zhao J, Ladisch MR, Mosier MS (2007) In situ visualization of celluloses in maize mutants with enhanced biomass conversion properties. In: 29th symposium on biotechnology for fuels and chemicals, Denver, CO, USA. Poster 1A-21Google Scholar
  34. Vignols F, Rigau J, Torres MA, Capellades M, Puigdomènech P (1995) The brown midrib3 (bm3) mutation in maize occurs in the gene encoding caffeic acid O-methyltransferase. Plant Cell 7:407–416PubMedGoogle Scholar
  35. Xu J, Pemberton GH, Almira EC, McCarty DR, Koch KE (1995) The Ivr 1 gene for invertase in maize. Plant Physiol 108:1293–1294PubMedCrossRefGoogle Scholar
  36. Zhang K, Qian Q, Huang Z, Wang Y, Li M, Hong L, Zeng D, Gu M, Chu C, Cheng Z (2006) GOLD HULL AND INTERNODE2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase in rice. Plant Physiol 140:972–983PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Wei Chen
    • 1
  • Nathan VanOpdorp
    • 1
  • Dennis Fitzl
    • 1
  • Jagdish Tewari
    • 1
  • Peter Friedemann
    • 1
  • Tom Greene
    • 2
  • Steve Thompson
    • 1
  • Siva Kumpatla
    • 1
  • Peizhong Zheng
    • 1
    Email author
  1. 1.Dow AgroSciences LLCIndianapolisUSA
  2. 2.Pioneer Hi-Bred International, Inc.JohnstonUSA

Personalised recommendations