Over-expression of the cucumber expansin gene (Cs-EXPA1) in transgenic maize seed for cellulose deconstruction

Abstract

Plant cell wall degradation into fermentable sugars by cellulases is one of the greatest barriers to biofuel production. Expansin protein loosens the plant cell wall by opening up the complex of cellulose microfibrils and polysaccharide matrix components thereby increasing its accessibility to cellulases. We over-expressed cucumber expansin in maize kernels to produce enough protein to assess its potential to serve as an industrial enzyme for applications particularly in biomass conversion. We used the globulin-1 embryo-preferred promoter to express the cucumber expansin gene in maize seed. Expansin protein was targeted to one of three sub-cellular locations: the cell wall, the vacuole, or the endoplasmic reticulum (ER). To assess the level of expansin accumulation in seeds of transgenic kernels, a high throughput expansin assay was developed. The highest expressing plants were chosen and enriched crude expansin extract from those plants was tested for synergistic effects with cellulase on several lignocellulosic substrates. Activity of recombinant cucumber expansin from transgenic kernels was confirmed on these pretreated substrates. The best transgenic lines (ER-targeted) can now be used for breeding to increase expansin expression for use in the biomass conversion industry. Results of these experiments show the success of expansin over-expression and accumulation in transgenic maize seed without negative impact on growth and development and confirm its synergistic effect with cellulase on deconstruction of complex cell wall substrates.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. An G, Mitra A, Choi HK et al (1989) Functional analysis of the 3′ control region of the potato wound-inducible proteinase inhibitor II gene. Plant Cell 1:115–122. doi:10.1105/tpc.1.1.115

    PubMed Central  CAS  PubMed  Google Scholar 

  2. Anzai H, Yoneyama K, Yamaguchi I (1989) Transgenic tobacco resistant to a bacterial disease by the detoxification of a pathogenic toxin. MGG Mol Gen Genet 219:492–494

    Article  CAS  Google Scholar 

  3. Cho H-T, Cosgrove DJ (2000) Altered expression of expansin modulates leaf growth and pedicel abscission in Arabidopsis thaliana. Proc Natl Acad Sci USA 97:9783–9788. doi:10.1073/pnas.160276997

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  4. Choi D, Lee Y, Cho H-T, Kende H (2003) Regulation of expansin gene expression affects growth and development in transgenic rice plants. Society 15:1386–1398. doi:10.1105/tpc.011965.1998

    CAS  Google Scholar 

  5. Cosgrove DJ (1989) Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls. Planta 177:121–130. doi:10.1007/BF00392162

    Article  CAS  Google Scholar 

  6. Cosgrove DJ (2001) Enhancement of accessibility of cellulose by expansins. US Patent 6326470 B1. http://www.google.com/patents/US6326470

  7. Cosgrove DJ (2005) Growth of the plant cell wall. Nat Rev Mol Cell Biol 6:850–861. doi:10.1038/nrm1746

    Article  CAS  PubMed  Google Scholar 

  8. Cosgrove DJ, Li LC, Cho H-T et al (2002) The growing world of expansins. Plant Cell Physiol 43:1436–1444

    Article  CAS  PubMed  Google Scholar 

  9. Goh HH, Sloan J, Malinowski R, Fleming A (2014) Variable expansin expression in Arabidopsis leads to different growth responses. J Plant Physiol 171:329–339. doi:10.1016/j.jplph.2013.09.009

    Article  CAS  PubMed  Google Scholar 

  10. 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 6:271–282

    Article  CAS  PubMed  Google Scholar 

  11. Holwerda BC, Padgett HS, Rogers JC (1992) Proaleurain vacuolar targeting is mediated by short contiguous peptide interactions. Plant Cell 4:307–318. doi:10.1105/tpc.4.3.307

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  12. Hood EE, Howard JA (2014) Commerl Plant Prod Recomb Protein Prod 68:15–26. doi:10.1007/978-3-662-43836-7

    Google Scholar 

  13. Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168:1291–1301

    PubMed Central  CAS  PubMed  Google Scholar 

  14. Hood EE, Witcher DR, Maddock S et al (1997) Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification. Mol Breed 3:291–306. doi:10.1023/A:1009676322162

    Article  CAS  Google Scholar 

  15. Hood EE, Bailey MR, Beifuss K et al (2003) Criteria for high-level expression of a fungal laccase gene in transgenic maize. Plant Biotechnol J 1:129–140

    Article  CAS  PubMed  Google Scholar 

  16. Hood EE, Love R, Lane J et al (2007) Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed. Plant Biotechnol J 5:709–719. doi:10.1111/j.1467-7652.2007.00275.x

    Article  CAS  PubMed  Google Scholar 

  17. Hood EE, Nelson P, Powell R (2011) Plant Biomass Conversion. Wiley, New York

    Book  Google Scholar 

  18. Hood EE, Devaiah SP, Fake G et al (2012) Manipulating corn germplasm to increase recombinant protein accumulation. Plant Biotechnol J 10:20–30. doi:10.1111/j.1467-7652.2011.00627.x

    Article  CAS  PubMed  Google Scholar 

  19. Hu Z, Song N, Xing J et al (2013) Overexpression of three TaEXPA1 homoeologous genes with distinct expression divergence in hexaploid wheat exhibit functional retention in Arabidopsis. PLoS ONE 8:e63667. doi:10.1371/journal.pone.0063667

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  20. Ishida Y, Saito H, Ohta S et al (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750

    Article  CAS  PubMed  Google Scholar 

  21. Kang K, Wang S, Lai G et al (2013) Characterization of a novel swollenin from Penicillium oxalicum in facilitating enzymatic saccharification of cellulose. BMC Biotechnol 13:42. doi:10.1186/1472-6750-13-42

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  22. Klein-Marcuschamer D, Oleskowicz-Popiel P, Simmons BA, Blanch HW (2012) The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnol Bioeng 109:1083–1087. doi:10.1002/bit.24370

    Article  CAS  PubMed  Google Scholar 

  23. Kusnadi AR, Hood EE, Witcher DR et al (1998) Production and purification of two recombinant proteins from transgenic corn. Biotechnol Prog 14:149–155. doi:10.1021/bp970138u

    Article  CAS  PubMed  Google Scholar 

  24. Lee Y, Kende H (1997) Expression of β-expansins Is correlated with internodal elongation in deepwater rice. Plant Cell 9:1661–1671. doi:10.1105/tpc.9.9.1661

    Article  Google Scholar 

  25. Li F, Han Y, Feng Y et al (2013) Expression of wheat expansin driven by the RD29 promoter in tobacco confers water-stress tolerance without impacting growth and development. J Biotechnol 163:281–291. doi:10.1016/j.jbiotec.2012.11.008

    Article  CAS  PubMed  Google Scholar 

  26. Ma N, Wang Y, Qiu S et al (2013) Overexpression of OsEXPA8, a root-specific gene, improves rice growth and root system architecture by facilitating cell extension. PLoS One 8:e75997. doi:10.1371/journal.pone.0075997

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  27. McQueen-Mason S, Cosgrove DJ (1994) Disruption of hydrogen bonding between plant cell wall polymers by proteins that induce wall extension. Proc Natl Acad Sci USA 91:6574–6578

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  28. McQueen-Mason SJ, Cosgrove DJ (1995) Expansin mode of action on cell walls. Analysis of wall hydrolysis, stress relaxation, and binding. Plant Physiol 107:87–100

    PubMed Central  CAS  PubMed  Google Scholar 

  29. McQueen-Mason S, Durachko DM, Cosgrove DJ (1992) Two endogenous proteins that induce cell wall extension in plants. Plant Cell 4:1425–1433

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  30. Medrano G, Reidy MJ, Liu J et al (2009) Recombinant Proteins From Plants. In: Faye L, Gomord V (eds) Recombinant proteins from plants: methods and protocols. Humana Press, Totowa, pp 51–67

    Google Scholar 

  31. Perlack RD, Stokes BJ (2011) U.S. Billion–Ton update: biomass supply for a bioenergy and bioproducts industry. http://energy.gov/eere/bioenergy/downloads/us-billion-ton-update-biomass-supply-bioenergy-and-bioproducts-industry

  32. Rogers JC (1985) Two barley alpha-amylase gene families are regulated differently in aleurone cells. J Biol Chem 260:3731–37388

    CAS  PubMed  Google Scholar 

  33. Rose JK, Lee HH, Bennett AB (1997) Expression of a divergent expansin gene is fruit-specific and ripening-regulated. Proc Natl Acad Sci USA 94:5955–5960

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  34. Sack M, Hofbauer A, Fischer R, Stoger E (2015) The increasing value of plant-made proteins. Curr Opin Biotechnol 32:163–170. doi:10.1016/j.copbio.2014.12.008

    Article  CAS  PubMed  Google Scholar 

  35. Sampedro J, Cosgrove DJ (2005) The expansin superfamily. Genome Biol 6:242. doi:10.1186/gb-2005-6-12-242

    PubMed Central  Article  PubMed  Google Scholar 

  36. Sathitsuksanoh N, Zhu Z, Templeton N et al (2009) Saccharification of a Potential Bioenergy Crop, Phragmites australis (Common Reed), by Lignocellulose Fractionation Followed by Enzymatic Hydrolysis at Decreased Cellulase Loadings. Ind Eng Chem Res 48:6441–6447

    Article  CAS  Google Scholar 

  37. Streatfield SJ, Jilka JM, Hood EE et al (2001) Plant-based vaccines: unique advantages. Vaccine 19:2742–2748

    Article  CAS  PubMed  Google Scholar 

  38. Uchimiya H, Iwata M, Nojiri C et al (1993) Bialaphos treatment of transgenic rice plants expressing a bar gene prevents infection by the sheath blight pathogen (Rhizoctonia solani). Nat Biotechnol 11:835–836

    Article  CAS  Google Scholar 

  39. White J, Chang SY, Bibb MJ (1990) A cassette containing the bar gene of Streptomyces hygroscopicus: a selectable marker for plant transformation. Nucleic Acids Res 18:1062

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  40. Woodard SL, Mayor JM, Bailey MR et al (2003) Maize (Zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants. Biotechnol Appl Biochem 38:123–130

    Article  CAS  PubMed  Google Scholar 

  41. Xu Q, Xu X, Shi Y et al (2014) Transgenic tobacco plants overexpressing a grass PpEXP1 gene exhibit enhanced tolerance to heat stress. PLoS ONE 9:1–9. doi:10.1371/journal.pone.0100792

    Google Scholar 

  42. Yennawar NH, Li L-C, Dudzinski DM et al (2006) Crystal structure and activities of EXPB1 (Zea m 1), a β-expansin and group-1 pollen allergen from maize. Proc Natl Acad Sci U S A 103:14664–14671

    PubMed Central  Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by a grant from the Department of Energy DE FG36 GO88025. We gratefully acknowledge Dr. Daniel Cosgrove (The Pennsylvania State University) for providing the cucumber expansin gene and the anti-expansin antibodies used in this work.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Elizabeth E. Hood.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 797 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yoon, S., Devaiah, S.P., Choi, Se. et al. Over-expression of the cucumber expansin gene (Cs-EXPA1) in transgenic maize seed for cellulose deconstruction. Transgenic Res 25, 173–186 (2016). https://doi.org/10.1007/s11248-015-9925-1

Download citation

Keywords

  • Cucumber expansin
  • Over-expression
  • Transgenic maize
  • Expansin assay