Advertisement

Planta

, Volume 236, Issue 6, pp 1803–1815 | Cite as

Reuteran and levan as carbohydrate sinks in transgenic sugarcane

  • Rolene BauerEmail author
  • Carin E. Basson
  • Jan Bekker
  • Iban Eduardo
  • Johann M. Rohwer
  • Lafras Uys
  • Johannes H. van Wyk
  • Jens Kossmann
Original Article

Abstract

The present study reports the effect of high molecular weight bacterial fructan (levan) and glucan (reuteran) on growth and carbohydrate partitioning in transgenic sugarcane plants. These biopolymers are products of bacterial glycosyltransferases, enzymes that catalyze the polymerization of glucose or fructose residues from sucrose. Constructs, targeted to different subcellular compartments (cell wall and cytosol) and driven by the Cauliflower mosaic virus-35S: maize-ubiquitin promoter, were introduced into sugarcane by biolistic transformation. Polysaccharide accumulation severely affected growth of callus suspension cultures. Regeneration of embryonic callus tissue into plants proved problematic for cell wall-targeted lines. When targeted to the cytosol, only plants with relative low levels of biopolymer accumulation survived. In internodal stalk tissue that accumulate reuteran (max 0.03 mg/g FW), sucrose content (ca 60 mg/g FW) was not affected, while starch content (<0.4 mg/g FW) was increased up to four times. Total carbohydrate content was not significantly altered. On the other hand, starch and sucrose levels were significantly reduced in plants accumulating levan (max 0.01 mg/g FW). Heterologous expression resulted in a reduction in total carbohydrate assimilation rather than a simple diversion by competition for substrate.

Keywords

Glycosyltransferase Levan Reuteran Starch Sucrose Sugarcane 

Abbreviations

FW

Fresh weight

DP

Degree of polymerization

HoPS

Homopolysaccharide

FTF

Fructosyltransferase

GTF

Glucosyltransferase

PAS

Periodic acid-Schiff

MS

Murashige–Skoog

WT

Wild type

Notes

Acknowledgments

This work was supported by grants from the South African National Research Foundation and the South African Sugar Association.

Supplementary material

425_2012_1731_MOESM1_ESM.doc (106 kb)
Supplementary material 1 (DOC 105 kb)
425_2012_1731_MOESM2_ESM.pdf (12 kb)
Supplementary material 2 (PDF 12 kb)

References

  1. Abdel-Fattah AF, Mahmoud DAR, Esawy MAT (2005) Production of levansucrase from Bacillus subtilis NRC 33a and enzymic synthesis of levan and fructo-oligosaccharides. Curr Microbiol 51:402–407PubMedCrossRefGoogle Scholar
  2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1994) Current protocols in molecular biology. Wiley, New YorkGoogle Scholar
  3. Basnayake SW, Morgan TC, Wu L, Birch RG (2012) Field performance of transgenic sugarcane expressing isomaltulose synthase. Plant Biotechnol J 10:217–225Google Scholar
  4. Basson CE, Groenewald J-H, Kossmann J, Cronje C, Bauer R (2010) Sugar and acid-related quality attributes and enzyme activities in strawberry fruits: invertase is the main sucrose hydolysing enzyme. Food Chem 121:1156–1162CrossRefGoogle Scholar
  5. Bauer R, Volschenk H, Dicks LMT (2005) Cloning and expression of the malolactic gene of Pediococcus damnosus NCFB 1832 in Saccharomyces cerevisiae. J Biotechnol 118:353–362PubMedCrossRefGoogle Scholar
  6. Bauer R, van Bekker J, du Wyk N, Toit C, Dicks LMT, Kossmann J (2009) Exopolysaccharide production by lactose-hydrolyzing bacteria isolated from traditionally fermented milk. Int J Food Microbiol 131:260–264PubMedCrossRefGoogle Scholar
  7. Bower R, Birch RG (1992) Transgenic sugarcane plants via microprojectile bombardment. Plant J 2:409–416CrossRefGoogle Scholar
  8. Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein–dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  9. Caimi PG, McCole LM, Klein TM, Kerr PS (1996) Fructan accumulation and sucrose metabolism in transgenic maize endosperm expressing a Bacillus amyloliquefaciens SacB gene. Plant Physiol 110:355–363PubMedGoogle Scholar
  10. Caimi PG, McCole LM, Klein TM, Hershey HP (1997) Cytosolic expression of the Bacillus amyloliquefaciens SacB protein inhibits tissue development in transgenic tobacco and potato. New Phytol 136:19–28Google Scholar
  11. Cairns AJ (2003) Fructan biosynthesis in transgenic plants. J Exp Bot 54:549–567PubMedCrossRefGoogle Scholar
  12. Chang YC, Hsieh PW, Chang FR, Wu RR, Liaw CC, Lee KH, Wu YC (2003) Two new protopines argemexicaines A and B and the anti HIV alkaloid 6-acetonyldihydrochelerytrine from formosan Argemone mexicana. Planta Med 69:148–152PubMedCrossRefGoogle Scholar
  13. Esawy MA, Mahmoud DAR, Fattah AFA (2008) Immobilisation of Bacillus subtilis NRC33a levansucrase and some studies on its properties. Braz J Chem Eng 25:237–246CrossRefGoogle Scholar
  14. Gerrits N, Turk SCHJ, van Dun KPM, Hulleman SHD, Visser RGF, Weisbeek PJ, Smeekens SCM (2001) Sucrose metabolism in plastids. Plant Physiol 125:926–934PubMedCrossRefGoogle Scholar
  15. Hamerli D, Birch RG (2011) Transgenic expression of trehalulose synthase results in high concentrations of the sucrose isomer trehalulose in mature stems of field-grown sugarcane. Plant Biotech J 9:32–37CrossRefGoogle Scholar
  16. Hellwege EM, Czapla S, Jahnke A, Willmitzer L, Heyer AG (2000) Transgenic potato (Solanum tuberosum) tubers synthesize the full spectrum of inulin molecules naturally occurring in globe artichoke (Cynara scolymus) roots. Proc Nat Acad Sci USA 97:8699–8704PubMedCrossRefGoogle Scholar
  17. Hendry GAF, Wallace RK (1993) The origin, distribution and evolutionary significance of fructans. In: Suzuki M, Chatterton NJ (eds) Science and technology of fructans. CRC Press, Boca Raton, pp 119–139Google Scholar
  18. Jenkins LD, Snow AJ, Simpson RJ, Higgins TJ, Jacques NA, Pritchard J, Gibson J, Larkin PJ (2002) Fructan formation in transgenic white clover expressing a fructosyltransferase from Streptococcus salivarius. Funct Plant Biol 29:1287–1298CrossRefGoogle Scholar
  19. Jones MGK, Outlaw WH, Lowry OH (1977) Enzymic assay of 10−7 to 10−14 moles of sucrose in plant tissues. Plant Physiol 60:379–383PubMedCrossRefGoogle Scholar
  20. Klibanov AM (2003) Asymmetric enzymatic oxidoreductions in organic solvents. Curr Opin Biotechnol 14:427–431PubMedCrossRefGoogle Scholar
  21. Komor E, Thom M, Maretzki A (1981) The mechanism of sugar uptake by sugarcane suspension cells. Planta 153:181–192CrossRefGoogle Scholar
  22. Korakli M, Vogel RF (2006) Structure/function relationship of homopolysaccharide producing glycansucrases and therapeutic potential of their synthesised glycans. Appl Microbiol Biotechnol 71:790–803PubMedCrossRefGoogle Scholar
  23. Kossmann J, Lloyd J (2000) Understanding and influencing starch biochemistry. Crit Rev Plant Sci 19:171–226Google Scholar
  24. Kralj S, van Geel-Schutten GH, Rahaoui H, Leer RJ, Faber EJ, van der Maarel MJEC, Dijkhuizen L (2002) Molecular characterization of a novel glucosyltransferase from Lactobacillus reuteri strain 121 synthesizing a unique, highly branched glucan with α-(1 → 4) and α-(1 → 6) glucosidic bonds. Appl Environ Microbiol 68:4283–4291PubMedCrossRefGoogle Scholar
  25. Kralj S, van Geel-Schutten GH, van der Maarel MJEC, Dijkhuizen L (2004) Biochemical and molecular characterization of Lactobacillus reuteri 121 reuteransucrase. Microbiol 150:2099–2112Google Scholar
  26. Kralj S, Stripling E, Sanders P, van Gel-Schutten GH, Dijkhuizen L (2005) Highly hydrolytic reuteransucrase from probiotic Lactobacillus reuteri strain ATCC 55730. Appl Environ Microbiol 71:3942–3950PubMedCrossRefGoogle Scholar
  27. Moore PH (1987) Anatomy and morphology. In: Heinz DJ (ed) Sugarcane improvement through breeding. Elsevier Science Publisher, New York, pp 85–142Google Scholar
  28. Moore PH, Maretzki A (1996) Photoassimilate distribution in plants and crops. In: Zamski E, Schaffer AA (eds) Source–sink relationships in sugarcane. Marcel Dekker Inc, New York, pp 643–669Google Scholar
  29. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Plant Physiol 15:473–497CrossRefGoogle Scholar
  30. Pilon-Smits EAH, Ebskamp MJM, Paul MJ, Jeuken MJW, Weisbeek PJ, Smeekens SCM (1995) Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiol 107:125–130PubMedGoogle Scholar
  31. Pilon-Smits EAH, Ebskamp MJW, Jeuken IM, van der Meer RGF, Visser RGF, Weisbeek PJ, Smeekens SCM (1996) Microbial fructan production in transgenic potato plants and tubers. Ind Crop Prod 5:35–46CrossRefGoogle Scholar
  32. Rae AL, Perroux JM, Grof CLG (1996) Sucrose partitioning between vascular bundles and storage parenchyma in the sugarcane stem: a potential role for the ShSUT1 sucrose transporter. Planta 220:817–825CrossRefGoogle Scholar
  33. Raven JA (2005) Cellular location of starch synthesis and evolutionary origin of starch genes. J Phycol 41:1070–1072CrossRefGoogle Scholar
  34. Röber M, Geider K, Muller-Röber B, Willmitzer L (1996) Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates. Planta 199:528–536PubMedCrossRefGoogle Scholar
  35. Roberfroid MB (1999) What is beneficial for health? The concept of functional food. Food Chem Toxicol 37:1039–1041PubMedCrossRefGoogle Scholar
  36. Rossouw D, Bosch S, Kossmann J, Botha FC, Groenewald JH (2007) Downregulation of neutral invertase activity in sugarcane cell suspension cultures leads to a reduction in respiration and growth and an increase in sucrose accumulation. Funct Plant Biol 34:490–498CrossRefGoogle Scholar
  37. Schwab C, Gänzle MG (2006) Effect of membrane lateral pressure on the expression of fructosyltransferases in Lactobacillus reuteri. Syst Appl Microbiol 29:89–99PubMedCrossRefGoogle Scholar
  38. Sévenier R, Hall RD, van der Meer IM, Hakkert HJC, van Tunen AJ, Koops AJ (1998) High level fructan accumulation in a transgenic sugar beet. Nat Biotechnol 16:843–846PubMedCrossRefGoogle Scholar
  39. Smith AM (2008) Harnessing plant biomass for biofuels and biomaterials. Prospects for increasing starch and sucrose yields for bioethanol production. Plant J 54:546–558PubMedCrossRefGoogle Scholar
  40. Snyman SJ, Meyer GM, Carson DL, Botha FC (1996) Establishment of embryogenic callus and transient gene expression in selected sugarcane varieties. S Afr J Bot 62:151–154Google Scholar
  41. Taylor P, Ko H, Adkins S, Rathus C, Birch R (1992) Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum spp. hybrids). Plant Cell Tissue Organ Cult 28:69–78CrossRefGoogle Scholar
  42. Tieking M, Ehrmann MA, Vogel RF, Gänzle MG (2005) Molecular and functional characterization of a levansucrase from the sourdough isolate Lactobacillus sanfranciscensis TMW 1.392. Appl Micorbiol Biotech 66:655–663CrossRefGoogle Scholar
  43. Turk SCHJ, de Roos K, Scott PA, van Kun K, Weisbeek P, Smeekens SCM (1997) The vacuolar sorting domain of sporamin transports GUS, but not levansucrase, to the plant vacuole. New Phytol 136:29–38Google Scholar
  44. Uys L (2006) Computational systems biology of sucrose accumulation in sugarcane. M.Sc. (Biochemistry) thesis, University of Stellenbosch, South AfricaGoogle Scholar
  45. Uys L (2009) Coupling kinetic models and advection-diffusion equations to model vascular transport in plants, applied to sucrose accumulation in sugarcane. Ph.D. (Biochemistry) thesis, University of Stellenbosch, South AfricaGoogle Scholar
  46. van der Meer IM, Ebskamp MJM, Visser RGF, Weisbeek PJ, Smeekensa SCM (1994) Fructan as a new carbohydrate sink in transgenic potato plants. Plant Cell 6:561–570Google Scholar
  47. Wei H, Wang M-L, Moore PH, Albert HH (2003) Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants. J Plant Physiol 160:1241–1251PubMedCrossRefGoogle Scholar
  48. Weising K, Schell J, Kahl G (1988) Foreign genes in plants: transfer, structure, expression and applications. Annu Rev Genet 22:241–277CrossRefGoogle Scholar
  49. Weyens G, Ritsema T, Van Dun K, Meyer D, Lommel M, Lathouwers J, Rosquin I, Denys P, Tossens A, Marleen Nijs M, Turk T, Gerrits N, Bink S, Walraven B, Lefèbvre M, Smeekens S (2004) Production of tailor-made fructans in sugar beet by expression of onion fructosyltransferases. Plant Biotech J 2:321–327CrossRefGoogle Scholar
  50. Wu L, Birch RG (2007) Doubled sugar content in sugarcane plants modified to produce a sucrose isomer. Plant Biotech J 5:109–117CrossRefGoogle Scholar
  51. Zhang S, Dong JG, Wang T, Guo S, Glassman K, Ranch J, Nichols SE (2007) High level accumulation of a-glucan in maize kernels by expressing the gtfD gene from Streptococcus mutans. Transgenic Res 16:467–478PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Rolene Bauer
    • 1
    Email author
  • Carin E. Basson
    • 2
  • Jan Bekker
    • 3
  • Iban Eduardo
    • 3
  • Johann M. Rohwer
    • 4
  • Lafras Uys
    • 4
  • Johannes H. van Wyk
    • 5
  • Jens Kossmann
    • 3
  1. 1.Department of Biotechnology, Institute for Microbial Biotechnology and MetagenomicsUniversity of the Western CapeBellvilleSouth Africa
  2. 2.Department of Viticulture and Enology, Institute for Wine BiotechnologyStellenbosch UniversityMatielandSouth Africa
  3. 3.Genetics Department, Institute for Plant BiotechnologyStellenbosch UniversityMatielandSouth Africa
  4. 4.Department of Biochemistry, Triple-J Group for Molecular Cell PhysiologyStellenbosch UniversityMatielandSouth Africa
  5. 5.Department of Botany and ZoologyStellenbosch UniversityMatielandSouth Africa

Personalised recommendations