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Reuteran and levan as carbohydrate sinks in transgenic sugarcane

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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.

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Abbreviations

FW:

Fresh weight

DP:

Degree of polymerization

HoPS:

Homopolysaccharide

FTF:

Fructosyltransferase

GTF:

Glucosyltransferase

PAS:

Periodic acid-Schiff

MS:

Murashige–Skoog

WT:

Wild type

References

  • 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 51:402–407

    Article  PubMed  CAS  Google Scholar 

  • Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1994) Current protocols in molecular biology. Wiley, New York

    Google Scholar 

  • Basnayake SW, Morgan TC, Wu L, Birch RG (2012) Field performance of transgenic sugarcane expressing isomaltulose synthase. Plant Biotechnol J 10:217–225

    Google Scholar 

  • 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 121:1156–1162

    Article  CAS  Google Scholar 

  • Bauer R, Volschenk H, Dicks LMT (2005) Cloning and expression of the malolactic gene of Pediococcus damnosus NCFB 1832 in Saccharomyces cerevisiae. J Biotechnol 118:353–362

    Article  PubMed  CAS  Google Scholar 

  • 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 131:260–264

    Article  PubMed  CAS  Google Scholar 

  • Bower R, Birch RG (1992) Transgenic sugarcane plants via microprojectile bombardment. Plant J 2:409–416

    Article  CAS  Google Scholar 

  • Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein–dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • 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 110:355–363

    PubMed  CAS  Google Scholar 

  • 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 136:19–28

    CAS  Google Scholar 

  • Cairns AJ (2003) Fructan biosynthesis in transgenic plants. J Exp Bot 54:549–567

    Article  PubMed  CAS  Google Scholar 

  • 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 69:148–152

    Article  PubMed  CAS  Google Scholar 

  • Esawy MA, Mahmoud DAR, Fattah AFA (2008) Immobilisation of Bacillus subtilis NRC33a levansucrase and some studies on its properties. Braz J Chem Eng 25:237–246

    Article  CAS  Google Scholar 

  • Gerrits N, Turk SCHJ, van Dun KPM, Hulleman SHD, Visser RGF, Weisbeek PJ, Smeekens SCM (2001) Sucrose metabolism in plastids. Plant Physiol 125:926–934

    Article  PubMed  CAS  Google Scholar 

  • 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 9:32–37

    Article  CAS  Google Scholar 

  • 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 97:8699–8704

    Article  PubMed  CAS  Google Scholar 

  • 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 119–139

    Google Scholar 

  • 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 29:1287–1298

    Article  CAS  Google Scholar 

  • Jones MGK, Outlaw WH, Lowry OH (1977) Enzymic assay of 10−7 to 10−14 moles of sucrose in plant tissues. Plant Physiol 60:379–383

    Article  PubMed  CAS  Google Scholar 

  • Klibanov AM (2003) Asymmetric enzymatic oxidoreductions in organic solvents. Curr Opin Biotechnol 14:427–431

    Article  PubMed  CAS  Google Scholar 

  • Komor E, Thom M, Maretzki A (1981) The mechanism of sugar uptake by sugarcane suspension cells. Planta 153:181–192

    Article  CAS  Google Scholar 

  • Korakli M, Vogel RF (2006) Structure/function relationship of homopolysaccharide producing glycansucrases and therapeutic potential of their synthesised glycans. Appl Microbiol Biotechnol 71:790–803

    Article  PubMed  CAS  Google Scholar 

  • Kossmann J, Lloyd J (2000) Understanding and influencing starch biochemistry. Crit Rev Plant Sci 19:171–226

    CAS  Google Scholar 

  • 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 68:4283–4291

    Article  PubMed  CAS  Google Scholar 

  • Kralj S, van Geel-Schutten GH, van der Maarel MJEC, Dijkhuizen L (2004) Biochemical and molecular characterization of Lactobacillus reuteri 121 reuteransucrase. Microbiol 150:2099–2112

  • 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 71:3942–3950

    Article  PubMed  CAS  Google Scholar 

  • Moore PH (1987) Anatomy and morphology. In: Heinz DJ (ed) Sugarcane improvement through breeding. Elsevier Science Publisher, New York, pp 85–142

    Google Scholar 

  • 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 643–669

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Plant Physiol 15:473–497

    Article  CAS  Google Scholar 

  • 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 107:125–130

    PubMed  CAS  Google Scholar 

  • 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 5:35–46

    Article  CAS  Google Scholar 

  • 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 220:817–825

    Article  Google Scholar 

  • Raven JA (2005) Cellular location of starch synthesis and evolutionary origin of starch genes. J Phycol 41:1070–1072

    Article  Google Scholar 

  • 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 199:528–536

    Article  PubMed  Google Scholar 

  • Roberfroid MB (1999) What is beneficial for health? The concept of functional food. Food Chem Toxicol 37:1039–1041

    Article  PubMed  CAS  Google Scholar 

  • 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 34:490–498

    Article  CAS  Google Scholar 

  • Schwab C, Gänzle MG (2006) Effect of membrane lateral pressure on the expression of fructosyltransferases in Lactobacillus reuteri. Syst Appl Microbiol 29:89–99

    Article  PubMed  CAS  Google Scholar 

  • 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 16:843–846

    Article  PubMed  Google Scholar 

  • Smith AM (2008) Harnessing plant biomass for biofuels and biomaterials. Prospects for increasing starch and sucrose yields for bioethanol production. Plant J 54:546–558

    Article  PubMed  CAS  Google Scholar 

  • 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 62:151–154

    Google Scholar 

  • 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 28:69–78

    Article  Google Scholar 

  • 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 66:655–663

    Article  CAS  Google Scholar 

  • 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 136:29–38

    CAS  Google Scholar 

  • Uys L (2006) Computational systems biology of sucrose accumulation in sugarcane. M.Sc. (Biochemistry) thesis, University of Stellenbosch, South Africa

  • 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 Africa

  • 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 6:561–570

    Google Scholar 

  • 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 160:1241–1251

    Article  PubMed  CAS  Google Scholar 

  • Weising K, Schell J, Kahl G (1988) Foreign genes in plants: transfer, structure, expression and applications. Annu Rev Genet 22:241–277

    Article  Google Scholar 

  • 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 2:321–327

    Article  CAS  Google Scholar 

  • Wu L, Birch RG (2007) Doubled sugar content in sugarcane plants modified to produce a sucrose isomer. Plant Biotech J 5:109–117

    Article  CAS  Google Scholar 

  • 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 16:467–478

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

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

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Authors and Affiliations

  1. Department of Biotechnology, Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Private Bag X17, Bellville, 7535, South Africa

    Rolene Bauer

  2. Department of Viticulture and Enology, Institute for Wine Biotechnology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa

    Carin E. Basson

  3. Genetics Department, Institute for Plant Biotechnology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa

    Jan Bekker, Iban Eduardo & Jens Kossmann

  4. Department of Biochemistry, Triple-J Group for Molecular Cell Physiology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa

    Johann M. Rohwer & Lafras Uys

  5. Department of Botany and Zoology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa

    Johannes H. van Wyk

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  1. Rolene Bauer
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  2. Carin E. Basson
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  3. Jan Bekker
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Correspondence to Rolene Bauer.

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Bauer, R., Basson, C.E., Bekker, J. et al. Reuteran and levan as carbohydrate sinks in transgenic sugarcane. Planta 236, 1803–1815 (2012). https://doi.org/10.1007/s00425-012-1731-x

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