Skip to main content
SpringerLink
Log in

RNAi Mediated Down-Regulation of PDS Gene Expression in Sugarcane (Saccharum), a Highly Polyploid Crop

  • Published:
Tropical Plant Biology Aims and scope Submit manuscript

Abstract

Sugarcane is a crop with great potential for metabolic engineering, but progress has been limited by highly efficient transgene silencing. The potential exists to utilize efficient gene silencing in molecular improvement through down-regulation of sugarcane genes. However, sugarcane is highly polyploid and heterozygous, which might complicate efforts to employ transgene-mediated silencing of endogenous genes. To explore this issue, we tested in sugarcane a construct designed for hairpin-mediated silencing of the phytoene desaturase (PDS) gene in sugarcane. The hairpin construct was designed based on the sugarcane PDS EST collection containing multiple alleles, to down-regulate the suite of PDS alleles. Three out of four plants transformed using the PDS hairpin construct showed detectable hairpin transcripts when analyzed by northern analysis, and displayed the photo-bleaching phenotype characteristic of PDS knockout lines. There was near-complete reduction in the levels of endogenous PDS transcripts in leaf tissue, indicating efficient hairpin-mediated down-regulation despite the highly polyploid and heterozygous sugarcane genome. Hairpin-mediated gene silencing should therefore be a powerful tool for the molecular improvement of this important crop.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

CDS:

Coding sequence

cDNA:

complementary DNA

DNA:

Deoxyribonucleic acid

EST:

Expressed sequence tags

INDEL:

Insertion/deletion

PCR:

Polymerase chain reaction

PDS:

Phytoene desaturase

RNA:

Ribonucleic acid

RNAi:

RNA interference

RT-PCR:

Reverse transcription-polymerase chain reaction

SNP:

Single nucleotide polymorphism

UTR:

Untranslated region

References

  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    PubMed  CAS  Google Scholar 

  2. Bartley GE, Scolnik PA (1995) Plant carotenoids—pigments for photoprotection, visual attraction, and human health. Plant Cell 7:1027–1038

    Article  PubMed  CAS  Google Scholar 

  3. Birch RG (2007) Metabolic engineering in sugarcane: assisting the transition to a bio-based economy. In: Applications of plant metabolic engineering. Springer: Berlin, pp 249–281

  4. Botha F, Sawyer B, Birch R (2001) Sucrose metabolism in the culm of transgenic sugarcane with reduced soluble acid invertase activity. Proceedings of the International Society of Sugarcane Technologists XXIV Congress ASSCT: Mackay, pp 588–591

  5. Bower R, Elliott AR, Potier BAM, Birch RG (1996) High-efficiency, microprojectile-mediated cotransformation of sugarcane, using visible or selectable markers. Mol Breed 2:239–249

    Article  CAS  Google Scholar 

  6. Casu RE, Dimmock CM, Chapman SC, Grof CPL, McIntyre CL, Bonnett GD, Manners JM (2004) Identification of differentially expressed transcripts from maturing stem of sugarcane by in silico analysis of stem expressed sequence tags and gene expression profiling. Plant Mol Biol 54:503–517

    Article  PubMed  Google Scholar 

  7. Christensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18:675–689

    Article  PubMed  CAS  Google Scholar 

  8. Cordeiro GM, Taylor GO, Henry RJ (2000) Characterisation of microsatellite markers from sugarcane (Saccharum sp.), a highly polyploid species. Plant Sci 155:161–168

    Article  PubMed  CAS  Google Scholar 

  9. Davies MJ (2003) Singlet oxygen-mediated damage to proteins and its consequences. Biochem Biophys Res Commun 305:761–770

    Article  PubMed  CAS  Google Scholar 

  10. Fromm M, Taylor LP, Walbot V (1985) Expression of genes transferred into monocot and dicot plant-cells by electroporation. Proc Natl Acad Sci USA 82:5824–5828

    Article  PubMed  CAS  Google Scholar 

  11. Fusaro AF, Matthew L, Smith NA, Curtin SJ, Dedic-Hagan J, Ellacott GA, Watson JM, Wang MB, Brosnan C, Carroll BJ, Waterhouse PM (2006) RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway. EMBO Rep 7:1168–1175

    Article  PubMed  CAS  Google Scholar 

  12. Glaszmann JC, Fautret A, Noyer JL, Feldmann P, Lanaud C (1989) Biochemical genetic-markers in sugarcane. Theor Appl Genet 78:537–543

    Article  CAS  Google Scholar 

  13. Grivet L, Arruda P (2001) Sugarcane genomics: depicting the complex genome of an important tropical crop. Curr Opin Plant Biol 5:122–127

    Article  Google Scholar 

  14. Grivet L, Glaszmann JC, Arruda P (2001) Sequence polymorphism from EST data in sugarcane: a fine analysis of 6-phosphogluconate dehydrogenase genes. Genet Mol Biol 24:161–167

    Article  CAS  Google Scholar 

  15. Grivet L, Glaszmann J-C, Vincentz M, da Silva F, Arruda P (2003) ESTs as a source for sequence polymorphism discovery in sugarcane: example of the Adh genes. Theor Appl Genet 106:190–197

    PubMed  CAS  Google Scholar 

  16. Groenewald JH, Botha FC (2008) Down-regulation of pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity in sugarcane enhances sucrose accumulation in immature internodes. Transgenic Res 17:85–92

    Article  PubMed  CAS  Google Scholar 

  17. Hansom S, Bower R, Zhang L, Potier B, Elliot A, Basnayke S, Cordeiro G, Hogarth D, Cox M, Berding N, Birch R (1999) Regulation of transgene expression in sugarcane. In: Singh V (ed) Proc XXIII International Society of Sugarcane Technologies Congress. pp 278–290

  18. Jannoo N, Grivet L, Seguin M, Paulet F, Domaingue R, Rao PS, Dookun A, D’Hont A, Glaszmann JC (1999) Molecular investigation of the genetic base of sugarcane cultivars. Theor Appl Genet 99:171–184

    Article  CAS  Google Scholar 

  19. Lawrence RJ, Pikaard CS (2003) Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations. Plant J 36:114–121

    Article  PubMed  CAS  Google Scholar 

  20. Ma HM, Albert HH, Paull R, Moore PH (2000) Metabolic engineering of invertase activities in different subcellular compartments affects sucrose accumulation in sugarcane cells. Aust J Plant Physiol 27:1021–1030

    CAS  Google Scholar 

  21. Ma HM, Schulze S, Lee S, Yang M, Mirkov E, Irvine J, Moore P, Paterson A (2004) An EST survey of the sugarcane transcriptome. Theor Appl Genet 108:851–863

    Article  PubMed  Google Scholar 

  22. McIntyre CL, Jackson M, Cordeiro GM, Amouyal O, Hermann S, Aitken KS, Eliott F, Henry RJ, Casu RE, Bonnet GD (2006) The identification and characterisation of alleles of sucrose phosphate synthase gene family III in sugarcane. Mol Breed 18:39–50

    Article  CAS  Google Scholar 

  23. Mudge SR, Osabe K, Casu RE, Bonnett GD, Manners JM, Birch RG (2009) Efficient silencing of reporter transgenes coupled to known functional promoters in sugarcane, a highly polyploid crop species. Planta 229:549–558

    Article  PubMed  CAS  Google Scholar 

  24. Papini-Terzi FS, Rocha FR, Vencio RZN, Oliveira KC, Felix JD, Vicentini R, Rocha CD, Simoes ACQ, Ulian EC, Di Mauro SMZ, Da Silva AM, Pereira CAD, Menossi M, Souza GM (2005) Transcription profiling of signal transduction-related genes in sugarcane tissues. DNA Res 12:27–38

    Article  PubMed  CAS  Google Scholar 

  25. 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 

  26. Sambrook J, Russell DW (2001) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, third edition

  27. Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Gene expression—total silencing by intron-spliced hairpin RNAs. Nature 407:319–320

    Article  PubMed  CAS  Google Scholar 

  28. Travella S, Klimm TE, Keller B (2006) RNA interference-based gene silencing as an efficient tool for functional genomics in hexaploid bread wheat. Plant Physiol 142:6–20

    Article  PubMed  CAS  Google Scholar 

  29. Vettore AL, da Silva FR, Kemper EL, Arruda P (2001) The libraries that made SUCEST. Genet Mol Biol 24:1–7

    Article  CAS  Google Scholar 

  30. Vickers JE, Grof CPL, Bonnett GD, Jackson PA, Knight DP, Roberts SE, Robinson SP (2005) Overexpression of polyphenol oxidase in transgenic sugarcane results in darker juice and raw sugar. Crop Sci 45:354–362

    Article  CAS  Google Scholar 

  31. Wang T, Iyer LM, Pancholy R, Shi XY, Hall TC (2005) Assessment of penetrance and expressivity of RNAi-mediated silencing of the Arabidopsis phytoene desaturase gene. New Phytol 167:751–760

    Article  PubMed  CAS  Google Scholar 

  32. Watson JM, Fusaro AF, Wang MB, Waterhouse PM (2005) RNA silencing platforms in plants. FEBS Lett 579:5982–5987

    Article  PubMed  CAS  Google Scholar 

  33. Wetzel CM, Rodermel SR (1998) Regulation of phytoene desaturase expression is independent of leaf pigment content in Arabidopsis thaliana. Plant Mol Biol 37:1045–1053

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgement

This research was supported by a full scholarship provided by the Australian Sugar Research and Development Corporation for Kenji Osabe.

Author information

Authors and Affiliations

  1. School of Biological Science, The University of Queensland, Brisbane, QLD, 4072, Australia

    Kenji Osabe, Stephen R. Mudge, Michael W. Graham & Robert G. Birch

Authors
  1. Kenji Osabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stephen R. Mudge
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael W. Graham
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert G. Birch
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kenji Osabe.

Additional information

Communicated by: Paul Moore

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Figure 1

(DOC 503 kb)

Supplementary Figure 2

(DOC 192 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Osabe, K., Mudge, S.R., Graham, M.W. et al. RNAi Mediated Down-Regulation of PDS Gene Expression in Sugarcane (Saccharum), a Highly Polyploid Crop. Tropical Plant Biol. 2, 143–148 (2009). https://doi.org/10.1007/s12042-009-9036-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12042-009-9036-3

Keywords

Search

Navigation