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Functional Plant Biology Functional Plant Biology Society
Plant function and evolutionary biology
RESEARCH ARTICLE

Comparative efficiency of subcellular targeting signals for expression of a toxic protein in sugarcane

Mark A. Jackson A B C E , Kerry A. Nutt A D , Rachael Hassall A D and Anne L. Rae A B
+ Author Affiliations
- Author Affiliations

A Cooperative Research Centre for Sugar Industry Innovation through Biotechnology, The University of Queensland, St Lucia, Qld 4072, Australia.

B CSIRO Plant Industry, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Qld 4067, Australia.

C School of Molecular and Microbial Sciences, The University of Queensland, St Lucia, Qld 4072, Australia.

D David North Plant Research Centre, BSES Limited, PO Box 86, Indooroopilly, Qld 4068, Australia.

E Corresponding author. Email: m.jackson1@uq.edu.au

Functional Plant Biology 37(8) 785-793 https://doi.org/10.1071/FP09243
Submitted: 7 October 2009  Accepted: 12 March 2010   Published: 26 July 2010

Abstract

Transgenic sugarcane plants (Saccharum hybrid) have been proposed as a production platform for recombinant proteins, including those providing pathogen resistance as well as high value therapeutic proteins. For the in planta production of proteins that are potentially toxic, a careful consideration of subcellular location is required in order to optimise yield and to avoid detrimental interaction with plant cellular processes. In this study, avidin, a glycoprotein that is potentially toxic to cells because of its high affinity to the co-vitamin biotin, was used to test the effectiveness of a range of targeting signals. Accumulation of avidin was directed to the apoplast, endoplasmic reticulum and to the lytic and delta type vacuoles. Although targeting to the delta vacuole resulted in the highest yields of avidin, these plants developed a biotin deficient phenotype, indicating that this targeting was not fully effective in protecting cellular biotin pools. Similar problems were also observed when avidin was retained in the endoplasmic reticulum. When avidin was targeted to the lytic vacuole using the targeting signal from the sugarcane legumain, plants remained phenotypically normal; however, avidin was predominantly detected as a degraded product due to site-specific limited proteolysis in the vacuole. For avidin and other potentially toxic products, this lytic vacuole targeting signal may be useful if stability within this proteolytic environment can be improved.

Additional keywords: biofactory, endoplasmic reticulum, Saccharum, vacuole.


Acknowledgements

Mark Jackson was supported by a PhD research scholarship from the Cooperative Research Centre for Sugar Industry Innovation through Biotechnology. The authors thank Dr Colleen Murray (HortResearch, NZ) for the potato proteinase inhibitor I sequence and for helpful discussions. We are grateful to Dr Rosanne Casu (CSIRO Plant Industry) for ADF primers sequences and Associate Professor Don Maclean for helpful discussion and critical evaluation of this manuscript.


References


Abranches R, Arcalis E, Marcel S, Altmann F, Ribeiro-Pedro M, Rodriguez J, Stoger E (2008) Functional specialization of Medicago truncatula leaves and seeds does not affect the subcellular localization of a recombinant protein. Planta 227, 649–658.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Alban C, Job D, Douce R (2000) Biotin metabolism in plants. Annual Review of Plant Physiology and Plant Molecular Biology 51, 17–47.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Bower R, Elliott AR, Potier BAM, Birch RG (1996) High-efficiency, microprojectile-mediated cotransformation of sugarcane, using visible or selectable markers. Molecular Breeding 2, 239–249.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Burgess EPJ, Malone LA, Christeller JT, Lester MT, Murray C, Philip BA, Phung MM, Tregidga EL (2002) Avidin expressed in transgenic tobacco leaves confers resistance to two noctuid pests, Helicoverpa armigera and Spodoptera litura. Transgenic Research 11, 185–198.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Conrad U, Fiedler U (1998) Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Molecular Biology 38, 101–109.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Czechowski T, Bari RP, Stitt M, Scheible WR, Udvardi MK (2004) Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. The Plant Journal 38, 366–379.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Ellison D, Hinton J, Hubbard SJ, Beynon RJ (1995) Limited proteolysis of native proteins – the interaction between avidin and proteinase-K. Protein Science 4, 1337–1345.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Ginzberg I, Perl A, Genser M, Wininger S, Nemas C, Kapulnik Y (2004) Expression of streptavidin in tomato resulted in abnormal plant development that could be restored by biotin application. Journal of Plant Physiology 161, 611–620.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Gnanasambandam A, Birch RG (2004) Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and Arabidopsis. Plant Cell Reports 23, 435–447.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Hiller Y, Bayer EA, Wilchek M (1991) Studies on the biotin-binding site of avidin – minimized fragments that bind biotin. The Biochemical Journal 278, 573–585.
CAS | PubMed |
open url image1

Hoisington D (1992) ‘Laboratory protocols: CIMMYT Applied Molecular Genetics Laboratory.’ (CIMMYT: Mexico)

Hood EE, Witcher DR, Maddock S, Meyer T, Baszczynski C , et al . (1997) Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification. Molecular Breeding 3, 291–306.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Iskandar HM, Simpson RS, Casu RE, Bonnett GD, Maclean DJ, Manners JM (2004) Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression. Plant Molecular Biology Reporter 22, 325–337.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Jackson MA, Rae AL, Casua RE, Grof CPL, Bonnett GD, Maclean DJ (2007) A bioinformatic approach to the identification of a conserved domain in a sugarcane legumain that directs GFP to the lytic vacuole. Functional Plant Biology 34, 633–644.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Jauh GY, Fischer AM, Grimes HD, Ryan CA, Rogers JC (1998) delta-Tonoplast intrinsic protein defines unique plant vacuole functions. Proceedings of the National Academy of Sciences of the United States of America 95, 12 995–12 999.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Korpela J, Kulomaa M, Tuohimaa P, Vaheri A (1983) Avidin is induced in chicken-embryo fibroblasts by viral transformation and cell-damage. EMBO Journal 2, 1715–1719.
CAS | PubMed |
open url image1

Kramer KJ, Morgan TD, Throne JE, Dowell FE, Bailey M, Howard JA (2000) Transgenic avidin maize is resistant to storage insect pests. Nature Biotechnology 18, 670–674.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Markwick NP, Docherty LC, Phung MM, Lester MT, Murray C , et al . (2003) Transgenic tobacco and apple plants expressing biotin-binding proteins are resistant to two cosmopolitan insect pests, potato tuber moth and light brown apple moth, respectively. Transgenic Research 12, 671–681.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

McQualter RB, Dale JL, Harding RM, McMahon JA, Smith GR (2004) Production and evaluation of transgenic sugarcane containing a Fiji disease virus (FDV) genome segment S9-derived synthetic resistance gene. Australian Journal of Agricultural Research 55, 139–145.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Murray C, Sutherland PW, Phung MM, Lester MT, Marshall RK, Christeller JT (2002) Expression of biotin-binding proteins, avidin and streptavidin, in plant tissues using plant vacuolar targeting sequences. Transgenic Research 11, 199–214.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Peng RH, Yao QH, Xiong AS, Cheng ZM, Li Y (2006) Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola. Plant Cell Reports 25, 124–132.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Pinon V, Ravanel S, Douce R, Alban C (2005) Biotin synthesis in plants. The first committed step of the pathway is catalyzed by a cytosolic 7-keto-8-aminopelargonic acid synthase. Plant Physiology 139, 1666–1676.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neuroscience Letters 339, 62–66.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Rivard D, Anguenot R, Brunelle F, Le VQ, Vezina LP, Trepanier S, Michaud D (2006) An in-built proteinase inhibitor system for the protection of recombinant proteins recovered from transgenic plants. Plant Biotechnology Journal 4, 359–368.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Sardana R, Dudani AK, Tackaberry E, Alli Z, Porter S, Rowlandson K, Ganz P, Altosaar I (2007) Biologically active human GM-CSF produced in the seeds of transgenic rice plants. Transgenic Research 16, 713–721.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Sétamou M, Bernal JS, Legaspi JC, Mirkov TE, Legaspi BC (2002) Evaluation of lectin-expressing transgenic sugarcane against stalkborers (Lepidoptera: Pyralidae): effects on life history parameters. Journal of Economic Entomology 95, 469–477.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Tang W, Newton RJ, Weidner DA (2007) Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine. Journal of Experimental Botany 58, 545–554.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Vitale A, Boston RS (2008) Endoplasmic reticulum quality control and the unfolded protein response: insights from plants. Traffic 9, 1581–1588.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Vitale A, Pedrazzini E (2005) Recombinant pharmaceuticals from plants: the plant endomembrane system as bioreactor. Molecular Interventions 5, 216–225.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Wallen MJ, Laukkanen MO, Kulomaa MS (1995) Cloning and sequencing of the chicken egg-white avidin-encoding gene and its relationship with the avidin-related genes Avr1-Avr5. Gene 161, 205–209.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Wang ML, Goldstein C, Su W, Moore PH, Albert HH (2005) Production of biologically active GM-CSF in sugarcane: a secure biofactory. Transgenic Research 14, 167–178.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Yang JJ, Barr LA, Fahnestock SR, Liu ZB (2005) High yield recombinant silk-like protein production in transgenic plants through protein targeting. Transgenic Research 14, 313–324.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Yoza K, Imamura T, Kramer KJ, Morgan TD, Nakamura S, Akiyama K, Kawasaki S, Takaiwa F, Ohtsubo K (2005) Avidin expressed in transgenic rice confers resistance to the stored-product insect pests Tribolium confusum and Sitotroga cerealella. Bioscience, Biotechnology, and Biochemistry 69, 966–971.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1

Zhang L, Birch RG (1996) Biocontrol of sugar cane leaf scald disease by an isolate of Pantoea dispersa which detoxifies albicidin phytotoxins. Letters in Applied Microbiology 22, 132–136.
Crossref | GoogleScholarGoogle Scholar | CAS | open url image1

Zhou FY, Wang ML, Albert HH, Moore PH, Zhu YJ (2006) Efficient transient expression of human GM-CSF protein in Nicotiana benthamiana using potato virus × vector. Applied Microbiology and Biotechnology 72, 756–762.
Crossref | GoogleScholarGoogle Scholar | CAS | PubMed | open url image1