, Volume 251, Issue 3, pp 639–648 | Cite as

Chloroplast molecular farming: efficient production of a thermostable xylanase by Nicotiana tabacum plants and long-term conservation of the recombinant enzyme

  • Laura Pantaleoni
  • Paolo Longoni
  • Lorenzo Ferroni
  • Costanza Baldisserotto
  • Sadhu Leelavathi
  • Vanga Siva Reddy
  • Simonetta Pancaldi
  • Rino Cella
Original Article


The high cost of recombinant enzymes for the production of biofuel from ligno-cellulosic biomass is a crucial factor affecting the economic sustainability of the process. The use of plants as biofactories for the production of the suitable recombinant enzymes might be an alternative to microbial fermentation. In the case of enzyme accumulation in chloroplasts, it is fundamental to focus on the issue of full photosynthetic efficiency of transplastomic plants in the field where they might be exposed to abiotic stress such as high light intensity and high temperature. Xylanases (EC, a group of enzymes that hydrolyse linear polysaccharides of beta-1,4-xylan into xylose, find an application in the biofuel industry favouring biomass saccharification along with other cell-wall degrading enzymes. In the present study, we analysed how a high level of accumulation of a thermostable xylanase in tobacco chloroplasts does not impact on photosynthetic performance of transplastomic plants grown outdoors. The recombinant enzyme was found to be stable during plant development, ex planta and after long-term storage.


Chloroplast transformation Enzyme long-term storage Photosynthetic performance of tobacco transplastomic plants Plant biofactory Recombinant thermostable xylanase 



Bacillus sp. xylanase


Total soluble proteins






Maximum PSII fluorescence in the dark-adapted state


Minimum fluorescence in the dark-adapted state


Variable fluorescence


Pulse amplitude modulated fluorimetry


Photosystem II


  1. Allahverdiyeva T, Aro E-M (2012) Photosynthetic responses of plants to excess light: mechanisms and conditions for photoinhibition, excess energy dissipation and repair. In: Eaton JJ, Tripathy BC, Sharkey TD (eds) Photosynthesis: plastid biology, energy conversion and carbon assimilation. Advances in photosynthesis and respiration. Springer, Dordrecht, pp 275–297CrossRefGoogle Scholar
  2. Anderson JM (1986) Photoregulation of the composition, function, and structure of thylakoid membranes. Annu Rev Plant Physiol Plant Mol Biol 37:93–136CrossRefGoogle Scholar
  3. Baldisserotto C, Ferroni L, Anfuso E, Pagnoni A, Fasulo MP, Pancaldi S (2007) Responses of Trapa natans L. floating laminae to high concentrations of manganese. Protoplasma 231:65–82PubMedCrossRefGoogle Scholar
  4. Baldisserotto C, Ferroni L, Zanzi C, Marchesini R, Pagnoni A, Pancaldi S (2010) Morpho-physiological and biochemical responses in the floating lamina of Trapa natans exposed to molybdenum. Protoplasma 240:83–97PubMedCrossRefGoogle Scholar
  5. Bally J, Nadai M, Vitel M, Rolland A, Dumain R, Dubald M (2009) Plant physiological adaptations to the massive foreign protein synthesis occurring in recombinant chloroplasts. Plant Physiol 150:1474–1481PubMedCentralPubMedCrossRefGoogle Scholar
  6. Bhardwaj A, Leelavathi S, Mazumdar-Leighton S, Ghosh A, Ramakumar S, Reddy VS (2010) The critical role of N- and C-terminal contact in protein stability and folding of a family 10 xylanase under extreme conditions. Plos One 5:e11347PubMedCentralPubMedCrossRefGoogle Scholar
  7. Borisjuk NV, Borjsiuk LG, Logendra S, Petersen F, Gleba Y, Raskin I (1999) Production of recombinant proteins in plant root exudates. Nat Biotechnol 17:466–469PubMedCrossRefGoogle Scholar
  8. Borkhardt B, Harholt J, Ulvskov P, Ahring BK, Jorgensen B, Brinch-Pedersen H (2010) Autohydrolysis of plant xylans by apoplastic expression of thermophilic bacterial endo-xylanases. Plant Biotechnol J 8:363–374PubMedCrossRefGoogle Scholar
  9. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  10. Conley AJ, Joensuu JJ, Richman A, Manassa R (2011) Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants. Plant Biotechnol J 9:419–433PubMedCrossRefGoogle Scholar
  11. Doran PM (2006) Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol 24:426–432PubMedCrossRefGoogle Scholar
  12. Ghose TK (1987) Measurements of cellulase activities. Pure Appl Chem 59:257–268Google Scholar
  13. Gupta N, Reddy VS, Maiti S, Ghosh A (2000) Cloning, expression, and sequence analysis of the gene encoding the alkali-stable, thermostable endoxylanase from alkalophilic, mesophilic Bacillus sp. strain NG-27. Appl Environ Microbiol 66:2631–2635PubMedCentralPubMedCrossRefGoogle Scholar
  14. Hendrickson L, Furbank RT, Chow WS (2004) A simple alternative approach to assessing the fate of absorbed light energy using chlorophyll fluorescence. Photosynth Res 82:73–81PubMedCrossRefGoogle Scholar
  15. Horton P, Ruban AV, Walters RG (1996) Regulation of light harvesting in green plants. Annu Rev Plant Physiol Plant Mol Biol 47:655–684PubMedCrossRefGoogle Scholar
  16. Jahns P, Holzwarth AR (2012) The role of the xanthophyll cycle and of lutein in photoprotection of photosystem. Biochim Biophys Acta 1817:182–193PubMedCrossRefGoogle Scholar
  17. Juturu V, Wu JC (2012) Microbial xylanases: engineering, production and industrial applications. Biotechnol Adv 30:1219–1227PubMedCrossRefGoogle Scholar
  18. Kim JY, Kavas M, Fouad WM, Nong G, Preston JF, Altpeter F (2011) Production of hyperthermostable GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants enables complete hydrolysis of methylglucuronoxylan to fermentable sugars for biofuel production. Plant Mol Biol 76:357–369PubMedCrossRefGoogle Scholar
  19. Klughammer C, Schreiber U (2008) Complementary PSII quantum yields calculated from simple fluorescence parameters measured byPAM fluorometry and the saturation pulse method. PAM Appl Notes 1:27–35Google Scholar
  20. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  21. Leelavathi S, Gupta N, Maiti S, Ghosh A, Reddy VS (2003) Overproduction of an alkali- and thermo-stable xylanase in tobacco chloroplasts and efficient recovery of the enzyme. Mol Breed 11:59–67CrossRefGoogle Scholar
  22. Lichtenthaler HK, Buschmann C, Knapp M (2005) How to correctly determine the different chlorophyll fluorescence parameters and the chlorophyll fluorescence decrease ratio R-Fd of leaves with the PAM fluorometer. Photosynthetica 43:379–393CrossRefGoogle Scholar
  23. Magee AM, Coyne S, Murphy D, Horvath EM, Medgyesy P, Kavanagh TA (2004) T7 RNA polymerase-directed expression of an antibody fragment transgene in plastids causes a semi-lethal pale-green seedling phenotype. Transgenic Res 13:325–337PubMedCrossRefGoogle Scholar
  24. Matsubara S, Chow WS (2004) Populations of photo inactivated photosystem II reaction centers characterized by chlorophyll a fluorescence lifetime in vivo. PNAS 101:18234–18239PubMedCentralPubMedCrossRefGoogle Scholar
  25. Meyers B, Zaltsman A, Lacroix B, Kozlovsky SV, Krichevsky A (2010) Nuclear and plastid genetic engineering of plants: comparison of opportunities and challenges. Biotechnol Adv 28:747–756PubMedCrossRefGoogle Scholar
  26. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  27. Obembe OO, Popoola JO, Leelavathi S, Reddy SV (2011) Advances in plant molecular farming. Biotechnol Adv 29:210–222PubMedCrossRefGoogle Scholar
  28. Pauly M, Keegstra K (2010) Plant cell wall polymers as precursors for biofuels. Curr Opin Plant Biol 13:305–312PubMedCrossRefGoogle Scholar
  29. Pogorelko G, Fursova O, Lin M, Pyle E, Jass J, Zabotina OA (2011) Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast. Plant Mol Biol 77:433–445PubMedCrossRefGoogle Scholar
  30. Ruban AV, Johnson MP, Duffy CDP (2012) The photoprotective molecular switch in the photosystem II antenna. BBA Bioenergetics 1817:167–181PubMedCrossRefGoogle Scholar
  31. Wagner R, Dietzel L, Braeutigam K, Fischer W, Pfannschmidt T (2008) The long-term response to fluctuating light quality is an important and distinct light acclimation mechanism that supports survival of Arabidopsis thaliana under low light conditions. Planta 228:573–587PubMedCrossRefGoogle Scholar
  32. Wellburn AR (1994) The spectral determination of chlorophyll-a and chlorophyll-b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144:307–313CrossRefGoogle Scholar
  33. Xu J, Dolan MC, Medrano G, Cramer CL, Weathers PJ (2012) Green factory: plants as bioproduction platforms for recombinant proteins. Biotechnol Adv 30:1171–1184PubMedCrossRefGoogle Scholar
  34. Zhang Z, Donaldson AA, Ma X (2012) Advancements and future directions in enzyme technology for biomass conversion. Biotechnol Adv 30:913–919PubMedCrossRefGoogle Scholar
  35. Zieminski K, Romanowska I, Kowalska M (2012) Enzymatic pretreatment of lignocellulosic wastes to improve biogas production. Waste Manag 32:1131–1137PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2013

Authors and Affiliations

  • Laura Pantaleoni
    • 1
  • Paolo Longoni
    • 1
    • 4
  • Lorenzo Ferroni
    • 2
  • Costanza Baldisserotto
    • 2
  • Sadhu Leelavathi
    • 3
  • Vanga Siva Reddy
    • 3
  • Simonetta Pancaldi
    • 2
  • Rino Cella
    • 1
  1. 1.Department of Biology and BiotechnologyUniversity of PaviaPaviaItaly
  2. 2.Department of Life Sciences and BiotechnologiesUniversity of FerraraFerraraItaly
  3. 3.Plant Transformation GroupInternational Centre for Genetic Engineering and BiotechnologyNew DelhiIndia
  4. 4.Department of Botany and Plant BiologyUniversity of Geneva, Sciences IIIGeneve 4Switzerland

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