Advertisement

Biologia

, Volume 70, Issue 8, pp 1063–1069 | Cite as

cDNA cloning, heterologous expression and characterization of a cell wall invertase from copper tolerant population of Elsholtzia haichowensis

  • Chen LiuEmail author
  • Zhongrui Xu
  • Shenwen Cai
  • Luan Zhang
  • Zhiting Xiong
Article

Abstract

The main objective of the present study was to clone, heterologously express and characterize a novel cell wall invertase (FCWI) from a Cu tolerant population of Elsholtzia haichowensis. The full-length FCWI cDNA contained an open reading frame (ORF) of 1671 bp which encoded a 556-amino-acid protein. The theoretical molecular mass and pI of the deduced protein were 62.5 kDa and 9.29, respectively. Phylogenetic analysis showed that FCWI had a closer evolutionary relationship to cell wall invertase of dicot. FCWI was expressed in methylotrophic yeast Pichia pastoris and purified to near homogeneity. Recombinant FCWI enzyme had pH optima of 4.0 and temperature optima of 50 °C. Activity analyses in the presence of various metal cations indicated that FCWI was completely inhibited by Hg2+ (0%), while retained 77.4% activity when exposure to Cu2+. The Km and Kmax values of FCWI for hydrolyzing sucrose were 0.282 mM and 1.576 μkat/mg, respectively. This is the first report that the heterologous expression and characterization of a cell wall invertase from a Cu tolerant population of E. haichowensis. These results helped to understanding the characteristics of FCWI and its physiological role in the resistance mechanisms of Cu tolerant plants.

Key words

cell wall invertase copper enzyme kinetics Elsholtzia haichowensis heterologous expression Pichia pastoris 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Notes

Acknowledgements

The work was supported by the National Nature Science Foundation of China (Projects 20677046, 30870365, 31270432, 21477093). The authors declare that there is no conflict of interest.

References

  1. Albacete A., Grosskinsky D.K. & Roitsch T. 2011. Trick and treat: a review on the function and regulation of plant invertases in the abiotic stress response. Phyton 50: 181–204.Google Scholar
  2. Asthir B., Kaur A. & Basra A.S. 1998. Cultivar variation in heat stability and kinetic properties of soluble invertase in wheat grains. Acta Physiol. Plant. 20: 339–345.CrossRefGoogle Scholar
  3. Cai S., Xiong Z., Li L., Li M., Zhang L., Liu C. & Xu Z. 2014. Differential responses of root growth, acid invertase activity and transeript level to copper stress in two contrasting populations of Elsholtzia haichowensis. Ecotoxicology 23: 76–91.Google Scholar
  4. Canam T., Unda F. & Mans field S.D. 2008. Heterologous expression and functional characterization of two hybrid poplar cellwall invertases. Planta 228: 1011–1019.CrossRefGoogle Scholar
  5. Charng Y., Juang R., Su J. & Sung H. 1994. Partial puri fication and characterization of invertase isozymes from rice grains (Oryza sativa). Biochem. Mol. Biol. Int. 33: 607–615.Google Scholar
  6. CortésRomero C., MartínezHernández A., MelladoMojica E., López M.G. & Simpson J. 2012. Molecular and functional characterization of novel fructosyltransferases and invertases from Agave tequilana. PLOS ONE 7: e35878.Google Scholar
  7. Daly R. & Hearn M.T. 2005. Expression of heterologous proteins in Pichio, pastoris: a useful experimental tool in protein engineering and production. J. Mol. Recognit. 18: 119–138.CrossRefGoogle Scholar
  8. Goetz M. & Roitsch T. 2000. Identification of amino acids essential for enzymatic activity of plant invertases. J. Plant Physiol. 157: 581–585.CrossRefGoogle Scholar
  9. Hanson J. & Smeekens S. 2009. Sugar perception and signalingan update. Curr. Opin. Plant Biol. 12: 562–567.CrossRefGoogle Scholar
  10. Hsieh C., Liu L., Yeh S., Chen C., Lin H., Sung H. & Wang A. 2006. Molecular cloning and functional identi fication of invertase isozymes from green bamboo Bambusa oldhamii. J. Agr. Food Chem. 54: 3101–3107.CrossRefGoogle Scholar
  11. Ji X., Van den Ende W., Van Laere A., Cheng S. & Bennett J. 2005. Structure, evolution, and expression ofthetwo invertase gene families of rice. J. Mol. Evol. 60: 615–634.CrossRefGoogle Scholar
  12. Koch K. 2004. Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Curr. Opin. Plant Biol. 7: 235–246.CrossRefGoogle Scholar
  13. Lammens W., Le Roy K., Schroeven L., Van Laere A., Rabijns A. & Van den Ende W. 2009. Structural insights into glycoside hydrolase family 32 and 68 enzymes: functional implications. J. Exp. Bot. 60: 727–740.CrossRefGoogle Scholar
  14. Le Roy K., Lammens W., Verhaest M., De Coninck B., Rabijns A., Van Laere A. & Van den Ende W. 2007. Unraveling the difference between invertases and fructan exohydrolases: a single amino acid (Asp-239) substitution transforms Arabidopsis cell wall invertasel into a fructan 1-exohydrolase. Plant Physiol. 145: 616–625.CrossRefGoogle Scholar
  15. Liu J. & Xiong Z. 2005. Differences in accumulation and physiological response to copper stress in three populations of Elsholtzia haichowensis S. Water Air Soil Poli. 168: 5–16.CrossRefGoogle Scholar
  16. Proels R.K. & Roitsch T. 2009. Extracellular invertase LIN6 of tomato: a pivotal enzyme for integration of metabolic, hor-monal, and stress signals is regulated by a diurnal rhythm. J. Exp. Bot. 60: 1555–1567.CrossRefGoogle Scholar
  17. Roitsch T., Balibrea M.E., Hofmann M., Proels R. & Sinha A.K. 2003. Extracellular invertase: key metabolic enzyme and PR protein. J. Exp. Bot. 54: 513–524.CrossRefGoogle Scholar
  18. Roitsch T. & Gonzalez M.C. 2004. Function and regulation of plant invertases: sweet sensations. Trends Plant Sci. 9: 606–613.CrossRefGoogle Scholar
  19. Ruan Y., Jin Y., Yang Y., Li G. & Boyer J.S. 2010. Sugar input, metabolism, and signaling mediated by invertase: roles in development, yield potential, and response to drought and heat. Mol. Plant. 3: 942–955.CrossRefGoogle Scholar
  20. Smeekens S., Ma J., Hanson J. & Rolland F. 2010. Sugar signals and molecular networks controlling plant growth. Curr. Opin. Plant Biol. 13: 273–278.CrossRefGoogle Scholar
  21. Somogyi M. 1952. Notes on sugar determination. J. Biol. Chem. 195: 19–23.Google Scholar
  22. Sturm A. 1999. Invertases. Primary structures, functions, and roles in plant development and sucrose partitioning. Plant Physiol. 121: 1–8.PubMedGoogle Scholar
  23. Tang S., Wilke B. & Huang C. 1999. The uptake of copper by plants dominantly growing on copper mining spoils along the Yangtze River, the People’s Republic of China. Plant Soil 209: 225–232.CrossRefGoogle Scholar
  24. Xiong Z., Wang T., Liu K., Zhang Z., Gan J., Huang Y. & Li M. 2008. Differential invertase activity and root growth between Cutolerant and nontolerant populations in Kummerowia stipulacea under Cu stress and nutrient de ficiency. Environ. Exp. Bot. 62: 17–27.CrossRefGoogle Scholar
  25. Zhang L., Xiong Z., Xu Z., Liu C. & Cai S. 2014. Cloning and characterization of acid invertase genes in the roots of the metallophyte Kummerowia stipulacea (Maxim.) Makino from two populations: Differential expression under copper stress. Ecotox. Environ. Safe. 104: 87–95.CrossRefGoogle Scholar

Copyright information

© Slovak Academy of Sciences 2015

Authors and Affiliations

  • Chen Liu
    • 1
    Email author
  • Zhongrui Xu
    • 1
  • Shenwen Cai
    • 2
  • Luan Zhang
    • 3
  • Zhiting Xiong
    • 1
  1. 1.School of Resource and Environmental ScienceWuhan UniversityWuhan, HubeiPeople’s Republic of China
  2. 2.College of Resource and EnvironmentZunyi Normal CollegeZunyi, GuizhouPeople’s Republic of China
  3. 3.School of Resource and Environmental ScienceFujian Agriculture and Forestry UniversityFuzhou, FujianPeople’s Republic of China

Personalised recommendations