Whole cell solid-state NMR study of Chlamydomonas reinhardtii microalgae

  • Alexandre A. Arnold
  • Jean-Philippe Bourgouin
  • Bertrand Genard
  • Dror E. Warschawski
  • Réjean Tremblay
  • Isabelle Marcotte
Article

Abstract

In vivo or whole-cell solid-state NMR is an emerging field which faces tremendous challenges. In most cases, cell biochemistry does not allow the labelling of specific molecules and an in vivo study is thus hindered by the inherent difficulty of identifying, among a formidable number of resonances, those arising from a given molecule. In this work we examined the possibility of studying, by solid-state NMR, the model organism Chlamydomonas reinhardtii fully and non-specifically 13C labelled. The extension of NMR-based dynamic filtering from one-dimensional to two-dimensional experiments enabled an enhanced selectivity which facilitated the assignment of cell constituents. The number of resonances detected with these robust and broadly applicable experiments appears to be surprisingly sparse. Various constituents, notably galactolipids abundant in organelle membranes, carbohydrates from the cell wall, and starch from storage grains could be unambiguously assigned. Moreover, the dominant crystal form of starch could be determined in situ. This work illustrates the feasibility and caveats of using solid-state NMR to study intact non-specifically 13C labelled micro-organisms.

Keywords

In vivo NMR Magic-angle spinning Lipids Cell-wall Starch Isotope labelling Dynamic filters 

Notes

Acknowledgements

The authors would like to thank Dr. Francesca Zito (CNRS, France) for providing the Chlamydomonas strain and her insights on Chlamydomonas growth and physiology. This work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada (Grant 326750-2013 to I.M.) and the Centre National de la Recherche Scientifique (UMR 7099 to D.E.W.). J.-P.B. would like to acknowledge the Groupe de Recherche Axé sur la Structure des Protéines (GRASP) and the NSERC for the award of scholarships. B.G. would like to thank the Canadian Institutes of Health Research Strategic Training initiative in Chemical Biology and the Réseau Aquaculture Québec (RAQ) for the award of scholarships. IM and RT are members of the RAQ.

Supplementary material

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Supplementary material 1 (PDF 757 KB)

References

  1. Akhter M et al (2016) Identification of aquatically available carbon from algae through solution-state NMR of whole (13)C-labelled cells. Anal Bioanal Chem 408:4357–4370CrossRefGoogle Scholar
  2. Andronesi OC et al (2005) Determination of membrane protein structure and dynamics by magic-angle-spinning solid-state NMR spectroscopy. J Am Chem Soc 127:12965–12974CrossRefGoogle Scholar
  3. Arnold AA et al (2015) Identification of lipid and saccharide constituents of whole microalgal cells by 13C solid-state NMR. Biochim Biophys Acta 1848:369–377CrossRefGoogle Scholar
  4. Beal CM, Webber ME, Ruoff RS, Hebner RE (2010) Lipid analysis of Neochloris oleoabundans by liquid state NMR. Biotechnol Bioeng 106:573–583CrossRefGoogle Scholar
  5. Blaby IK et al (2014) The Chlamydomonas genome project: a decade on. Trends Plant Sci 19:672–680CrossRefGoogle Scholar
  6. Boender GJ, Raap J, Prytulla S, Oschkinat H, De Groot HJ (1995) MAS NMR structure refinement of uniformly 13C enriched chlorophyll a/water aggregates with 2D dipolar correlation spectroscopy. Chem Phys Lett 237:502–508ADSCrossRefGoogle Scholar
  7. Bollig K et al (2007) Structural analysis of linear hydroxyproline-bound O-glycans of Chlamydomonas reinhardtii—conservation of the inner core in Chlamydomonas and land plants. Carbohydr Res 342:2557–2566CrossRefGoogle Scholar
  8. Bonente G, Pippa S, Castellano S, Bassi R, Ballottari M (2012) Acclimation of Chlamydomonas reinhardtii to different growth irradiances. J Biol Chem 287:5833–5847CrossRefGoogle Scholar
  9. Boyle NR, Morgan JA (2009) Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii. BMC Syst Biol 3:4CrossRefGoogle Scholar
  10. Bradbury JH, Jenkins GA (1984) Determination of the structures of trisaccharides by 13C-n.m.r. spectroscopy. Carbohydr Res 126:125–156CrossRefGoogle Scholar
  11. Buleon A et al (1997) Starches from A to C. Chlamydomonas reinhardtii as a model microbial system to investigate the biosynthesis of the plant amylopectin crystal. Plant Physiol 115:949–957CrossRefGoogle Scholar
  12. Catalanotti C, Yang W, Posewitz MC, Grossman AR (2013) Fermentation metabolism and its evolution in algae. Front Plant Sci 4:150CrossRefGoogle Scholar
  13. Dick-Perez M et al (2011) Structure and interactions of plant cell-wall polysaccharides by two- and three-dimensional magic-angle-spinning solid-state NMR. Biochemistry 50:989–1000CrossRefGoogle Scholar
  14. Ferris PJ et al (2001) Glycosylated polyproline II rods with kinks as a structural motif in plant hydroxyproline-rich glycoproteins. Biochemistry 40:2978–2987CrossRefGoogle Scholar
  15. Fu R et al (2011) In situ structural characterization of a recombinant protein in native Escherichia coli membranes with solid-state magic-angle-spinning NMR. J Am Chem Soc 133:12370–12373CrossRefGoogle Scholar
  16. Guschina IA, Harwood JL Algal lipids and effect of the environment on their biochemistry. In: Lipids in aquatic ecosystems (Arts MT, Brett MT, Kainz MJ (eds)) pp 1–24 (Springer, 2009)Google Scholar
  17. Johns SR, Ralph Leslie D, Willing RI, Bishop DG (1977) Studies on chloroplast membranes. II 13C chemical shifts and longitudinal relaxation times of 1,2-di[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl]-3-galactosyl-sn-glycerol. Aust J Chem 30:823–834CrossRefGoogle Scholar
  18. Johns SR, Ralph Leslie D, Willing RI, Bishop DG (1978) Studies on chloroplast membranes. III 13C chemical shifts and longitudinal relaxation times of 1,2-diacyl-3-(6-sulpho-a-quinovosyl)-sn-glycerol. Aust J Chem 31:65–72CrossRefGoogle Scholar
  19. Kilz S, Waffenschmidt S, Budzikiewicz H (2000) Mass spectrometric analysis of hydroxyproline glycans. J Mass Spectrom 35:689–697ADSCrossRefGoogle Scholar
  20. Merchant SS et al (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245–250ADSCrossRefGoogle Scholar
  21. Merchant SS, Kropat J, Liu B, Shaw J, Warakanont J (2012) TAG, you’re it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation. Curr Opin Biotechnol 23:352–363CrossRefGoogle Scholar
  22. Renault M et al (2012) Cellular solid-state nuclear magnetic resonance spectroscopy. Proc Natl Acad Sci USA 109:4863–4868ADSCrossRefGoogle Scholar
  23. Rondeau-Mouro C, Veronese G, Buleon A (2006) High-resolution solid-state NMR of B-type amylose. Biomacromol 7:2455–2460CrossRefGoogle Scholar
  24. Siaut M et al (2011) Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves. BMC Biotechnol 11:7CrossRefGoogle Scholar
  25. Siemer AB et al (2006) Observation of highly flexible residues in amyloid fibrils of the HET-s prion. J Am Chem Soc 128:13224–13228CrossRefGoogle Scholar
  26. Specht E, Miyake-Stoner S, Mayfield S (2010) Micro-algae come of age as a platform for recombinant protein production. Biotechnol Lett 32:1373–1383CrossRefGoogle Scholar
  27. Tan L, Qiu F, Lamport DT, Kieliszewski MJ (2004) Structure of a hydroxyproline (Hyp)-arabinogalactan polysaccharide from repetitive Ala-Hyp expressed in transgenic Nicotiana tabacum. J Biol Chem 279:13156–13165CrossRefGoogle Scholar
  28. Tang H, Hills BP (2003) Use of 13C MAS NMR to study domain structure and dynamics of polysaccharides in the native starch granules. Biomacromol 4:1269–1276CrossRefGoogle Scholar
  29. van den Hoek C, Mann DG, Jahns HM Algae: an introduction to phycology, (Cambridge University Press, Cambridge, 1995)Google Scholar
  30. Vieler A, Wilhelm C, Goss R, Suss R, Schiller J (2007) The lipid composition of the unicellular green alga Chlamydomonas reinhardtii and the diatom Cyclotella meneghiniana investigated by MALDI-TOF MS and TLC. Chem Phys Lipids 150:143–155CrossRefGoogle Scholar
  31. Wang T, Hong M (2016) Solid-state NMR investigations of cellulose structure and interactions with matrix polysaccharides in plant primary cell walls. J Exp Bot 67:503–514CrossRefGoogle Scholar
  32. Warnet XL, Arnold AA, Marcotte I, Warschawski DE (2015) In-cell solid-state NMR: an emerging technique for the study of biological membranes. Biophys J 109:2461–2466CrossRefGoogle Scholar
  33. Warschawski DE, Devaux PF (2000) Polarization transfer in lipid membranes. J Magn Reson 145:367–372ADSCrossRefGoogle Scholar
  34. Wijffels RH, Barbosa MJ (2010) An outlook on microalgal biofuels. Science 329:796–799ADSCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of ChemistryUniversité du Québec à MontréalMontrealCanada
  2. 2.Institut des Sciences de la Mer de RimouskiUniversité du Québec à RimouskiRimouskiCanada
  3. 3.Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099, CNRSUniversité Paris Diderot and IBPCParisFrance

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