Skip to main content

Creating Leaf Cell Suspensions for Characterization of Mesophyll and Bundle Sheath Cellular Features

  • Protocol
  • First Online:
Photosynthesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1770))

Abstract

Imaging of mesophyll cell suspensions prepared from Arabidopsis has been pivotal for forming our current understanding of the molecular control of chloroplast division over the past 25 years. In this chapter, we provide a method for the preparation of leaf cell suspensions that improves upon a previous method by optimizing cellular preservation and cell separation. This technique is accessible to all researchers and amenable for use with all plant species. The leaf suspensions can be used for imaging chloroplast features within a cell that are important for photosynthesis such as size, number, and distribution. However, we also provide examples to illustrate how the cells in the suspensions can be easily stained to image other features, for example pit fields where plasmodesmata are located and organelles such as mitochondria, to improve our understanding of traits that are important for photosynthetic physiology.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Hanson MR, Köhler RH (2001) GFP imaging: methodology and application to investigate cellular compartmentation in plants. J Exp Bot 52:529–539

    Article  CAS  PubMed  Google Scholar 

  2. Sankaranarayanan S, Samuel MA (2015) Guiding principles for live cell imaging of plants using confocal microscopy. In: Yeung ECT, Stasolla C, Sumner MJ, Huang BQ (eds) Plant microtechniques and protocols. Springer, New York, pp 213–224

    Chapter  Google Scholar 

  3. Khoshravesh R, Lundsgaard-Nielsen V, Sultmanis S, Sage TL (2017) Light microscopy, transmission electron microscopy and immunohistochemistry protocols for study of the role of photorespiration. In: Fernie A, Bauwe H, Weber APM (eds) Photorespiration: methods and protocols. Springer, New York, pp 243–270

    Chapter  Google Scholar 

  4. Mackinder LCM, Meyer MT, Mettler-Altmann T et al (2016) A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle. PNAS 113:5958–5963

    Article  CAS  PubMed  Google Scholar 

  5. Gao H, Sage TL, Osteryoung KW (2006) FZL, an FZO-like protein in plants, is a determinant of thylakoid and chloroplast morphology. PNAS 103:6759–6764

    Article  CAS  PubMed  Google Scholar 

  6. Armbruster U, Labs M, Pribil M et al (2013) Arabidopsis CURVATURE THYLAKOID1 proteins modify thylakoid architecture by inducing membrane curvature. Plant Cell 25:2661–2678

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Rosnow J, Yerramsetty P, Berry JO et al (2014) Exploring mechanisms linked to differentiation and function of dimorphic chloroplasts in the single cell C4 species Bienertia sinuspersici. BMC Plant Biol 14:34

    Article  PubMed  PubMed Central  Google Scholar 

  8. Danila F, Quick WP, White RG et al (2016) The metabolite pathway between bundle sheath and mesophyll: quantification of plasmodesmata in leaves of C3 and C4 monocots. Plant Cell 28:1461–1471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Larkin RM, Stefano G, Ruckle ME et al (2016) REDUCED CHLOROPLAST COVERAGE genes from Arabidopsis thaliana help to establish the size of the chloroplast compartment. PNAS 113:E1116–E1125

    Article  CAS  PubMed  Google Scholar 

  10. Pyke KA, Leech RM (1991) Rapid image analysis screening procedure for identifying chloroplast number mutants in mesophyll mells of Arabidopsis thaliana (L.) Heynh. Plant Physiol 96:1193–1195

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Osteryoung KW, Stokes KD, Rutherford SM et al (1998) Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ. Plant Cell 10:1991–2004

    PubMed  PubMed Central  CAS  Google Scholar 

  12. Colletti KS, Tattersall EA, Pyke KA et al (2000) A homologue of the bacterial cell division site-determining factor MinD mediates placement of the chloroplast division apparatus. Curr Biol 10:507–516

    Article  CAS  PubMed  Google Scholar 

  13. Miyagishima SY, Froehlich JE, Osteryoung KW (2006) PDV1 and PDV2 mediate recruitment of the dynamin-related protein ARC5 to the plastid division site. Plant Cell 18:2517–2530

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Glynn JM, Yang Y, Vitha S et al (2009) PARC6, a novel chloroplast division factor, influences FtsZ assembly and is required for recruitment of PDV1 during chloroplast division in Arabidopsis. Plant J 59:700–711

    Article  CAS  PubMed  Google Scholar 

  15. Stata M, Sage TL, Rennie TD, Khoshravesh R, Sultmanis S, Khaikin Y, Ludwig M, Sage RF (2014) Mesophyll cells of C4 plants have fewer chloroplasts than those of closely related C3 plants. Plant Cell Environ 37:2587–2600

    Article  CAS  PubMed  Google Scholar 

  16. Stata M, Sage TL, Hoffman N et al (2016) Mesophyll chloroplast investment in C3, C4 and C2 species of the genus Flaveria. Plant Cell Physiol 57:904–918

    Article  CAS  PubMed  Google Scholar 

  17. Busch FA, Sage TL, Cousins AB, Sage RF (2013) C3 plants enhance rates of photosynthesis by reassimilating photorespired and respired CO2. Plant Cell Environ 36:200–212

    Article  CAS  PubMed  Google Scholar 

  18. Zavaliev R, Epel BL (2015) Imaging callose at plasmodesmata using aniline blue: quantitative confocal microscopy. In: Heinlein M (ed) Plasmodesmata: methods and protocols. Springer New York, New York, NY, pp 105–119

    Google Scholar 

  19. Zhu H, Fan J, Du J, Peng X (2016) Fluorescent probes for sensing and imaging within specific cellular organelles. Acc Chem Res 49:2115–2126

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Tammy L. Sage .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Khoshravesh, R., Sage, T.L. (2018). Creating Leaf Cell Suspensions for Characterization of Mesophyll and Bundle Sheath Cellular Features. In: Covshoff, S. (eds) Photosynthesis. Methods in Molecular Biology, vol 1770. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7786-4_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7786-4_15

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7785-7

  • Online ISBN: 978-1-4939-7786-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics