Skip to main content
SpringerLink
Log in

Chloroplast functionality assessment by flow cytometry: Case study with pea plants under Paraquat stress

  • Published:
Photosynthetica

Abstract

Photosynthesis is one of the most important processes in plant biology and in the development of new methodologies that allow a better understanding and characterization of the photosynthetic status of organisms, which is invaluable. Flow cytometry (FCM) is an excellent tool for measuring fluorescence and physical proprieties of particles but it has seldom been used in photosynthetic studies and thus the full extent of its potentialities, in this field of research, remains unknown. To determine the suitability of FCM in photosynthesis studies, pea plants were exposed to Paraquat and their status was analyzed during 24 h. FCM was used to evaluate the integrity (volume and internal complexity) and the relative fluorescence intensity (FL) of chloroplasts extracted from those plants. To elucidate which type of information the FL conveys, FL values were correlated with the minimum fluorescence level (F0), maximum fluorescence level (Fm) and maximum photochemical efficiency of PSII (Fv/Fm), obtained by using Pulse-Amplitude-Modulation (PAM) fluorometry. Results indicate that: (1) the biomarkers used to evaluate the structural integrity of the chloroplasts were more sensitive to Paraquat exposure than the ones related to fluorescence; (2) the variation of the chloroplast’s structure, as time progressed, pointed to a swelling and subsequent burst of the chloroplast which, in turn, compromised fluorescence emission; (3) FL presented a high and significant correlation with the Fv/Fm and to a lesser degree with Fm but not with F0; (4) pigment content did not reveal significant changes in response to Paraquat exposure and is in agreement with the proposed model, suggesting that the cause for fluorescence decrease is due to chloroplast disruption. In sum, FCM proved to be an outstanding technique to evaluate chloroplastidal functional and structural status and therefore it should be regarded as a valuable asset in the field of photosynthetic research.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

Chl:

chlorophyll

F0 :

minimum fluorescence level of dark-adapted state

FCM:

flow cytometry

Fm :

maximum fluorescence level of dark-adapted state

FL:

relative fluorescence intensity

FS:

forward light scatter

Fv/Fm :

maximum photochemical efficiency of PSII

HS:

Hepes-Sorbitol buffer

LED:

light-emitting diode

PS:

photosystem

Pop:

population

PAM:

Pulse Amplitude Modulated

SS:

side light scatter

References

  • Ali, N.A., Dewez, D., Didur, O., Popovic, R.: Inhibition of photosystem II photochemistry by Cr is caused by the alteration of both D1 protein and oxygen evolving complex. — Photosynth. Res. 89: 81–87, 2006.

    Article  PubMed  CAS  Google Scholar 

  • Ashcroft, R.G., Preston, C., Cleland, R.E., Critchley, C.: Flowcytometry of isolated-chloroplasts and thylakoids. — Photobiochem. Photobiophys. 13: 1–14, 1986.

    CAS  Google Scholar 

  • Azevedo, H., Pinto, C.G.G., Fernandes, J., Loureiro, S., Santos, C.: Cadmium effects on sunflower growth and photosynthesis. — J. Plant Nutr. 28: 2211–2220, 2005.

    Article  CAS  Google Scholar 

  • Bromilow, R.H.: Paraquat and sustainable agriculture. — Pest. Manag. Sci. 60: 340–349, 2004.

    Article  PubMed  CAS  Google Scholar 

  • Dias, M., Brüggemann, W.: Limitations of photosynthesis in Phaseolus vulgaris under drought stress: gas exchange, chlorophyll fluorescence and Calvin cycle enzymes. — Photosynthetica 48: 96–102, 2010.

    Article  CAS  Google Scholar 

  • Dubelaar, G.B.J, Jonker, R.R: Flow cytometry as a tool for the study of phytoplankton. — Sci. Mar. 64: 135–156, 2000.

    Google Scholar 

  • Ekmekci, Y., Terzioglu, S.: Effects of oxidative stress induced by paraquat on wild and cultivated wheats. — Pestic. Biochem. Physiol. 83: 69–81, 2005.

    Article  CAS  Google Scholar 

  • Franqueira, D., Cid, A., Torres, E., Orosa, M., Herrero, C.: A comparison of the relative sensitivity of structural and functional cellular responses in the alga Chlamydomonas eugametos exposed to the herbicide paraquat. — Arch. Environ. Contam. Toxicol. 36: 264–269, 1999.

    Article  PubMed  CAS  Google Scholar 

  • Hattab, S., Dridi, B., Chouba, L., Kheder, M.B., Bousetta, H.: Photosynthesis and growth responses of pea (Pisum sativum L.) under heavy metals stress. — J. Environ. Sci-China 21: 1552–1556, 2009.

    Article  PubMed  CAS  Google Scholar 

  • Iturbe-Ormaetxe, I., Escuredo, P.R., Arrese-Igor, C., Becana, M.: Oxidative damage in pea plants exposed to water deficit or Paraquat. — Plant Physiol. 116: 173–181, 1998.

    Article  CAS  Google Scholar 

  • Juneau, P., Dewez, D., Matsui, S., Kim, S.G., Popovic, R.: Evaluation of different algal species sensitivity to mercury and metolachlor by PAM-fluorometry. — Chemosphere 45: 589–598, 2001.

    Article  PubMed  CAS  Google Scholar 

  • Krause, G.H., Weis, E.: Chlorophyll Fluorescence and Photosynthesis - the Basics. — Annu. Rev. Plant Physiol. Plant Mol. Biol. 42: 313–349, 1991.

    Article  CAS  Google Scholar 

  • Loureiro, J., Rodriguez, E., Dolezel, J., Santos, C.: Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content. — Ann. Bot. 98: 515–527, 2006.

    Article  PubMed  CAS  Google Scholar 

  • Marwood, C.A., Smith, R.E.H., Furgal, J.A., Charlton, M.N., Solomon, K.R., Greenberg, B.M.: Photoinhibition of natural phytoplankton assemblages in Lake Erie exposed to solar ultraviolet radiation. — Can. J. Fish Aquat. Sci. 57: 371–379, 2000.

    Article  CAS  Google Scholar 

  • Mascher, R., Fischer, S., Scheiding, W., Neagoe, A., Bergmann, H.: Exogenous 2-aminoethanol can diminish Paraquat induced oxidative stress in barley (Hordeum vulgare L.). — Plant Growth Regul. 45: 103–112, 2005.

    Article  CAS  Google Scholar 

  • Maxwell, K., Johnson, G.N.: Chlorophyll fluorescence — a practical guide. — J. Exp. Bot. 51: 659–668, 2000.

    Article  PubMed  CAS  Google Scholar 

  • Neuhaus, H.E., Stitt, M.: Perturbation of photosynthesis in spinach leaf-disks by low concentrations of methyl viologen. Influence of increased thylakoid energization on ATP synthesis, electron-transport, energy-dissipation, light-activation of the Calvin-cycle enzymes, and control of starch and sucrose synthesis. — Planta 179: 51–60, 1989.

    Article  CAS  Google Scholar 

  • Pfundel, E., Nagel, E., Meister, A.: Analyzing the light energy distribution in the photosynthetic apparatus of C4 plants using highly purified mesophyll and bundle-sheath thylakoids. — Plant Physiol. 112: 1055–1070, 1996.

    PubMed  Google Scholar 

  • Po, E.S.M., Ho, J.W.: Paraquat affects light-induced proton transport through chloroplast membranes in spinach. — Compt. Biochem Physiol. Part C: Pharmacol. Toxicol. Endocrinol. 118: 65–69, 1997.

  • Robinson, C., Mant, A.: Import of proteins into isolated chloroplasts and thylakoid membranes. — In: Gilmartin, P, Bowler, C. (ed.): Molecular Plant Biology A Practical Approach. Vol 2: 123–146. Oxford Univ. Press, Oxford 2002.

    Google Scholar 

  • Schreiber, U., Schliwa, U., Bilger, W.: Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. — Photosynth. Res. 10: 51–62, 1986.

    Article  CAS  Google Scholar 

  • Schröder, W.P., Petit, P.X.: Flow cytometry of spinach chloroplasts. Determination of intactness and lectin-binding properties of the envelope and the thylakoid membranes. — Plant Physiol. 100: 1092–1102, 1992.

    Article  PubMed  Google Scholar 

  • Sims, D., Gamon, J.: Relationships between leaf pigment content and spectral reflectance across a wide range of species, leaf structures and developmental stages. — Rem. Sens. Environ. 81: 337–354, 2002.

    Article  Google Scholar 

  • Váňová, L., Kummerová, M., Klemš, M., Zezulka, Š.: Fluoranthene influences endogenous abscisic acid level and primary photosynthetic processes in pea (Pisum sativum L.) plants in vitro. — Plant Growth Regul. 57: 39–47, 2009.

    Article  Google Scholar 

  • Varadi, G., Darko, E., Lehoczki, E.: Changes in the xanthophyll cycle and fluorescence quenching indicate light-dependent early events in the action of paraquat and the mechanism of resistance to paraquat in Erigeron canadensis (L.) Cronq. — Plant Physiol. 123: 1459–1469, 2000.

    Article  PubMed  CAS  Google Scholar 

  • Vernay, P., Gauthier-Moussard, C., Hitmi, A.: Interaction of bioaccumulation of heavy metal chromium with water relation, mineral nutrition and photosynthesis in developed leaves of Lolium perenne L. — Chemosphere 68: 1563–1575, 2007.

    Article  PubMed  CAS  Google Scholar 

  • Xu, C.H., Auger, J., Govindjee.: Chlorophyll a fluorescence measurements of isolated spinach thylakoids obtained by using single-laser-based flow-cytometry. — Cytometry 11: 349–358, 1990.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Biotechnology and Cytomics, CESAM & Departamento de Biologia, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal

    E. Rodriguez, R. Azevedo, A. Costa & C. Santos

  2. Phycology Laboratory, CESAM & Departamento de Biologia, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal

    J. Serôdio

Authors
  1. E. Rodriguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. Azevedo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Costa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Serôdio
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C. Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C. Santos.

Additional information

Acknowledgments: The Portuguese Foundation for Science and Technology, FCT is thanked for supporting the work of E.R. with the fellowship: FCT/BD/27467/2006.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rodriguez, E., Azevedo, R., Costa, A. et al. Chloroplast functionality assessment by flow cytometry: Case study with pea plants under Paraquat stress. Photosynthetica 50, 197–205 (2012). https://doi.org/10.1007/s11099-012-0022-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11099-012-0022-x

Additional keywords

Search

Navigation