Photoautotrophic suspension cultures have been established from various model and crop plants and proved to be valuable and robust experimental system to assess coordinated responses of primary and secondary metabolism to metabolic and stress related signals. The use of suspension cultures combines the ease of handling microalgae in microtiter plates with the advantage of testing physiological responses of higher plants, notably in combination with the assessment of the response of photosynthetic activity by PAM chlorophyll fluorescence imaging as well as monitoring changes in secondary metabolite production and ROS formation by steady state fluorescence of plant fluorophores or introduced fluorescent probes. Photoautotrophic cultures provide various advantages as fast, highly sensitive, robust and high-through-put experimental system for screening and characterization of the impact of toxic compounds on higher plants. This opinion article discusses and critically evaluates the potential of photoautotrophic cultures of higher plants in combination with fluorescence imaging assays in microtiter plates as a complement to existing guidelines for testing the toxicity of chemicals in plants.
OECD (2006) Test No. 208: OECD Guidelines for the Testing of Chemicals. Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test. Organisation for Economic Co-Operation and Development, Paris.
OECD (2006) Test No. 227: OECD Guidelines for Testing of Chemicals. Terrestrial Plant Test: Vegetative Vigour Test. Organisation for Economic Co-Operation and Development, Paris.
OECD (2006) Test No. 221: Lemna sp. Growth Inhibition Test. Organisation for Economic Co-Operation and Development, Paris.
OECD (2011) Test No. 201: OECD Guidelines for the Testing of Chemicals. Freshwater Alga and Cyanobacteria, Growth Inhibition Test. Organisation for Economic Co-operation and Development, Paris.
Klaine, S., Lewis, M. and Knuteson, S. (2003) Phytotoxicity. In: Hoffman, D., Rattner, B. and Burton, G., Eds., Handbook of Ecotoxicology, Lewis Publishers, 191-218.
Grimme, L.H., Riess, M.H., Manthey, M., Faust, M. and Altenburger, R. (1993) Cell Physiological Parameters to Detect Ecotoxicological Risks. Science of the Total Environment, 134, 741-748.
Inderjit, Saini, M. and Kaur, H. (2005) Experimental Complexities in Evaluating the Comparative Phytotoxicity of Chemicals with Different Modes of Action. Environmental and Experimental Botany, 53, 97-104.
Kawabata, Y. and Takeda, S. (2014) Regulation of Xanthophyll Cycle Pool Size in Response to High Light Irradiance in Arabidopsis. Plant Biotechnology, 31, 229-240. https://doi.org/10.5511/plantbiotechnology.14.0609a
Sato, F. (2013) Characterization of Plant Functions Using Cultured Plant Cells, and Biotechnological Applications. Bioscience, Biotechnology, and Biochemistry, 77, 1-9. https://doi.org/10.1271/bbb.120759
Roitsch, T. and Sinha, A. (2002) Application of Photoautotrophic Suspension Cultures in Plant Science. Photosynthetica, 40, 481-492. https://doi.org/10.1023/A:1024332430494
Gómez-Torres, L.M., Moreno-Gómez, B., Velásquez-Lozano, M.E., Aguirre-Mancilla, C. and Aguado-Santacruz, G.A. (2014) Plant Cell Photoautotrophic Suspension Cultures. Establishment and Application Perspectives. Revista Fitotecnia Mexicana, 37, 165-179.
Doran, P.M. (2009) Application of Plant Tissue Cultures in Phytoremediation Research: Incentives and Limitations. Biotechnology and Bioengineering, 103, 60-76. https://doi.org/10.1002/bit.22280
Santos, R.B., Abranches, R., Fischer, R., Sack, M. and Holland, T. (2016) Putting the Spotlight Back on Plant Suspension Cultures. Frontiers in Plant Science, 7, 1-13. https://doi.org/10.3389/fpls.2016.00297
Harms, H.H. (1992) In Vitro Systems for Studying Phytotoxicity and Metabolic Fate of Pesticides and Xenobiotics in Plants. Pesticide Science, 35, 277-281. https://doi.org/10.1002/ps.2780350313
Schwenger-Enger, C., Barz, W. and Weber, N. (2001) Fatty Acid Alteration of Plastidic and Extra-Plastidic Membrane Lipids in Metribuzin-Resistant Photoautotrophic Chenopodium rubrum Cells as Compared to Wild-Type Cells. Verlag Der Zeitschrift Für Naturforschung, 56, 1047-1056.
Olofsdotter, M., Olesen, A., Andersen, S.B. and Streibig, J.C. (1994) A Comparison of Herbicide Bioassays in Cell Cultures and Whole Plants. Weed Research, 34, 387-394. https://doi.org/10.1111/j.1365-3180.1994.tb02034.x
Thiemann, J., Nieswandt, A. and Barz, W. (1989) A Microtest System for the Serial Assay of Phytotoxic Compounds Using Photoautotrophic Cell Suspension Cultures of Chenopodium rubrum. Plant Cell Reports, 8, 399-402. https://doi.org/10.1007/BF00270078
Zilkah, S. and Gressel, J. (1977) Cell Cultures vs. Whole Plants for Measuring Phytotoxicity I. The Establishment and Growth of Callus and Suspension Cultures; Definition of Factors Affecting Toxicity on Calli. Plant and Cell Physiology, 18, 641-655.
Roitsch, T. and Sinha, A. (2001) Effect of Different Sugars on Photosynthesis and Chlorophyll Fluorescence in Photoautotrophic Tomato Suspension Cell Cultures. Photosynthetica, 39, 611-614. https://doi.org/10.1023/A:1015624600607
Hüsemann, W. (1985) Photoautotrophic Growth of Cells in Culture. In: Vasil, I.K., Ed., Cell Culture and Somatic Cell Genetics of Plants, Vol. 2, Academic Press, New York, 213-251.
Widholm, J.M. (1992) Properties and Uses of Photoautotrophic Plant Cell Cultures. International Review of Cytology, 132, 109-175.
Hofmann, M., Ehneß, R., Lee, T.K. and Roitsch, T. (1999) Intracellular Protons Are Not Involved in Elicitor Dependent Regulation of mRNAs for Defense Related Enzymes in Chenopodium rubrum. Journal of Plant Physiology, 155, 527-532.
Sano, T., Higaki, T., Handa, K., Kadota, Y., Kuchitsu, K., Hasezawa, S., Hoffmann, A., Zimmermann, U., Hedrich, R. and Roitsch, T. (2006) Calcium Ions Are Involved in the Delay of Plant Cell Cycle Progression by Abiotic Stresses. FEBS Letters, 580, 597-602.
Ehness, R., Ecker, M., Godt, D.E. and Roitsch, T. (1997) Glucose and Stress Independently Regulate Source and Sink Metabolism and Defense Mechanisms via Signal Transduction Pathways Involving Protein Phosphorylation. The Plant Cell, 9, 1825-1841. https://doi.org/10.1105/tpc.9.10.1825
Berger, S., Sinha, A.K. and Roitsch, T. (2007) Plant Physiology Meets Phytopathology: Plant Primary Metabolism and Plant-Pathogen Interactions. Journal of Experimental Botany, 58, 4019-4026. https://doi.org/10.1093/jxb/erm298
Fai, P.B., Grant, A. and Reid, B. (2007) Chlorophyll a Fluorescence as a Biomarker for Rapid Toxicity Assessment. Environmental Toxicology and Chemistry/SETAC, 26, 1520-1531. https://doi.org/10.1897/06-394R1.1
Chaerle, L., Lenk, S., Leinonen, I., Jones, L., Van Der Straeten, D. and Buschmann, C. (2009) Multi-Sensor Imaging of Plant Stresses: Towards the Development of a Stress-Catalogue. Biotechnology Journal, 4, 1152-1167. https://doi.org/10.1002/biot.200800242
Baker, N.R. (2008) Chlorophyll Fluorescence: A Probe of Photosynthesis in Vivo. Annual Review of Plant Biology, 59, 89-113. https://doi.org/10.1146/annurev.arplant.59.032607.092759
Maxwell, K. and Johnson, G.N. (2000) Chlorophyll Fluorescence—A Practical Guide. Journal of Experimental Botany, 51, 659-668.
Lichtenthaler, H.K., Langsdorf, G., Lenk, S. and Buschmann, C. (2005) Chlorophyll Fluorescence Imaging of Photosynthetic Activity with the Flash-Lamp Fluorescence Imaging System. Photosynthetica, 43, 355-369. https://doi.org/10.1007/s11099-005-0060-8
Barbagallo, R., Oxborough, K., Pallett, K. and Baker, N. (2003) Rapid, Noninvasive Screening for Perturbations of Metabolism and Plant Growth Using Chlorophyll Fluorescence Imaging. Plant Physiology, 132, 485-493. https://doi.org/10.1104/pp.102.018093
Nedbal, L., Soukupová, J., Kaftan, D., Whitmarsh, J. and Trtílek, M. (2000) Kinetic Imaging of Chlorophyll Fluorescence Using Modulated Light. Photosynthesis Research, 66, 3-12. https://doi.org/10.1023/A:1010729821876
Harbinson, J., Prinzenberg, A.E., Kruijer, W. and Aarts, M.G.M. (2012) High Throughput Screening with Chlorophyll Fluorescence Imaging and Its Use in Crop Improvement. Current Opinion in Biotechnology, 23, 221-226.
Baker, N.R. and Rosenqvist, E. (2004) Applications of Chlorophyll Fluorescence Can Improve Crop Production Strategies: An Examination of Future Possibilities. Journal of Experimental Botany, 55, 1607-1621. https://doi.org/10.1093/jxb/erh196
Gorbe, E. and Calatayud, A. (2012) Applications of Chlorophyll Fluorescence Imaging Technique in Horticultural Research: A Review. Scientia Horticulturae, 138, 24-35.
Wilkinson, A.D., Collier, C.J., Flores, F., Mercurio, P., O’Brien, J., Ralph, P.J. and Negri, A.P. (2015) A Miniature Bioassay for Testing the Acute Phytotoxicity of Photosystem II Herbicides on Seagrass. PLoS ONE, 10, e0117541. https://doi.org/10.1371/journal.pone.0117541
Lichtenthaler, H.K., Lang, M., Sowinska, M., Heisel, F. and Miehé, J.A. (1996) Detection of Vegetation Stress via a New High Resolution Fluorescence Imaging System. Journal of Plant Physiology, 148, 599-612.
Buschmann, C. and Lichtenthaler, H.K. (1998) Principles and Characteristics of Multi-Colour Fluorescence Imaging of Plants. Journal of Plant Physiology, 152, 297-314.
Morales, F., Cerovic, Z.G. and Moya, I. (1994) Characterization of Blue-Green Fluorescence in the Mesophyll of Sugar Beet (Beta vulgaris L.) Leaves Affected by Iron Deficiency. Plant Physiology, 106, 127-133. https://doi.org/10.1104/pp.106.1.127
Karlicky, V., Nezval, J., Stroch, M. and Spunda, V. (2013) Response of Epidermal Blue-Green Fluorescence Emission from Barley Leaves to UV Radiation Stress. In: Stojanov, R., Ed., Global Change and Resilience: From Impacts to Responses, Global Change Research Centre, Academy of Sciences of the Czech Republic, Brno, 231-235.
Kristiansen, K.A., Jensen, P.E., Møller, I.M. and Schulz, A. (2009) Monitoring Reactive Oxygen Species Formation and Localisation in Living Cells by Use of the Fluorescent Probe CM-H2DCFDA and Confocal Laser Microscopy. Physiologia Plantarum, 136, 369-383. https://doi.org/10.1111/j.1399-3054.2009.01243.x
Swanson, S.J., Choi, W., Chanoca, A. and Gilroy, S. (2011) In Vivo Imaging of Ca2+, pH, and Reactive Oxygen Species Using Fluorescent Probes in Plants. Annual Review of Plant Biology, 62, 273-297. https://doi.org/10.1146/annurev-arplant-042110-103832
Hideg, E., Barta, C., Kálai, T., Vass, I., Hideg, K. and Asada, K. (2002) Detection of Singlet Oxygen and Superoxide with Fluorescent Sensors in Leaves under Stress by Photoinhibition or UV Radiation. Plant & Cell Physiology, 43, 1154-1164. https://doi.org/10.1093/pcp/pcf145
Jambunathan, N. (2010) Determination and Detection of Reactive Oxygen Species (ROS), Lipid Peroxidation, and Electrolyte Leakage in Plants. In: Sunkar, R., Ed., Plant Stress Tolerance: Methods and Protocols, Springer, Berlin, 291-297. https://doi.org/10.1007/978-1-60761-702-0_18
Hideg, E., Kálai, T., Kós, P.B., Asada, K. and Hideg, K. (2006) Singlet Oxygen in Plants—Its Significance and Possible Detection with Double (Fluorescent and Spin) Indicator Reagents. Photochemistry and Photobiology, 82, 1211-1218. https://doi.org/10.1562/2006-02-06-RA-797
Hideg, é. (2008) A Comparative Study of Fluorescent Singlet Oxygen Probes in Plant Leaves. Central European Journal of Biology, 3, 273-284. https://doi.org/10.2478/s11535-008-0018-5
Kálai, T., Hideg, é., Ayaydin, F. and Hideg, K. (2013) Synthesis and Potential Use of 1,8-Naphthalimide Type (1) O2 Sensor Molecules. Photochemical & Photobiological Sciences: Official Journal of the European Photochemistry Association and the European Society for Photobiology, 12, 432-438. https://doi.org/10.1039/C2PP25253H
Owusu-Ansah, E., Yavari, A. and Banerjee, U. (2008) A Protocol for in Vivo Detection of Reactive Oxygen Species. Protocol Exchange, Nature Publishing Group.
Snyrychová, I., Ayaydin, F. and Hideg, é. (2009) Detecting Hydrogen Peroxide in Leaves in Vivo—A Comparison of Methods. Physiologia Plantarum, 135, 1-18. https://doi.org/10.1111/j.1399-3054.2008.01176.x
Srivastava, R.K., Pandey, P., Rajpoot, R., Rani, A. and Dubey, R.S. (2014) Cadmium and Lead Interactive Effects on Oxidative Stress and Antioxidative Responses in Rice Seedlings. Protoplasma, 251, 1047-1065. https://doi.org/10.1007/s00709-014-0614-3
Hideg, é. and Schreiber, U. (2007) Parallel Assessment of ROS Formation and Photosynthesis in Leaves by Fluorescence Imaging. Photosynthesis Research, 92, 103-108. https://doi.org/10.1007/s11120-007-9146-4
Dewez, D., Marchand, M., Eullaffroy, P. and Popovic, R. (2002) Evaluation of the Effects of Diuron and Its Derivatives on Lemna Gibba Using a Fluorescence Toxicity Index. Environmental Toxicology, 17, 493-501. https://doi.org/10.1002/tox.10084
Link, V.L., Hofmann, M.G., Sinha, A.K., Ehness, R., Strnad, M. and Roitsch, T. (2002) Biochemical Evidence for the Activation of Distinct Subsets of Mitogen-Activated Protein Kinases by Voltage and Defense-Related Stimuli 1. Plant Physiology, 128, 271-281. https://doi.org/10.1104/pp.010569
Großkinsky, D.K., Svensgaard, J., Christensen, S. and Roitsch, T. (2015) Plant Phenomics and the Need for Physiological Phenotyping across Scales to Narrow the Genotype-to-Phenotype Knowledge Gap. Journal of Experimental Botany, 66, 5429-5440. https://doi.org/10.1093/jxb/erv345
Kristen, U. (1997) Use of Higher Plants as Screens for Toxicity Assessment. Toxicology in Vitro, 11, 181-191.
Edwards, R. and Owen, W.J. (1986) Comparison of Glutathione S-Transferases of Zea mays Responsible for Herbicide Detoxification in Plants and Suspension-Cultured Cells. Planta, 169, 208-215. https://doi.org/10.1007/BF00392316