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Lecturer(s)
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Fellner Martin, prof. RNDr. Ph.D.
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Nožková Vladimíra, Mgr. Ph.D.
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Husičková Alexandra, Mgr. Ph.D.
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Plíhal Ondřej, RNDr. Ph.D.
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Balarynová Jana, Mgr. Ph.D.
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Karady Michal, Mgr. Ph.D.
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Course content
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1) Basis of photomorphogenesis - physiological methdos Study of photomorphogenesis - effect of blue and red light on hypocotyl and root elongation in Arabidopsis thaliana and tomato Solanum lycopersicum in conditions in vitro. Basic photomorphogenic responses of plants during transfer from the dark to light will be studied. Hypocotyl and root length will be measured in dependence on light conditions and final effect (inhibition, stimulation) of light quality (continuous blue and red light) on the organ elongation will be determined. Study of photomorphogenic mutants will be performed (mutants in phytochromes or cryptochromes) 2) Gasometric methods Paralel determination of the speed of CO2 assimilation, transpiration, respiration, stomata conductance in leaves of the experimental plants. Measurement of light curves of photosynthesis on the leaf of plants. Measurement of basic fluorescence parameters of the sample adapted for light or dark (FV/FM, FV´/FM´, PSII, qP, qN, NPQ). 3) Analysis of photosynthetic apparatus Analysis of very fast changes during induction of chlorophyll fluorescence. Kinetics of extinguishing of fluorescence of chlorophyll. Measurement of chlorophyll content analytically and by nondestructive methods using chlorophyllmeters (SPAD vs. NDVI). 4) Measurement of content of the selected plant hormones using mass spectroscopy. Extraction of planz material, adding of labeled standard, transfer of the sample to the final form, measurements on mass spectrometer. Preparation of calibraion curve and measurement of evaluation. 5) Basis of fluorescent and electron microscopy - methods for quantification of gene expression. Subcelullar localization of GFP-labelled proteins. 6) Quantitative PCR Preparation of plate and starting the plate measuremet (primer efficiency and gene expression), evaluation of obtained results. Electrophoretic methods Preparation of agarose gel, sample application, evaluation of results, preparation and start of native acrylamide gel, activity detectionPříprava agarósového gelu, nanesení vzorku, vyhodnocení výsledku, příprava a spuštění nativního akrylamidového gelu, detectioon of polyphenoloxidase activity.
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Learning activities and teaching methods
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Work with Text (with Book, Textbook), Observation, Demonstration, Laboratory Work
- Attendace
- 24 hours per semester
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Learning outcomes
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The aim of the laboratory practice is to acquaint students with basic of some experimental methods used in study of processes in plants on cell and molecular level.
After completing the course, the student should be able to: - perform basic experimental methods used in study of plant photomorphogenesis - use gasometric methods - perform basic methods for study of chlorophyll fluorescence in vivo - use basic methods of determination of simple chemicals in biological material - use of fluorescent and electron microscopy, perform basics of cloning experiments, use experimental methods for quantification of gene expression - perform quantitative PCR (qPCR) - perform gel electrophoresis
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Prerequisites
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Attendance at the course is conditioned by graduation of courses KBB/MBIO1, KBC/BCH, LRR/FZRSB
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Assessment methods and criteria
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Systematic Observation of Student
full attendance, credit
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Recommended literature
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Arndt C et al. (2012). Native Polyacrylamide Gels. In: Kurien B., Scofield R. (eds) Protein Electrophoresis. Methods in Molecular Biology (Methods and Protocols), vol 869. Humana Press, Totowa, NJ.
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Bustin SA et al. (2009). The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.
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Dunst S, Tomancak P. (2019). Imaging Flies by Fluorescence Microscopy: Principles, Technologies, and Applications. Genetics 211: 15?34.
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Holčapek M. Univerzita Pardubice, http://holcapek.upce.cz/teaching/01_Uvod.pdf.
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Jones R et al. (2013). The molecular life of plants; Wiley-Blackwell, American Society of Plant Biologists. USA.
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Lazár D. (2015). Parameters of photosynthetic partitioning. J. Plant Physiol. 175, 131?147..
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Procházka S., Macháčková I., Krekule J., Šebánek J. a kol. (1998). Fyziologie rostlin. Academia, Praha.
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Šmarda J. (2008). Metody molekulární biologie. Brno: Masarykova univerzita, ISBN 978-80-210-3841-7.
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Von Caemmerer S and Farquhar GD. (1981). Some relationships between the biochemistry of photosynthesis and the gas-exchange of leaves. Planta 153: 376-387.
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