Abstract
Tetrapyrroles and carotenoids are required for many indispensable functions in photosynthesis. Tetrapyrroles are essential metabolites for photosynthesis, redox reaction, and detoxification of reactive oxygen species and xenobiotics, while carotenoids function as accessory pigments, in photoprotection and in attraction to animals. Their branched metabolic pathways of synthesis and degradation are tightly controlled to provide adequate amounts of each metabolite (carotenoids/tetrapyrroles) and to prevent accumulation of photoreactive intermediates (tetrapyrroles). Many Arabidopsis mutants and transgenic plants have been reported to show variations in steady-state levels of tetrapyrrole intermediates and contents of different carotenoid species. It is a challenging task to determine the minute amounts of these metabolites to assess the metabolic flow and the activities of both pigment-synthesising and degrading pathways, to unravel limiting enzymatic steps of these biosynthetic pathways, and to characterise mutants with accumulating intermediates. In this chapter, we present a series of methods to qualify and quantify anabolic and catabolic intermediates of Arabidopsis tetrapyrrole metabolism, and describe a common method for quantification of different plant carotenoid species. Additionally, we introduce two methods for quantification of non-covalently bound haem. The approach of analysing steady-state levels of tetrapyrrole intermediates in plants, when applied in combination with analyses of transcripts, proteins, and enzyme activities, enables the biochemical and genetic elucidation of the tetrapyrrole pathway in wild-type plants, varieties, and mutants. Steady-state levels of tetrapyrrole intermediates are only up to 1/1,000 of the amounts of the accumulating end-products, chlorophyll, and haem. Although present in very low amounts, the accumulation and availability of tetrapyrrole intermediates have major consequences on the physiology and activity of chloroplasts due to their additional photoreactive and possible signalling functions. Although adjusted for Arabidopsis tetrapyrrole metabolites, the presented methods can also be applied for analysis of cyanobacterial and other plant tetrapyrroles.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Mock, H. P., and Grimm, B. (1997) Reduction of uroporphyrinogen decarboxylase by antisense RNA expression affects activities of other enzymes involved in tetrapyrrole biosynthesis and leads to light-dependent necrosis. Plant Physiol. 113, 1101–1112.
Moulin, M., and Smith, A. G. (2008) A robust method for determination of chlorophyll intermediates by tandem mass spectrometry. In, Photosynthesis. Energy from the Sun(Allen, J.F., Gantt, E., Golbeck, J.H. and Osmond, B., eds.) Springer, Dordrecht, Netherlands, pp. 1215–1222.
Lorenzen, C. J., and Downs, J. N. (1986) The specific absorption-coefficients of chlorophyllide-a and pheophorbide-a in 90-percent acetone, and comments on the fluorometric-determination of chlorophyll and pheopigments. Limnol. Oceanogr. 31, 449–452.
Mauzerall, D., and Granick, S. (1956) The occurrence and determination of delta-amino-levulinic acid and porphobilinogen in urine. J. Biol. Chem. 219, 435–446.
Kruse, E., Mock, H. P., and Grimm, B. (1995) Reduction of coproporphyrinogen oxidase level by antisense RNA synthesis leads to deregulated gene expression of plastid proteins and affects the oxidative defense system. EMBO J. 14, 3712–3720.
Jensen, P. E., Gibson, L. C. D., Shephard, F., Smith, V., and Hunter, C. N. (1999) Introduction of a new branchpoint in tetrapyrrole biosynthesis in Escherichia coli by co-expression of genes encoding the chlorophyll-specific enzymes magnesium chelatase and magnesium protoporphyrin methyltransferase. FEBS Lett. 455, 349–354.
de Rooij, F. W. M., Edixhoven, A., and Wilson, J. H. P. (2003) Porphyria: A diagnostic approach. In, The Porphyrin Handbook, Vol. 14(Kadish, K.M., Smith, K.M. and Guilard, R., eds.) Academic Press, Amsterdam, Netherlands, pp. 211–245.
Langmeier, M., Ginsburg, S., and Matile, P. (1993) Chlorophyll breakdown in senescent leaves: demonstration of Mg-dechelatase activity. Physiol. Plant. 89, 347–353.
Koski, V. M., and Smith, J. H. (1948) The isolation and spectral absorption properties of protochlorophyll from etiolated barley seedlings. J. Am. Chem. Soc. 70, 3558–3562.
Dawson, R. C. M., Elliott, D. C., Elliot, W. H., and Jones, K. M. (1986) Data for Biochemical Research, 3rdedn. Oxford University Press, Oxford, UK.
Lichtenthaler, H. K. (1987) Chlorophylls and carotenoids: pigments of photosynthetic membranes. Meth. Enzymol. 148, 350–383.
Porra, R. J., Thompson, W. A., and Kriedemann, P. E. (1989) Determination of accurate extinction coefficients and simultaneous-equations for assaying chlorophyll-a and chlorophyll-b extracted with 4 different solvents - verification of the concentration of chlorophyll standards by atomic-absorption spectroscopy. Biochim. Biophys. Acta 975, 384–394.
Britton, G., Liaaen-Jensen, S., and Pfander, H. (2004) Carotenoids, Handbook, 1stedn. Birkhäuser Verlag, Basel, Switzerland.
Rowan, K. S. (1989) Photosynthetic Pigments of Algae. Cambridge University Press, Cambridge, UK.
Stillman, L. C., and Gassman, M. L. (1978) Protoheme extraction from plant-tissue. Anal. Biochem. 91, 166–172.
Berry, E. A., and Trumpower, B. L. (1987) Simultaneous determination of hemes-a, hemes-b, and hemes-c from pyridine hemochrome spectra. Anal. Biochem. 161, 1–15.
Weinstein, J. D., and Beale, S. I. (1983) Separate physiological roles and subcellular compartments for two tetrapyrrole biosynthetic pathways in Euglena gracilis. J. Biol. Chem. 258, 6799–6807.
Masuda, T., and Takahashi, S. (2006) Chemiluminescent-based method for heme determination by reconstitution with horseradish peroxidase apo-enzyme. Anal. Biochem. 355, 307–309.
Takahashi, S., and Masuda, T. (2009) High throughput heme assay by detection of chemiluminescence of reconstituted horseradish peroxidase. Comb. Chem. High. Throughput Screen. 12, 532–535.
Tamura, M., Yonetani, T., and Asakura, T. (1972) Heme-modification studies on horseradish-peroxidase. Biochim. Biophys. Acta 268, 292–304.
Teale, F. W. J. (1959) Cleavage of the haem-protein link by acid methylethylketone. Biochim. Biophys. Acta 35, 543–543.
Breslow, E. (1964) Changes in side chain reactivity accompanying binding of heme to sperm whale apomyoglobin. J. Biol. Chem. 239, 486–496.
Schelbert, S., Aubry, S., Burla, B., Agne, B., Kessler, F., Krupinska, K., and Hörtensteiner, S. (2009) Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. Plant Cell 21, 767–785.
Pruzinska, A., Tanner, G., Aubry, S., Anders, I., Moser, S., Muller, T., Ongania, K. H., Kräutler, B., Youn, J. Y., Liljegren, S. J., and Hortensteiner, S. (2005) Chlorophyll breakdown in senescent Arabidopsis leaves. Characterization of chlorophyll catabolites and of chlorophyll catabolic enzymes involved in the degreening reaction. Plant Physiol. 139, 52–63.
Richter, A., Peter, E., Pörs, Y., Lorenzen, S., Grimm, B., and Czarnecki, O. (2010) Rapid dark repression of 5-aminolevulinic acid synthesis in green barley leaves. Plant Cell Physiol. 51, 670–681.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Czarnecki, O., Peter, E., Grimm, B. (2011). Methods for Analysis of Photosynthetic Pigments and Steady-State Levels of Intermediates of Tetrapyrrole Biosynthesis. In: Jarvis, R. (eds) Chloroplast Research in Arabidopsis. Methods in Molecular Biology, vol 775. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-237-3_20
Download citation
DOI: https://doi.org/10.1007/978-1-61779-237-3_20
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-236-6
Online ISBN: 978-1-61779-237-3
eBook Packages: Springer Protocols