Abstract
The most obvious event of leaf senescence is the loss of chlorophyll. Chlorophyll degradation proceeds in a well-characterized pathway that, although being common to higher plants, yields a species-specific set of chlorophyll catabolites, termed phyllobilins. Analysis of chlorophyll degradation and phyllobilin accumulation by high-performance liquid chromatography (HPLC) is a valuable tool to investigate senescence processes in plants. In this chapter, methods for the extraction, separation, and quantification of chlorophyll and its degradation products are described. Because of their different physicochemical properties, chlorin-type pigments (chlorophylls and magnesium-free pheo-pigments) and phyllobilins (linear tetrapyrroles) are analyzed separately. Specific spectral properties and polarity differences allow the identification of the different classes of known chlorins and phyllobilins. The methods provided facilitate the analysis of chlorophyll degradation and the identification of chlorophyll catabolites in a wide range of plant species, in different tissues, and under a variety of physiological conditions that involve loss of chlorophyll.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Hörtensteiner S, Kräutler B (2011) Chlorophyll breakdown in higher plants. Biochim Biophys Acta 1807:977–988
Christ B, Hörtensteiner S (2014) Mechanism and significance of chlorophyll breakdown. J Plant Growth Regul 33:4–20
Sakuraba Y, Schelbert S, Park S-Y et al (2012) STAY-GREEN and chlorophyll catabolic enzymes interact at light-harvesting complex II for chlorophyll detoxification during leaf senescence in Arabidopsis. Plant Cell 24:507–518
Christ B, Schelbert S, Aubry S et al (2012) MES16, a member of the methylesterase protein family, specifically demethylates fluorescent chlorophyll catabolites during chlorophyll breakdown in Arabidopsis. Plant Physiol 158:628–641
Christ B, Süssenbacher I, Moser S et al (2013) Cytochrome P450 CYP89A9 is involved in the formation of major chlorophyll catabolites during leaf senescence in Arabidopsis. Plant Cell 25:1868–1880
Kräutler B (2014) Phyllobilins—the abundant bilin-type tetrapyrrolic catabolites of the green plant pigment chlorophyll. Chem Soc Rev 43:6227–6238
Oberhuber M, Berghold J, Breuker K et al (2003) Breakdown of chlorophyll: a nonenzymatic reaction accounts for the formation of the colorless “nonfluorescent” chlorophyll catabolites. Proc Natl Acad Sci U S A 100:6910–6915
Moser S, Müller T, Ebert MO et al (2008) Blue luminescence of ripening bananas. Angew Chem Int Ed 47:8954–8957
Kräutler B, Banala S, Moser S et al (2010) A novel blue fluorescent chlorophyll catabolite accumulates in senescent leaves of the peace lily and indicates a divergent path of chlorophyll breakdown. FEBS Lett 584:4215–4221
Ulrich M, Moser S, Müller T et al (2011) How the colourless ‘nonfluorescent’ chlorophyll catabolites rust. Chem Eur J 17:2330–2334
Shioi Y, Fukae R, Sasa T (1983) Chlorophyll analysis by high-performance liquid chromatography. Biochim Biophys Acta 722:72–79
Perkins HJ, Roberts DWA (1962) Purification of chlorophylls, pheophytins and pheophorbides for specific activity determinations. Biochem Biophys Acta 58:486–498
Curty C, Engel N (1996) Detection, isolation and structure elucidation of a chlorophyll a catabolite from autumnal senescent leaves of Cercidiphyllum japonicum. Phytochemistry 42:1531–1536
Pružinská A, Tanner G, Aubry S et al (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
Mazaki H, Watanabe T, Takahashi T et al (1992) Pheophytination of eight chlorophyll derivatives in aqueous acetone. Bull Chem Soc Jpn 65:3212–3214
Hu XY, Tanaka A, Tanaka R (2013) Simple extraction methods that prevent the artifactual conversion of chlorophyll to chlorophyllide during pigment isolation from leaf samples. Plant Methods 9:19
Oberhuber M, Berghold J, Kräutler B (2008) Chlorophyll breakdown by a biomimetic route. Angew Chem Int Ed 47:3057–3061
Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Meth Enzymol 148:350–382
Strain HH, Cope BT, Svec WA (1971) Analytical procedures for the isolation, identification, estimation and investigation of the chlorophylls. Methods Enzymol 23:452–476
Kräutler B, Jaun B, Bortlik K-H et al (1991) On the enigma of chlorophyll degradation: the constitution of a secoporphinoid catabolite. Angew Chem Int Ed Engl 30:1315–1318
Berghold J, Eichmüller C, Hörtensteiner S et al (2004) Chlorophyll breakdown in tobacco: on the structure of two nonfluorescent chlorophyll catabolites. Chem Biodivers 1:657–668
Scherl M, Müller T, Kräutler B (2012) Chlorophyll catabolites in senescent leaves of the lime tree (Tilia cordata). Chem Biodivers 9:2605–2617
Schenk N, Schelbert S, Kanwischer M et al (2007) The chlorophyllases AtCLH1 and AtCLH2 are not essential for senescence-related chlorophyll breakdown in Arabidopsis thaliana. FEBS Lett 581:5517–5525
Guyer L, Schelbert Hofstetter S, Christ B et al (2014) Different mechanisms are responsible for chlorophyll dephytylation during fruit ripening and leaf senescence in tomato. Plant Physiol 166:44–56
Guzman I, Yousef GG, Brown AF (2012) Simultaneous extraction and quantitation of carotenoids, chlorophylls, and tocopherols in Brassica vegetables. J Agric Food Chem 60:7238–7244
Acknowledgments
This work was supported by grants from the Swiss National Science Foundation and by CropLife, an EU Marie-Curie Initial Training Network. Aditi Das and Luzia Guyer contributed equally to this work.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Das, A., Guyer, L., Hörtensteiner, S. (2018). Chlorophyll and Chlorophyll Catabolite Analysis by HPLC. In: Guo, Y. (eds) Plant Senescence. Methods in Molecular Biology, vol 1744. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7672-0_18
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7672-0_18
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7670-6
Online ISBN: 978-1-4939-7672-0
eBook Packages: Springer Protocols