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Chlorophyll breakdown: analysis of fruit ripening, evolution and regulation

English title Chlorophyll breakdown: analysis of fruit ripening, evolution and regulation
Applicant Hörtensteiner Stefan
Number 132603
Funding scheme Project funding
Research institution Institut für Pflanzen- und Mikrobiologie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Botany
Start/End 01.01.2011 - 30.04.2014
Approved amount 422'233.85
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Keywords (7)

chlorophyll breakdown; leaf senescence; fruit ripening; evolution; protein-protein interaction; phytol hydrolysis; protein crystallization

Lay Summary (English)

Lead
Lay summary
During leaf senescence and fruit ripening chlorophyll is degraded to colorless tetrapyrroles termed NCCs. Structures of NCCs from different species point to a common pathway of chlorophyll breakdown (termed PAO pathway), whose key enzyme, PAO, is responsible for the opening of the macrocycle ring of chlorophyll. Most of the intermediates of the PAO pathway are devoid of the phytyl-moiety characteristic for chlorophyll, indicating that dephytylation is an early reaction in the pathway. Pheophytinase (PPH) was recently shown to catalyze the dephytylation of Mg-free chlorophyll (pheophytin) to pheophorbide. PPH is specific for pheophytin and does not accept chlorophyll as substrate. One aim of this project is to biochemically characterize PPH in more detail and to crystallize the protein to get an insight into its intriguing specificity. We, furthermore, aim to elucidate the mechanism of chlorophyll breakdown during fruit ripening in detail, in particular with respect to the involvement of PPH. Finally we plan to investigate evolutionary aspects of the PAO pathway.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Hydroxymethylated phyllobilins: a puzzling new feature of the dioxobilin branch of chlorophyll breakdown.
Süssenbacher Iris, Christ Bastien, Hörtensteiner Stefan, Kräutler Bernhard (2014), Hydroxymethylated phyllobilins: a puzzling new feature of the dioxobilin branch of chlorophyll breakdown., in Chemistry Eur. J., 20(1), 87-92.
Mechanism and significance of chlorophyll breakdown
Christ Bastien, Hörtensteiner Stefan (2014), Mechanism and significance of chlorophyll breakdown, in Journal of Plant Growth Regulation, 33, 4-20.
7-Hydroxymethyl chlorophyll a reductase functions in metabolic channeling of chlorophyll breakdown intermediates during leaf senescence.
Sakuraba Yasuhito, Kim Ye-Sol, Yoo Soo-Cheul, Hörtensteiner Stefan, Paek Nam-Chon (2013), 7-Hydroxymethyl chlorophyll a reductase functions in metabolic channeling of chlorophyll breakdown intermediates during leaf senescence., in Biochemical and Biophysical Research Communications, 430, 32-37.
Chlorophyll breakdown: chemistry, biochemistry and biology
Kräutler Bernhard, Hörtensteiner Stefan (2013), Chlorophyll breakdown: chemistry, biochemistry and biology, in Ferreira G.C. (ed.), World Scientific Publishing, Hackensack, NJ, USA, 117-185.
Cytochrome P450 CYP89A9 is involved in the formation of major chlorophyll catabolites during leaf senescence in Arabidopsis.
Christ Bastien, Süssenbacher Iris, Moser Simone, Bichsel Nicole, Egert Aurelie, Müller Thomas, Kräutler Bernhard, Hörtensteiner Stefan (2013), Cytochrome P450 CYP89A9 is involved in the formation of major chlorophyll catabolites during leaf senescence in Arabidopsis., in The Plant Cell, 25(5), 1868-80.
Update on the biochemistry of chlorophyll breakdown.
Hörtensteiner Stefan (2013), Update on the biochemistry of chlorophyll breakdown., in Plant Molecular Biology, 82, 505-517.
Accelerated cell death 2 suppresses mitochondrial oxidative bursts and modulates cell death in Arabidopsis.
Pattanayak Gopal K, Venkataramani Sujatha, Hörtensteiner Stefan, Kunz Lukas, Christ Bastien, Moulin Michael, Smith Alison G, Okamoto Yukihiro, Tamiaki Hitoshi, Sugishima Masakazu, Greenberg Jean T (2012), Accelerated cell death 2 suppresses mitochondrial oxidative bursts and modulates cell death in Arabidopsis., in The Plant Journal, 69(4), 589-600.
Early events in plastid protein degradation in stay-green Arabidopsis reveal differential regulation beyond the retention of LHCII and chlorophyll.
Grassl Julia, Pružinská Adriana, Hörtensteiner Stefan, Taylor Nicolas L, Millar A Harvey (2012), Early events in plastid protein degradation in stay-green Arabidopsis reveal differential regulation beyond the retention of LHCII and chlorophyll., in Journal of Proteome Research, 11(11), 5443-52.
MES16, a member of the methylesterase protein family, specifically demethylates fluorescent chlorophyll catabolites during chlorophyll breakdown in Arabidopsis.
Christ Bastien, Schelbert Silvia, Aubry Sylvain, Süssenbacher Iris, Müller Thomas, Kräutler Bernhard, Hörtensteiner Stefan (2012), MES16, a member of the methylesterase protein family, specifically demethylates fluorescent chlorophyll catabolites during chlorophyll breakdown in Arabidopsis., in Plant Physiology, 158(2), 628-41.
STAY-GREEN and chlorophyll catabolic enzymes interact at light-harvesting complex II for chlorophyll detoxification during leaf senescence in Arabidopsis.
Sakuraba Yasuhito, Schelbert Silvia, Park So-Yon, Han Su-Hyun, Lee Byoung-Doo, Andrès Céline Besagni, Kessler Felix, Hörtensteiner Stefan, Paek Nam-Chon (2012), STAY-GREEN and chlorophyll catabolic enzymes interact at light-harvesting complex II for chlorophyll detoxification during leaf senescence in Arabidopsis., in The Plant Cell, 24(2), 507-18.
Chlorophyll breakdown in higher plants.
Hörtensteiner Stefan, Kräutler Bernhard (2011), Chlorophyll breakdown in higher plants., in Biochimica et Biophysica Acta, 1807(8), 977-88.
Arabidopsis STAY-GREEN2 Is a Negative Regulator of Chlorophyll Degradation during Leaf Senescence.
Sakuraba Yasuhito, Park So-Yon, Kim Ye-Sol, Wang Seung-Hyun, Yoo Soo-Cheul, Hörtensteiner Stefan, Paek Nam-Chon, Arabidopsis STAY-GREEN2 Is a Negative Regulator of Chlorophyll Degradation during Leaf Senescence., in Molecular Plant.
Delayed degradation of chlorophylls and photosynthetic proteins in Arabidopsis autophagy mutants during stress-induced leaf yellowing.
Sakuraba Yasuhito, Lee Sang-Hwa, Kim Ye-Sol, Park Ohkmae K, Hörtensteiner Stefan, Paek Nam-Chon, Delayed degradation of chlorophylls and photosynthetic proteins in Arabidopsis autophagy mutants during stress-induced leaf yellowing., in Journal of Experimental Botany.
Water deficit induces chlorophyll degradation via the 'PAO/phyllobilin' pathway in leaves of homoio- (Craterostigma pumilum) and poikilochlorophyllous (Xerophyta viscosa) resurrection plants.
Christ Bastien, Egert Aurélie, Süssenbacher Iris, Kräutler Bernhard, Bartels Dorothea, Peters Shaun, Hörtensteiner Stefan, Water deficit induces chlorophyll degradation via the 'PAO/phyllobilin' pathway in leaves of homoio- (Craterostigma pumilum) and poikilochlorophyllous (Xerophyta viscosa) resurrection plants., in Plant, Cell & Environment.

Collaboration

Group / person Country
Types of collaboration
Universität Seoul Korean Republic (South Korea) (Asia)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Volcani Center, Rehovot Israel (Asia)
- in-depth/constructive exchanges on approaches, methods or results
Hokkaido Universität Japan (Asia)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Universität Neuchatel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Universität Sao Paulo Brazil (South America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Universität Innsbruck Austria (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
University of Chicago United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Swiss Plant Science Web Meeting Talk given at a conference Analysis of catabolite modifying reactions during chlorophyll breakdown 29.01.2014 Meiringen, Switzerland Hörtensteiner Stefan;
Japan-Switzerland Workshop on Adaptation of the plastids to various environmental conditions: Evolution, acclimation and regulation Talk given at a conference Chlorophyll degradation in Arabidopsis thaliana: modification of catabolites 21.11.2013 Zürich, Switzerland Hörtensteiner Stefan; Guyer Luzia Barbara; Sheng Song;
6th European Workshop on Leaf Senescence, Versailles, France Poster Dephytylation during chlorophyll breakdown differs between tomato fruits and leaves 14.10.2013 Versailles, France Hörtensteiner Stefan; Guyer Luzia Barbara;
International Conference on Tetrapyrroles of Photosynthetic Organisms Poster Analysis of dephytylation and deformylation reactions during chlorophyll breakdown 11.09.2013 Wuhan, China Hörtensteiner Stefan;
Instituts Seminar Individual talk Molecular biology of chlorophyll degradation in Arabidopsis thaliana 10.09.2013 Beijing, China Hörtensteiner Stefan;
18th International Conference on Cytochrome P450 Poster Cytochrome P450 CYP89A9 is involved in the formation of novel major chlorophyll catabolites during leaf senescence in Arabidopsis thaliana 18.06.2013 Seattle, United States of America Hörtensteiner Stefan;
Instituts Seminar Individual talk The pathway of chlorophyll breakdown 10.01.2013 Göttingen, Germany Hörtensteiner Stefan;
Instituts Seminar Individual talk The pathway of chlorophyll breakdown 13.11.2012 München, Germany Hörtensteiner Stefan;
Instituts Seminar Individual talk A cytochrome P450 monooxygenase is involved in chlorophyll breakdown in Arabidopsis thaliana 02.11.2012 Bonn, Germany Hörtensteiner Stefan;
Gordon Research Conference on the Chemistry and Biology of Tetrapyrroles Talk given at a conference Chlorophyll breakdown: A cytochrome P450 monooxygenase is involved in chlorophyll breakdown in Arabidopsis thaliana 22.07.2012 Newport, RI, United States of America Hörtensteiner Stefan;
Instituts Seminar Individual talk New aspects of an old story: A cytochrome P450 monooxygenase is involved in chlorophyll breakdown in Arabidopsis thaliana 22.05.2012 Cambridge, Great Britain and Northern Ireland Hörtensteiner Stefan;
1st Annual Meeting CropLife Talk given at a conference Chlorophyll catabolism and senescence: new aspects of an old story 06.05.2012 Kiel, Germany Hörtensteiner Stefan;
Instituts Seminar Individual talk The pathway of chlorophyll breakdown 24.01.2012 Grenoble, France Hörtensteiner Stefan;
Swiss Plant Science Web Meeting Talk given at a conference A cytochrome P450 monooxygenase is involved in chlorophyll breakdown in Arabidopsis thaliana 18.01.2012 Meiringen, Switzerland Hörtensteiner Stefan;
Symposium on Plant Senescence and Postharvest Crop Biology Talk given at a conference Molecular biology of leaf yellowing in Arabidopsis thaliana 11.11.2011 Guangzhou, China Hörtensteiner Stefan;
International Conference on Tetrapyrroles of Photosynthetic Organisms Talk given at a conference Molecular biology of leaf yellowing in Arabidopsis thaliana 24.07.2011 Berlin, Germany Hörtensteiner Stefan; Christ Bastien; Schelbert Silvia; Guyer Luzia Barbara;
Annual Meeting of the French Photosynthesis Society Talk given at a conference Chlorophyll breakdown: protein-protein interaction of chlorophyll catabolic enzymes 16.05.2011 Paris, France Hörtensteiner Stefan;
International Conference on Plant Science in the Post Genomic Era, Sambalpur University Talk given at a conference Pheophytinase cleaves phytol during chlorophyll breakdown and forms a multiprotein complex with other chlorophyll catabolic enzymes 17.02.2011 Sambalpur, India Hörtensteiner Stefan;


Communication with the public

Communication Title Media Place Year
Talks/events/exhibitions Herbstliche Blattverfärbung German-speaking Switzerland 2013

Awards

Title Year
Cozzarelli Preis der US Academy of Sciences 2011

Associated projects

Number Title Start Funding scheme
145020 Plant metabolite analysis by liquid chromatography-mass spectrometry 01.10.2013 R'EQUIP
149389 Chlorophyll breakdown: catabolite modification and transport, and the relation to stoma function and cell death signalling 01.05.2014 Project funding
117940 Identification and characterization of key components of chlorophyll breakdown in Arabidopsis 01.11.2007 Project funding
172977 Diversity and evolution of the PAO/phyllobilin pathway of chlorophyll breakdown 01.07.2017 Project funding

Abstract

Title: Chlorophyll breakdown: analysis of fruit ripening, evolution and regulation.Project summary1. Background. Loss of green color during leaf senescence and fruit ripening is the result of chlorophyll degradation to colorless catabolites. Whereas most of the enzymes and intermediates of leaf senescence-related chlorophyll breakdown are known, fruit ripening has only partially been analyzed in this respect. In particular, inconsistent data exist about the enzyme(s) being responsible for phytol removal, an early reaction in the pathway. Pheophytinase was shown to be involved during leaf senescence and to catalyze dephytylation of pheophytin (Mg-free chlorophyll), in contrast to chlorophyllase, which acts on chlorophyll. It is unclear whether pheophytinase is involved in fruit ripening. In addition, the specificity of pheophytinase is intriguing and calls for further biochemical analysis. Most of the reactions of chlorophyll breakdown occur in the chloroplast and recent, preliminary, results in our lab indicate that different enzymes form a protein complex, yet this remains to be verified. In recent years, several chlorophyll catabolic genes mainly from Arabidopsis thaliana and rice were cloned. Homologous sequences are also present in lower plants, including eu- and prokaryotic photosynthetic organisms, but none of these proteins has functionally been characterized yet. 2. Working hypothesis. Although the pathway of chlorophyll breakdown during leaf senescence in higher plants is rather well understood, questions remain concerning the pathway in fruit ripening, its presence in lower plants and the mode of pathway regulation. The following questions can be raised:- Is the pathway of chlorophyll breakdown during fruit ripening identical to the one in leaf senescence?- What are the biochemical properties of pheophytinase?- Do chlorophyll catabolic enzymes inside the chloroplast form a multiprotein complex?- To what extend is the pathway of chlorophyll breakdown evolutionary conserved?- What are the regulatory mechanisms of chlorophyll breakdown?3. Specific aims. To address some of these chlorophyll breakdown-related questions, we have the following aims:- establishing tomato as a model for both leaf senescence and fruit ripening, with a particular focus on pheophytinase- analyzing biochemical properties of pheophytinase, including crystal structure analysis of the protein - analyzing interaction of different chl catabolic proteins- analyzing evolutionary aspects by characterizing potential catabolic enzymes from lower plants and algae- identifying regulatory factors by isolating suppressors of pao1, a catabolic mutant exhibiting a cell death phenotype4. Methods. Carrying out the proposed research involves a variety of molecular, biochemical and analytical methods, such as (i) production of transgenic plants, (ii) mutant identification, crossing and analysis, (iii) expression and enzymatic analysis of enzymes, (iv) chromatographic methods, (v) pigment and protein structure analysis techniques, and (vi) confocal microscopy.5. Expected value of the proposed project. The research proposed here will allow new insight into different aspects of chl breakdown, which are so far unexplored. It will answer questions about the evolution of chlorophyll breakdown and will allow elucidating the pathway in ripening fruit. This together with the elucidation of regulatory factors might provide interesting targets for modulating fruit ripening for economic purposes.
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