Project

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

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.

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Last update: 21.02.2013

Active participation

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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.