Chemical Properties of Chlorophyll Catabolites

Intellectual Merit: Chlorophyll, because of its role in photosynthesis in plants, is one of the most important and best studied molecules in all of the natural sciences. A signature of chlorophyll in plants is its characteristic brilliant green color. This color, along with the chlorophyll molecule, is transformed in leaves in the Fall season, as evidenced by their spectacular color changes. These color changes are triggered by metabolic processes that produce chlorophyll breakdown products, termed catabolites. The chlorophyll catabolism process also occurs in common fruit, such as bananas. The conversion of the peel of a banana from green (unripe) to yellow (ripe) is a common visual observation of the transformation of chlorophyll molecules through their light absorption properties. Several years ago Professor B. Kraeutler`s group at Innsbruck discovered a new phenomenon that was found to parallel the characteristic change in the absorption spectrum of banana peels: yellow bananas were found to glow blue when excited with UV light and intensity of the photoluminescence was at a maximum when the ripening was at its peak. The catablolites responsible for this striking photoluminescence were isolated and their structures characterized. The photophysics behind the photoluminescence of the catabolites was examined in preliminary exploratory and discovery oriented experiments by the research group at Columbia, lead by Professor Nicholas J. Turro. This proposal describes a continuation of this collaboration, in which the project now builds a transition from mainly discovery driven to mainly hypothesis driven chemical research, that will make important contributions to a fascinating biological phenomenon. Research at Innsbruck be directed towards the synthesis of selected samples of pure and well defined chlorophyll catabolites and include studies of their redox chemistry and antioxidant activities. The photophysical properties of these catabolites will be examined in detail at Columbia. When sufficient information on the structures of the catabolites is established by research at Innsbruck and Columbia, the current speculations concerning "the why" of chlorophyll catabolism will be translated into testable hypothesis concerning the possible role of these catabolites as antioxidants, as the source of "cell poisons" through photosensitized oxidations and as "sun screens" through the absorption of light. The role of catabolites in the presumed photosensitized production of singlet oxygen (1O2) will be directly tested by spectroscopic means at Columbia. The products of photooxidation and autooxidation will be characterized at Innsbruck. Broader Impact: The proposed chemical research and collateral activities will directly advance our knowledge on the impact of chlorophyll catabolites in nature, and will promote and assist the exploration of new paths for the discovery of their relevance in plant physiology. All of this research is driven by a change of paradigm concerning the role of chlorophyll breakdown in higher plants. It will also engage graduate students and postdoctoral associates at a new forefront of research, and promote their intellectual and professional development. Likewise, the proposed activities will enhance the infrastructure for research and education, e.g., by developing internet based cyber infrastructure to boost the effectiveness of the Innsbruck-Columbia collaboration. The PI at Columbia has initiated design of an iCollaboratory (internet collaboration laboratory) that employs commonly available laptop computers and free software to integrate the scientific knowledge exchange between the collaborators. For example, the Innsbruck-Columbia team uses available audio-video tools coupled with free internet connections to discuss on- going research in a face-to-face manner. These cyber tools will be extended to the laboratory where researchers can see experiments being executed in real time. These tools will be especially powerful in preparing for visits of researchers between the laboratories. Both PIs will explore the "glowing banana" photoluminescence phenomena to develop experiments that allow high school students and college undergraduate to experience this stunning visual discovery. The chemistry and biochemistry of pigments related to chlorophyll and their chemical changes, that underlie cell death, provide a stimulating context for inspiring the study of the ripening of bananas and other fruit. Professor Pat O`Hara of Amherst College has used our publications on the phenomenon to develop experiments for high school teachers to design experiments employing fluorescence to monitor the ripening of bananas.

Chlorophyll (Chl) is the characteristic green pigment of plants. It is degraded in the fall leaves of deciduous trees that undergo fascinating changes of their colors. Only about 25 years ago, the surprising formation of colorless Chl-catabolites was clearly documented in yellow, senescent leaves. Until that time, Chl-breakdown was a remarkable enigma. In part thanks to groundbreaking contributions from our group, important insights into Chl-breakdown have been gained in the meantime, which have already found their way into plant biological textbooks. The discovery of the accumulation of unusual fluorescent Chl-catabolites (FCCs) in ripe bananas, as well as the observation of the blue luminescence of these fruit, due to these FCCs, have made us propose to consider important physiological roles of some Chl-catabolites (now called phyllobilins) in plants. The present project deals with the chemical properties of colored phyllobilins and of FCCs, all of which are only transiently existent intermediate products of Chl-breakdown, and generally are found in trace amounts only in senescent leaves and in ripening fruit. Experimental studies with the red chlorophyll catabolite (RCC), with FCCs and with yellow chlorophyll catabolites (YCCs), all of which were prepared by partial synthesis, were conducted applying photo-physical, structural and redox-chemical methods. These studies revealed some extraordinary properties of RCCs (as easily reducible phyllobilins), of FCCs (as effective sensitizers for the generation of singlet oxygen) and of YCCs (as excellent antioxidants and effective bidentate ligands for transition metal ions), which may also be physiologically relevant in plants.Our studies have reinforced the view that phyllobilins (the open chain tetrapyrrolic catabolites of Chl) should not be considered any more as (mere) waste products resulting from a biological detoxification process, as presumed earlier. The hypothetical main effects of phyllobilins in higher plants may be derived, primarily, from their anti-oxidative properties, as well as in the context of their ability to absorb solar light and, for FCCs, to emit light (as fluorescence), as well as in sensitizing the formation of singlet oxygen. Some of the photochemical properties of the Chl-catabolites may be directly useful for studies of intact plant tissues. These findings provide a basis for further experimental studies with plants concerning physiological effects of Chl-catabolites, some of which may also be relevant for plant eating mammals and humans.