Nonfluorescent Chlorophyll Catabolites Research Articles

Profile of Chlorophyll Catabolites in Senescent Leaves of Epipremnun aureum Includes a Catabolite Esterified with Hydroxytyrosol 1-O-Glucoside.

Despite the fact that chlorophyll degradation is a physiological phenomenon occurring daily in all photosynthetic tissues, chlorophyll catabolites are not fully identified. Three new forms (1, 3, and 4) of linear chlorophyll catabolites (phyllobilins) have been characterized in senescent leaves of Epipremnun aureum with spectroscopic data. Compound 1 is a hypermodified blue fluorescent chlorophyll catabolite (hmFCC) esterified with the potent antioxidant hydroxytyrosol. The sequestration of this phenol by a chlorophyll catabolite could explain the physiological meaning of the persistence of hmFCCs in some senescent plants. Compound 3, a yellow chlorophyll catabolite (YCC) originated from the oxidation at C-15 of 1. YCCs have been identified previously and are exclusively formed in the plant vacuole from the final nonfluorescent chlorophyll catabolites (NCCs). The presence of 3 in leaves implies a new reaction in chlorophyll catabolism, as the characterization of 3 implies that YCCs can be also be oxidized in the cytosol from FCCs. Finally, phyllobilin 4 represents a new type of YCC characterized by an inflexible bicyclo glucosyl moiety linked through an intramolecular esterification of the propionic acid residue with the C-3 hydroxy group. The corresponding NCC precursor was recently identified and now the characterization of 4 shows that even this rigid structure can be further oxidized. Undoubtedly, the characterization of phyllobilins is essential to completely comprehend chlorophyll degradation.

Fractionation and origin of NyOx and Ox compounds in the Barnett Shale sequence of the Marathon 1 Mesquite well, Texas

Abstract We have previously reported the origin and fractionation of pyrrolic nitrogen-containing compounds (Ny) during the primary migration of oil within the Barnett Shale sequence of the Marathon 1 Mesquite Well, Texas. Here, we provide insights into the oxygen-containing NyOx and Ox compounds. In negative-ion electrospray (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), Ny and NyOx are the predominant elemental species, and the remaining Ox, SzOx, Sz, NySz and OxNySz compounds are generally less than 5% relative to the total monoisotopic ion abundance (TMIA). In contrast to Ny compounds, which were preferentially retained in source rocks during expulsion, the NyOx compounds were selectively expelled out of source rocks. Among the NyOx compounds, N1O1, N1O2, and N2O1 are the dominant species, which exhibit different fractionation effects. Despite this observation, very similar carbon number (CN) distributions were illustrated, i.e., the predominance of C0-5 alkylated homologues maximizing at C2 or C3 substitutes. Additionally, all NyOx compounds fall in the range of 10–30 DBE (double bond equivalents), with the major classes illustrating an interval of three DBE units. Notably, this is also the case for Ny compounds. The overall similar distributions of CN and DBE suggest that Ny and NyOx compounds have restricted precursors and common mechanisms of formation. Nonfluorescent chlorophyll catabolites (NCCs), the final breakdown products of chlorophyll, were tentatively proposed as possible precursors. For Ox compounds, O2 is the dominant species spread over a DBE range of 1–17, with a maximum at 1 DBE class. The predominance of even-carbon-number molecules, particularly C16H32O2 and C18H36O2, suggests the possible origin of O2 compounds with 1 DBE from fatty acids.

The fluorescent blue glow of banana fruits is not due to symplasmic plastidial catabolism but arises from insoluble phenols estherified to the cell wall

Banana fruits are firstly green due to chlorophyll, then yellow due to carotenoids and finally turn black due to polyphenols. However, bananas glow blue when observed under UV light. It has been reported that chlorophylls fade to give rise to fluorescent chlorophyll catabolites (FCCs) in senescent banana leaves and in ripening banana peels. FCCs are short lived catabolic intermediates that ultimately lead to non-fluorescent chlorophyll catabolites (NCCs). FCCs are abundant in bananas due to hypermodification; therefore, it was concluded that FCC caused yellow bananas to glow blue. Experiments were performed in order to shed new light into the autofluorescence phenomenon. Microscopy performed on living plant samples contradict the interpretation that the fluorescent blue glow is mainly caused by FCC inside the cell. Blue fluorescence in banana emerges from the cell wall, not from the symplasm. It is not primarily caused by soluble chlorophyll catabolites in the vacuoles or senescing plastids. Insoluble phenolics from the apoplast make bananas shine strongly blue under black light. Chlorophyll is a light trap that generates black holes of blue fluorescence, and therefore cells with chloroplasts glow less blue. The white pulp of banana fruits shine more strongly than the outer peel. In both tissues autofluorescence arises from insoluble phenols that are estherified to the cell wall. In monocot species (banana, maize, sugarcanne), blue fluorescense was strongest in the cell wall, whereas in dicots (e.g. arabidopsis, spearmint, hibiscus), blue fluorescence may be dominant from cytosolic, vacuolar or plastidial compartments.

On the Nature of Isomeric Nonfluorescent Chlorophyll Catabolites in Leaves and Fruit - A Study with a Ubiquitous Phylloleucobilin and its Main Isomerization Product.

The typical main products of chlorophyll (Chl) breakdown in higher plants are non-fluorescent, colorless phyllobilins, named phylloleucobilins. These long elusive Chl-catabolites are linear tetrapyrroles, whose structure elucidation has required thorough spectroscopic analyses. Interestingly, in recent LC/MS studies of leaf extracts, isomeric forms of phylloleucobilins were detected. The existence of isomeric phyllobilins may suggest incomplete stereo-selectivity of catabolic processes, or isomerization processes in plant cells or in the analytes. Here we report a study with the phylloleucobilin NCC-1, a basic Chl-catabolite in extracts of leaves and fruit. NCC-1 and its main isomerization product in aqueous solution were identified as 82 -epimers. Formation of 82 -epi-NCC-1 from NCC-1 implies an unstable enol(ate)-intermediate, which reverts to NCC-1 or converts to 82 -epi-NCC-1. Such reversible epimerization reactions are a non-biological in vitro feature of typical phylloleucobilins, and probably also take place in vivo.

Non-fluorescent and yellow chlorophyll catabolites in Japanese plum fruits (Prunus salicina, Lindl.)

Although several chlorophyll metabolites have been shown to exert prominent benefits to human health when consumed, the battery of linear chlorophyll derivatives (phyllobilins) presents in fruits is poorly understood. Yellow chlorophyll catabolites (YCCs) are a new kind of phyllobilins recently identified in senescent leaves, probably arising from an oxidative process of the terminal chlorophyll catabolites, NCCs (non-fluorescent chlorophyll catabolites). This work deals with the characterization by first time of this kind of phytochemicals in edible fruits. Two YCCs have been identified in yellow Japanese plums, one (Ps-YCC1) previously described in the senescent leaves of Cercidiphyllum japonicum Siebold & Zucc. and Ps-YCC2, a chlorophyll catabolite structure described by first time in the edible parts of Japanese plum fruits. These YCCs were characterized by high-resolution MS/MS, describing the specific fragmentation (ring A) and the absence of the typical cleavage of phyllobilins (ring D), as a consequence of the unsaturated bond at C15–16 typical of YCCs, allowing the differentiation from NCCs. To the already known array of phenolic acids, anthocyanins and carotenoids, NCCs and YCCs may contribute to the antioxidant potential of these fruits, a potential that deserves attention and future research, considering the photochemical and photophysical behaviour of this group of tetrapyrrolic breakdown products.

Chlorophyll Catabolites in Senescent Leaves of the Plum Tree (Prunus domestica).

In cold extracts of senescent leaves of the plum tree (Prunus domestica ssp. domestica), six colorless non‐fluorescent chlorophyll catabolites (NCCs) were characterized, named Pd‐NCCs. In addition, several minor NCC fractions were tentatively classified. The structure of the most polar one of the NCCs, named Pd‐NCC‐32, featured an unprecedented twofold glycosidation pattern. Three of the NCCs are also functionalized at their 32‐position by a glucopyranosyl group. In addition, two of these glycosidated NCCs carry a dihydroxyethyl group at their 18‐position. In the polar Pd‐NCC‐32, the latter group is further glycosidated at the terminal 182‐position. Four other major Pd‐NCCs and one minor Pd‐NCC were identified with five NCCs from higher plants known to belong to the ‘epi’‐series. In addition, tentative structures were derived for two minor fractions, classified as yellow chlorophyll catabolites, which represented (formal) oxidation products of two of the observed Pd‐NCCs. The chlorophyll catabolites in leaves of plum feature the same basic structural pattern as those found in leaves of apple and pear trees.

Investigating the Control of Chlorophyll Degradation by Genomic Correlation Mining.

Chlorophyll degradation is an intricate process that is critical in a variety of plant tissues at different times during the plant life cycle. Many of the photoactive chlorophyll degradation intermediates are exceptionally cytotoxic necessitating that the pathway be carefully coordinated and regulated. The primary regulatory step in the chlorophyll degradation pathway involves the enzyme pheophorbide a oxygenase (PAO), which oxidizes the chlorophyll intermediate pheophorbide a, that is eventually converted to non-fluorescent chlorophyll catabolites. There is evidence that PAO is differentially regulated across different environmental and developmental conditions with both transcriptional and post-transcriptional components, but the involved regulatory elements are uncertain or unknown. We hypothesized that transcription factors modulate PAO expression across different environmental conditions, such as cold and drought, as well as during developmental transitions to leaf senescence and maturation of green seeds. To test these hypotheses, several sets of Arabidopsis genomic and bioinformatic experiments were investigated and re-analyzed using computational approaches. PAO expression was compared across varied environmental conditions in the three separate datasets using regression modeling and correlation mining to identify gene elements co-expressed with PAO. Their functions were investigated as candidate upstream transcription factors or other regulatory elements that may regulate PAO expression. PAO transcript expression was found to be significantly up-regulated in warm conditions, during leaf senescence, and in drought conditions, and in all three conditions significantly positively correlated with expression of transcription factor Arabidopsis thaliana activating factor 1 (ATAF1), suggesting that ATAF1 is triggered in the plant response to these processes or abiotic stresses and in result up-regulates PAO expression. The proposed regulatory network includes the freezing, senescence, and drought stresses modulating factor ATAF1 and various other transcription factors and pathways, which in turn act to regulate chlorophyll degradation by up-regulating PAO expression.

Chlorophyll catabolism in olive fruits (var. Arbequina and Hojiblanca) during maturation

The central reaction of chlorophyll (chl) breakdown pathway occurring during olive fruits maturation is the cleavage of the macrocycle pheophorbide a to a primary fluorescent chl catabolite (pFCC) and it is catalyzed by two enzymes: pheophorbide a oxygenase (PaO) and red chl catabolite reductase (RCCR). In subsequent steps, pFCC is converted to different fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs). This work demonstrated that RCCR activity of olive fruits is type II. During the study of evolution of PaO and RCCR activities through the olive fruits maturation in two varieties: Hojiblanca and Arbequina, a significant increase in PaO and RCCR activity was found in ripening stage. In addition, the profile and structure of NCCs present in epicarp of this fruit was studied using HPLC/ESI-TOF-MS. Five different NCCs were defined and for the first time the enzymatic reactions implied in chlorophyll degradations in olive fruits elucidated.

Chlorophyll Catabolites in Fall Leaves of the Wych Elm Tree Present a Novel Glycosylation Motif.

Fall leaves of the common wych elm tree (Ulmus glabra) were studied with respect to chlorophyll catabolites. Over a dozen colorless, non‐fluorescent chlorophyll catabolites (NCCs) and several yellow chlorophyll catabolites (YCCs) were identified tentatively. Three NCC fractions were isolated and their structures were characterized by spectroscopic means. Two of these, Ug‐NCC‐27 and Ug‐NCC‐43, carried a glucopyranosyl appendage. Ug‐NCC‐53, the least polar of these NCCs, was identified as the formal product of an intramolecular esterification of the propionate and primary glucopyranosyl hydroxyl groups of Ug‐NCC‐43. Thus, the glucopyranose moiety and three of the pyrrole units of Ug‐NCC‐53 span a 20‐membered ring, installing a bicyclo[17.3.1]glycoside moiety. This structural motif is unprecedented in heterocyclic natural products, according to a thorough literature search. The remarkable, three‐dimensional bicyclo[17.3.1]glycoside architecture reduces the flexibility of the linear tetrapyrrole. This feature of Ug‐NCC‐53 is intriguing, considering the diverse biological effects of known bicyclo[n.3.1]glycosidic natural products.

Zn-complex of a natural yellow chlorophyll catabolite

“Non-fluorescent” chlorophyll catabolites (NCCs) were named “rusty pigments” originally, as they easily oxidized to yellow chlorophyll catabolites (YCCs) and other colored natural “phyllobilins.” In the present work, binding of Zn(II)-ions by YCC and its methyl ester YCC-Me, and structural investigations of the resulting Zn(II)-complexes are reported. Binding of Zn-ions to the weakly luminescent YCC or YCC-Me in DMSO produces orange-yellow complexes that exhibit strong green emission. The Zn-complex of YCC-Me was isolated and characterized by UV-vis-, fluorescence-, mass- and NMR-spectra. The data revealed a 2:1 complex, Zn(YCC-Me)[Formula: see text], in which YCC-Me serves as bidentate ligand. The Zn(II)-center in Zn(YCC-Me)[Formula: see text] is, thereby, deduced to be coordinated in a pseudo tetrahedral fashion. Formation of Zn(YCC-Me)[Formula: see text] (and of Zn(YCC)[Formula: see text] is compatible with an isomerization of the lactam form of ring D to the corresponding lactim tautomer in these neutral Zn(II)-complexes.

Systematic HPLC/ESI-High Resolution-qTOF-MS Methodology for Metabolomic Studies in Nonfluorescent Chlorophyll Catabolites Pathway.

Characterization of nonfluorescent chlorophyll catabolites (NCCs) and dioxobilane-type nonfluorescent chlorophyll catabolite (DNCC) in peel extracts of ripened lemon fruits (Citrus limon L.) was performed by HPLC/ESI-high resolution-qTOF-MS method. Compounds were identified in samples on the basis of measured accurate mass, isotopic pattern, and characteristic fragmentation profile with an implemented software postprocessing routine. Three NCC structures already identified in other vegetal tissues were present in the lemon fruit peels (Cl-NCC1; Cl-NCC2; Cl-NCC4) while a new structure not defined so far was characterized (Cl-NCC3). This catabolite exhibits an exceptional arrangement of the peripheral substituents, allowing concluding that the preferences for the NCC modifications could be a species-related matter.

Stereo- and regioselective phyllobilane oxidation in leaf homogenates of the peace lily (Spathiphyllum wallisii): hypothetical endogenous path to yellow chlorophyll catabolites.

In senescent leaves, chlorophyll typically is broken down to colorless and essentially photo-inactive phyllobilanes, which are linear tetrapyrroles classified as “nonfluorescent” chlorophyll catabolites (NCCs) and dioxobilane-type NCCs (DNCCs). In homogenates of senescent leaves of the tropical evergreen Spathiphyllum wallisii, when left at room temperature and extracted with methanol, the major endogenous, naturally formed NCC was regio- and stereoselectively oxidized (in part) to a mixture of its 15-hydroxy and 15-methoxy derivative. In the absence of methanol in the extract, only the 15-OH-NCC was observed. The endogenous oxidation process depended upon molecular oxygen. It was inhibited by carbon monoxide, as well as by keeping the leaf homogenate and extract at low temperatures. The remarkable “oxidative activity” was inactivated by heating the homogenate for 10 min at 70 °C. Upon addition of a natural epimeric NCC (epiNCC) to the homogenate of senescent or green Sp. wallisii leaves at room temperature, the exogenous epiNCC was oxidized regio- and stereoselectively to 15-OH-epiNCC and 15-OMe-epiNCC. The identical two oxidized epiNCCs were also obtained as products of the oxidation of epiNCC with dicyanodichlorobenzoquinone (DDQ). Water elimination from 15-OH-epiNCC occurred readily and gave a known “yellow” chlorophyll catabolite (YCC). The endogenous oxidation process, described here, may represent the elusive natural path from the colorless NCCs to yellow and pink coloured phyllobilins, which were found in (extracts of) some senescent leaves.

Nonfluorescent chlorophyll catabolites in loquat fruits (Eriobotrya japonica Lindl.).

Nonfluorescent chlorophyll catabolites (NCCs) and nonfluorescent dioxobilane chlorophyll catabolites (NDCCs) are the terminal compounds of the chlorophyll degradation pathway that may display beneficial properties to human health related to their antioxidant properties, which were recently shown. A profile of NCCs/NDCC of the loquat fruit Eriobotrya japonica Lindl. is described. From the 13 known different NCC structures described to date, three have been identified in loquats. Two new structures not defined so far were characterized in loquat fruits: Ej-NCC2, which corresponds to the methyl ester at C13(2) of Bn-NCC1 and in very low amount Ej-NDCC1, the only NDCC found in loquats. Keto-enol tautomerism at the C13(1) position in NCCs is described for the first time as a regular process in chlorophyll catabolism, probably through a nonspecific mechanism since almost all the chlorophyll catabolites structures detected in fruits of loquat present keto and enol tautomers. The results obtained have been possible through a high-performance liquid chromatography coupled with electrospray ionization ion trap and quadropole time-of-flight mass spectrometry fitted with a powerful postprocessing software.

Non-fluorescent chlorophyll catabolites in quince fruits

During senescence of leaf tissues and fruits, chlorophylls are degraded following a catabolic process that transforms the molecule of chlorophyll into final colorless, non-fluorescent chlorophyll catabolites (NCCs) and non-fluorescent dioxibilane chlorophyll catabolites (NDCCs). These compounds have been characterized in different vegetal species showing several structural constraints and defined functional groups at three peripheral positions. Information regarding the NCC profile of fruits is scarce, and at the moment NDCCs have not been described in fruits. Characterization of NCCs and NDCCs in ripened quince fruits (Cydonia oblonga, Miller) was analyzed by HPLC–ESI-MS, describing four chlorophyll breakdown products (two NCCs and two NDCCs) by considering their chromatographic properties, UV-spectrum and MS profile, and was compared, if possible, with NCC/NDCC standards. Co-NCC1 and Co-NDCC2 are equal to the catabolites identified in senescence leaves of maize and barley, respectively, while the catabolites Co-NCC2 and Co-NDCC1 are new structures but closely related to the previous ones. This is the first time that NDCCs are tentatively identified in a fruit tissue, which presents a NCC/NDCC profile dominated by the series of dioxibilin-type chlorophyll catabolites, an additional proof that the chlorophyll pathway is similar in leaves and fruits.

Hydroxymethylated phyllobilins: a puzzling new feature of the dioxobilin branch of chlorophyll breakdown.

Colorless nonfluorescent chlorophyll (Chl) catabolites (NCCs) are formyloxobilin-type phyllobilins, which are considered the typical products of Chl breakdown in senescent leaves. However, in degreened leaves of some plants, dioxobilin-type Chl catabolites (DCCs) predominate, which lack the formyl group of the NCCs, and which arise from Chl catabolites by oxidative removal of the formyl group by a P450 enzyme. Here a structural investigation of the DCCs in the methylesterase16 mutant of Arabidopsis thaliana is reported. Eight new DCCs were identified and characterized structurally. Strikingly, three of these DCCs carry stereospecifically added hydroxymethyl groups, and represent bilin-type linear tetrapyrroles with an unprecedented modification. Indeed, DCCs show a remarkable structural parallel, otherwise, to the bilins from heme breakdown.

Chlorophyll Breakdown in Senescent Banana Leaves: Catabolism Reprogrammed for Biosynthesis of Persistent Blue Fluorescent Tetrapyrroles

Chlorophyll breakdown is a visual phenomenon of leaf senescence and fruit ripening. It leads to the formation of colorless chlorophyll catabolites, a group of (chlorophyll-derived bilin-type) linear tetrapyrroles. Here, analysis and structure elucidation of the chlorophyll breakdown products in leaves of banana (Musa acuminata) is reported. In senescent leaves of this monocot all chlorophyll catabolites identified were hypermodified fluorescent chlorophyll catabolites (hmFCCs). Surprisingly, nonfluorescent chlorophyll catabolites (NCCs) were not found, the often abundant and apparently typical final chlorophyll breakdown products in senescent leaves. As a rule, FCCs exist only fleetingly, and they isomerize rapidly to NCCs in the senescent plant cell. Amazingly, in the leaves of banana plants, persistent hmFCCs were identified that accounted for about 80 % of the chlorophyll broken down, and yellow leaves of M. acuminata display a strong blue luminescence. The structures of eight hmFCCs from banana leaves were analyzed by spectroscopic means. The massive accumulation of the hmFCCs in banana leaves, and their functional group characteristics, indicate a chlorophyll breakdown path, the downstream transformations of which are entirely reprogrammed towards the generation of persistent and blue fluorescent FCCs. As expressed earlier in related studies, the present findings call for attention, as to still elusive biological roles of these linear tetrapyrroles.

Cytochrome P450 CYP89A9 is involved in the formation of major chlorophyll catabolites during leaf senescence in Arabidopsis.

Nonfluorescent chlorophyll catabolites (NCCs) were described as products of chlorophyll breakdown in Arabidopsis thaliana. NCCs are formyloxobilin-type catabolites derived from chlorophyll by oxygenolytic opening of the chlorin macrocycle. These linear tetrapyrroles are generated from their fluorescent chlorophyll catabolite (FCC) precursors by a nonenzymatic isomerization inside the vacuole of senescing cells. Here, we identified a group of distinct dioxobilin-type chlorophyll catabolites (DCCs) as the major breakdown products in wild-type Arabidopsis, representing more than 90% of the chlorophyll of green leaves. The molecular constitution of the most abundant nonfluorescent DCC (NDCC), At-NDCC-1, was determined. We further identified cytochrome P450 monooxygenase CYP89A9 as being responsible for NDCC accumulation in wild-type Arabidopsis; cyp89a9 mutants that are deficient in CYP89A9 function were devoid of NDCCs but accumulated proportionally higher amounts of NCCs. CYP89A9 localized outside the chloroplasts, implying that FCCs occurring in the cytosol might be its natural substrate. Using recombinant CYP89A9, we confirm FCC specificity and show that fluorescent DCCs are the products of the CYP89A9 reaction. Fluorescent DCCs, formed by this enzyme, isomerize to the respective NDCCs in weakly acidic medium, as found in vacuoles. We conclude that CYP89A9 is involved in the formation of dioxobilin-type catabolites of chlorophyll in Arabidopsis.

Chlorophyll catabolites in senescent leaves of the lime tree (Tilia cordata).

In cold extracts of senescent leaves of the Lime tree (Tilia cordata), two colorless nonfluorescent chlorophyll catabolites (NCCs) were identified, named Tc-NCC-1 and Tc-NCC-2, as well as a polar yellow chlorophyll catabolite (YCC), named Tc-YCC. The constitution of the two NCCs was determined by spectroscopic means. In addition, a tentative structure was derived for Tc-YCC. The three chlorophyll degradation products exhibited tetrapyrrolic structures, as are typical of NCCs or YCCs, and turned out to be rather polar, due to a glucopyranosyl group at their 82-position. At their 3-positions, the more polar Tc-NCC-1 carried a 1,2-dihydroxyethyl group and the less polar Tc-NCC-2 a vinyl group. Tc-YCC was identified as the product of an oxidation of Tc-NCC-1.

Inside Cover: Structures of Chlorophyll Catabolites in Bananas (Musa acuminata)Reveal a Split Path of Chlorophyll Breakdown in a Ripening Fruit (Chem. Eur. J. 35/2012)

The accumulation of blue-fluorescing tetrapyrroles in the peels of ripe bananas is reported by S. Moser, B. Kräutler et al. in the Full Paper on page 10873 ff. The analysis and structure elucidation of ripening banana peels reveals an unprecedented structural variety of catabolites, indicating two chlorophyll-breakdown pathways. The inside cover shows a vertical cut through the epidermal section of a ripening banana (as seen by fluorescence microscopy), which is superimposed by the structures of chlorophyll a and of representative fluorescent- and nonfluorescent chlorophyll catabolites.

Structures of chlorophyll catabolites in bananas (Musa acuminata) reveal a split path of chlorophyll breakdown in a ripening fruit.

The disappearance of chlorophyll is a visual sign of fruit ripening. Yet, chlorophyll breakdown in fruit has hardly been explored; its non-green degradation products are largely unknown. Here we report the analysis and structure elucidation of colorless tetrapyrrolic chlorophyll breakdown products in commercially available, ripening bananas (Musa acuminata, Cavendish cultivar). In banana peels, chlorophyll catabolites were found in an unprecedented structural richness: a variety of new fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs) were detected. As a rule, FCCs exist only "fleetingly" and are hard to observe. However, in bananas several of the FCCs (named Mc-FCCs) were persistent and carried an ester function at the propionate side-chain. NCCs were less abundant, and exhibited a free propionic acid group, but functional modifications elsewhere. The modifications of NCCs in banana peels were similar to those found in NCCs from senescent leaves. They are presumed to be introduced by enzymatic transformations at the stage of the mostly unobserved, direct FCC-precursors. The observed divergent functional group characteristics of the Mc-FCCs versus those of the Mc-NCCs indicated two major "late" processing lines of chlorophyll breakdown in ripening bananas. The "last common precursor" at the branching point to either the persistent FCCs, or towards the NCCs, was identified as a temporarily abundant "secondary" FCC. The existence of two "downstream" branches of chlorophyll breakdown in banana peels, and the striking accumulation of persistent Mc-FCCs call for attention as to the still-elusive biological roles of the resulting colorless linear tetrapyrroles.