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.

Abstract: 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.
Keywords: biosynthesis · chlorophyll · fluorescence · structures · tetrapyrroles uncharacterized FCCs. [23] As reported here, we now have analyzed the major chlorophyll catabolites in such leaves in order to elucidate their structures and to help resolve the puzzle of their accumulation.

Results
Chlorophyll catabolites in senescent banana leaves: In freshly prepared extracts of yellow senescent leaves from bananas (Musa acuminata, Cavendish cultivar, short Ma) about a dozen of blue fluorescent fractions were detected by high performance liquid chromatography (HPLC), all of which were tentatively identified as fluorescent chlorophyll catabolites (FCCs) on the basis of their fluorescence and their absorbance characteristics. [11] Surprisingly, nonfluorescent chlorophyll catabolites (NCCs), the typically more abundant chlorophyll catabolites in leaves, [6] could not be detected in fresh extracts of yellow banana leaves ( Figure 2 and the Experimental Section).

Quantification of tetrapyrroles in senescent banana leaves:
In M. acuminata leaves, freshly harvested at different stages of senescence, the amounts of chlorophylls (a and b) and of fluorescent chlorophyll catabolites (FCCs) were determined quantitatively ( Figure 3). For this purpose, green, greenishyellow, yellow-greenish, shiny yellow and yellow-brownish areas were cut out from different banana leaves and extracted with methanol. Quantitative UV/Vis spectroscopic measurements of the filtered extracts of green leaves and analysis for their chlorophyll content [27] indicated the leaves to contain 61.3A C H T U N G T R E N N U N G (AE5.3) nmol cm À2 of chlorophyll a and b (n = 3).
Likewise, the overall amounts of FCCs were determined by analyzing the complete set of fractions containing FCCs by semipreparative HPLC and quantification of their FCC content by UV/Vis spectroscopy (see below and ref. [11]). Yellow senescent banana leaves were found to contain 47.2-A C H T U N G T R E N N U N G (AE3.1) nmol cm À2 of FCCs (and still about 0.9-A C H T U N G T R E N N U N G (AE0.5) nmol cm À2 of chlorophylls). These data indicated conversion of chlorophyll a and b to FCCs of at least 77 % and a total recovery of chlorophyll catabolites in apparently viable (shiny) yellow senescent leaves of bananas of about 80 %. In such yellow leaves the FCCs observed clearly accounted for the major part of the chlorophylls broken down during senescence, and NCCs were not detected.
Identification of major FCC fractions in extracts of senescent banana leaves: From the extracts of 60 g of yellow Figure 1. Abbreviated structural outline of chlorophyll breakdown in senescent leaves and ripening fruits. [5] Chlorophylls (Chl a and b) are degraded to primary fluorescent chlorophyll catabolites (pFCC, or its C-1 epimer, epi-pFCC). [9,24] FCCs with free propionic acid groups isomerize spontaneously by an acid-catalyzed reaction to the corresponding nonfluorescent chlorophyll catabolites (NCCs), [12] such as Hv-NCC-1. [4,25] FCCs esterfied at the propionic acid group are persistent, such as Mc-FCC-56, a hypermodified FCC (hmFCC) in peels of ripe banana. [20][21][22] In an alternative path, dioxobilin-type nonfluorescent chlorophyll catabolites result from deformylation at ring B, such as Ap-DNCC from senescent leaves of Norway maple. [18,26] Figure 2. HPLC analysis of chlorophyll catabolites in an extract of yellow senescent banana leaves (Musa acuminata, Cavendish cultivar). The chromatogram was recorded with online detection of absorbance at 320 nm (lower trace) and fluorescence emission at 450 nm (upper trace, excitation at 350 nm). Fractions classified as fluorescent chlorophyll catabolites (FCCs) are highlighted and indexed according to the retention times (t R ) observed under conditions of a standard analytical HPLC experiment. [20,23] FCCs from banana leaves were thus named Ma-FCC-t R .
M. acuminata leaves the fractions of the most abundant four FCCs were separated by preparative and semipreparative HPLC and used for further spectroscopic characterization (see the Experimental Section). Cooling with liquid nitrogen during the extraction procedure was used to prevent eventual further reactions of the FCCs. [28] NCCs and yellow chlorophyll catabolites (YCCs) [29,30] were not observed in these extracts.
The FCC fraction with a retention time of 25.6 min (under our conditions of the HPLC-experiment; Figure 2) was identified with the previously described Ma-FCC-61, [23] first by mass spectrometry (molecular formula C 50 H 66 N 4 O 20 ) and then by one-and two-dimensional NMR spectroscopy studies. The new data confirmed the earlier deduced structure of this FCC as a 3 1 , 1,4,5,10,17,18,20,22-octahydro-4,5-seco-(22H)-phytoporphyrin. From 60 g of the yellow banana leaves 2.35 mg (2.26 mmol) of Ma-FCC-61 were obtained as a dry white powder. The UV/Vis spectroscopic characteristics of Ma-FCC-61 were determined quantitatively, and were consistent with data for the related hmFCC, named Mc-FCC-56. [20] The two slightly less polar FCCs, Ma-FCC-63 and Ma-FCC-64, could be separated (in part) by preparative HPLC in MeOH/H 2 O (see the Experimental Section). The first separation was incomplete due to partial interconversion of these isomeric hmFCCs (see below). For the second, final purification step corresponding precautions were taken to prevent isomerization and possible transesterification (with methanol) by changing the solvent system to ACN/H 2 O and by running HPLC with shorter on-column times. The FCC containing fractions were directly frozen in liquid nitrogen, stored at À80 8C to avoid isomerization. The samples were lyophilized, to yield 0.79 mg (0.98 mmol) of analytical pure Ma-FCC-63, and 0.63 mg (0.77 mmol) of Ma-FCC-64, to be used for spectroscopic analyses. The UV/Vis spectra of the two FCCs as well as their CD spectra showed considerable similarities; these spectra were also comparable to those of Ma-FCC-69 ( Figure 4). showed each a set of the characteristic signals of the tetrapyrrole moiety, among them signals at low field due to a formyl and a vinyl group, three singlets and one doublet of four methyl groups at high field, as well as a sharp singlet of the methyl ester group at 3.65 ppm ( Figure 5).
Indeed, when samples of both isomers were stored in a 1:1 (v/v) mixture of methanol and water under argon atA C H T U N G T R E N N U N G mo-A C H T U N G T R E N N U N G sphere at room temperature in the dark, analysis by HPLC indicated complete equilibration to a 1:1 mixture after approximately 24 h in both cases (see Figure 7). Thus, the equilibration of the two FCCs occurred at a rate comparable to that of the free anomeric glycopyranoses in aqueous solution. [36] The UV/Vis and CD spectra of the less polar FCC, named Ma-FCC-69, were similar to those of the more polar analogues ( Figure 4). Its ESI-MS showed a signal for the  bold lines refer to COSY spectra, arrows to ROESY spectra. Right: Assigned signals of 13 C atoms from heteronuclear 1 H, 13 C correlations; shadowed boxes indicate 13 C assignments from direct correlations (HSQC spectra), arrows point to 13 C assignments from long-range couplings with H atoms, as seen in HMBC spectra. The signals of the 35 non-exchangeable protons of the tetrapyrrole core structure could be assigned in the same way as described above (see also the Experimental Section). [27] The 600 MHz 1 H NMR spectrum of Ma-FCC-69 in CD 3 CN showed the signals of seven additional protons in the intermediate field range, consistent with a hexopyranosyl group. The heteronuclear correlation between the 6'-methylene protons of the sugar unit and the carbonyl carbon of the propionate side chain (in an 1 H, 13 C HMBC spectrum) indicated an ester linkage, that is, similar to that in Ma-FCC-63. The sugar moiety was identified as a b-glycopyranosyl unit by analysis of the coupling pattern in 1 H, 1 H COSY and ROESY NMR spectra, as well as by correlations to six carbons in the 1 H, 13 C HSQC and HMBC spectra. 1 H, 1 H coupling constants together with 1 H and 13 C shifts in comparison to reference compounds provided further support for a glucopyranosyl moiety. [34] Three additional signals in the 1 H NMR spectrum showed 1 H, 1 H ROESY correlations to the hydrogen at the anomeric center, supported by a heteronuclear correlation between the 1'-hydrogen of the pyranose and carbon 1'' of the (unknown) aglycon moiety. The latter was identified as a 2-(3,4-dihydroxyphenyl)-ethyl group, and Ma-FCC-69 was thus a 3 1 , ]-1,4,5,10,17,18,20,22-octahydro-4,5seco-(22H)-phytoporphyrin. Interestingly, a chlorophyll catabolite with the same molecular constitution is already known as an FCC in senescent leaves of the Peace Lily, Spathiphyllum wallisii (Sw). [37] Comparison of Ma-FCC-69 with Sw-FCC-62, an hmFCC from senescent leaves of the Peace Lily: Ma-FCC-69 was deduced to have the same molecular constitution as Sw-FCC-62, isolated from senescent leaves of the tropical evergreen S. wallisii. [37] To test the eventual identity of the two hmFCCs, their elution properties were compared in HPLC experiments. Solutions of Ma-FCC-69 and Sw-FCC-62 in MeOH/H 2 O were separately analyzed by HPLC, and as a 1:1 mixture of both in a co-injection experiment (see Figure 9). Ma-FCC-69 was found to elute later in a reversed-phase system than Sw-FCC-62, and the two FCCs were shown to be non-identical. Their stereochemical difference was assigned to the configuration of the 1-position, which was indicated to be of the epi-type in catabolites from bananas, since it is of the normal-type in the leaves from the Peace Lily. [37] Selected spectroscopic data of minor FCC fractions in extracts of senescent banana leaves: Four additional, minor FCC fractions were analyzed by HPLC and mass spectrome-

Discussion
Nonfluorescent chlorophyll catabolites (NCCs) are a typical result of chlorophyll breakdown in senescent leaves. [5,8,38] A recent study with apples and pears also revealed the formation of NCCs in ripening fruit. These NCCs, furthermore, were the same as those in leaves of the corresponding fruit trees. [39] This suggested the existence of a common path of chlorophyll breakdown in leaf senescence and fruit ripening, that led to NCCs. [5,39] The discovery of the striking accumulation of blue fluorescent chlorophyll catabolites (FCCs) in the peels of ripening bananas (Musa acuminata, Cavendish cultivar) [20,21] stimulated us to also study the nature of the corresponding catabolites in senescent (yellow) banana leaves. A fluorescent chlorophyll catabolite was identified in banana leaves, which was non-identical to those from the banana fruit. [23] In senescent M. acuminata leaves FCCs accumulate, and NCCs are not found: Indeed, as was shown here, the structures of the leaf hmFCCs differ characteristically from those in the peels of banana fruit, a result, apparently, from processes that occur at the stage of the FCCs. In senescent banana leaves, hypermodified FCCs (hmFCCs) accumulate and their amounts come up to as much as about 80 % of the degraded chlorophylls. Thus, hmFCCs are, by far, the major product from chlorophyll breakdown, and they induce senescent M. acuminata leaves to fluoresce blue ( Figure 10). Remarkably, the formation of NCCs appears to be com-pletely inhibited due to efficient esterification of FCCs, and formation of the persistent hmFCCs.
The banana plant (M. acuminata) belongs to the Zingiberales, an order of monocotyledons that includes bananas, gingers, and their relatives. [40] In this (tropical) evergreen chlorophyll breakdown may have a major purpose other than (is suspected) in deciduous plants. This may be reflected by the striking accumulation of hmFCCs in senescent banana leaves, and their structural variety. A somewhat related situation was found recently in the senescent leaves of another tropical evergreen, the Peace Lily (S. wallisii), in which a persistent hmFCC accumulated (called Sw-FCC-62) besides lesser amounts of NCCs. [23,37] In both of these monocotyls a particular hmFCC has now been identified (Sw-FCC-62 and Ma-FCC-69), in which the glucosyl moiety carries a dihydroxyphenylethyl aglycon. As was shown here, the hmFCCs from the two evergreens had the same molecular constitution, and still were non-identical. A stereochemical difference was indicated that could be assigned to the C-1 position. This stereodivergence is due to the action of two lines of (RCC) reductases during chlorophyll breakdown. [41] RCC reductase in banana leaves is thus of type-2, providing the epi-series of colorless chlorophyll catabolites, as likewise found in banana fruit. [28] M. acuminata leaf FCCs are esterified with two types of hexopyranoses: In an exploratory earlier study of senescent (yellow) leaves of the banana plant an hmFCC was characterized, named Ma-FCC-61. [23] The persistent Ma-FCC-61 was esterified with a 6-a-galactopyranosyl-(1!6)-b-galactopyranosyl-(1!1)-glycerol moiety. In the present investigation, the structures of three slightly less polar and similarly abundant hmFCCs were determined, Ma-FCC-63, Ma-FCC-64 and Ma-FCC-69 (Figures 6 and 8), and they were compared with the structure of Ma-FCC-61 ( Figure 11). In contrast to Ma-FCC-61, these three slightly less polar hmFCCs are all esterified by a glucopyranosyl group, attached at the critical propionate with its primary 6'-OH-group. Ma-FCC-  The remarkable 6-a-galactopyranosyl-(1!6)-b-galactopyranosyl-(1!1)-glycerol moiety found in Ma-FCC-61 from senescent M. acuminata leaves relates chlorophyll catabolites with the ubiquitous membrane components of the thylakoids and elsewhere in plant leaves, which carry digalactosyl diacylglycerides as their polar head. [42] Ma-FCC-61 may thus be a building block for further assembly of more complex, so far unidentified tetrapyrrolic (bilin-type) pigments, or it could represent an adventitious cleavage product, carrying the polar remains of a membrane component. [42,43] Increased lipophilic character (and membrane affinity) of hmFCCs in the banana leaves could be relevant for binding to cell membranes, and the intraand intercellular transport through them. [44] In this context, the structure of the light-harvesting porphyrinoids (chlorophylls c) from the marine photoautotroph Emiliania huxleyi (a coccolithophore) is of interest, in which lipophilic digalactosyl diacylglyceride esters replace the phytol ester of the chlorophylls (apparently in a functional way). [43] Indeed, extracts of senescent banana leaves do contain several very minor fractions that have been tentatively identified as FCCs. These presumed FCCs are less polar even than Ma-FCC-69, and will be the subject of further studies to characterize their structures.
The strikingly different dihydroxyphenylethyl aglycon in Ma-FCC-69 appears to be among the typical constituents of low-molecular-weight natural products isolated from dicotyls, and suggested to be useful as taxonomic markers for this class of higher plants. [34] It was found in hmFCCs from two distantly related tropical evergreens, encouraging the consideration of physiological roles in these monocots. Clearly, at present, the role of such phenylethyl glycosides for the further fate and possible use of hmFCCs in the plants is unknown.
Chlorophyll breakdown in M. acuminata leaves is reprogrammed by efficient esterification of FCCs: Esterification at their propionyl group appears to be a general feature of the FCCs detected in senescent leaves of M. acuminata. When carrying a free propionic acid function, FCCs are typically only fleetingly existent, and are programmed to undergo isomerization to NCCs. [12,13] In contrast, esterification of the propionyl side chain generates persistent hmFCCs, and provides the chemical basis for the accumulation of these hmFCCs. Thus, it stalls chlorophyll breakdown at the stage of FCCs, and inhibits the further isomerization of the latter to the NCCs. The observed esterification may be rationalized as a catabolic intervention [20] and purposeful reprog- Figure 11. Structural outline of the proposed later part of chlorophyll degradation in senescent leaves of bananas, beginning with epi-pFCC, the C1 epimer of the primary FCC (pFCC). [2,9,13]  Selective attachment of b-glucopyranosyl units via their anomeric center to the terminal oxygen of the hydroxylethyl side chain at their 8-position is a typical feature of a variety of NCCs (X in Figure 12) and of some hmFCCs, as well. [21] Indeed, the glycosidation observed in NCCs is (currently) presumed to already occur at the stage of the corresponding FCC precursors in the cytosol, where it would be catalyzed by still unidentified glycosidases. [8,45] This type of glycosidation is reminiscent of the glucuronidation of bilirubine (in mammals), [15] and has also been rationalized on the basis of the hypothetical requirements for the transport of FCCs into the vacuoles (where they isomerize to the corresponding NCCs). In contrast, esterification of FCCs by a gluco-or galactopyranosyl group at the critical propionate with the primary 6'-OH-group of the sugar units, as found here, provides sugar esters that may have (different) basic physiological roles: the sugar esters stabilize hmFCCs against their acid induced isomerization to NCCs (as also achieved, similarly, by a daucic acid residue [20] ), and they simulA C H T U N G T R E N N U N G taA C H T U N G T R E N N U N G neousA C H T U N G T R E N N U N G ly provide linkers for attachment of further groups. Thus, (at least) two further types of hypothetical cytosolic enzymes are suggested, which wait for identification.
The question of physiological roles of chlorophyll catabolites in higher plants: Chlorophyll breakdown in higher plants may be considered, first of all, to be a detoxification process, helpful, indirectly, in permitting the remobilization of nitrogen from chlorophyll-binding proteins to proceed during senescence. [8,46] Indeed, a physiological function of the ubiquitous chlorophyll catabolites is still unknown. This is striking, as it contrasts the important and well-known physiological functions in plants and in algae of the structurally related heme catabolites (e.g., of biliverdin and the phycobilins), as well as those of biliverdin and bilirubin in animals. [14,15,47,48] The colorless NCCs exhibit the properties of remarkable antioxidants, which may be of particular relevance in senes-cent leaves, as well as in ripening fruit. [5] Possibly, the related (but less well studied) FCCs could have similar beneficial properties that may help to extend the viability of senescent or ripened plant tissue. [24] Indeed, in senescent M. acuminata leaves chlorophyll breakdown appears to be reprogrammed to furnish the persistent blue luminescent hmFCCs. Clearly, it deviates from the pathway towards colorless and photoA C H T U N G T R E N N U N G in-A C H T U N G T R E N N U N G active tetrapyrroles, such as NCCs (and the related DNCCs), typical of senescent leaves of higher plants. [5,10] The extraordinary biosynthetic esterifications of FCCs in yellow banana leaves (and fruit) provides uniquely structured hmFCCs, which suggests the possible existence of endogenous physiological roles of FCCs, other than those of NCCs. Specific hmFCCs are indicated, for example, to accumulate selectively in the senescent tissue surrounding dark necrotic parts of banana peels. [21,22] FCCs absorb UV light very effectively and may represent, for example, a type of sun screen against UV light for the plant. [10] They also emit blue light, that is, they act as optical brighteners to the human eye. [20] In a related sense, the hmFCCs are natural endogenous fluorescence signals that may be useful as noninvasive, molecular tools for biochemical investigations for studying cellular (senescence) processes in plants. [21,23] Indeed, in bright yellow bananas, [20,28] and even more so, in senescent banana leaves, persistent hmFCCs accumulate, [23] and are the molecular origin of their blue luminescence. In a broader sense, the fascinating colors that appear in fall leaves and in ripening fruit, due to the degradation of chlorophyll, stimulate further considerations with respect to the biological [49] and ecological [50,51] relevance of such color changes. In a few exceptional cases, degreening leaves are, indeed, known to develop strong luminescence, one example being the fall leaves of Ginkgo biloba. [52] The luminescence of the senescent leaves of this tree is mainly caused by an unsaturated alkaloid. [52] Clearly, the bright colors of fruit are primarily seen as signals for frugivorous animals, helping to attract these for the purpose of increasing the local distribution of the seeds. [53,54] Fruits and plants in tropical regions furnish indispensable feed for domestic animals. [55] This underscores the relevance of chlorophyll breakdown in providing suitable visual signals to mark ripening fruits. The blue luminescence observed in bananas has also been rationalized on this basis. [20] Possibly, similar arguments may also apply to the leaves of fruit-bearing plants. "Fruit flagging" could be an additional optical signal of fruit-bearing plants, relayed by the help of colorful and possibly luminescent leaves in the surrounding of ripe fruit. [56] The development of bright and distinct colors of fruit and of leaves, even in the UV-and blue-light regions, [57] as well as the complementary capacity for their perception by insects, birds and other animals may contribute significantly to the means of communication between plants and animals. [58,59] Often, but not always, this type of communication is beneficial for both sides: as recently reported, insect eating plants make use of a still uncharacterized blue luminescent compound to attract insects into their deadly fly traps. [60]

Conclusion
We describe here the identification and structure elucidation of a group of FCCs in extracts of senescent Musa acuminata leaves. These leaves accumulate hypermodified FCCs (hmFCCs) massively, and luminesce blue, when excited by UV light, due to these chlorophyll catabolites. The persistent hmFCCs may be useful as natural endogenous luminescent signals of cell death that may open up access to new, noninvasive observations of cellular processes in leaves and fruit. In contrast to the situation in other known senescent leaves, the Ma-FCCs are not further processed in the banana leaves to NCCs or related nonfluorescent tetrapyrroles. This discovery indicates chlorophyll breakdown in banana leaves to be completely reprogrammed. It provides further examples to contrast the view, [24,49] that chlorophylls are degraded in senescent leaves by a general and common pathway to NCCs, which were presumed earlier to be the typical end products of chlorophyll breakdown in leaves. Ma-FCCs represent new variants of unique and linear tetrapyrroles. The exploration of their possible physiological functions could lead to a fundamental expansion of our views on why chlorophyll is broken down in higher plants.

Experimental Section Materials
Plant material: Yellow-greenish senescent leaves of bananas (Musa acuminata, Cavendish cultivar) for standard analysis and preparative extractions were harvested at the plantation Malpais Trece (Garachico, Island of Tenerife, Spain) and transported at ambient temperature to Innsbruck, where they were frozen in liquid N 2 and stored at À80 8C, until they were analyzed. Leaves at different stages of senescence for determination of chlorophyll content and total amount of FCCs were collected from plants grown at the University of Innsbruck (Center for Chemistry and Biomedicine, Institute of Organic Chemistry) and picked directly before use. The batches were shown to contain the same catabolites, but in slightly different distributions.
Chemicals: Commercially available solvents (reagent grade) were redistilled and in the case of dichloromethane (CH 2 Cl 2 ) filtered over Alox before use for extractions. HPLC grade methanol (MeOH) was from VWR (West Chester, USA); acetonitrile (ACN) from Sigma-Aldrich (St. Louis, USA), ultrapure water (18 MW cm À1 ) from a Millipore apparatus. The 1 and 5 g SepPak C18 cartridges were from Waters Associates (Milford, USA).

Methods
Analytical and semipreparative HPLC:   [31,32] 10 8C; residual solvent peaks (CD 2 HOD: d H = 3.31 ppm, d C = 49.00 ppm; CD 2 HCN: d H = 1.94 ppm, d C = 1.32 ppm) were used as internal reference [61] signals are classified as singlet (s), doublet (d), doubled doublet (dd), double doubled doublet (ddd), triplet (t) and multiplet (m); apparent = app.; broad = br. ESI-MS: [62]  Analysis of chlorophyll catabolites in senescent leaves by analytical HPLC: Yellow senescent banana leaves (200 mg wet weight, M. acuminata, Cavendish cultivar) were frozen in liquid N 2 and pulverized frozen in a mortar. After extraction with cold MeOH (400 mL) and centrifugation of the resulting suspension for 3 min at 12 700g, the supernatant was diluted 2:1 (v/v) with water and centrifuged (3 min, 12 700g) once more. The resulting yellow extract was injected into the analytical HPLC system by using absorbance and fluorescence detection ( Figure 2). (Figure 3): From freshly harvested banana leaves three samples each (leaf areas between 9 and 26 cm 2 ) at five different senescence stages were cut out (green, greenishyellow, yellow-greenish, yellow, yellow-brownish; n = 3; 15 samples in total). Each sample was frozen in liquid N 2 , pulverized in a mortar with sea sand and extracted with MeOH. The slurry was centrifuged for 2 min at 12 700g, the supernatant was removed and the residue was ground and extracted a second time. The procedure was repeated four to five times (until the solid residue was colorless).

Quantification of tetrapyrroles in banana leaves
Determination of chlorophyll content in banana leaves: The obtained methanolic extracts were combined and diluted with MeOH to 10.0 mL (or to 20.0 mL) in a volumetric flask. From UV/Vis spectroscopic analysis of these fractions the concentration of chlorophyll was calculated first, [27] and from this the amount of chlorophyll per cm 2 leaf section.
Determination of the total amount of FCCs in banana leaves: An aliquot (7.0 mL) of each obtained methanolic fractions from the chlorophyll content measurements was diluted 1:4 (v/v) with H 2 O and (if necessary after centrifugation for 2 min at 12 700g) applied to a preconditioned 1 g SepPak cartridge. After being washed with H 2 O (15 mL) the FCC containing fraction was eluted with MeOH (5 mL). The solvents were removed by using a rotary evaporator and the remaining residue was dissolved in 500 mL MeOH/H 2 O 1:1 (v/v). An aliquot (200 mL) of the solution was separated by semipreparative HPLC, all FCC containing fractions were collected. The unified solutions of each run were diluted with MeOH to 5.0 mL in a volumetric flask and analyzed by UV/Vis spectroscopy. From the absorbance at 317 nm the total concentration of FCCs was calculated. Isolation of chlorophyll catabolites: Yellow-greenish senescent banana leaves (60 g wet weight; Musa acuminata, Cavendish cultivar) were frozen in liquid N 2 , mixed with sea sand (15 g) and ground to a fine powder. To the collected fine powder ice-cooled CH 2 Cl 2 (100 mL) were added and the cold slurry was filtered through a Buchner funnel. Extraction with CH 2 Cl 2 was repeated four times with 80 mL each to remove chlorophyll and unpolar carotinoides. Afterwards ice-cooled MeOH (100 mL) was added to the plant material and the cold slurry was filtered once more. This procedure was repeated four times with MeOH (50 mL each). The collected methanolic extracts were concentrated to 50 mL at reduced pressure by using a rotary evaporator. After being diluted with H 2 O (200 mL) the yellow clear solution was applied to a preconditioned 5 g SepPak cartridge and rinsed with MeOH/H 2 O 1:3 (v/v; 80 mL). The FCC-containing fraction was eluted with MeOH (30 mL), dried under reduced pressure and redissolved in MeOH/H 2 O 2:1 (v/v; 3 mL). Further purification by three preparative HPLC runs gave crude samples of the Spectroanalytical data