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Breakdown of chlorophyll is the characteristic visual sign of leaf senescence.[1,2] In the past two decades it has been shown that the chlorophyll in degreening leaves is degraded to colorless and nonfluorescent chlorophyll catabolites (NCCs).[2-5] In apples and pears chlorophylls are also broken down to NCCs that are identical to those from senescent leaves of the corresponding fruit trees. Chlorophyll breakdown was thus suggested to follow a common path in senescence and fruit ripening, and to yield NCCs as its “final” product (Scheme 1).[4,7]
In contrast, fluorescent chlorophyll catabolites (FCCs) accumulate in senescent, yellow banana leaves, and NCCs occur only in traces in their extracts (Figure 1 and Figure S1 in the Supporting Information). Indeed, FCCs (such as pFCC) were observed earlier in senescent leaves, but only as short-lived precursors of NCCs.[8,9] In ripening bananas, “persistent” FCCs such as Mc-FCC-56 (Scheme 1) have been discovered only recently; these are the compounds responsible for bananas’ blue luminescence. The FCCs from banana leaves (Ma-FCCs) differ from their relatives in fruit peels (Figure 1), but appear to be “persistent” also and to feature complex propionyl ester functions. The structure of one of them, Ma-FCC-61, was elucidated as a tetrapyrrole with an unprecedented digalactosylglyceryl moiety (Scheme 1).
HPLC analysis of an extract of senescent leaves of bananas (Musa acuminata, “cavendish” cultivar) indicated a variety of (Ma-)FCCs. An abundant and relatively polar FCC was observed at a retention time of 61 min, and was provisionally termed Ma-FCC-61. From a 42 g sample of a yellow banana leaf we isolated 4.2 mg of Ma-FCC-61 by HPLC which we characterized by UV/Vis and fluorescence spectra (see Figure S6 in the Supporting Information and Figure 1). The molecular formula of Ma-FCC-61 (C50H66N4O20) was deduced by HRMS from the observed molecular ion [M+H]+ at m/z 1043.394 (calcd for C50H67N4O20+: 1043.434).
The 600 MHz 1H NMR spectrum of Ma-FCC-61 showed the 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 a methyl ester at 3.71 ppm (see Figure S2 in the Supporting Information). Detailed information on the constitution of Ma-FCC-61 was gained from multidimensional NMR spectroscopy (1H,1H COSY and ROESY spectra (Figure 2), as well as 1H,13CHSQC and HMBC-spectra (see the Supporting Information). The signals of all of the 35 non-exchangeable protons of the tetrapyrrole unit could be assigned, establishing the core structure of the functionalized tetrapyrrole (see, for example, Ref. ). Additional signals of 19 hydrogen atoms were observed in the intermediate field region of the 1H NMR spectrum of Ma-FCC-61, and were also analyzed by extensive 1H,1H and 1H,13C correlations. In this way we could identify (two) pyranose units and a glycerol moiety as the intricate modification of Ma-FCC-61.
A 1H,13C correlation between the carbonyl carbon and one of the 6′-hydrogens of the directly bound pyranose unit indicated that the propionyl side chain of the tetrapyrrole core is the attachment site of the novel functionalized disaccharide unit. The assignments of both pyranose units as derived from galactose, as well as the relative configuration of their intermodular linkages at the anomeric centers (as 6′-α and 6″-β), were based on analysis of the values of the 1H,1H coupling constants within the two pyranose units, and comparison of the 1H and 13C chemical shift data with that of reference compounds (see the Supporting Information). The assignment was supported by data from 1H,1H ROESY spectra. Ma-FCC-61 was found to bear a propionyl ester function with a 6-α-galactopyranosyl-(1→6)-β-galactopyranosyl-(1→1)-glyceryl unit as well as a hydroxy group at the 82 position of its tetrapyrrolic FCC core. Thus, Ma-FCC-61 is a 31,32-didehydro-82-hydroxy-132-(methoxycarbonyl)-173-[6′-α-galactopyranosyl-(1′→6″)-β-galactopyranosyl-(1″→1′″)-glyceryl)]-1,4,5,10,17,18,20,22-octahydro-4,5-seco-(22H)-phytoporphyrin (Figure 2 and Scheme 1).
Ma-FCC-61 is a new type of multiply functionalized natural tetrapyrrole and a new representative of the “persistent” and “hypermodified” FCCs. These unique tetrapyrroles carry complex ester functions at the propionyl side chain and have turned up, so far, in ripening or senescent parts of the banana plant (for a second new case, see below).[5,10,13] The presence of ester functions at the propionyl side chain in the “persistent” Ma-FCCs explains the stunning lack of NCCs in the extracts of the banana leaves (see Figure S1 in the Supporting Information), since a free propionic acid function is required for the rapid formation of NCCs from FCCs by a stereoselective, acid-catalyzed isomerization.[9,12]
While there is no precedence for an esterification of a chlorophyll catabolite with a (di)galactosyl unit, as observed here for Ma-FCC-61, related chlorophyll analogues occur in the coccolithophore Emiliana huxleyi. The light-harvesting porphyrinoids (“chlorophylls c”) from this ubiquitous marine photo-autotroph display a galactosyldiacylglyceride ester, which appears to replace the phytol ester of the chlorophylls in a functional way. In Ma-FCC-61 the 6-α-galactopyranosyl-(1→6)-β-galactopyranosyl-(1→1)-glyceryl moiety represents the core structure of digalactosyldiacylglycerides, ubiquitous membrane components of the thylakoids and elsewhere in plant leaves. This digalactosylglyceryl unit is bound strongly by lipases that hydrolyze the ester functions of digalactosyldiacylglycerides with loss of the polar head. Ma-FCC-61 may thus be a building block for further assembly (or an adventitious cleavage product) of more complex, so far unidentified pigments.
At present, a physiological function of the ubiquitous linear tetrapyrroles that arise from breakdown of chlorophyll is unknown. This is remarkable, as the structurally related tetrapyrroles from heme breakdown have important roles (e.g. biliverdin and phycobilins in plants and algae, biliverdin, and bilirubin in higher animals).[18-20] Chlorophyll breakdown may, first of all, be a detoxification process. However, the ubiquitous, but invisible NCCs have been shown to be effective antioxidants of possible physiological benefit in fruit. For the structurally similar FCCs, beneficial effects on the viability of the banana peels have also been taken into consideration. The biosynthetic esterification of typical FCCs of banana plants and the intriguing disaccharide moiety in Ma-FCC-61, in particular, now draw attention to possible physiological roles of FCCs and to their further endogenous use. Clearly, as FCCs absorb UV irradiation efficiently and emit blue light instead, they are “optical brighteners” to the human eye, and they may also have a role as endogenous “sun screens” for UV light.
In senescent banana leaves chlorophyll breakdown deviates from the apparently “common” path in higher plants and appears to stop at the stage of the blue-luminescent FCCs. FCCs were found to accumulate likewise and to induce blue luminescence in bright yellow bananas and in senescent sections of the peels of overripe bananas. However, banana leaf FCCs (Ma-FCCs) differ from their analogues in banana peels, and (most) Ma-FCCs are less polar. Mass spectrometric analysis of several Ma-FCCs also suggested complex modifications that involve the propionyl ester functions (see the Supporting Information). New variants of the natural “persistent” FCCs thus accumulate in senescent banana leaves and make them luminesce blue. This remarkable discovery with banana leaves is not unique: accumulation of FCCs was also found (recent preliminary observation) in yellow leaves of the peace lily (Spathiphyllum wallisii), a subject of ongoing work in our labs.
To learn about the spatial distribution of Ma-FCCs in senescent banana leaves, studies by light and fluorescence microscopy were carried out. Analysis of a leaf cross section revealed blue-luminescent material in senescent palisade parenchyma cells (Figure 3c). These cells thus appear to be the most likely location for the accumulation of the FCCs. Thicker cell walls, for example, of the epidermis, xylem tracheids, and fibers, also show blue fluorescence, which is generally ascribed to cell wall components.[23,24] Likewise, green banana leaves showed blue fluorescence in cell walls, but they lacked the marked blue luminescence in the lumen of the green cells (Figure S4 in the Supporting Information).
In the yellow banana leaf, red chlorophyll fluorescence was found in the largely intact chloroplasts of guard cells (of the stomata) as well as in some parenchyma cells (Figure 3b). Additional yellow spots are assumed to be due to putative lipid droplets, as also depicted in Figure 3b. Indeed, osmiophilic globules were seen in senescent yellow banana leaves in transmission electron microscopic images, supporting this assumption (Figure S5 in the Supporting Information). In senescent leaves, chloroplasts transform into gerontoplasts in a process characterized by loss of thylakoid membranes and by massive accumulation of lipid-containing plastoglobules. Eventual rupture of the gerontoplast envelope leads to a release of plastoglobules to the cytoplasm, causing lipid droplets, as observed in this study. A similar process may occur in peels of ripe bananas during chromoplast differentiation.
When leaves of plants de-green and when fruits ripen, they develop fascinating colors (fall colors of deciduous trees and bushes), provoking basic biological and ecological questions. However, strongly luminescent senescent leaves are known in only a few plants, one of which is Ginkgo biloba. Luminescence of its leaves is due to an unsaturated alkaloid. Bright colors of fruit are believed to have evolved as valuable signals to attract frugivores, needed for seed dispersal.[32,33] Indeed, the blue luminescence of ripe bananas may fulfill such a role. “Fruit flagging” by colorful (and possibly by luminescent) leaves may be an additional optical signal of fruit-bearing plants. Perception of the bright and distinctive colors of both, leaves and fruit, may thus help guide the crucial interactions of plants with insects, birds, and other animals.[32,33]
We describe the observation of blue luminescence of senescent banana leaves. Its main contributors were identified as new types of “persistent” FCCs and uniquely “hypermodified” tetrapyrroles. This discovery, first of all, contrasts the earlier notion of a “common” basic path of chlorophyll breakdown in senescent leaves, in which NCCs were seen as the final products of a rapid and directed metabolic detoxification process.[4,21] It also draws attention to possible physiological roles of FCCs and other chlorophyll catabolites. Furthermore, as endogenous products and luminescent signals of cells undergoing programmed death, “persistent” FCCs commend themselves as natural molecular markers of senescence, which open a new noninvasive view into cellular processes in leaves and fruit.
Isolation and spectroscopic characterization of Ma-FCC-61: Fresh yellow sections of de-greened banana leaves were collected (sample size about 42 g) and frozen in liquid nitrogen. Extraction and further purification by semipreparative HPLC (as described in the Supporting Information) gave crude Ma-FCC-61, which was desalted and purified further by semipreparative HPLC. The fraction containing Ma-FCC-61 was again desalted and dried in vacuo. A pure sample of Ma-FCC-61 (4.2 mg) was obtained, which was used for recording spectra: UV/Vis (MeOH): λmax (Irel.): 379 sh (0.482), 358.5 (0.692), 316.5 (1.0). Fluorescence (MeOH, excitation at 360 nm): λmax at 446 nm (see Figure 1). High-resolution mass (HRMS, see the Supporting Information for details).
Photographs were taken with a mounted digital camera (Canon EOS 450D) and irradiation with day light (exposure time 0.167 s) or UV light (UV lamp Benda NU-15 KL, 15 W, nominal emission at 366 nm, exposure time 10 s). Light and fluorescence microscopy: Cross sections of a senescent yellow part of a banana leaf were cut with a razor blade. Sections were viewed with a Zeiss Axiovert 200 M microscope equipped with a Zeiss Axiocam MRc5 (Carl Zeiss AG, Jena, Germany). For fluorescence images either filter set 01 (excitation BP 365/12 nm, emission LP 397 nm) or filter set 09 (excitation BP 450–490 nm, emission LP 515 nm) was used.
[**]We thank Prof. Stefan H rtensteiner (University of Z rich) and Prof. Nicholas J. Turro (Columbia University) for helpful discussions. This work was supported by the Austrian National Science Foundation (FWF, Project no. 19596).
Dr. Srinivas Banala, Institute of Organic Chemistry and Centre of Molecular Biosciences University of Innsbruck, 6020 Innsbruck (Austria)
Dr. Simone Moser, Institute of Organic Chemistry and Centre of Molecular Biosciences University of Innsbruck, 6020 Innsbruck (Austria)
Dr. Thomas Müller, Institute of Organic Chemistry and Centre of Molecular Biosciences University of Innsbruck, 6020 Innsbruck (Austria)
Dr. Christoph Kreutz, Institute of Organic Chemistry and Centre of Molecular Biosciences University of Innsbruck, 6020 Innsbruck (Austria)
Priv.-Doz. Dr. Andreas Holzinger, Institute of Botany, Department of Physiology and Cell Physiology of Alpine Plants University of Innsbruck, 6020 Innsbruck (Austria)
Prof. Cornelius Lütz, Institute of Botany, Department of Physiology and Cell Physiology of Alpine Plants University of Innsbruck, 6020 Innsbruck (Austria)
Prof. Bernhard Kräutler, Institute of Organic Chemistry and Centre of Molecular Biosciences University of Innsbruck, 6020 Innsbruck (Austria)