Journal of Experimental Botany, Vol. 51, No. 346, pp. 873-884,
May 2000
© 2000 Oxford University Press
Carotenoid and ultrastructure variations in plastids of Arum italicum Miller fruit during maturation and ripening
Department of Biology, Section of Botany, University of Ferrara, C.so Porta Mare, 2, I–44100 Ferrara, Italy
Received 19 July 1999; Accepted 24 January 2000
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionConclusionsReferences |
|---|
The changes in the pigment pattern and composition occurring in the Arum italicum berry during the various steps of maturation (ivory to deep-green stages) and ripening (yellow and red-orange stages) were studied and correlated to the ultrastructural modifications of plastids. Transmission electron microscopy showed that each stage was characterized by a specific plastidial type following the unusual sequence amyloplast
chloroplastchromoplast. Plastidial transitions were accompanied by profound modifications in the pigmental composition, in particular, in the pattern of carotenoids and their precursors. The HPLC analysis of the carotenoids showed that, besides the two usual all-trans metabolic pathways leading to lutein through 
-carotene and to auroxanthin through ß-carotene, an additional cis-isomeric biosynthetic pathway leading to cis-neoxanthin through cis-ß-carotene exists. During the pre-ripening stages, the three pathways were present even if with qualitative and quantitative variations. When complete ripening was reached, a block occurred at the cyclization level causing the accumulation of both all-trans (i.e. 
-carotene and neurosporene) and cis-isomer (i.e. lycopene and 
-carotene) carotene precursors. Because of the occurrence of unusual pigments and the presence of the three main plastidial types, the fruit of A. italicum may constitute a most instructive model for the study of carotenogenesis. Key words: Arum italicum Miller, carotenoid pattern, chromoplasts, fruit maturation and ripening, plastid ultrastructure.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
|---|
Fruit ontogenesis develops in two phases: a first period called ‘maturation’ which begins with early carpel modifications and ends when maximum organ expansion is reached, and a second period ‘ripening’, which is characterized by striking modifications in the structure and chemical composition of the organ (Goldschmidt, 1980
). In fresh fruits these changes often consist of the transformation of chloroplasts into chromoplasts with the concomitant loss of chlorophylls and the accumulation of carotenoids, tissue softening and alterations in the metabolism of organic acids and monosaccharides (Brady, 1987). These fruits, at least in their exterior layers, are similar to senescent leaves in that they begin their development as green, photosynthetic tissues (Rhodes, 1980). The similarity is maintained to some extent during maturation and ripening. The disappearance of chlorophylls and the accumulation of yellow-red pigments are typical of both leaves and fruits. The pigment changes associated with fruit development are generally more intense than those occurring during leaf senescence. The structural and biochemical differentiation of chromoplasts, for example, is usually more advanced and complete in ripe fruits than in senescent leaves of the same species (for literature on the argument, see Rhodes, 1980). Certainly, these changes are adequately programmed in order to ensure the successful dispersal of the mature seeds. The ultrastructural, biochemical, and genetical aspects of the process, known as chloroplast–chromoplast transformation, have been studied in great detail in Lycopersicon, Capsicum and Citrus fruits (for reviews see: Gross, 1987; Gillapsy et al., 1993). From these studies it emerges that carotenoids are usually massively synthesized during the ripening period, but that the complement of carotenoid pigments differs greatly among species so that the pathways of either biosynthesis, breakdown or interconversion of carotenoids cannot, at present, be developed into a single scheme which fits all plant species. Moreover, numerous aspects of carotenoid metabolism are still unclear. On the other hand, a knowledge of carotenoid metabolism should be useful for a better understanding of flowering and fruit biogenesis and of their environmental and developmental regulation. A series of studies was undertaken on the structural and pigmental variations occurring in plastids during fruit maturation and ripening in a plant species that has not been studied in this regard. This study focused on the fruit of Arum italicum Miller (Araceae), a geophyte which is common in the shady damp habitats of southern and western Europe. This species was chosen since it forms an infructescence constituted by numerous spherical berries which, during ontogenesis, change in colour from white to red-orange, passing through green and yellow stages. The authors believe that this singular behaviour may be useful for acquiring more information on carotenogenesis to complement the scanty data reported on the better known A. maculatum species, where the yellow stage is lacking (Valadon and Mummery, 1974). |
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
|---|
Plant material
Plants of Arum italicum Miller (Araceae), grown in their natural environment in the Po plain area around Ferrara (Italy) were used in this study. The fruits were harvested at the following five stages of development: (I) immature ivory fruits; (II) immature pale-green fruits; (III) mature dark-green fruits; (IV) partially ripe yellow fruits; (V) fully ripe red-orange fruits. The berries were collected over a period of 4 months from April to the beginning of August, 1998. Since in each berry the distal cap (with respect to the insertion on the infructescence axis) was the zone most advanced developmentally during the whole of ontogenesis, these five stages were established on the basis of the colour of the distal cap. Ultrastructural observations of the plastids and spectrophotometric and chromatographic determinations of pigment patterns were carried out on this region of pericarp. In each stage of development five fruits were used for each parameter.
Transmission electron microscopy
For electron microscopy, small pieces (2 mm3) of fruits were cut from the outer sub-epidermal region of the distal caps of the fruits at progressive stages of development and immediately fixed with 3% glutaraldehyde in 0.1 M Na-K-phosphate buffer (pH 7.2) for 2 h at 4 °C. The pieces were then incubated overnight in 1% OsO4 in the same buffer at 4 °C, dehydrated in a graded series of ethanol, and embedded in Araldite-Epon 812. Sections, obtained with a diamond knife, were stained with uranyl acetate and lead citrate and then observed with a Hitachi H800 electron microscope (Electron Microscopy Center, Ferrara University, Italy) (Pancaldi et al., 1998).
X-ray microanalysis
X-ray microanalysis for iron (K=6.4 KeV) was performed on the same samples prepared for TEM. Thin sections were observed by a scanning electron microscope Cambridge S 360 equipped with a Link Analytical W.D.S. spectrometer (Electron Microscopy Center, Ferrara University, Italy).
Pigment extraction
Sub-epidermal portions of fruit distal caps were ground with a tissue mixer (Sorvall Omni-Mixer) in cold absolute ethanol. After centrifugation at 3000 g for 5 min, the residues were re-extracted to remove the pigments completely. To verify the effectiveness of the ethanolic extraction, further extraction of the residues with cold n-hexane was made. UV-spectrophotometric analysis (250–400 nm) of this extract showed the effectiveness of ethanolic extraction of all pigments, including non-polar pigments. After mixing, the crude ethanolic fractions were purified through an octadecyl silica cartridge (Waters C-18 Sep-Pak) and eluted with a few ml of ethyl acetate. The solvents were removed on a rotatory evaporator (Buchi 461 rotavapor) at 30 °C and the residues dissolved in ethanol. The solutions were filtered through a 0.45 µm HVLP Millipore filter. The samples were stored at -30 °C under N2, until analysis. The ethanol used, contained butylated hydroxytoluene as an antioxidant (Gut et al., 1987). All operations were carried out on ice under a dim green safe light to prevent photodegradation, isomerization and structural changes of the pigments. To verify the introduction of artefacts due to the presence of organic acids, some unripe fruits were also extracted with ethanol-TRIS-buffer (Gerdol et al., 1994). No pigment isomerization was observed after this operation.
Spectrophotometric analysis
Absorption spectra of total fruit extract and of isolated compounds were recorded at room temperature, 280–720 nm range, with a Perkin-Elmer model 554 UV-Vis double beam spectrophotometer. For chlorophyll and carotenoid determinations, the ethanolic extracts were measured at 664 nm (chlorophyll a), 648 nm (chlorophyll b) and 470 nm (carotenoids) (Lichtenthaler, 1987).
HPLC analysis
The liquid chromatograph apparatus consisted of a Varian 5020 system equipped with a Rheodyne 7126 injector and Varian UV-100 visible-UV-variable wavelength detector set at 287 nm (phytoene), 348 nm (phytofluene) and 425 nm (according to Minguez-Mosquera et al., 1992). This wavelength represents a good compromise for the detection of carotenoids and chlorophylls together with the phaeoderivatives which can not be detected at the greater wavelengths (440–455 nm) usually employed (Minguez-Mosquera et al., 1992). Chromatograms were recorded on a Merk-Hitachi D-2000 chromato-integrator. Portions of the ethanolic extracts, from 10 µl to 100 µl (analytical and micropreparative modes), were chromatographed by using Hibar-Lichrosorb Rp 18 column (5 µm, 4x250 mm) with a precolumn Lichrocart-Lichrosorb Rp 18 (5 µm, 4x4 mm). A Gilson 201 fraction collector was employed for collecting the pigment peaks. Each analytical determination was replicated three times. The mobile phase composition, at a flow rate of 1 ml m-1, was: A=water; B=acetonitrile, methanol, and 2-propanol (80:15:5; by vol.). Gradient mode elution was: from 0 min to 17 min=92% B; from 17 min to 23 min=92–97% B; from 23 min to 35 min=97–100% B. The various carotenoids and porphyrins were identified by comparing retention times and spectral characteristics of separated peaks with reference standards (see below).
Preparation of carotenoid reference compounds
Individual carotenoids were isolated by rp-HPLC from crude ethanolic extracts obtained from sunlight laminae of Quercus robur and mature fruits of Lycopersicon esculentum and Zea mays (Britton, 1991; De Las Rivas et al., 1991). The identification and quantification of the separated pigments are based on both spectroscopic and chromatographic properties and chemical tests (Table 1) as reported in Pancaldi et al. (Pancaldi et al., 1998).
|
Saponification procedure
In order to remove chlorophyll and lipid contaminant materials which could interfere with the chromatographic separation of pure carotenoids, the ethanolic fruit extracts were concentrated under reduced pressure and the residue was dissolved in methanol and saponified with 60% aqueous KOH (Goodwin and Goad, 1970). Saponification was also useful to verify the presence of carotenol esters since these compounds are hydrolysed by this procedure. The alkaline mixture was left in the dark at 5 °C under N2 overnight and then extracted with ethyl ether. The ethered layer was washed, dehydrated with Na2SO4, reduced to dryness, and the pigments taken up in ethyl acetate and then used for HPLC analysis.
Epoxide test
The epoxide test was performed directly in a spectrophotometric cuvette by adding a small drop of concentrated HCl to an ethanolic solution of the carotenoid fraction (Davies, 1976). Six carotenoids containing epoxidic groups were detected. These compounds were identified as 5–6 monoepoxides (neoxanthin, cis-neoxanthin, luteoxanthin, anteraxanthin, and taraxanthin) and 5–6, 5'-6' diepoxide (cis-violaxanthin) on the basis of hypsochromic shifts of about 20 nm and 40 nm, respectively.
Iodine-catalysed photoisomerization test
In order to verify the cis or trans nature of carotenoids, n-hexane carotenoid solutions were illuminated in the presence of iodine (1–2% of pigment weight) and isomerizations were monitored periodically by spectrophotometric examination. Cis-carotenoids (cis-neoxanthin, cis-violaxanthin, cis-lycopene, prolycopene, cis-neurosporene, cis--carotene, and cis-ß-carotene) were identified with respect to the all-trans-related compounds on the basis of the shifts of the visible absorption maxima to lower wavelengths (hypsochromic effect) and of the appearance of a new peak in the UV region of the spectrum (cis-peak). Moreover, the cis and trans isomers of OH-phytoene and phytofluene having the same UV absorbance maxima were discriminated on the basis of the ratio of heights of the middle absorption band versus the longest wavelength band (Moss and Weedon, 1976; Davies, 1976).
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
|---|
Ultrastructural aspects of plastids
The sequential steps of fruit maturation (from the ivory to the deep green stage) and ripening (yellow and red-orange stages) were characterized by the presence of different plastidial types following the unusual sequence amyloplast
chloroplastchromoplast.
Stage I:
The pericarp in the ivory stage of maturation was characterized by the presence of colourless plastids having the typical feature of amyloplasts. The organelles were oval- or round-shaped and were sorrounded by a double membrane envelope enclosing a granular stroma occupied to a large extent by conspicuous starch grains, and by a few small plastoglobules. The internal membrane system was hardly developed. Only a few long, single perforated and usually concentrically ordered thylakoids were present (Fig. 1a).
|
Stage II:
In the immature pale-green fruit, transition forms of amyloplasts into chloroplasts were always observed. In the organelles the stroma region was still partially occupied by starch grains, but also by a quite abundant membrane system already differentiated in stromal and granal thylakoids (Fig. 1b
).
Stage III:
When the Arum fruit reached the mature green stage, most of plastids presented the normal chloroplast morphology observed previously in Arum italicum leaves (Pancaldi et al., 1998). The organelles were lens-shaped and had a well-developed thylakoid membrane system with many granal stacks. Small plastoglobules and some starch grains were normally scattered in the stroma (Fig. 1c). However, at this stage, 5–10% of the chloroplasts showed the first signs of transition into chromoplasts. Electron-dense lines associated with granal and intergranal membranes appeared. This aspect was observed in tomato and was interpreted as early carotene crystalloids (Harris and Spurr, 1969). Furthermore, individual granal compartments began swelling, particularly those at or near the ends of a granal stack. Sometimes grana lost their stacked organization and appeared to slide partially apart at the partitions. Plastoglobules increased in number and size (Fig. 1d).
Stage IV:
In the yellow stage, plastids developed more and more into mature chromoplasts and were characterized by the degradation of the photosynthetic machinery and the lost of starch deposits (Fig. 2).
|
Among the features of membrane degradation, tightly stuck thylakoids were frequently observed (Fig. 2a
). Similarly, degraded thylakoids have been described in some young chromoplasts of ripening pumpkin fruits (Ljubesic, 1977) and in leaf chloroplasts of plants treated with the herbicide aminotriazole (Wrischer and Ljubesic, 1989). These structures are attributed to granal thylakoids which adhere after losing their lumina and seem to be related to the block of carotene cyclization and the consequent accumulation of acyclic carotenes. In fact, HPLC analysis of the yellow fruit revealed the conspicuous presence of these carotene precursors.
Simultaneously with the degradation of the thylakoid system, special carotenoid-bearing substructures were developed. These consisted of globules and tubules that were scattered in the stroma. The globules lay singly or in groups (Fig. 2b). The tubules were found either by themselves or connected with the globules, and sometimes grew out of the globules, assuming the shape of tadpoles (Fig. 2c). A similar aspect was observed in ripe Cucurbita fruit (Ljubesic, 1977; Knoth et al., 1986). Structures resembling prolamellar bodies were frequently formed (Fig. 2b). These membrane plexes, usually lacking order, have been described by Spurr and Harris (1968) and Harris and Spurr (1969) in tomato, when fruits contained little or no chlorophylls and large amounts of carotenoids. In some plastids long single perforated thylakoids were observed which seem to develop by a ‘remodelling’ of degrading photosynthetic membranes (Ljubesic et al., 1991).
Other membrane systems corresponding to ‘chromoplast internal structures’ (CIMs) described by other authors (Sitte et al., 1980; Ljubesic et al., 1991) were observed in plastids. They consisted of many concentrically arranged layers in the stroma lying usually at the periphery of the organelle (Fig. 2a) or in the two or more peripheral linear membranes following the outline of the double external envelope (Fig. 2b). The presence of connections of these membraneous structures with the inner membrane confirm that these structures develop de novo by invagination of the inner membrane of the envelope and not directly from degraded photosynthetic membranes (Ljubesic et al., 1991).
The chemical content of CIMs was studied in certain flowers. In addition to lipid and special proteins (Hansmann and Sitte, 1984), they consist of carotenoids, but never chlorophylls. In CIMs carotenogenic enzymes have been found (Kreuz et al., 1982) and these membranes are the site of secondary carotenoid synthesis (Ljubesic et al., 1991).
The CIM abundance reflects the amount of transport activity of the plastid envelope (Wrischer et al., 1986). The inner membrane of the envelope in chromoplasts has a 2-fold function as in the other plastid types: first in the transport of different metabolites into and out of the organelle, and secondly as the site of synthesis of new membrane structures. At this stage of ripening, in some chromoplasts a conspicuous granular osmiophilic body was often present in the stroma (Fig. 2c). This body was identified as phytoferritin, a non-toxic complex of protein and iron that has been found in the stroma of some chromoplasts (Ljubesic, 1976) where it builds crystalloid aggregates. In fact, the X-ray spectrum analysis of these aggregates showed a peak at 6.4 KeV specific for the K line of iron (Fig. 2e). It is generally thought that phytoferritin derives from the cytochromes and the ferredoxin of the degraded thylakoids. Very large phytoferritin aggregates are characteristic of mature and senescent chromoplasts (gerontoplasts) (Ljubesic, 1976). Division profiles of chromoplasts could frequently be observed at this stage (Fig. 2c), as already reported in the early stages of ripening of other fruits (Sitte et al., 1980; Knoth et al., 1986; Ljubesic et al., 1991). This is in accordance with the recognized active metabolism of the chromoplasts in fruits, compared to the gerontoplasts appearing in senescent cells that always develop from fully green, i.e. old chloroplasts and are unable to multiply (Ljubesic et al., 1991).
Stage V:
In the completely ripe red-orange fruit, mature chromoplasts were present. In the organelles membraneous structures almost completely disappeared; however, some wavy membrane remnants could still be present beside one or two linear membranes lying under the external envelope. Chromoplasts were studed with numerous small osmiophylic globules rendering their fine structure extremely minute. Elliptical carotenoid crystalloids were also abundant in the stroma (Fig. 2d). At the red-orange stage of ripening no phytoferritin deposits, nor signs of plastid multiplication were observed, in accordance with the last stage of development of the chromoplast.
Spectrophotometric determinations
The spectral profiles of the ethanolic extracts obtained in the wavelength range from 380 nm to 720 nm (Fig. 3) reflected the different colour stages characterizing the progressive developmental phases of the fruit, and the different plastidial types.
|
In ivory fruit (stage I), chlorophylls were lacking and only traces of carotenoids were present (Fig. 4
). This was obviously due to the virtual absence of light in the young infructescence which was still enclosed in the enfolding spate. In the pale-green and deep-green fruits (stages II and III of maturation) a very great increase of chlorophylls and a modest accumulation of carotenoids were noted with spectral profiles characterized by the peaks of chlorophyll a (663 nm) and chlorophyll a plus carotenoids (435 nm) (Figs 3, 4). Spectrophotometric analysis showed that at stage IV (first stage of ripening) the yellow colour of the fruit was the result of a striking degradation of chlorophylls instead of a substantial encrease of carotenoids. Furthermore, the main component of carotenoids had a shift at 440 nm (Figs 3, 4).
|
...), car (—
—) and Chl (a+b)/car molar ratio (—
—) were determined by spectrophotometry; xanthophyll/carotene molar ratio (—
—) was monitored by rp-HPLC. The bars represent the arithmetic means of five replicates ±SD. Chl, chlorophyll; car, carotenoid; SD, standard deviation.Strong qualitative and quantitative changes in the absorption spectra of the extracts occurred at stage V (red-orange fruit) where a new peak at 466 nm and a shoulder at 493 nm appeared besides a peak at 440 nm. The presence of absorption maxima above 460 nm indicates the predominance of carotenoid precursors with 11 conjugated double bonds which are related to the change of the berries from yellow to red-orange. This stage was characterized by the complete disappearance of chlorophylls and by a 100-fold increase of total carotenoids with respect to stage I (ivory fruit) (Figs 3
, 4).
Qualitative and quantitative variations of the individual carotenoids
Elution profiles and percentage composition of pigments extracted from Arum fruits during ontogenesis are reported in Fig. 5 and Table 2, respectively. In the ivory fruit (stage I), xanthophylls were represented by lutein, auroxanthin, cis-neoxanthin, and neoxanthin, while carotenes, by ß-carotene and by cis-ß-carotene. Carotenoid precursors were absent. Quantitatively, more than 50% of the total carotenoids were composed of lutein and auroxanthin. This latter compound was not an artefact since during the first stages (II and III) of maturation it was found either by itself or together with cis-violaxanthin, and always in different quantities. On the other hand, auroxanthin was found in many flowers and in some fruits although it is not clear whether it is constitutive or an isomerization derivative of violaxanthin (Gross, 1987). It should be stressed that in the A. maculatum ivory fruit, auroxanthin is lacking while 20% of the total carotenoids is represented by the isomer violaxanthin. Unusual amounts of neoxanthin and cis-ß-carotene were evident. On the basis of the fruit classification of Goodwin and Goad (Goodwin and Goad, 1970) which is based on the pigment pattern type, the ivory fruit may be included in the I category because of: (1) the high trans/cis molar ratio of carotenoids; (2) the presence of similar concentrations of alcoholic and epoxidic xanthophylls; and (3) the absence of carotenoid precursors (Fig. 6).
|
|
|
—) Alcoholic/epoxidic xanthophylls; (...In pale-green fruits (stage II) and mainly in deep-green fruits (stage III), the pigment pattern was modified in accordance with the transition of amyloplasts into chloroplasts. This was inferred by the increase of both total chlorophyll/total carotenoid and xanthophyll/ß-carotene molar ratios (Fig. 4
). With the persistence of lutein and the appearance of antheraxanthin and cis-violaxanthin (Table 2), the pattern of carotenoids at these two stages of maturation was similar to that found in the antenna pigments of Arum italicum leaves (Pancaldi et al., 1998). However, some qualitative and quantitative differences were noted with respect to the leaf. Among the qualitative differences are the presence of cis-violaxanthin instead of its all-trans isomer and the persistence of neoxanthin, auroxanthin and cis-ß-carotene. Among the quantitative differences are: (1) lower trans/cis molar ratio (3.2 in fruit and 6.4 in leaf); (2) lower alcoholic/epoxidic xanthophyll molar ratio (1.6 in fruit and 3.7 in leaf); and (3) 50% of trans-ß-carotene as related to total carotenoids (Fig. 6; Table 2). The coexistence of both isomeric forms of ß-carotene was consistent with the different functional roles of the two molecular types, the all-trans form being mainly associated with the reaction centres of both photosystems and also with the internal LHC CP47 (CP-1) and CP43 (CP-2) (Green and Durnford, 1996), while the cis-form could be a ‘secondary’ carotenoid localized in the plastoglobules rather than in the thylakoids (Steinmüller and Tevini, 1985; Ben-Amotz et al., 1988). In these two stages carotenoid precursors were also lacking. This behaviour differentiates A. italicum from the A. maculatum fruit where the absence of an intermediate yellow stage of ripening is reflected in a different pigment pattern, with the accumulation of carotenoid precursors (-carotene, lycopene, -carotene, phytofluene beta;-zeacarotene, and -carotene) in the green stage (Valadon and Mummery, 1974).
Owing to the predominance of a pigment pattern resembling that in photosynthetic tissues, the green fruit may normally be included in the II category of fruits (Goodwin and Goad, 1970).
In yellow fruit (stage IV, first stage of ripening), the pigment pattern changed greatly according to the disorganization of the thylakoid system and the appearance of the above-mentioned special carotenoid-bearing substructures. The total chlorophyll/carotenoid molar ratio greatly decreased. The xanthophyll/carotene molar ratio diminished to the same extent, owing to the lowering of xanthophylls and the doubling of carotenes (Fig. 4). At this stage of ripening, lutein, cis-neoxanthin, cis-violaxanthin, and antheraxanthin which are the usual components of photosystems in photosynthetic tissues decreased, while zeaxanthin, its diepoxidic derivative luteoxanthin, and the monoepoxidic lutein derivative, taraxanthin, which are characteristic of chromoplasts (Britton, 1991; Toth and Szabolcs, 1981), appeared (Table 2). Therefore, the alcoholic/epoxidic xanthophyll molar ratio rose (Fig. 6). The carotene fraction was characterized by the presence of acyclic and monocyclic precursors. From this stage onwards, these carotenes will predominate over the other pigments. Among the acyclic precursors, mono-cis lycopene (neolycopene) and tetra-cis lycopene (prolycopene) were prevalent (Table 2). These pigments, which are rather unusual, have been found in fruits of Prunus armeniaca L., Cytrullus vulgaris L., Cytrus paradisi Macfald. and Lycopersicon esculentum L. cv. tangerine (Curl, 1960; Morgan, 1967; Poling et al., 1980; Frecknall and Pattenden, 1984). Analogous to other fruits, (Gross, 1987) cis-neurosporene, neurosporene, -carotene, and cis--carotene were also present among the visible light-absorbing compounds, while cis-phytofluene was only detected among UV-absorbing precursors. Among mono-cyclic precursors only -carotene was detected (Table 2).
The red-orange berry (stage V, second stage of ripening) was characterized by the complete disappearance of chlorophylls, by the rapid synthesis of carotenoids (100 fold with respect to the green stage), and by the predominance of carotene linear precursors in the cis configuration. In particular, lycopene and neurosporene isomers constituted about 55% of the total carotenoids (Fig. 4; Table 2). A low concentration of ß-carotene was found. Xanthophylls were mainly present in the alcoholic form and represented 10% of total carotenoids (Table 2). Moreover, cis-phytofluene increased and cis-hydroxyphytoene appeared.
Therefore, acyclic/cyclic and trans/cis carotenoid molar ratios reached their maximum and minimum values, respectively, during stage V (Fig. 6). The presence of cis-carotenoid precursors differentiates the red berry of A. italicum from that of A. maculatum where only trans-isomer precursors have been detected (Valadon and Mummery, 1974).
Owing to the presence of a pigment pattern in which various precursors of carotenoids were predominant, the yellow and red-orange fruits of Arum should be classified in the III and VII representative groups of fruits, respectively (Goodwin and Goad, 1970).
On the basis of the analytical data obtained, a scheme of the possible biosynthetic steps of carotenoids in A. italicum fruit was drawn (Fig. 7). Analogous to the other plant species, in A. italicum fruit cis-isomers are normal intermediates in the first metabolic steps of carotenoid synthesis leading to -carotene via mono-cis-phytoene and mono-cis-phytofluene (Davies, 1980; Norris et al., 1995). Then, the initial pathways flow into carotene from which two all-trans metabolic pathways leading to lutein through -carotene and to auroxanthin through ß-carotene occur (Britton and Powls, 1977; Taylor, 1996). Besides the two usual all-trans pathways, an additional cis-isomeric pathway leading to cis-neoxanthin through cis-ß-carotene seems to be active in A. italicum fruit, analogous to some Scenedesmus and tomato mutants (Powls and Britton, 1977; Clough and Pattenden, 1979). In the ivory, pale-green and deep-green fruits (stages I–III of maturation) the three metabolic pathways are fully functional with the all-trans ones predominating, as shown by the high xanthophyll/carotene and all-trans/cis carotenoid molar ratios. However, it cannot be excluded that the cis-pathway is impoverished, possibly because of the presence of cis-trans isomerase activity furnishing precursors to the all-trans-pathways.
|
In the yellow and red-orange ripe fruits (stages IV and V) the cis-isomeric pathway predominates over the all-trans pathways, but with the prevalence of the linear and monocyclic cis-isomers. It is likely that insufficient or lack of activity of cis-trans isomerases and cis-ß-cyclases (whose existence was shown by Britton, 1986
; Lütke-Brinkhaus and Kleining, 1987; and Shaish et al., 1990) does not permit the intermediates from cis-phytofluene to cis-lycopene to feed the anabolic all-trans pathways or to form cis--carotene and then isomeric xanthophylls. The conversion of the cis- into the all-trans configuration of carotenoids is a light-regulated step (photoisomerization). These photodependent structural changes should be due to triplet-to-triplet energy transfer from chlorophyll a to cis-carotenoids (Fong and Schiff, 1979; Ashikawa et al., 1986; Sandmann, 1991). Consequently, it is likely that in the red-orange fruit, the absence of sensitizing molecules (i.e. chlorophyll a sensitizer) prevents the activation of the photoisomerization reactions with the consequent accumulation of cis-isomers.
Distinct from the cis pathway, in the all-trans biosynthetic sequence the monocyclic -carotene accumulates instead of the linear lycopene. This suggests that a trans-ß-cyclase is expressed which is unable to form the second iononic ring with the consequent decrease of the levels of ß-carotene, lutein and their derivatives. The presence of considerable concentrations of -carotene was also found in the fruit in the last stage of ripening of A. maculatum L. (Valadon and Mummery, 1974), Diospyros kaki L. fil.cv. Triumph (Ebert and Gross, 1985), and Lycopersicon esculentum L. High-Bet Tomato genotype (Raymundo et al., 1976).
|
Conclusions |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
|---|
Chlorophylls and carotenoids are synthesized in a qualitatively and quantitatively co-ordinated manner in chloroplasts. Whenever this balance is strongly changed in favor of carotenoids, the plastid ultrastructure is also changed and, concomitantly, chlorophylls are degraded. The resulting chromoplasts are morphologically characterized by the absence of thylakoids and by the presence of newly formed structures in which the overproduced carotenoids are sequestered (for a recent review of chromoplast development, see Camara et al., 1995
). The carotenoid-bearing structures may be plastoglobules (lipid droplets in most carotenoid-bearing flower petals), crystals (e.g. in Lycopersicon esculentum fruit), fibrils/tubules (e.g. in Capsicum annuum fruit), or membranes (e.g. in Narcissus pseudonarcissus petals). These structures probably prevent products from overloading the chromoplast membranes, the site of carotenoid formation (Rabbani et al., 1998). In the Arum italicum fruit all these substructures appear during the two successive stages of ripening (yellow and red-orange fruit) and are related to different qualitative and quantitative carotenoid pigment compositions. In this plant species the individual stages of fruit maturation and ripening are easily recognizable by the different colours assumed by the berries during development. Each stage is characterized by a specific plastidial type following the unusual sequence amyloplast chloroplastchromoplast. Plastidial transitions are accompanied by profound modifications in the pigmental composition, in particular, in the pattern of carotenoids and their precursors. The HPLC analysis of the carotenoids showed that, besides the two usual all-trans metabolic pathways leading to lutein through -carotene and to auroxanthin through ß-carotene, an additional cis-isomeric biosynthetic pathway leading to cis-neoxanthin through cis-ß-carotene exists. During the pre-ripening stages, the three pathways were present even if with qualitative and quantitative variations. When the complete ripening was reached, a block occurred at the cyclization level causing the accumulation of both all-trans (i.e. -carotene and neurosporene) and cis-isomer (i.e. lycopene and -carotene) carotene precursors. Because of the occurrence of unusual pigments and the presence of the three main plastidial types, A. italicum fruit seems to constitute an ideal tissue in which to investigate carotenoid biosynthesis in relation to plastid differentiation during ontogenesis.
|
Acknowledgments |
|---|
This work was supported by grants from Consiglio Nazionale delle Ricerche (CNR) and Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST) of Italy.
|
Notes |
|---|
1 To whom correspondence should be addressed. Fax: +39 532 208561. E-mail:fsm{at}dns.unife.it
|
Abbreviations |
|---|
CIMs, chromoplast internal structures; rp-HPLC, reverse-phase high-performance liquid chromatography; TEM, transmission electron microscope; UV, ultraviolet light.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
|---|
Ashikawa I, Miyato A, Koike H, Inoue J, Kayama Y.1986. Light-induced structural changes of ß-carotene in thylakoid membranes. Biochemistry 25, 6154–6160.
Ben-Amotz A, Lers A, Avron M.1988. Stereoisomers of ß-carotene and phytoene in the alga Dunaliella bardawii. Plant Physiology 86, 1286–1291.
Brady CJ.1987. Fruit ripening. Annual Review of Plant Physiology 38, 155–178.[Web of Science]
Britton G.1986. Biosynthesis of chloroplast carotenoids. In: Akoyunoglou G, Benger H, Liss AR, eds. Regulation of chloroplast differentiation. New York: Academic Press, 125–134.
Britton G.1991. Carotenoids. In: Dey PE, Harborne JB, eds. Methods in plant biochemistry, Vol. 7. New York: Academic Press, 473–518.
Britton G, Powls R.1977. Phytoene, phytofluene and -carotene isomers from a Scenedesmus obliquus mutant. Phytochemistry 16, 1253–1255.[Web of Science]
Camara B, Hugueney P, Bouvier F, Kuntz M, Moneger R.1995. Biochemistry and molecular biology of chromoplast development. International Review of Cytology 163, 175–247.[Web of Science][Medline]
Clough JM, Pattenden G.1979. Naturally-occurring poly-cis-carotenoids. Stereochemistry of poly-cis-lycopene and its congenes in ‘tangerine’ tomato fruit. Journal of the Chemical Society Chemical Communications 616–619.
Curl AL.1960. The carotenoids of apricots. Food Research 25, 190–196.
Davies BH.1976. Carotenoids. In: Goodwin TW, ed. Chemistry and biochemistry of plant pigments, Vol. II. New York: Academic Press, 38–165.
Davies BH.1980. Analysis of carotenoid pigments. In: Czygan FC, ed. Chemistry and biochemistry of plant pigment. Stuttgard: Fischer-Verlag, 31–56.
De Las Rivas J, Milicua JCG, Gomez R.1991. Determination of carotenoid pigments in several tree leaves by reversed-phase high-performance liquid chromatography. Journal of Chromatography 585, 168–172.
Ebert G, Gross J.1985. Carotenoid changes in the peel of ripening persimmon (Diospyros kaki) cv. Triumph. Phytochemistry 24, 29–32.
Fong F, Schiff JA.1979. Blue-light induced absorbance changes associated with carotenoids in Euglena. Planta 146, 119–127.
Frecknall EA, Pattenden G.1984. Carotenoid differences in isogenic lines of tomato fruit colour mutants. Phytochemistry 23, 1707–1710.
Gerdol R, Bonora A, Poli F.1994. The vertical pattern of pigment concentrations in chloroplasts of Sphagnum capillifolium. Bryologist 97, 158–161.
Gillaspy G, Ben-David H, Gruissem W.1993. Fruits: a developmental perspective. The Plant Cell 5, 1439–1451.
Goldschmit EE.1980. Pigment changes associated with fruit maturation and their control. In: Thimann KV, ed. Senescence in plants. Boca Raton: CRC Press Inc. 207–217.
Goodwin TW, Goad L.1970. Carotenoids and triterpenoids. In: Hulme AC, ed. The biochemistry of fruits and their products, Vol. 1. London, New York: Academic Press, 305–368.
Green BR, Durnford DG.1996. The chlorophyll-carotenoid protein of oxygenic photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 47, 685–714.[Web of Science][Medline]
Gross J.1987. Carotenoids. In: Schweigert BS, ed. Pigments in fruits. London, New York: Academic Press, 87–186.
Gut H, Rutz C, Matile P, Thomas H.1987. Leaf senescence in a non-yellowing mutant Festuca pratensis: degradation of carotenoids. Physiologia Plantarum 70, 659–663.
Hansmann P, Sitte P.1984. Comparison of the polypeptide complement of different plastid types and mitochondria of Narcissus pseudonarcissus. Zeitschrift für Naturforschung 39c, 758–766.
Harris WM, Spurr AR1969. Chromoplasts of tomato fruits. II. The red tomato. American Journal of Botany 56, 380–389.
Knoth R, Hansmann P, Sitte P.1986. Chromoplasts of Palisota barteri, and the molecular structure of chromoplast tubules. Planta 168, 167–174.
Kreuz K, Beyer P, Kleinig H.1982. The site of carotenogenic enzymes in chromoplasts from Narcissus pseudonarcissus.L. Planta 154, 66–69.
Lichtenthaler HK.1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods in Enzimology 148, 350–382.
Ljubesic N.1976. Phytoferritin in plastids of blackberry leaves. Acta Botanica Croatica 35, 51–55.
Ljubesic N.1977. The formation of chromoplasts in fruits of Cucurbita maxima Duch. ‘turbaniformis’. The Botanical Gazette 138, 286–290.
Ljubesic N, Wrischer M, Zvonimir D.1991. Chromoplasts—the last stages in plastid development. International Journal of Developmental Biology 35, 251–258.[Medline]
Lütke-Brinkhaus F, Kleining H.1987. Carotenoid and chlorophyll biosynthesis in isolated plastids from mustard seedling cotyledons (Sinapis alba L.) during etioplast–chloroplast conversion. Planta 170, 121–129.
Minguez-Mosquera MI, Gandul-Rojas B, Gallardo-Guerrero ML.1992. Rapid method of quantification of chlorophylls and carotenoids in virgin olive oil by high-performance liquid chromatography. Journal of Agricultural and Food Chemistry 40, 60–63.
Morgan RC.1967. The carotenoids of Queensland fruits. Carotenes of the watermelon (Citrullus vulgaris). Journal of Food Sciences 32, 275–278.
Moss GP, Weedon BC.1976. Chemistry of carotenoids. In: Goodwin TW, ed. Chemistry and biochemistry of plant pigments, Vol. I. London, New York: Academic Press, 149–224.
Norris SR, Barrette TR, Della Penna D.1995. Genetic dissection of carotenoid synthesis in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation. The Plant Cell 7, 2139–2149.[Abstract]
Pancaldi S, Bonora A, Gualandri R, Gerdol R, Manservigi R, Fasulo MP.1998. Intra-tissue characteristics of chloroplasts in the lamina and petiole of mature winter leaf of Arum italicum Miller. Botanica Acta 111, 261–272.
Poling SM, Hsu WJ, Yokoyama H.1980. Chemical induction of poly-cis-carotenoid biosynthesis. Phytochemistry 19, 1677–1680.
Powls R, Britton G.1977. The role of isomers of phytoene, phytofluene and -carotene by a mutant strain of Scenedesmus obliquus. Archives of Microbiology 115, 175–179.[Web of Science][Medline]
Rabbani S, Beyer P, Lintig JV, Hugueney P, Kleinig H.1998. Induced ß-carotene synthesis driven by triacylglycerol deposition in the unicellular alga Dunaliella bardawii. Plant Physiology 116, 1239–1248.
Raymundo LC, Chichester CO, Simpson KL.1976. Light-dependent carotenoid synthesis in the tomato fruit. Journal of Agricultural and Food Chemistry 24, 59–64.[Web of Science][Medline]
Rhodes MJC.1980. The maturation and ripening of fruits. In: Thimann KV, ed. Senescence in plants. Boca Raton: CRC Press Inc, 157–205.
Sandmann G.1991. Light-dependent switch from formation of poly-cis-carotenes to all-trans carotenoids in the Scenedesmus mutant C-6D. Archives of Microbiology 155, 229–233.
Shaish A, Avro M, Ben-Amotz A.1990. Effect of inhibitors on the formation of stereoisomers in the biosynthesis of ß-carotene in Dunaliella bardawii. Plant and Cell Physiology 31, 689–696.
Sitte P, Falk H, Liedvogel B.1980. Chromoplasts. In: Czygan FCG, ed. Pigments in plants. Stuttgard and New York: Fischer-Verlag, 117–148.
Spurr AR, Harris WM.1968. Ultrastructure of chloroplasts and chromoplasts in Capsicum annuum. I. Thylakoid membrane changes during fruit ripening. American Journal of Botany 55, 1210–1224.
Steinmüller D, Tevini M.1985. Composition and function of plastoglobuli. I. Isolation and purification from chloroplasts and chromoplasts. Planta 163, 201–207.[Web of Science]
Taylor CB.1996. Control of cyclic carotenoid biosynthesis: no lutein, no problem! The Plant Cell 8, 1447–1450.[Web of Science]
Toth G, Szabolcs J.1981. Occurrence of some mono-cis-isomers of asymmetric C40-carotenoids. Phytochemistry 20, 2411–2415.
Valadon LRG, Mummery RS.1974. Carotenoids of the floral parts of the spadix of Arum maculatum. Zeitschrift für Pflanzenphysiologie 75, 88–94.
Wrischer M, Ljubesic N, Marcenko E, Kunst LJ, Hlousek-Radojcic A.1986. Fine structural studies of plastids during their differentiation and dedifferentiation. Acta Botanica Croatica 45, 43–54.
Wrischer M, Ljubesic N.1989. Some structural aspects of degradational processes in photosynthetic membranes. 3rd Balkan Congress of Electron Microscopy, 171.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. A. Maresca, J. E. Graham, M. Wu, J. A. Eisen, and D. A. Bryant Identification of a fourth family of lycopene cyclases in photosynthetic bacteria PNAS, July 10, 2007; 104(28): 11784 - 11789. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Conti, S. Pancaldi, M. Fambrini, V. Michelotti, A. Bonora, M. Salvini, and C. Pugliesi A Deficiency at the Gene Coding for {zeta}-Carotene Desaturase Characterizes the Sunflower non dormant-1 Mutant Plant Cell Physiol., April 15, 2004; 45(4): 445 - 455. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.V. Milborrow The pathway of biosynthesis of abscisic acid in vascular plants: a review of the present state of knowledge of ABA biosynthesis J. Exp. Bot., June 1, 2001; 52(359): 1145 - 1164. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||