Journal of Experimental Botany, Vol. 54, No. 383, pp. 727-738,
February 1, 2003
© 2003 Oxford University Press
Characterization of Pinalate, a novel Citrus sinensis mutant with a fruit-specific alteration that results in yellow pigmentation and decreased ABA content
Received 19 July 2002; Accepted 21 October 2002
Departamento de Ciencia de Alimentos, Instituto de Agroquímica y Tecnología de Alimentos (IATA)-CSIC, Apartado de Correos 73, Burjassot, 46100 Valencia, Spain
1 To whom correspondence should be addressed. Fax: +34 96 363 63 01. E-mail: lzacarias{at}iata.csic.es
Abbreviations: ABA, abscisic acid; HPLC, high-performance liquid chromatography.
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Abstract |
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The characterization of a novel mutant, named Pinalate, derived from the orange (Citrus sinensis L. Osbeck) Navelate, which produces distinctive yellow fruits instead of the typical bright orange colouration, is reported. The carotenoid content and composition, and ABA content in leaf and flavedo tissue (coloured part of the skin) of fruits at different developmental and maturation stages were analysed. No important differences in leaf carotenoid pattern of both phenotypes were found. However, an unusual accumulation of linear carotenes (phytoene, phytofluene and
- carotene) was detected in the flavedo of Pinalate. As fruit maturation progressed, the flavedo of mutant fruit accumulated high amounts of these carotenes and the proportion of cyclic and oxygenated carotenoids was substantially lower than in the parental line. Full-coloured fruit of Pinalate contained about 44% phytoene, 21% phytofluene, 25% -carotene, and 10% of xanthophylls, whereas, in Navelate, 98% of total carotenoids were xanthophylls and apocarotenoids. The ABA content in the flavedo of Pinalate mature fruit was 3–6 times lower than in the corresponding tissue of Navelate, while no differences were found in leaves. Other maturation processes were not affected in Pinalate fruit. Taken together, the results indicate that Pinalate is a fruit-specific alteration defective in -carotene desaturase or in - carotene desaturase-associated factors. Possible mechanisms responsible for the Pinalate phenotype are discussed. Because of the abnormal fruit-specific carotenoid complement and ABA deficiency, Pinalate may constitute an excellent system for the study of carotenogenesis in Citrus and the involvement of ABA in fruit maturation and stress responses.
Key words: ABA, carotenoid, Citrus sinensis L. Osbeck, colour, fruit maturation, xanthophylls, -carotene desaturase.
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Introduction |
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Carotenoids are terpenoids synthesized in plastids as hydrocarbons (carotenes) and their oxygenated derivatives (xanthophylls) (Bramley, 1997), and serve essential roles in plants as components of the photosynthetic apparatus and protectors against oxidation derived from excess light energy (Demmig-Adams and Adams, 1996). They also provide the yellow, orange or red colouration characteristic of many flowers and fruits. Their importance is also recognized as nutritional components, vitamin A precursors, in the prevention of human diseases such as cancer, and from an industrial perspective (Bramley et al., 1993; Mayne, 1996; Olsen, 1989; Hirschberg, 1999; Sandmann, 2001).
The biochemistry of carotenoid biosynthesis has been well established. Genes and cDNAs encoding some of the carotenogenic enzymes have been isolated and characterized in bacteria, algae, fungi and, more recently, in higher plants (reviewed in Cunningham and Gantt, 1998; Hirschberg, 2001; Sandmann, 2001). The first committed step in the carotenoid pathway (Fig. 1) is the synthesis of phytoene, catalysed by the enzyme phytoene synthase (PSY). Subsequently, the colourless phytoene undergoes four consecutive symmetrical desaturation steps. In plants and cyanobacteria these four desaturations are catalysed by two related enzymes, postulated to act co-ordinately (Cunningham and Gantt, 1998), phytoene desaturase (PDS) and -carotene desaturase (ZDS), yielding the red carotene lycopene through the intermediate -carotene (pale-yellow). These desaturase reactions require plastoquinone (Norris et al., 1995) and a plastid terminal oxidase as electron acceptors (Carol and Kuntz, 2001). In the next step of the pathway, cyclation of lycopene yields ß-carotene and/or 
-carotene, and subsequent substitutions by hydroxyl, oxo, and/or epoxy groups produce xanthophylls with bright orange/yellow colours.
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Carotenoid biosynthesis in plants is connected with that of the plant growth regulator abscisic acid (ABA), which is produced through C15 intermediates after oxidative cleavage of specific xanthophylls (Marin et al., 1996; Schwartz et al., 1997; Milborrow, 2001). Indeed, many ABA-deficient mutants identified are related to carotenoid synthesis (Taylor et al., 2000).
Carotenoid complements of fruits and flowers vary considerably among species (Bramley, 1997), thus conferring their characteristic colour. The peel and pulp of citrus fruits are among the richest source of carotenoids, with hundreds of micrograms per gram of tissue and more than 100 different carotenoids identified (Bramley et al., 1993; Stewart and Wheaton, 1973). Carotenoid content and composition may vary greatly among citrus species, and also depend on the growing conditions (Gross, 1987). In coloured citrus fruits, such as oranges and mandarins, epoxy and hydroxylated carotenoids are the major components and account for up to 80% of total carotenoids. By contrast, the content of linear carotenes in full mature fruits is relatively low (less than 20% of total carotenoids). Besides the common carotenoids, citrus fruits accumulate genus-specific C30-apocarotenoids, formed by cleavage of C40 precursors (Farin et al., 1983); ß-citraurin, ß-citraurinene and ß-apo-8'-carotenal being the most abundant in the peel and responsible for the orange-reddish colouration of some citrus fruits (Gross, 1987). Despite the importance of citrus fruits as a carotenogenic source, little is known about carotenoid biosynthetic regulation during their maturation. Recently, cDNAs encoding PSY, PDS and ß-carotene hydroxylase from Satsuma mandarin have been isolated. The accumulation of PSY mRNA increased in the peel and juice sacs with the onset of coloration (Ikoma et al., 2001; Kim et al., 2001a), whereas the levels of PDS and ß-carotene hydroxylase mRNAs remained constant once fruit is fully developed (Kita et al., 2001; Kim et al., 2001b).
The characterization of mutants altered in the carotenoid biosynthetic pathway is a useful experimental system to identify molecular mechanisms regulating the process. This approach, however, is limited to a small number of plant species, mainly Arabidopsis and tomato (DellaPenna, 1999; Hirschberg, 2001). There is great interest in the study of carotenoid biosynthesis in citrus fruits in order to gain knowledge on the regulation of this process in citrus and also as a first step to address the improvement of their nutritional and commercial quality. In this work an interesting novel mutant of the ‘Navelate’ orange, named Pinalate, which produces fruits of a distinctive yellow colour, is characterized. This remarkable phenotype suggests an alteration of the mechanism(s) regulating the accumulation of carotenoids and may provide new insights of how these processes are co-ordinated in citrus. It is shown that Pinalate is partially blocked at -carotene desaturation. This alteration produces an accumulation of early linear carotenes, a reduction in xanthophylls and thus, a deficiency in ABA content in the peel of the fruit, whereas leaf tissue is unaffected.
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Plant material
Fruits of Navelate and Pinalate oranges (C. sinensis [L.] Osbeck) at different developmental stages were harvested from trees grafted on Citrange carrizo rootstocks. Experiments were conducted with adult trees grown in two locations: San Pedro del Pinatar (Murcia, Spain), where Pinalate was originally identified, and The Citrus Germplasm Bank at Instituto Valenciano de Investigaciones Agrarias (Moncada, Valencia, Spain). In both locations, the trees of each variety were of the same age, grown in the same orchard and subjected to standard cultural practices.
Fruit colour was measured using a Minolta CR-330 on three locations around the equatorial plane of the fruit. Hunter parameters a (negative to positive correspond from green to red, respectively), b (negative to positive, from blue to yellow, respectively) and L (0 to 100, black to white) were used and colour was expressed as the a/b Hunter ratio, a classical relationship for colour measurement in citrus fruits (Stewart and Whitaker, 1972). The a/b ratio is negative for green fruits, the zero value correspond to yellow fruits at the midpoint of the colour break period and is positive for orange fruit.
Leaves and flavedo tissue (the outer coloured part of the fruit peel) were frozen in liquid nitrogen, ground to a fine powder and stored at –70 °C until analysis.
Carotenoid extraction and quantification
For carotenoid analysis three developmental/maturation fruit stages were selected: (1) immature green fruit harvested in July–August with an average diameter of 4.52±0.25 cm and 3.95±0.22 cm and a/b ratios of –0.79±0.01 and –0.77±0.01 for Navelate and Pinalate, respectively, (2) mature green fruit harvested in October–November with an average diameter of 5.38±0.35 cm and 5.21±0.42 cm and a/b ratios of –0.47±0.02 and –0.56±0.02 for Navelate and Pinalate, respectively, and (3) full-coloured fruit harvested in February–March, with an average diameter 5.50±0.25 cm and 5.10±0.32 cm and a/b ratios of 0.64±0.02 and 0.10±0.01 for Navelate and Pinalate, respectively.
Freeze-ground material (500 mg) of leaves or flavedo was weighed in screw-capped Pyrex tubes (15 ml) and 2 ml of MeOH were added. The suspension was stirred for 5 min at 4 °C. Tris-HCl (50 mM, pH 7.5) (containing 1 M NaCl) was then added (1.5 ml) and further stirred for 5 min at 4 °C. Chloroform (4 ml) was added to the mixture, stirred for 5 min at 4 °C and centrifuged at 3000 g for 5 min at 4 °C. The hypophase was removed with a Pasteur pipette and the aqueous phase re-extracted with chloroform until it was colourless. The pooled chloroform extracts were dried on a rotary evaporator at 40 °C. For saponification the dried residue was completely dissolved in 1.8 ml of MeOH and 200 µl of 60% (w/v) KOH. Prior to capping, the flask was gently blanketed with nitrogen, closed, and placed in the dark overnight at room temperature. Saponified carotenoids were recovered from the upper phase after adding 2 ml of MilliQ water and 6 ml of solution A (petroleum ether:diethyl ether, 9:1, v:v) to the mixture. Repeated re-extractions by adding 3 ml of solution A were carried out until the hypophase was colourless. The volume recovered was transferred to a volumetric flask and adjusted to 10 ml with solution A. An aliquot of solution A extract was used for the quantification of total carotenoid content.
The extracts were reduced to dryness by rotary evaporation at 40 °C and quantitatively transferred to a Pyrex tube (15 ml) with acetone. In order to precipitate the sterols present in the samples, the acetone extracts were kept overnight at –20 °C and centrifuged at 3000 g for 15 min at 4 °C. The supernatant was transferred to a 1.5 ml vial, dried under N2 and kept at –20 °C until HPLC analysis. All operations were carried out on ice under dim light to prevent photo degradation, isomerizations and structural changes of carotenoids. Each sample was extracted at least twice.
Absorption spectra (300–550 nm) of saponified extracts (see above) of leaves and flavedo from Navelate and Pinalate were recorded at room temperature with a Diode array spectrophotometer (model 8452A Hewlett Packard, Germany). The maximum absorbance peaks were registered and total carotenoid content was calculated by measuring the absorbance at 450 nm according to Davies (1976), using an extinction coefficient of ß-carotene, E1%=2500.
HPLC analysis of carotenoids
Samples were prepared for HPLC by dissolving the dried residues in MeOH: acetone (2:1, v:v). Volume of the sample was adjusted to 75 µl for flavedo extracts and to 225 µl for leaf extracts. Chromatography was carried out with a Waters liquid chromatography system equipped with a 600E pump and a model 996 photodiode array detector, and Millenium Chromatography Manager (version 2.0). A C30 carotenoid column (250x4.6 mm, 5 µm) coupled to a C30 guard column (20x4.0 mm, 5 µm) (YMC Europe GMBH, Germany) were used with MeOH, water and methyl tert-butyl ether (MTBE). Carotenoid pigments were analysed by HPLC using a ternary gradient elution reported in a previous work (Rouseff et al., 1996). Briefly, the initial solvent composition consisted of 90% MeOH, 5% water and 5% MTBE. The solvent composition changed in a linear fashion to 95% MeOH and 5% MTBE at 12 min. During the next 8 min the solvent composition was changed to 86% MeOH and 14% MTBE. After reaching this concentration the solvent was gradually changed to 75% MeOH and 25% MTBE at 30 min. The final composition was reached at 50 min and consisted of 50% MeOH and 50% MTBE. The initial conditions were re-established in 2 min and re-equilibrated for 15 min before the next injection. The flow rate was 1 ml min–1, column temperature was set to 25 °C and the injection volume was 25 µl. Each analytical determination was replicated at least twice. The photodiode array detector was set to scan from 250 to 540 nm.
The ß-carotene, -carotene and lycopene standards were obtained from Sigma-Aldrich (Spain). The standards ß-cryptoxanthin, lutein and zeaxanthin were obtained from Extrasynthese (France).
ABA analysis
The quantification of ABA in flavedo and leaf tissue was performed by indirect enzyme-linked immunosorbent assay as reported previously (Zacarías et al., 1995; Lafuente et al., 1997).
Maturity index
Juice from fruits at different developmental/maturation stages (immature green, mature green and full-coloured) was extracted with a household electric hand reamer, filtered through a metal sieve with a pore size of 0.8 mm and analysed immediately. The acidity of the juice was determined by titration with 0.1 N NaOH and is expressed as mg citric acid per 100 ml and the soluble solid content (as °Brix) by refractrometry, using an Atago model PR32. The maturity index is expressed as the ratio of °Brix/acidity.
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Identification of Pinalate: a novel citrus with altered fruit colour
A citrus mutant was originally found in an orchard in ‘San Pedro del Pinatar’ (Murcia, Spain) in the mid-eighties and named Pinalate. Pinalate occurred spontaneously from the commercial variety of orange Navelate, and the novel tissue was identified as tree branches originated from previously grafted buds. The more remarkable phenotypic effect of Pinalate fruits is the yellow colouration of the flavedo (outer coloured tissue of the fruit peel) that is easily distinguishable from the orange colour in the parental (wild-type) variety (Fig. 2A). The internal juice vesicles of Pinalate are likewise yellow (not shown).
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Grafting is the common propagation procedure in the seedless Navel oranges. The Pinalate phenotype has been propagated by grafting onto different rootstocks and remained stable under field conditions and in a germplasm collection (IVIA, Moncada, Spain); and to the authors’ knowledge, it never reverted to the parental orange-coloured phenotype. Occasionally, individual fruits of the outer part of the canopy showed narrow sectors of pale-orange coloration in the peel (not shown). Appearance and agronomical behaviour of Pinalate trees were normal and indistinguishable from Navelate. During four consecutive seasons, fruit colour was periodically measured (from June to March) to determine changes through maturation and ripening. Quantitative differences were observed between seasons, probably due to environmental changes, although the patterns of change and the differences between Navelate and Pinalate were consistently maintained (Fig. 2B).
The onset of fruit colouration started at the same time (September–October) in Pinalate and Navelate, and the time span to complete full colouration (in January–February) is very similar in both varieties (Fig. 2B). However, the time required to reach complete chlorophyll disappearance (Hunter a/b ratios equal to 0) was longer in Pinalate. In addition, Pinalate fruits reach a plateau at lower a/b ratios when full matured, due to their distinct yellow colour. Other parameters assayed that were indicative of the ripening process, such as shape, size, weight, and flavour were not altered in Pinalate (not shown). Fruit acidity and soluble solid content were also similar in both phenotypes and, as a result, the internal maturity index was not significantly different (Fig. 3).
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Spectrum profiles of total carotenoid from flavedo of Navelate and Pinalate fruits
Spectra of total carotenoids extracts from the flavedo of Navelate and Pinalate were substantially different (Fig. 4). Typical spectra of flavedo extracts from Navelate showed two maximum peaks at 436 nm and 464 nm, while in Pinalate extracts new maxima appeared at approximately 400 nm and 425 nm. Moreover, Pinalate spectra showed higher absorbance values at lower wavelengths, which might be indicative of the presence of colourless carotenes.
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The total carotenoid content of flavedo extracts from Navelate and Pinalate fruits at different developmental stages was calculated by measuring the absorbance at 450 nm, according to Davies (1976), and expressed as µg of ß-carotene g–1 of fresh weight of flavedo. The flavedo from fruits of Navelate and Pinalate showed a similar content of total carotenoids at immature green (48.3±2.1 and 42.3±1.1 for Navelate and Pinalate, respectively) and mature green stage (15.9±2.8 and 18.3±3.3 for Navelate and Pinalate, respectively). Full-coloured flavedo from Navelate fruit contained 56.6±4.9 µg of ß-carotene g–1 fresh weight, while the estimated total carotenoid content for Pinalate flavedo at this stage was significantly lower (24.3±0.4) than in Navelate. However, this value is likely to be underestimated, since the content of total carotenoids is based in the absorbance of the extract at 450 nm and Pinalate showed a clear shift of the spectrum to lower wavelengths (Fig. 4), but it may be well indicative of the differences in carotenoid content between both phenotypes.
Qualitative and quantitative variations of carotenoids in the leaf and fruit from Navelate and Pinalate
HPLC carotenoid profiles from leaves and flavedo from fruits at three developmental stages (immature green, mature green and full-coloured) from both Navelate and Pinalate were analysed. Twenty-three carotenoid pigments were resolved and their spectral characteristics are shown in Table 1. Lutein (peak no. 13), zeaxanthin (peak no. 15), ß-cryptoxanthin (peak no. 19), -carotene (peak no. 21), and ß-carotene (peak no. 22) were identified by comparison of the spectra and retention time with those of authentic standards. Sixteen more peaks were tentatively identified by matching the observed versus literature spectral data and retention times under identical chromatographic conditions (Lee, 2001; Lee et al., 2001; Rouseff et al., 1996). A numerical notation (%III/II), which describes the ratio of the peak height of the longest wavelength absorption band (band III) to that of the middle absorption band (band II) as a percentage, was also used for identification. In general, values for 
max spectra and %III/II obtained from this study agree well with the values reported in the literature. Nine remaining peaks, whose spectroscopic characteristics are also described in Table 1, were not assigned to a defined carotenoid. A more exhaustive analysis would be required to identify these peaks.
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The profiles and composition of the carotenoids found in leaves and flavedo at different developmental stages from Navelate and Pinalate were compared. Carotenoid analysis of leaf samples did not reveal qualitative or quantitative significant differences between Navelate and Pinalate (Fig. 5; Table 2). Xanthophylls were represented by lutein and neoxanthin, while carotenes by
- and ß-carotene. Smalls amounts of isolutein (lutein-5,6-epoxide) and one unidentified carotenoid (peak no. 23) were also found. Quantitatively, around 50% of the total carotenoid content was lutein.
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When carotenoid HPLC profiles from Navelate fruits at different developmental stages were compared, significant quantitative and qualitative differences were observed (Fig. 5; Table 2). In immature green Navelate fruit the pattern and carotenoid composition was similar to that found in leaves, being lutein and neoxanthins the main carotenoids. The pigment pattern was modified at the beginning of chlorophyll degradation and transition from chloroplast into chromoplast. The noticeable decrease in total carotenoid content observed in mature green fruits was mainly due to a large reduction in lutein and neoxanthins. It is important to note the presence of violaxanthin (peak no. 12) in mature green fruit, which was not detectable in earlier stages of development. Analysis of the HPLC profile of flavedo from full-coloured Navelate fruit showed a remarkable increment in the percentage of violaxanthin (more than 50% of total carotenoid content, Table 2) as compared to the mature green stage. At this stage, other characteristic pigments from Citrus chromoplast were also found, as ß-cryptoxanthin (peak no. 19) and some apocarotenoid-like compounds (peaks no. 7, 9, and 14), representing all together around 12% of the total carotenoid content.
The HPLC profile of Pinalate extracts from flavedo of immature fruit pointed to the accumulation of the linear carotenes phytoene, phytofluene and -carotene (peaks no. 11, 18 and 20, respectively), which were not detected, or only in trace amounts, in Navelate (Fig. 5; Table 2). Therefore, the relative proportions of the main carotenoids lutein and neoxanthins (peaks no. 13 and 8, respectively) were lower in Pinalate than in the corresponding tissue of Navelate. At the mature green stage, the Pinalate carotenoid profile was substantially different from Navelate because of the presence of phytoene, phytofluene and -carotene. The proportion of these linear carotenes accounted for up to 50% of total carotenoids, whereas in Navelate they were not detected. By contrast, the percentage of neoxanthin, violaxanthin and lutein was significantly reduced in Pinalate. In full-coloured Pinalate flavedo, massive amounts of phytoene were detected, as compared with Navelate. A 4-times dilution of this sample was necessary to obtain a linear response of the phytoene peak area (peak no. 11') versus absorbance. Phytofluene and -carotene were also accumulated to a great extent (Fig. 5; Table 2). Several isomers of phytoene, phytofluene and -carotene were identified in HPLC profiles of flavedo extracts from Pinalate (see Table 1 for spectral properties). Concomitantly with the accumulation of linear carotenes in mature flavedo of Pinalate fruit, the amount of downstream products in the carotenoid pathway, such as the xanthophylls violaxanthin or neoxanthins, dramatically decreased compared to Navelate. It is interesting to note that apocarotenoid-like compounds or ß-cryptoxanthin were not identified in Pinalate at any stage of development. Consistently, peaks in the more polar diol-polyol region (early elution times) (peaks no. 2, 3 and 4) were detected in Pinalate chromatograms, which were not present in Navelate.
In the context of this study, it is important to stress that neurosporene, lycopene and their all-cis-isomers (proneurosporene and prolycopene) were not detected in either Navelate or, most importantly, in Pinalate, as already known for orange fruit (Gross, 1987).
ABA content in leaves and flavedo of fruits from Navelate and Pinalate
Carotenoids are also precursors of the hormone ABA (Fig. 1). To investigate whether the alteration in carotenoid composition found in Pinalate flavedo had also impaired ABA accumulation, the content of this hormone in the flavedo at different ripening stages was analysed through two consecutive seasons (Table 3). In Navelate, ABA content increased with the onset of colouration and remained at high levels at later stages. In Pinalate, however, ABA content was lower and the flavedo of full-coloured fruits contained between 3 and 6-times lower ABA than the parental fruit (Table 3). It is noticeable that leaf ABA amounts were similar in both phenotypes, suggesting, again, that the pathway is not affected in Pinalate vegetative tissue.
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Mutants with altered carotenoid synthesis and accumulation are useful for the study of the regulation of these processes (Bramley et al., 1993). Most of the information on the regulation of carotenogenesis in plants arises from studies on Arabidopsis and tomato plants, because of the availability of collection mutants and the convenience of these plants for genetic analysis (DellaPenna, 1999; Hirschberg, 2001). Citrus are plants prone to develop spontaneous mutations in the field and many of the cultivars currently available in the market have been obtained by the agronomical and nutritional selection of naturally occurring mutants. Navel oranges (Citrus sinensis) are among the citrus species more sensitive to develop spontaneous bud mutations (Saunt, 2000). However, and despite the importance of carotenoids in the commercial value of citrus fruits, only a few biochemical studies on mutants with altered fruit colouration have been reported. The spontaneous Navel mutant Cara Cara has recently been characterized and accumulates high amounts of ß-carotene, lycopene and colourless carotenes (phytoene and phytofluene) in the pulp, resulting in red pigmentation (Lee, 2001). The Sarah variety, a Shamouti orange with pink colour, has been also described and contains lycopene but not ß-carotene (Monselise and Halevy, 1961).
In the present work the authors have investigated the biochemical basis of a novel fruit colour mutant of Navel orange, named Pinalate. The most striking feature of Pinalate is the yellow colour of the peel of mature fruits. Data presented demonstrate that this phenotype results from an apparent partial blockage at the -carotene desaturation step of the carotenogenic pathway, the substrate -carotene and upstream compounds, phytoene and phytofluene, accumulate, whereas downstream products, such as xanthophylls and ABA, are decreased (Fig. 5; Tables 2, 3).
Although the colour of Pinalate fruits is similar to that of the so-called ‘colourless’ Citrus species, such as lemon (C. limon) and white grapefruit (C. paradisi), its pigment complement and biochemical properties are distinctive. Lemon accumulates phytofluene and -carotene, about 18% and 17% of total carotenoids, respectively, but not phytoene (Yokoyama and Vandercook, 1967; Gross, 1987). The peel of white grapefruit contains high relative levels of the earlier carotenes phytoene and phytofluene (accounting for up to 60–70% of total carotenoids), but the proportion of -carotene hardly reaches 1% (Romojaro et al., 1979), in contrast to Pinalate composition. Moreover, in these yellow species, ABA in the peel increases during maturation and ripening reaching, in some examples, concentrations even higher than that of oranges (Aung et al., 1991). In Pinalate, by contrast, the ABA content remains low throughout fruit maturation (Table 3). These observations suggest differences in the regulation of carotenoid composition between Pinalate and lemons and white grapefruits, despite a similarity in fruit colour, and indirectly reinforce the partial blockage of ZDS in Pinalate.
Mutations affecting carotenoid biosynthesis in green tissues may originate pleiotropic phenotypes. In contrast, mutations affecting later stages of chromoplast development of fruits may provide a more unambiguous phenotype (Bartley et al., 1994). This analyses of Pinalate did not reveal any other perturbation in the fruit maturation process (Fig. 3), besides those related with its altered pigmentation and ABA deficiency in fruits (Figs 4, 5, Tables 2, 3), thus suggesting that the mutation in Pinalate has not affected the general developmental regulation of maturation. Thus, it can be speculated that reduced ABA content in the flavedo may not be detrimental for fruit development and maturation or alternatively that the low level of ABA in Pinalate fruits is still above the threshold required for normal development. In fruit of different citrus species, the onset of fruit degreening has been found to be associated with an important increase in the ABA content (Table 3; Aung et al., 1991; Lafuente et al., 1997; Richardson and Cowan, 1995). Since fruits of the ABA-deficient mutant exhibit a delay in the rate of degreening (Fig. 2B) it is suggested that ABA may play a role in the regulation of the rate of fruit colouration in citrus fruits.
To the best of these analyses, the Pinalate phenotype is fruit-specific, a property exemplified by the carotenoid composition and ABA content, which were substantially distinct in the peel of Pinalate fruits, although unaffected in leaves (Fig. 5; Tables 2, 3). Differences in carotenoid composition are observed as early as immature green fruits from Pinalate, where a minor accumulation of the linear carotenes phytoene, phytofluene and -carotene becomes detectable, although the peel of immature green fruits is a chloroplast-containing tissue. In mature green fruits, a substantial decrease in carotenoid content, with respect to immature green fruits, is observed in both Navelate and Pinalate. A reduced level of carotenoids in the peel of citrus fruits at mid-season coincides with the beginning of the degreening process and may reflect the conversion of chloroplasts to chromoplasts (Farin et al., 1983; Eilati et al., 1975; Gross, 1987). Again, at the mature green stage, Pinalate fruits contain a relatively high proportion of colourless carotenes, which are not detectable in Navelate. In Navelate fruit chromoplasts new carotenoid biosynthetic activities appeared, resulting in a massive accumulation of violaxanthin, which is the more abundant pigment of the peel of full-coloured fruits. Other pigments, although in minor amounts, were tentatively identified as apocarotenoids and ß-cryptoxanthin, which have a significant contribution to the bright-orange colour of the peel. In full-coloured fruits of the mutant, the carotenoid biosynthetic pathway is severely altered at the level of ZDS, since phytoene, phytofluene and -carotene accumulate, and the content of downstream products in the pathway such as violaxanthin, neoxanthin and ABA were substantially lower. Deficiency of the characteristic orange-red apocarotenoid-like compounds and ß-cryptoxanthin is also found in Pinalate. The biosynthesis of citrus apocarotenoids is probably due to the enzymatic oxidative cleavage of certain xanthophylls (Farin et al., 1983; Gross, 1987), and therefore low xanthophyll content in Pinalate might also prevent the formation of apocarotenoids.
The results presented show that two properties define the Pinalate phenotype: (1) an accumulation of the linear carotenes phytoene, phytofluene and -carotene, and a reduction of xanthophylls and downstream products in the carotenoid pathway, and (2) these alterations are fruit-specific, without affecting other fruit maturation events. Taking these two properties together, several possibilities can be suggested to explain the origin of the Pinalate phenotype.
An interesting possibility would be the presence of two different isoforms of ZDS in Citrus sinensis: a leaf-associated isoform functional in both Pinalate and Navelate, and a fruit isoform, which would be defective in Pinalate. This fruit-specific ZDS isoform would have a minor role in the chloroplasts of fruits, but would be essential for the carotenoid accumulation in chromoplasts. Similar situations have been reported for other steps of tomato carotenoid biosynthesis, where chloroplastic and chromoplastic isoforms of the corresponding enzymes are differentially regulated in the chloroplast and chromoplast-containing tissues. Tomato contains two differentially expressed phytoene synthase genes, Psy-2 predominates in chloroplastic tissue, while Psy-1 shows a chromoplast-specific expression (Fraser et al., 1994, 1999). Two ß-lycopene cyclases have been also found in tomato, CYC-B and LCY-B. CYC-B does play a role in chromoplast-containing tissue, but not in vegetative tissues, whereas LCY-B is not functional in fruits, but it is in flowers and green tissues (Ronen et al., 2000). Two ß-carotene hydroxylases are also present in tomato plants, one is expressed in green tissues while the other is exclusively expressed in the flower (Hirschberg, 2001).
Alternatively, the fruit-specific nature of the Pinalate alteration might be explained by the presence of additional regulatory factors specific to fruits and related to carotenoid synthesis or accumulation, as occurs in the tomato Delta mutant. Delta fruits accumulate 
-carotene at the expense of lycopene and strong evidence suggests that the Del locus encodes a lycopene-
-cyclase. The amino acid sequence of the lycopene--cyclase is identical between Delta and the wild type and both are equally functional. It is suggested that Delta contains the allele of lycopene--cyclase from the green-fruited L. pennellii that could differ from the wild-type allele in the promoter region. In the Delta mutant lycopene--cyclase mRNA fails to be down-regulated during ripening, most probably because of the failure of the wild-type regulatory factor to interact with the L. pennellii allele (Ronen et al., 1999). Neither carotenoid content nor the mRNA levels of the involved cyclase gene are affected in leaves and petals (Ronen et al., 1999). This mutant illustrates the presence of additional fruit-specific factors that might operate to regulate, in a tissue-dependent manner, the expression of carotenoid biosynthetic genes.
Another different possibility to explain the nature of Pinalate would take into consideration an alteration in desaturase-associated factors. A similar situation has been found in mutants with a blockage in phytoene desaturation that do not map at the pds structural gene, like immutants, pds1 and pds2 in Arabidopsis (Norris et al., 1995; Wetzel and Rodermel, 1998), ghost in tomato (Giuliano et al., 1993), or víviparous2 in maize (Hable et al., 1998). Some of these mutations have been shown to target additional functions required for desaturation. For instance, pds1 codes for an enzyme required for the biosynthesis of plastoquinones, which would act as electron carriers in the desaturation reaction (Norris et al., 1995). Immutans and ghost define a plastid terminal oxidase (PTOX) as a component of a redox chain in phytoene desaturation (Carol et al., 1999; Wu et al., 1999; Josse et al., 2000). However, alterations in these factors, necessary for efficient desaturation, would probably impair both PDS and ZDS activities, given the similarity between the reactions catalysed by both enzymes (Cunningham and Gantt, 1998). Since in Pinalate leaves the carotenoid composition and ABA content are not affected and PDS activity in fruit appears not to be impaired, it, therefore, seems unlikely that the Pinalate phenotype is due to an alteration in desaturase associated factors.
Recently, the isolation and characterization of a new carotenoid isomerase (CRTISO) in Arabidopsis and tomato has revealed a novel enzymatic activity necessary for the cis to trans isomerizations of carotenes and the conversion to lycopene which takes place at the level of the -carotene desaturation (Isaacson et al., 2002; Park et al., 2002). Tomato, Arabidopsis, Scenedesmus, and Synechocystis mutants which have lost or impaired CRTISO function, accumulate poly-cis-carotenes, mainly poly-cis lycopene, and indicates that an all-trans-conformation of lycopene is necessary for its cyclation (Breitenbach et al., 2001; Isaacson et al., 2002; Park et al., 2002). It also seems that isomerase activity is not critical in photosynthetic tissues in the presence of light, but is only required in the dark or in chromoplast-containing tissues resulting, in some circumstances, in a fruit-specific alteration. Results of HPLC analysis of flavedo from Pinalate do not support the possibility that a defective isomerase may be the cause of the Pinalate phenotype. Minor amounts of different -carotene isomers (Table 1; Fig. 5) have been identified in Pinalate fruits. Moreover, prolycopene or proneurosporene, which would be the final products of the pathway in the absence of isomerase, have never been found in any of the samples analysed. Authors believe that cis isomers tentatively identified are probably the result of a photostationary mixture between cis and trans isomers, due to the high accumulation of carotenes in Pinalate.
Finally, an attractive possibility is that the altered carotene complement of Pinalate may be due to an alteration of carotenoid-associated proteins/lipids, which have a role in carotenoid accumulation, and also unique mechanisms specialized for chromoplasts (Vishnevetsky et al., 1999), which explain the fruit-specific nature of Pinalate.
In conclusion, Pinalate is a fruit-specific alteration with a high content of early carotenes (phytoene, phytofluene and -carotene) and a reduced proportion of downstream xanthophylls and ABA, which result in distinctive yellow-coloured mature fruits. These alterations presume a defect in ZDS or ZDS-associated activities. Pinalate, thus, provides an interesting experimental system to reveal molecular mechanisms regulating the synthesis and the accumulation of carotenoids in Citrus. Pinalate, because of its ABA deficiency, also provides excellent material with which to study the involvement of this hormone in fruit maturation and stress responses.
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Acknowledgements |
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We acknowledge Dr T Lafuente (IATA-CSIC) for the synthesis of the ABA conjugate used for ABA quantification, and for her help and comments during the course of this work. We acknowledge Dr L Navarro (IVIA, Moncada) for allowing us the use of the Citrus Germplasm Bank of the IVIA. We also thank J Cervera and J Gil for providing fruits from commercial orchards. The technical assistance of A Beneyto and I Chilet is acknowledged. This work was supported by grants ALI96-0506-C03-01 and ALI99-0954-C03-02 from ‘Comisión Interministerial de Ciencia y Tecnología’ (CICYT, Spain), GV-CAPA97-01-C2 from Conselleria de Agricultura (Generalitat Valenciana) and a post-doctoral contract from Ministerio of Ciencia y Tecnología (to MJR).
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