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Plant Physiol. Feb 2004; 134(2): 824–837.
PMCID: PMC344557

Accumulation of Carotenoids and Expression of Carotenoid Biosynthetic Genes during Maturation in Citrus Fruit1

Abstract

The relationship between carotenoid accumulation and the expression of carotenoid biosynthetic genes during fruit maturation was investigated in three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.). We cloned the cDNAs for phytoene synthase (CitPSY), phytoene desaturase (CitPDS), ζ-carotene (car) desaturase (CitZDS), carotenoid isomerase (CitCRTISO), lycopene β-cyclase (CitLCYb), β-ring hydroxylase (CitHYb), zeaxanthin (zea) epoxidase (CitZEP), and lycopene ε-cyclase (CitLCYe) from Satsuma mandarin, which shared high identities in nucleotide sequences with Valencia orange, Lisbon lemon, and other plant species. With the transition of peel color from green to orange, the change from β,ε-carotenoid (α-car and lutein) accumulation to β,β-carotenoid (β-car, β-cryptoxanthin, zea, and violaxanthin) accumulation was observed in the flavedos of Satsuma mandarin and Valencia orange, accompanying the disappearance of CitLCYe transcripts and the increase in CitLCYb transcripts. Even in green fruit, high levels of β,ε-carotenoids and CitLCYe transcripts were not observed in the juice sacs. As fruit maturation progressed in Satsuma mandarin and Valencia orange, a simultaneous increase in the expression of genes (CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP) led to massive β,β-xanthophyll (β-cryptoxanthin, zea, and violaxanthin) accumulation in both the flavedo and juice sacs. The gene expression of CitCRTISO was kept low or decreased in the flavedo during massive β,β-xanthophyll accumulation. In the flavedo of Lisbon lemon and Satsuma mandarin, massive accumulation of phytoene was observed with a decrease in the transcript level for CitPDS. Thus, the carotenoid accumulation during citrus fruit maturation was highly regulated by the coordination of the expression among carotenoid biosynthetic genes. In this paper, the mechanism leading to diversity in β,β-xanthophyll compositions between Satsuma mandarin and Valencia orange was also discussed on the basis of the substrate specificity of β-ring hydroxylase and the balance of expression between upstream synthesis genes (CitPSY, CitPDS, CitZDS, and CitLCYb) and downstream synthesis genes (CitHYb and CitZEP).

Carotenoids are essential components of the photosynthetic apparatus in plants, algae, and cyanobacteria, in which they protect against photooxidative damage and contribute to light harvesting for photosynthesis (Goodwin, 1980). In higher plants, the bright yellow, orange, and red colors provided by carotenoids accumulate in the chromoplasts of flowers and fruits. In these tissues, plants exploit carotenoids as colorants to attract pollinators and agents of seed dispersal. In addition, epoxy-carotenoids, violaxanthin, and neoxanthin are precursors for plant hormone abscisic acid (Rock and Zeevaart, 1991). Some carotenoids serve as precursors for vitamin A, which is essential to human and animal diets, and as antioxidants, which play a role in reducing the risk of certain forms of cancer (Olson, 1989).

Citrus is a complex source of carotenoids, with the largest number of carotenoids found in any fruit (Gross, 1987). Carotenoid concentration and composition vary greatly among citrus varieties and depend on the growing conditions (Gross, 1987). During citrus fruit development, massive accumulation of carotenoids occurred concomitantly with the degradation of chlorophyll. Mandarin varieties, such as Satsuma mandarin (Citrus unshiu Marc.), accumulated β-cryptoxanthin (β-cry) predominantly in the flavedo and juice sacs in mature fruit (Goodner et al., 2001; Ikoma et al., 2001). In contrast, mature sweet orange (Citrus sinensis Osbeck) accumulated violaxanthin isomers predominantly in fruit (Molnár and Szabolcs, 1980; Lee and Castle, 2001), in which 9-cis-violaxanthin was found to be the principal carotenoid (Molnár and Szabolcs, 1980). Because β-cry was detected to a minor extent in sweet orange varieties, the difference in β-cry concentration can be used as a discriminating factor among mandarin, orange, and their hybrids (Goodner et al., 2001). Mature lemon (Citrus limon Burm.f.) showed light yellow color in the flavedo and juice sacs. This light coloration was primarily because of a small concentration of total carotenoids, which was much lower than that in navel orange (Citrus sinensis Osbeck; Yokoyama and Vandercook, 1967). Thus, Satsuma mandarin (variety accumulating β-cry), Valencia orange (variety accumulating violaxanthin), and Lisbon lemon (variety accumulating small amounts of carotenoids) are useful experimental materials to investigate the molecular mechanism regulating carotenoid concentration and composition in fruit because the carotenoid profiles in mature fruit were much more diverse among these three citruses. These materials are especially useful for undertanding the mechanism of xanthophyll accumulation in fruit because massive xanthophyll accumulation does not occur in common experimental materials, such as tomato (Lycopersicon esculentum) and Arabidopsis.

The pathway of carotenoid biosynthesis in plants is illustrated in Figure 1 (Cunningham and Gantt, 1998; Ronen et al., 1999; Isaacson et al., 2002; Park et al., 2002). The first committed step in carotenoid biosynthesis is a head-to-head condensation of two molecules of geranylgeranyl pyrophosphate (C20) to form colorless phytoene (phy; C40) catalyzed by phytoene synthase (PSY). Phytoene desaturase (PDS) and ζ-carotene (car) desaturase (ZDS) introduce four double bonds into phy to yield lycopene (lyc) via phytofluene, and neurosporene. Recently, Park et al. (2002) and Isaacson et al. (2002) isolated the gene encoding carotenoid isomerase (CRTISO) from Arabidopsis and tomato, respectively, by map-based cloning. CRTISO functions as the isomerization of poly-cis-carotenoids to all-trans-carotenoids. The cyclization of lyc is a crucial branching point in this pathway, yielding α-car with one ε-ring and one β-ring and β-car with two β-rings, in which two cyclases, namely, lycopene β-cyclase (LCYb) and lycopene ε-cyclase (LCYe), are responsible for these reactions (Cunningham et al., 1996). α-Car is converted into lutein (lut) by sequential hydroxylations, which are catalyzed by ε-ring hydroxylase and β-ring hydroxylase (HYb). β-Car is converted to zeaxanthin (zea) via β-cry by two-step hydroxylation, which is catalyzed by HYb. Furthermore, zea is converted to violaxanthin via antheraxanthin by zea epoxidase (ZEP).

Figure 1.
Carotenoid biosynthetic pathway in plants. Eight cDNAs, CitPSY, CitPDS, CitZDS, CitCRTISO, CitLCYb, CitHYb, CitZEP, and CitL-CYe, were cloned from the Satsuma mandarin flavedo and used for RNA probes for northernblot analyses in this study. GGPP, Geranylgeranyl ...

Carotenoid biosynthesis and its regulation have been studied in various plant species, such as Arabidopsis (Pogson et al., 1996; Park et al., 2002), tomato (Giuliano et al., 1993; Fraser et al., 1994; Ronen et al., 1999; Isaacson et al., 2002), pepper (Capsicum annuum; Bouvier et al., 1996, 1998), tobacco (Nicotiana tabacum; Busch et al., 2002), and alga (Steinbrenner and Linden, 2001). Bramley (2002) reviewed carotenoid biosynthesis and regulation during ripening and development in tomato fruit. During tomato fruit ripening, the expression of PSY and PDS increased (Giuliano et al., 1993; Fraser et al., 1994; Ronen et al., 1999; Isaacson et al., 2002), whereas the expression of both LCYb and LCYe disappeared (Pecker et al., 1996; Ronen et al., 1999), leading to massive accumulation of lyc. Genes involved in carotenoid biosynthesis were induced by various environmental stimuli. It has been reported that PSY was induced by light in tomato seedlings (Giuliano et al., 1993). Moreover, oxidative stress induced chromoplast-specific carotenoid genes in pepper fruit (Bouvier et al., 1996, 1998). In green alga, high light and salt stress elicited the gene expression of PSY and carotenoid hydroxylase, resulting in a rapid accumulation of astaxanthin (Steinbrenner and Linden, 2001).

Also in citrus fruit, the gene expression of some carotenoid biosynthetic enzymes was investigated previously. These investigations showed that, in Satsuma mandarin, the gene expression of PSY increased in the peel and juice sacs with the onset of coloration (Ikoma et al., 2001; Kim et al., 2001), whereas the gene expression of PDS and HYb remained constant once fruit was fully developed (Kim et al., 2001; Kita et al., 2001). To elucidate the regulation steps for carotenoid synthesis in citrus fruit, these investigations were insufficient because at least six genes (PSY, PDS, ZDS, LCYb, HYb, and ZEP) participate in xanthophyll accumulation, which occurs massively in Satsuma mandarin and Valencia orange. Therefore, in the present study, the expression of genes, including the six genes mentioned above, was investigated simultaneously.

In this study, during fruit maturation, the concentration and composition of carotenoid and the expression of carotenoid biosynthetic genes were investigated in the flavedos and juice sacs of three varieties, Satsuma mandarin, Valencia orange, and Lisbon lemon. The cDNAs related to linear car biosynthesis (PSY, PDS, and ZDS), the cDNA related to the isomerization of poly-cis-carotenoids (CRTISO), the cDNAs related to cyclization (LCYb and LCYe), and the cDNAs related to hydroxylation and epoxidation (HYb and ZEP) were cloned. The expression of these genes was analyzed during fruit maturation in the three varieties. The results were the first of their kind, to our knowledge, to show the simultaneous expression of genes participating in the synthesis of xanthophylls in citrus fruit. This study was also the first, to our knowledge, to provide information on differences among the three varieties in the profiles of gene expression of carotenoid biosynthetic enzymes. On the basis of the comparison of these profiles, the mechanism causing the diversity of carotenoid accumulation in citrus fruit was discussed. The mechanism of xanthophyll accumulation in fruit, especially β-cry and violaxanthin accumulation, was also discussed.

RESULTS

Identification of Carotenoids

To identify and quantify carotenoids, three different gradient elution schedules of HPLC, methods A to C, were used. Method A was optimized for all-trans-violaxanthin (t-vio), cis-violaxanthin (c-vio), lut, phy, β-cry, and α-car analyses. Method B was optimized to separate β-car and ζ-car peaks because the peak of β-car overlapped with that of ζ-car by method A. Method C was optimized for zea analysis because the peak of zea overlapped with that of an unknown carotenoid by method A (Table I).

Table I.
Identification of carotenoids found in citrus fruit

Peaks 1, 3, 5 to 7, and 9 were identified as t-vio, lut, β-cry, α-car, β-car, and zea, respectively, by comparing their absorption spectra and retention times with those of purchased authentic standards (Table I). Although the corresponding authentic standard was obtained, lyc was not detected in our experimental samples.

We prepared the standard for phy. The acetone extract obtained from phy-producing E. coli cells was separated by HPLC (method A). The peak eluted at 56 min was isolated. The mass spectrum of the eluent showed the molecular ion at mass-to-charge ratio (m/z) 545 ([M + H]+). The eluent exhibited the typical absorption spectrum of phy. Thus, we used the eluent as a phy standard. Peak 4 was identified as phy by comparing its absorption spectrum and retention time with those of the phy standard (Table I).

Also, we prepared the standard for ζ-car. The acetone extract obtained from ζ-car-producing E. coli cells was separated by HPLC (method B). The peak eluted at 12 min was isolated. The mass spectrum of the eluent showed the molecular ion at m/z 541 ([M + H]+). The eluent exhibited the typical absorption spectrum of ζ-car. Thus, we used the eluent as a ζ-car standard. Peak 8 was identified as ζ-car by comparing its absorption spectrum and retention time with those of the ζ-car standard (Table I).

The standard for c-vio was prepared from the flavedo of Satsuma mandarin fruit. The crude extract of carotenoids from the tissue was separated by HPLC (method A). The peak eluted at 31 min was isolated. The absorption maxima of the eluent (414, 436, and 464 nm) were close to those of c-vio reported previously (Tai and Chen, 2000; Table I). The epoxide test indicated that the eluent was 5,6,5′,6′-diepoxide because the absorption maxima shifted from 414, 436, and 464 nm to 380, 401, and 427 nm, respectively, after the addition of HCl. The mass spectrum of the eluent showed the molecular ion at m/z 601 ([M + H]+). These results indicated that the eluent was c-vio. Therefore, the eluent was used as a c-vio standard. Peak 2 was identified as c-vio by comparing its absorption spectrum and retention time with those of the c-vio standard (Table I).

Isolation and Identification of the cDNA Fragments of Carotenoid Biosynthetic Genes

On the basis of the conserved amino acid sequences among plant species in carotenoid biosynthetic genes, eight sets of degenerated primers were designed for each of PSY, PDS, ZDS, CRTISO, LCYb, HYb, ZEP, and LCYe (Table II). Reverse transcriptase-PCRs were performed with the flavedos of Satsuma mandarin, Valencia orange, and Lisbon lemon. In the case of Satsuma mandarin, for each carotenoid biosynthetic gene, we sequenced at least 12 cDNAs among which the nucleotide sequences were completely identical except for the primer regions. Thus, a cDNA was selected for each carotenoid biosynthetic gene.

Table II.
Primers for reverse transcriptase-PCR, lengths of amplified products, and comparison of nucleotide sequences of carotenoid biosynthetic genes from Satsuma mandarin with those from Valencia orange, Lisbon lemon, and non-citrus plant species

Eight cDNA fragments from Satsuma mandarin for carotenoid biosynthetic genes were designated as CitPSY (accession no. AB114648), CitPDS (accession no. AB114649), CitZDS (accession no. AB114650), Cit-CRTISO (accession no. AB114651), CitLCYb (accession no. AB114652), CitHYb (accession no. AB114653), CitZEP (accession no. AB114654), and CitLCYe (accession no. AB114655; Table II). The nucleotide sequences of all cDNAs isolated from Satsuma mandarin showed high identity (>97.4% at nucleotide sequence level) to those of corresponding cDNAs isolated from Valencia orange (accession nos. AB114656-AB114663) and Lisbon lemon (accession nos. AB114664-AB114671; Table II). Because the identity of cDNAs among the three citrus varieties was very high, the RNA probes were synthesized from Satsuma mandarin cDNA for northern-blot analyses, which were used to compare gene expression among the three varieties. These high identities suggested that the differences among varieties in the hybridizing intensity of the probe to mRNA were negligible. The cDNAs isolated from Satsuma mandarin also shared high similarities with other citrus species, such as Citrus × paradisi and Citrus maxima (>97.6% at the nucleotide sequence level; data not shown) and non-citrus species (>73.4% at the nucleotide sequence level; Table II).

Changes in the Concentration of Carotenoid and Expression of Carotenoid Biosynthetic Genes in the Flavedo

The color of the flavedo changed from green to orange during fruit maturation. The green stages in Satsuma mandarin, Valencia orange, and Lisbon lemon were from August to September, from August to October, and from August to October, respectively.

During the green stage, β-car (<8.4 μg g–1), t-vio (<4.3 μg g–1), α-car (<5.0 μg g–1), and lut (<14.8 μg g–1) were predominant, although the amounts of these carotenoids were low in the three varieties (Fig. 2). Phy, ζ-car, β-cry, zea, and c-vio were barely detected in the three varieties. During this stage, high gene expression of CitLCYe and CitCRTISO and low gene expression of CitPSY, CitZDS, and CitHYb were observed in the three varieties (Fig. 3). The gene expression increased noticeably in CitLCYb of Satsuma mandarin and Valencia orange and in CitZEP of the three varieties. The gene expression of CitPDS increased rapidly in Satsuma mandarin but slowly in Valencia orange and Lisbon lemon.

Figure 2.
Carotenoid concentration in the flavedos of three citrus varieties, Satsuma mandarin, Valencia orange, and Lisbon lemon, during fruit maturation. Columns and bars represent the means and se (n = 3), respectively. Total car, Total carotenoids. The value ...
Figure 3.
Expressions of carotenoid biosynthetic genes in the flavedos of three citrus varieties, Satsuma mandarin, Valencia orange, and Lisbon lemon, during fruit maturation. A, Chemiluminescent images of northern-blot analyses. B, Quantification of mRNA abundance ...

After the green stage, the concentration of β-car, α-car, and lut decreased or remained constant at a low level with a concomitant decrease in the gene expression of CitLCYe in the three varieties (Figs. (Figs.22 and and3).3). In contrast, β,β-xanthophylls (β-cry, zea, t-vio, and c-vio) accumulated massively in Satsuma mandarin and Valencia orange. In Satsuma mandarin, β-cry and c-vio became abundant (in January, 48.7 μg g–1 and 48.1 μg g–1, respectively). In Valencia orange, c-vio became abundant (in February, 50.7 μg g–1). With the transition of the peel color from green to orange, the gene expression of CitPSY, Cit-PDS, CitZDS, CitLCYb, CitHYb, and CitZEP, which make up a necessary set of genes to produce β,β-xanthophylls, increased to maximum levels or remained high in Satsuma mandarin and Valencia orange. In Lisbon lemon, the concentration of β,β-xanthophylls remained low, although the expression of a gene set to produce β,β-xanthophylls increased slightly or remained at near a maximum level with the transition of the peel color. The increased levels of gene expression in Satsuma mandarin were higher than those in Valencia orange and Lisbon lemon.

After β,β-xanthophylls increased, massive accumulation of phy started in Satsuma mandarin (December–January). The concentration of phy (110.7 μg g–1) was much higher than that of ζ-car (6.8 μg g–1) in January, when phy became the most abundant carotenoid (Fig. 2). In Lisbon lemon, instead of massive accumulation of β,β-xanthophylls, accumulation of phy was observed (December–February). Phy became the most abundant carotenoid in February, when the phy concentration (77.2 μg g–1) was 36.6-fold of ζ-car. In Valencia orange, the concentration of phy was much lower than those in Satsuma mandarin and Lisbon lemon even in the latter maturation stage (January–February). The gene expression of CitPSY remained at near maximum levels in Satsuma mandarin and Lisbon lemon during the periods in which phy increased (Fig. 3). In contrast, the gene expression of CitPDS clearly decreased in Satsuma mandarin and Lisbon lemon during this period. The gene expression of CitCRTISO was kept low or decreased in the three varieties, whereas β,β-xanthophylls and phy were accumulating.

Changes in the Concentration of Carotenoid and Expression of Carotenoid Biosynthetic Genes in the Juice Sacs

During the green stage of the flavedo (August–September in Satsuma mandarin and August–October in Valencia orange and Lisbon lemon), the concentration of total carotenoids was low in the juice sacs in the three varieties (Fig. 4). However, in Satsuma mandarin, the concentration of β-cry in the juice sacs increased (5.1 μg g–1 in September) and was much higher than that in the flavedo (16.5-fold in September). In Valencia orange and Lisbon lemon, no noticeable accumulation of β-cry was detected during this stage (undetectable level in Valencia orange and 0.2 μg g–1 in Lisbon lemon in September). No gene expression of CitLCYe was detected in the three varieties (Fig. 5). In Satsuma mandarin, accompanying the accumulation of β-cry, the gene expression of CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP, which make up a set of genes to produce β,β-xanthophylls, increased simultaneously. In Valencia orange, the gene expression of CitPDS, CitHYb, and CitZEP increased clearly, whereas no noticeable increase in the gene expression of CitPSY, CitZDS, and CitLCYb was observed. No noticeable increase in the gene expression of CitPSY, CitZDS, and CitLCYb was observed in Lisbon lemon either. The gene expression of CitCRTISO increased in the three varieties.

Figure 4.
Carotenoid concentration in the juice sacs of three citrus varieties, Satsuma mandarin, Valencia orange, and Lisbon lemon, during fruit maturation. Columns and bars represent the means and se (n = 3), respectively. Total car, Total carotenoids. The value ...
Figure 5.
Expressions of carotenoid biosynthetic genes in the juice sacs of three citrus varieties, Satsuma mandarin, Valencia orange, and Lisbon lemon, during fruit maturation. A, Chemiluminescent images of northern-blot analyses. B, Quantification of mRNA abundance ...

After the green stage, massive accumulation of carotenoids, especially the accumulation of β,β-xanthophylls, occurred in Satsuma mandarin and Valencia orange (Fig. 4). In January, Satsuma mandarin accumulated predominantly a β,β-xanthophyll, β-cry (15.9 μg g–1), which accounted for 59.6% of the total identified carotenoids. Other β,β-xanthophylls, t-vio (4.7%) and c-vio (4.4%), were at low concentrations during this month. In February, Valencia orange accumulated predominantly t-vio and c-vio (2.4 and 9.6 μg g–1, respectively), which accounted for 65.4% of the total identified carotenoids. The concentration of the other β,β-xanthophyll, β-cry (15.1%), was low in February. In Lisbon lemon, the concentration of carotenoids was extremely low, although phy, β-car, and β-cry accumulated slightly (0.2, 0.1, and 0.4 μg g–1 in February, respectively). Clearly, the gene expression of a set of genes to produce β,β-xanthophylls (CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP) increased, reaching a maximum after the green stage in Satsuma mandarin and Valencia orange (Fig. 5). In Lisbon lemon, the gene expression of a set of genes to produce β,β-xanthophylls also increased. However, the increased levels of gene expression were lower than those in Satsuma mandarin and Valencia orange. The gene expression of Cit-CRTISO increased to a maximum and subsequently decreased in the three varieties after the green stages.

Xanthophyll Composition and Gene Expression Related to car and β,β-Xanthophyll Syntheses in Satsuma Mandarin and Valencia Orange Fruits

During 2 months of sampling just after the green stage (October and November for Satsuma mandarin and November and December for Valencia orange), β,β-xanthophylls accumulated massively in both the flavedo and juice sacs, which showed a considerable difference in xanthophyll composition. In the flavedo, the ratios of the predominant xanthophylls, β-cry to violaxanthin (t-vio + c-vio), were 0.43 in Satsuma mandarin (average of October and November) and 0.11 in Valencia orange (average of November and December). In the juice sacs, the ratios were 5.17 in Satsuma mandarin (average of October and November) and 0.10 in Valencia orange (average of November and December).

To compare gene expression related to the syntheses of car and β,β-xanthophyll between Satsuma mandarin and Valencia orange during a period of massive β,β-xanthophyll accumulation (October and November for Satsuma mandarin and November and December for Valencia orange), the transcript levels for CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP during these 2 months were averaged from the results of Figures Figures3B3B and and5B5B (Fig. 6). In the flavedo, the levels for transcripts involved in car synthesis (CitPSY, Cit-PDS, CitZDS, and CitLCYb) and β,β-xanthophyll synthesis (CitHYb and CitZEP) were higher in Satsuma mandarin than in Valencia orange. In the juice sacs, the levels for CitPSY, CitPDS, CitZDS, and CitLCYb transcripts involved in the biosynthesis from phy to β-car were higher in Satsuma mandarin. In contrast, the levels for CitHYb and CitZEP transcripts involved in the biosynthesis from β-cry to violaxanthin were much higher in Valencia orange than in Satsuma mandarin.

Figure 6.
Comparisons of mRNA abundance for carotenoid biosynthetic genes between Satsuma mandarin and Valencia orange in the flavedos and juice sacs during massive xanthophyll accumulations (October and November for Satsuma mandarin and November and December for ...

DISCUSSION

Carotenoid Accumulation and Gene Expression during the Green Stage

In the flavedo of the green stage, the low gene expression of CitPSY and CitZDS, which produce linear cars, was responsible for the low concentration of carotenoids in Satsuma mandarin, Valencia orange, and Lisbon lemon. It seems that a rapid increase in the gene expression of CitPDS of Satsuma mandarin did not lead to an increase in the concentration of carotenoids in this stage because the expression of the upstream gene CitPSY remained low in this variety (Fig. 3). In contrast, in the juice sacs of Satsuma mandarin, simultaneous increases in the expression of the CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP genes, which make up a set of genes to produce β,β-xanthophylls, led to the accumulation of β-cry in this stage (Fig. 5). In juice sacs of Valencia orange and Lisbon lemon, no noticeable accumulation of β,β-xanthophylls was detected during this stage because the gene expression of CitPSY, CitZDS, and CitLCYb did not increase noticeably.

In the flavedos of the three varieties, the gene expression of CitLCYe in the green stage was higher than that in the orange stage (Fig. 3). The cyclization of lyc is a key branch point in the pathway of carotenoid biosynthesis in plants and algae, in which the lycopene cyclases, LCYe and LCYb, are key enzymes (Cunningham et al., 1996). LCYe adds only one ε-ring to form the monocyclic δ-car, leading to the synthesis of β,ε-carotenoids (α-car and lut) after LCYb introduces one β-ring to δ-car (Ronen et al., 1999; Fig. 1), whereas LCYb introduces two β-rings, leading to the synthesis of β,β-carotenoids (β-car, β-cry, zea, and violaxanthin). In the flavedo, the high expression of the CitLCYe gene suggested that cyclization to the ε-ring was more active than that in the orange stage, resulting in the predominant accumulation of β,ε-carotenoids in the green stage. Thus, it was thought that the pathway changing from β,ε-carotenoid synthesis to β,β-carotenoid synthesis did not occur in the flavedo in the green stage. In the juice sacs, no transcripts for CitLCYe in the green and orange stages were detected in the three varieties (Fig. 5). Ikoma et al. (2001) reported that the chlorophyll content in the juice sacs of Satsuma mandarin was high in June, subsequently decreasing to undetectable levels in August. These results suggested that, in the juice sacs, β,ε-carotenoid synthesis occurred before August because the LCYe gene was expressed exclusively in chloroplast-containing photosynthetic tissues (Ronen et al., 1999). Thus, it was thought that the pathway changing from β,ε-carotenoid synthesis to β,β-carotenoid synthesis occurred in the juice sacs earlier than in the flavedo.

Xanthophyll Accumulation and Gene Expression during the Orange Stage

After the green stage of the flavedos in the three varieties, the increase in the gene expression of CitL-CYb and decrease in the gene expression of CitLCYe suggested that pathway changing from β,ε-carotenoid synthesis to β,β-carotenoid synthesis occurred in the flavedo with the transition from the green stage to the orange stage (Fig. 3).

During the orange stage, simultaneous increases in the expression of genes to participate in β,β-xanthophyll synthesis (CitPSY, CitPDS, CitZDS, CitL-CYb, CitHYb, and CitZEP) led to the massive accumulation of β,β-xanthophylls in the flavedos and juice sacs of Satsuma mandarin and Valencia orange (Figs. (Figs.33 and and5).5). In Lisbon lemon, the gene expression of a set of genes to produce β,β-xanthophylls also increased. However, the levels of gene expression in Lisbon lemon were much lower than those in Satsuma mandarin and Valencia orange. Low gene expression may lead to an extremely low concentration of β,β-carotenoids in the flavedo and juice sacs of Lisbon lemon.

These gene expression profiles in Satsuma mandarin and Valencia orange were different from those in tomato observed previously during fruit ripening. Previous studies showed that, in tomato fruit accumulating lyc, the gene expression of PSY and PDS increased and that of LCYe decreased at the breaker stage of ripening (Pecker et al., 1996; Giuliano et al., 1993; Ronen et al., 1999). These gene expression profiles of tomato fruit were consistent with those of citrus fruit observed in the present study. In contrast, the expression profiles of the LCYb gene were different between tomato and citrus. The transcripts for LCYb in the tomato fruit disappeared at this stage (Pecker et al., 1996; Ronen et al., 1999), although those for LCYb obviously increased in Satsuma mandarin and Valencia orange (Figs. (Figs.33 and and5).5). This difference in the gene expression profile of LCYb seems to be the primary determinant of the difference in the carotenoid profile between tomato and citrus fruit. In tomato fruit, the disappearances in the gene expression of LCYe and LCYb led to massive accumulation of a final upstream product, lyc. In contrast, in Satsuma mandarin and Valencia orange fruit, disappearances in the gene expression of LCYe and increases in the gene expression of LCYb led to the massive accumulation of β,β-xanthophylls. β,β-Xanthophyll accumulation, as observed in citrus, also was reported in tomato petal, in which the expression of the LCYb gene was up-regulated, whereas the LCYe gene was not expressed, resulting in the accumulations of β,β-xanthophylls, such as violaxanthin and neoxanthin (Ronen et al., 1999). Recently, Ronen et al. (2000) identified another type of LCYb gene, Beta, by map-based cloning from the tomato mutants. The gene was expressed exclusively in the chromoplast-containing tissues of flowers and fruits. In citrus fruit, whether the gene plays an important role in β-car formation is unknown.

Isaacson et al. (2002) found a gene, CRTISO, which encoded an authentic CRTISO that is required for carotenoid desaturation. In the report, the gene was expressed in all green tissues, but it was up-regulated during fruit ripening and in the flowers of tomato. In citrus, the gene expression of CitCRTISO remained low or decreased in the flavedo during the massive β,β-xanthophyll accumulation (Fig. 3). Thus, it is not clear whether the gene expression of CitCRTISO contributed to massive β,β-xanthophyll accumulation in citrus.

phy Accumulation and Gene Expression during the Orange Stage

The accumulation of upstream carotenoids, such as phytofluene and ζ-car, was reported previously in lemon (Yokoyama and Vandercook, 1967; Gross, 1987). Recently, a mutant Pinalate orange, which has a distinct yellow fruit instead of the typical orange (Citrus sinensis Osbeck), was characterized (Rodrigo et al., 2003). In this mutant, linear cars (phy, phytofluene, and ζ-car) had accumulated. In lemon fruit of the present study, we observed a high level of phy rather than phytofluene, which was high in previous reports (Yokoyama and Vandercook, 1967; Gross, 1987; Fig. 2). The result of carotenoid analysis suggested that lack of action in carotenoid desaturases led to the massive accumulation of phy and a limited accumulation of β,β-xanthophylls in Lisbon lemon. Actually, the transcript levels for CitPDS decreased remarkably in February in Lisbon lemon (Fig. 3). Massive accumulation of phy was also observed in the flavedo of Satsuma mandarin after the accumulation of β,β-xanthophyll with a decrease in transcript levels for CitPDS (in December and January; Figs. Figs.22 and and3).3). In these varieties, the accumulation of phy could be explained primarily by the levels of gene expression of CitPDS. phy accumulation was also reported in genetically transformed tobacco plants. Busch et al. (2002) reported that the accumulation of 15-cis-phy was detected in tobacco plants expressing antisense RNA to PDS. This result supports the present speculation that decreases in the gene expression of PDS lead to accumulation of phy.

Previously, Al-Babili et al. (1996) showed that PDS was regulated posttranscriptionally in the chromoplasts of N. pseudonarcissus. In the present study, the same mechanism that is related to the posttranscriptional regulation of PDS also may be responsible for the massive accumulation of phy.

Mechanism Leading to Diversity in Xanthophyll Compositions between Satsuma Mandarin and Valencia Orange

A clear difference in the β,β-xanthophyll composition between Satsuma mandarin and Valencia orange fruits was observed in the juice sacs. The juice sacs of Satsuma mandarin accumulated β-cry as a major carotenoid in mature fruit, whereas those of Valencia orange mainly accumulated violaxanthin isomers (Molnár and Szabolcs, 1980; Goodner et al., 2001; Ikoma et al., 2001; Lee and Castle, 2001; Fig. 4).

We isolated cDNAs encoding complete coding regions for HYb from Satsuma mandarin and Valencia orange (M. Kato and Y. Ikoma, unpublished data). The deduced amino acid sequences between these cDNAs were identical except for one amino acid residue, which was located in the transit peptide. This result suggested that the amino acid sequences between these cDNAs were completely identical in the regions related to enzyme activity. Thus, we thought that the difference in the amino acid sequences between these cDNAs was not responsible for the difference in the β,β-xanthophyll composition between Satsuma mandarin and Valencia orange.

A previous study demonstrated that β-cry instead of zea was mainly accumulated in E. coli cells carrying the truncated Arabidopsis HYb gene (Sun et al., 1996). The report speculated that HYb hydroxylated the β-rings of β-car with greater efficiency than the not-yet hydroxylated β-ring of β-cry. Because of the high substrate specificity of HYb to β-car, HYb would prefer the first step conversion from β-car to β-cry rather than the second step conversion from β-cry to zea under excessive β-car supply and/or low HYb activity.

In citrus fruits, the substrate specificity of HYb and expression balance between upstream synthesis genes (CitPSY, CitPDS, CitZDS, and CitLCYb) and downstream synthesis genes (CitHYb and CitZEP) seem important to determine the ratios of β-cry/violaxanthin. The higher expression of upstream synthesis genes and lower expression of the HYb gene suggested a higher supply of β-car and lower activity of CitHYb in the juice sacs of Satsuma mandarin than in those of Valencia orange (Fig. 6). The increased level of β-car in the juice sacs of Satsuma mandarin also suggested a higher supply of β-car and lower activity of CitHYb than in those of Valencia orange (Fig. 4). Therefore, it is thought that, under a balance of high gene expression of upstream synthesis and low gene expression of CitHYb (high supply of β-car and low activity of CitHYb), CitHYb catalyzes predominantly the first step conversion by the high substrate specificity of HYb to β-car, leading to massive accumulation of β-cry. Moreover, because β-car was rarely converted to zea via β-cry, and the gene expression of CitZEP was lower in the juice sacs of Satsuma mandarin than in those of Valencia orange, the accumulation of violaxanthin may be retarded in this tissue (Fig. 6). In contrast, in the juice sacs of Valencia orange, CitHYb is likely to sufficiently catalyze the reaction to zea via β-cry because lower gene expression of upstream synthesis and higher gene expression of CitHYb (low supply of β-car and high activity of CitHYb) were observed in the juice sacs of Valencia orange than in those of Satsuma mandarin (Fig. 6). Moreover, the intensity of gene expression related to epoxidation, CitZEP, were much higher in the juice sacs of Valencia orange than in those of Satsuma mandarin (Fig. 6). Thus, zea would be rapidly converted into violaxanthin in the juice sacs of Valencia orange.

In the flavedo, the gene expression involved in carotenoid biosynthesis (both upstream and downstream syntheses) was much higher in Satsuma mandarin than in Valencia orange (Fig. 6). The varietal difference in expression balance between upstream synthesis genes and downstream synthesis genes was much smaller in the flavedo than in the juice sacs. Thus, a small difference between Satsuma mandarin and Valencia orange in the expression balance of the genes resulted in a small difference in the β,β-xanthophyll composition in the flavedo.

CONCLUSION

In this paper, we investigated the relationship between carotenoid accumulation and the expression of carotenoid biosynthetic genes during fruit maturation in three citrus varieties. Our results clearly showed that pathway changing from β,ε-carotenoids to β,β-carotenoid synthesis was caused in the flavedos of Satsuma mandarin and Valencia orange by the disappearance of transcripts for CitLCYe and the increase in transcripts for CitLCYb with the transition of peel color from green to orange. In the juice sacs, pathway changing seems earlier than in flavedo because transcripts for CitLCYe were not detected even in green fruit. As fruit maturation progressed, in Satsuma mandarin and Valencia orange, a simultaneous increase in the expression of genes (CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP) led to massive β,β-xanthophyll accumulation in both the flavedo and juice sacs. In the flavedo of Lisbon lemon and Satsuma mandarin, massive accumulation of phy was observed with a decrease in the transcript level for CitPDS. The mechanism leading to diversity in β,β-xanthophyll compositions between Satsuma mandarin and Valencia orange also was discussed. The substrate specificity of HYb and expression balance between upstream synthesis genes (CitPSY, Cit-PDS, CitZDS, and CitLCYb) and downstream synthesis genes (CitHYb and CitZEP) seem responsible for the considerable difference in the β,β-xanthophyll compositions of the juice sacs between Satsuma mandarin and Valencia orange. Thus, the carotenoid accumulation during citrus fruit maturation was highly regulated by the coordination among the expression of the carotenoid biosynthetic genes.

MATERIALS AND METHODS

Plant Materials

Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.) cultivated at the National Institute of Fruit Tree Science, Department of Citrus Research, Okitsu (Shizuoka, Japan) were used as materials. Fruit samples were collected periodically from August to January for Satsuma mandarin and from August to February for Valencia orange and Lisbon lemon. The flavedos and juice sacs were separated from sampled fruits, immediately frozen in liquid nitrogen, and kept at –80°C until use.

Carotenoid Identification in Citrus Fruit

α-car, lut, and t-vio were obtained from DHI Water and Environment (Horsholm, Denmark). β-car and zea were obtained from Extrasynthese (Genay, France). β-cry was obtained from Sokenkagaku (Tokyo). These carotenoids were used for authentic standards.

A standard for phy was prepared from phy-producing Escherichia coli BL 21 (DE3) cells carrying plasmid pACCRT-EB (Misawa et al., 1995). A standard for ζ-car was also prepared from ζ-car-producing E. coli BL 21 (DE3) cells carrying plasmids pACCRT-EB and pET 30 (Novagen, Darmstadt, Germany), which harbors coding region of PDS cDNA from Satsuma mandarin (accession no. AB046992). These cells were grown in a Luria-Bertani medium at 27°C to OD600 = 0.6 and induced with 1 mm isopropyl-β-d-thiogalactoside for 24 h at 27°C. For the preparation of phy and ζ-car, cells were pelleted, frozen in liquid nitrogen, and suspended in acetone. After the acetone extract was partitioned into a diethyl ether phase, the phase was recovered and evaporated to dryness. Subsequently, the residue was dissolved in a methyl tert-butyl ether (MTBE):methanol (1:1 [v/v]) solution. Phy and ζ-car were separated by HPLC using methods A and B, respectively. Details of methods A and B are presented in the next section. For the identification of eluents, the absorption spectrum (220–550 nm) was monitored by a photodiode array detector (MD-910, Jasco, Tokyo) on an HPLC system. Moreover, fast atom bombardment mass spectrometry analysis was performed with a JMS-700 mass spectrometer (JEOL, Tokyo).

The standard of c-vio was prepared from the flavedo of Satsuma mandarin. The details of the carotenoid extraction method are presented in the next section. Extract was separated by HPLC (method A). For the identification of the eluent, the absorption spectrum and the mass spectrum were analyzed as mentioned above. Moreover, an epoxide test was performed to detect the epoxy groups of carotenoids. The spectrum was recorded before and after the addition of 50 μL of 0.1 n HCl into a 100-μL carotenoid solution dissolved in ethanol. A shift of about 20 nm to the shorter wave-lengths indicated the presence of a monoepoxy group, whereas a shift of approximately 40 nm was indicative of a diepoxy (Davies, 1976).

Carotenoid Quantification in Citrus Fruit

For the analysis of carotenoid contents, samples were homogenized in 40% (v/v) methanol containing 10% (w/v) magnesium carbonate basic. Pigments were extracted from the residues using an acetone:methanol (7:3 [v/v]) solution containing 0.1% (w/v) 2,6-di-tert-butyl-4-methylphenol and partitioned into diethyl ether. The extracts containing carotenoids esterified to fatty acids were saponified with 20% (w/v) methanolic KOH. After the saponification, water-soluble extracts were removed from the extract by adding NaCl-saturated water. The pigments repartitioned into the diethylether phase were recovered and evaporated to dryness. Subsequently, the residue was redissolved in 5 mL of an MTBE:methanol (1:1 [v/v]) solution.

An aliquot (20 μL) was separated by a reverse-phase HPLC system (Jasco) fitted with a YMC Carotenoid S-5 column of 250- × 4.6-mm-i.d. (Waters, Milford, MA) at a flow rate of 1 mL min–1. The eluent was monitored by a photodiode array detector (MD-910, Jasco). The sample was analyzed by three different gradient elution schedules. To assay t-vio, c-vio, lut, β-cry, and α-car, the gradient elution schedule consisted of an initial 30 min of 95% (v/v) methanol, 1% (v/v) MTBE, and 4% (v/v) water followed by a linear gradient of 6% (v/v) methanol, 90% (v/v) MTBE, and 4% (v/v) water for 60 min (method A). To assay ζ-car and β-car, the initial solvent composition consisted of 50% (v/v) methanol, 46% (v/v) MTBE, and 4% (v/v) water followed by a linear gradient of 6% (v/v) methanol, 90% (v/v) MTBE, and 4% (v/v) water for 60 min (method B). zea was assayed by the gradient elution schedule of Rouseff and Raley (1996). The initial composition consisted of 90% (v/v) methanol, 5% (v/v) MTBE, and 5% (v/v) water followed by a linear gradient of 95% (v/v) methanol and 5% (v/v) MTBE for 12 min, 86% (v/v) methanol, and 14% (v/v) MTBE for 8 min, 75% (v/v) methanol and 25% (v/v) MTBE for 10 min, and 50% (v/v) methanol and 50% (v/v) MTBE for 20 min (method C).

The peaks were identified by comparing their specific retention times and absorption spectra with the authentic standards. Standard curves for the carotenoid quantification were prepared with those of the authentic standards at 286 nm for phy, 400 nm for ζ-car, 452 nm for t-vio, c-vio, lut, β-cry, α-car, and zea, and 453 nm for β-car. The carotenoid concentration was estimated by the standard curves and expressed as milligrams per gram fresh weight. Carotenoid quantification was performed in three replicates.

Isolation and Sequence Analysis of Citrus Carotenoid Biosynthetic Genes

Total RNA was extracted from the green and orange flavedos according to the method described by Ikoma et al. (1996). The first strand cDNA was synthesized from 5 μg of the total RNA with the Ready-To-Go T-Primed First Strand Kit (Amersham Bioscience, Little Chalfont, UK). CitPSY, Cit-PDS, CitZDS, CitCRTISO, CitLCYb, CitHYb, CitZEP, and CitLCYe were amplified by PCR with the primers for these cDNAs designed by common sequences that have been reported (Table II). The amplified cDNAs were cloned with a TA Cloning Kit (Invitrogen, San Diego). The sequences were determined using the Taq Dye Primer Cycle Sequencing Kit (Perkin Elmer Applied Biosystems, Foster City, CA).

Total RNA Extraction and Northern-Blot Hybridization

Total RNA was extracted from the flavedos and juice sacs of Satsuma mandarin, Valencia orange, and Lisbon lemon fruits at different growing stages according to the method described by Ikoma et al. (1996). Aliquots of total RNA (10 μg) were separated on 1.2% (w/v) agarose-denaturing formaldehyde gels containing 20 mm MOPS (pH 7.0), 0.5 mm Na-acetate, and 1 mm EDTA. After electrophoresis for 2 h, the RNA was visualized with ethidium bromide under UV light to ensure equal loading of RNA in each lane. The RNA was transferred to nylon membranes (Roche Diagnostics GmbH, Mannheim, Germany) with 20× SSC, and the blots were baked for 3 h at 80°C.

CitPSY, CitPDS, CitZDS, CitCRTISO, CitLCYb, CitHYb, CitZEP, and CitL-CYe were labeled with a DIG RNA labeling kit (Roche Diagnostics GmbH) using T7 or SP6 RNA polymerase to synthesize the RNA probes. The blots were prehybridized at 68°C in a solution containing 5× SSC, 50% (v/v) formamide, 0.1% (w/v) N-lauroylsarcosine, 0.02% (w/v) SDS, and 2% (w/v) blocking regent (Roche Diagnostics GmbH) for 3 h. Hybridization was performed at 68°C in the same solution overnight. After the hybridization, the blots were washed twice at room temperature in 2× SSC and 0.1% (w/v) SDS for 5 min and then washed twice at 68°C in 0.1× SSC and 0.1% (w/v) SDS for 15 min. After the equilibration in buffer A (0.1 m maleic acid and 0.15 mm NaCl [pH 7.5]) for 5 min at room temperature, the blots were blocked with a 2% (w/v) blocking regent (Roche Diagnostics GmbH) in buffer A for 30 min. Subsequently, the blots were incubated with Anti-Digoxygenin-AP Fab fragments (Roche Diagnostics GmbH) in the blocking buffer for 30 min. The blots were washed four times for 8 min each in buffer A containing 0.3% (v/v) Tween 20 and equilibrated with a 0.1 m Tris-HCl buffer (pH 9.5) containing 0.1 m NaCl for 5 min. Chemiluminescence reactions were carried out with CDP-Star (Roche Diagnostics GmbH), a chemiluminescent substrate. Chemiluminescent images of the blots were acquired using a CCD camera (Night Owl LB 981, Berthold Technologies, Bad Wildbad, Germany). The pixel intensities of the bands on the chemiluminescent images were estimated by subtracting the background signals with the software analyzing the band intensity (Phoretix 1D Gel Analysis Software, Nonlinear Dynamics, Newcastle upon Tyne, UK).

Acknowledgments

We are grateful to Dr. Norihiko Misawa (Marine Biotechnology Institute, Iwate, Japan) for the gift of plasmids.

Notes

Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.031104.

1This work was supported by the Research and Development Program for New Bio-industry Initiative of the Bio-oriented Technology Research Advancement Institution.

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