RESEARCH PAPER |
A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck)
1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, PR China
2Guangxi Citrus Research Institute, Guangxi 541004, PR China
* To whom correspondence should be addressed. E-mail: xxdeng{at}mail.hzau.edu.cn
Received 15 August 2007; Revised 2 October 2007 Accepted 8 October 2007
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Abstract |
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A novel, pleiotropic sweet orange (Citrus sinensis L. Osbeck) mutant, ‘Hong Anliu’, is described. This mutation causes carotenoid accumulation, high sugar, and low acid in the fruits. Gas chromatographic analysis revealed that high sugar and low acid in the fruit were caused by the accumulation of sucrose and the deficiency of citric acid. The dominant carotenoid accumulated in albedo, segment membranes, and juice sacs is lycopene, which can reach levels that are a 1000-fold higher than those in comparable wild-type fruits. This mutation does not affect the carotenoid composition of leaves. Carotenoid concentration and biosynthetic gene expression of albedo, segment membranes, and juice sacs were dramatically altered by the mutation. Lycopene accumulation in the juice sacs was regulated by co-ordinate expression of carotenoid biosynthetic genes. However, in albedo and segment membranes, the expression of downstream carotenogenic genes seems to be feedback induced by lycopene accumulation. This implies that there must be at least two modes regulating lycopene accumulation in ‘Hong Anliu’ fruit. Taken together, these results suggest that massive amounts of lycopene might be synthesized in the juice sacs and then transported to the segment membrane and the albedo, which leads to lycopene accumulation there.
Key words: Bud mutation, carotenoid biosynthesis, Citrus, gene expression, lycopene, transport
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Introduction |
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Carotenoids play essential roles in plants as components of the photosynthetic apparatus and protectors against oxidation derived from excess light energy (Demmig-Adams and Adams, 1996). In higher plants they fulfil an additional important purpose as colorants of flowers and fruits. In these tissues, they accumulate in chromoplasts and render bright yellow, orange, or red colours. Carotenoids also serve as precursors of other biologically important compounds (Milborrow, 2001; Fester et al., 2002; Giuliano et al., 2003). Among the carotenoids, lycopene exhibits the highest physical quenching rate constant with singlet oxygen, and in humans the level is slightly higher than that of β-carotene (Dimascio et al., 1989; Lindshield et al., 2007; Roldan-Gutierrez and de Castro, 2007).
Carotenoids are synthesized in plastids by enzymes that are nuclear encoded (Sandmann, 2001). The pathway of carotenoid biosynthesis in plants is illustrated in Fig. 1 (Cunningham and Gantt, 1998; Ronen et al., 1999; Hirschberg, 2001; Sandmann, 2001; Isaacson et al., 2002; Kato et al., 2004). In plants, the C40-carotenoid skeleton is formed by a head-to-head condensation of two molecules of the C20-precursor geranylgeranyl pyrophosphate (GGPP) to form colourless phytoene under the action of phytoene synthase (PSY). Then, phytoene desaturase (PDS) and 
-carotene desaturase (ZDS) catalyse four consecutive desaturation steps to convert phytoene into the red lycopene. Park et al. (2002) and Isaacson et al. (2002) isolated the gene encoding carotenoid isomerase (CRTISO), which catalyses the isomerization of poly-cis-carotenoids to all-trans-carotenoids. The cyclization of lycopene is a branching point in this pathway, yielding 
-carotene with one 
-ring and one β-ring, and β-carotene with two β-rings, in which two cyclases, namely lycopene β-cyclase (LCYb) and lycopene 
-cyclase (LCYe), are responsible for these reactions (Cunningham et al., 1996). -Carotene is converted into lutein by sequential hydroxylations, which are catalysed by -ring hydroxylase and β-ring hydroxylase (HYb). β-Carotene is converted to zeaxanthin via β-cryptoxanthin by two-step hydroxylation, which is catalysed by HYb. Furthermore, zeaxanthin is converted to violaxanthin via antheraxanthin by zeaxanthin epoxidase (ZEP).
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Citrus is an important species for studies of plant carotenoids metabolism, because of the richness of the composition, with
115 kinds of carotenoids (Rouseff et al., 1996). Carotenoid content and composition vary greatly among citrus varieties and also depend on the growing conditions (Gross, 1987). Lycopene is absent in common citrus fruit, but lycopene-accumulating citrus mutants have drawn the attention of researchers for their attractive red pulp colour and their ability to provide protection against carcinogenesis and cardiovascular disease (Clinton, 1998). Mutants with alteration in the carotenoid biosynthetic pathway have proven to be useful experimental materials to identify molecular mechanisms regulating the process (Rodrigo et al., 2003). An interesting mutant is ‘Hong Anliu’ sweet orange (Citrus sinensis Osbeck), which causes carotenoid accumulation, high sugar, and low acid in the fruits. It was discovered in China as a bud mutation of ‘Anliu’ sweet orange. The structure of citrus fruit allows it to be separated into four parts: flavedo, albedo, segment membrane, and juice sacs (Fig. 2). The gene expression of carotenoid biosynthetic enzymes was investigated in these tissues of the citrus fruit. Expression of the PSY gene increased in the peel and juice sacs during the ripening of fruits (Ikoma et al., 2001; Kim et al., 2001). Expression of PDS increased during maturation of the juice sacs but remained constant once fruits were fully developed (Kim et al., 2001; Kita et al., 2001). Recently, the relationship between carotenoid accumulation and the expression of carotenoid biosynthetic genes during fruit maturation of three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.), has been reported, concluding that carotenoid accumulation in citrus is highly regulated by the co-ordinated expression of the different carotenoid biosynthetic genes (Kato et al., 2004). Although significant advances have been made in our understanding of the molecular biology of carotenogenesis in citrus, a clear understanding of the signals and mechanisms that dictate the regulation of carotenogenesis is still lacking.
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In this study, the concentration and composition of carotenoids and the expression of carotenoid biosynthetic genes in the flavedo, albedo, segment membranes, and juice sacs were comparatively analysed for ‘Hong Anliu’ and its wild type (WT) during fruit development and maturation. The expression of carotenoid biosynthetic genes, i.e. PSY, PDS, ZDS, CRTISO, LCYb, LCYe, HYb, and ZEP, was analysed. The concentrations of phytoene, lycopene,
-carotene, lutein, β-carotene, β-cryptoxanthin, and violaxanthin were also determined. The results were the first, to our knowledge, to provide information comparing the profiles of gene expression of carotenoids biosynthetic enzymes in citrus fruit of a lycopene-producing mutant and the original WT. Molecular and biochemical characterization of the mutant may provide new insights into the mechanism causing the lycopene accumulation in this mutant citrus selection. |
Materials and methods |
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Plant materials
‘Anliu’ sweet orange (C. sinensis L. Osbeck) and its red-flesh mutant, ‘Hong Anliu’, cultivated at the Institute of Citrus Research located in Guilin, Guangxi Province, China, were used as materials. Both of them were of the same age, grown in the same orchard, and subjected to standard cultivation. Fruit of each genotype were collected from three different trees, 10 representative fruit from each tree, for a total of 30 fruit per genotype. These samples were collected at five time points from August to December: 120, 150, 170, 190, and 220 days after flowering (DAF). The flavedo, albedo, segment membranes, and juice sacs were separated from sampled fruits, immediately frozen in liquid nitrogen, and kept at –80 °C until analysed.
Analysis of sugar and organic acid
Soluble sugar and organic acid contents and concentration were determined with gas chromatography using 3 g of frozen powder as described in Bartolozzi et al. (1997) with modifications. Three independent extractions were performed per sample.
Carotenoid quantification in citrus fruit
For analysis of carotenoid contents and concentration, samples were ground to a powder in liquid nitrogen. Carotenoids pigments were analysed by reversed phase high-performance liquid chromatography (RP-HPLC) with modification from a previous work (Lee, 2001). Chromatography was carried out with a Waters liquid chromatography system equipped with a model 600E solvent delivery system, a model 2996 photodiode array detection (DAD) system, a model 717 plus autosampler, and an empower Chromatography Manager. Carotenoids were eluted with MeOH–MTBE–H2O [81:15:4 (v/v), eluent A] and MeOH–MTBE–H2O [10:90:4 (v/v), eluent B] by a C30 carotenoid column (150x4.6 mm ID, 3 µm) from Waters (Wilmington, NC, USA). The linear gradient program was performed as follows: initial condition was 100% A to 100% B in 90 min, and back to the initial condition for re-equilibration. Analysis was conducted under subdued light to avoid carotenoid degradation during analysis.
The HPLC grade β-carotene and lycopene standards were obtained from Sigma (St Louis, MO, USA), while phytoene, -carotene, lutein, β-cryptoxanthin, and violaxanthin were obtained from CaroteNature (Lupsingen, Switzerland).
Real-time PCR quantification
Total RNA was extracted from the flavedo, albedo, segment membranes, and juice sacs of ‘Anliu’ and ‘Hong Anliu’ fruits collected at five different development stages according to Liu et al. (2006). Primer pairs (Table 1) were designed with the Primer Express software (Applied Biosystems, Foster City, CA, USA) and following the manufacturer's guidelines for primer design. Isolated RNA was treated with DNase I at 37 °C for 1 h to remove genomic DNA contamination, and first-strand cDNA was then synthesized from the DNase I-treated RNA using the RevertAidTM M-MuLV KIT (MBI, Lithuania). Real-time PCR was performed using the ABI 7500 Real Time System (PE Applied Biosystems, Foster City, CA, USA). Actin was amplified along with the target gene as an endogenous control to normalize expression between different samples. The control primers were: forward 5'-CCAAGCAGCATGAAGATCAA-3' and reverse 5'-ATCTGCTGGAAGGTGCTGAG-3'. The primers for the target gene were designed according to the sequence in GenBank. Specific details of these primers are shown in Table 1. The primers for the target gene and actin were diluted in the SYBER GREEN PCR Master Mix (PE Applied Biosystems) and 15 µl of the reaction mix were added to each well. Reactions were performed by an initial incubation at 50 °C for 2 min and at 95 °C for 1 min, and then cycled at 95 °C for 15 s and 60 °C for 1 min for 40 cycles. Output data generated by the instrument on-board software Sequence Detector Version 1.3.1 (PE Applied Biosystems) were transferred to a custom-designed Microsoft Excel macro for analysis.
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Results |
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Identification of mutant and the phenotypic changes associated with the mutation
The citrus mutant ‘Hong Anliu’ was originally found in an orchard in Guilin City (Guangxi, China) in the 1990s. It occurred spontaneously from the commercial variety of sweet orange ‘Anliu’ [C. sinensis (L.) Osbeck] as a bud mutation. The mutant has been propagated by grafting onto different rootstocks and remained stable under field conditions, and no reversion to the parental phenotype has been found so far. Tree habit, leaf morphology, and agronomical behaviour of the mutant trees were normal and indistinguishable from those of WT trees.
No acidity could be tasted in either the young or mature fruit of ‘Hong Anliu’, which is quite different from the situation in WT and most other sweet oranges. A more obvious change due to the mutation is the inner colour of fruit. Whereas the albedo, segment membranes, and juice sacs of mature WT fruit were slightly orange or yellow coloured, those of ‘Hong Anliu’ were pink-red (Fig. 3A, B). Both fruits are seedy, and mature in late November.
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The carotenoid concentrations of leaves were analysed by HPLC in ‘Hong Anliu’ and WT. Little difference in carotenoid concentration or composition was detected in the leaves (Fig. 4), with the major carotenoids identified present in both genotypes. Lutein was present in the highest concentration, and was higher in the WT than in the mutant, while the concentrations of
-carotene, β-carotene, and violaxanthin were almost the same in both genotypes.
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A comparison of organic acid and soluble sugar concentrations between ‘Hong Anliu’ and WT
Mature fruits of ‘Hong Anliu’ and WT, collected at five time points, were separated into four parts: flavedo, albedo, segment membrane, and juice sac. Gas chromatography was used to determine the concentrations of malic acid, citric acid, quinic acid, fructose, glucose, and sucrose in all the four parts of both ‘Hong Anliu’ and WT fruits.
The most obvious difference in organic acid between ‘Hong Anliu’ and WT was citric acid content (Fig. 5). In segment membrane and juice sacs, the citric acid content of ‘Hong Anliu’ was about a quarter of that of the WT. The concentrations of malic acid and quinic acid were almost at the same level, except that the malic acid content of WT in the flavedo and albedo was slightly higher than that in ‘Hong Anliu’. In the juice sac of ‘Hong Anliu’, the concentration of malic acid was higher than that of citric acid, which is in contrast to the finding that citric acid is the dominant acid in most citrus fruit juice.
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In flavedo and juice sac, the concentration of all the three identified soluble sugars was higher in ‘Hong Anliu’ than in WT. In albedo, the content of fructose and glucose was lower in ‘Hong Anliu’ than in WT, and the sucrose content was the same in the two genotypes. The concentration of fructose and glucose was at the same level in the segment membrane of ‘Hong Anliu’ and WT. Yet, the sucrose content of ‘Hong Anliu’ was much higher in the segment membrane than that in the WT.
Carotenoid concentration and biosynthetic gene expression profile in the flavedo
The colour of the flavedo changed from green to orange during fruit maturation. The green stages in ‘Hong Anliu’ and ‘Anliu’ were from August to September (120–150 DAF). To characterize the differences in carotenoid composition and content in ‘Hong Anliu’ versus WT, HPLC was performed (Table 2). The effect of the mutation on carotenogenic gene expression was examined by real-time PCR. Citrus cDNAs encoding PSY, PDS, ZDS, CRTISO, LCYb, LCYe, HYb, and ZEP were used as probes.
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The total carotenoid concentration of ‘Hong Anliu’ and WT was almost the same during fruit maturation (Fig. 6). During the green stage (120–150 DAF),
-carotene (<4.0 µg g–1 FW), β-carotene (<9.1 µg g–1 FW), lutein (<23.4 µg g–1 FW), and violaxanthin (<7.8 µg g–1 FW) were predominant in both ‘Hong Anliu’ and WT; phytoene, lycopene, and β-cryptoxanthin were barely detected in either genotype. At the same time, the expression of all the carotenogenic genes (PSY, PDS, ZDS, CRTISO, LCYb, LCYe, HYb, and ZEP) was low in both genotypes.
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After the green stage, the concentration of
-carotene, β-carotene, and lutein decreased with a concomitant decrease in the expression of the LCYe gene in ‘Hong Anliu’ and WT. In contrast, β-cryptoxanthin and violaxanthin massively accumulated in both genotypes. Violaxanthin became abundant in December (8.5 µg g–1 FW in ‘Hong Anliu’ and 33.2 µg g–1 FW in WT). With the transition of the peel colour from green to orange, the expression of the genes for PSY, PDS, ZDS, CRTISO, LCYb, HYb, and ZEP, which make up the necessary set of genes to produce β, β-xanthophylls, increased to maximum levels or remained high in both genotypes (Fig. 6). Lycopene (1.6 µg g–1 FW) was detected in ‘Hong Anliu’ in December. At this time, the expression of upstream carotenogenic genes was much higher in ‘Hong Anliu’ than in WT, while the LCYe expression level of both genotypes was at the same level.
Carotenoid concentration and biosynthetic gene expression profile in the albedo
At the green stage (120–150 DAF), the concentration of total carotenoids in the albedo was low in both ‘Hong Anliu’ and WT (Fig. 7). A trace amount of lycopene was detected in ‘Hong Anliu’, yet no lycopene was detected in ‘Anliu’ at that stage. The concentrations of β-carotene and violaxanthin were higher in WT than in ‘Hong Anliu’. The expression of PSY and PDS decreased in WT yet remained constant in ‘Hong Anliu’. The expression of ZDS and LCYe increased in WT yet decreased in ‘Hong Anliu’.
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After the green stage, dramatic accumulation of carotenoids occurred in both ‘Hong Anliu’ and WT, and the increase in total was much greater in ‘Hong Anliu’. WT albedo contained such a low level of lycopene that it was difficult to quantify accurately. In contrast, the comparable tissues of ‘Hong Anliu’ were found to contain nearly 22.82 µg g–1 FW lycopene, a level several hundred-fold higher than that detected in WT tissues. The concentrations of
-carotene and lutein decreased in both ‘Anliu’ and ‘Hong Anliu’. However, the amounts of violaxanthin and β-carotene increased or remained at a high level in both genotypes. The concentration of β-cryptoxanthin increased in WT, yet was undetectable in ‘Hong Anliu’. However, phytoene accumulated in ‘Hong Anliu’, but not in WT. In both ‘Anliu’ and ‘Hong Anliu’, the gene expression level of PSY, PDS, and HYb increased after the green stage, while the expression of ZDS, CRTISO, and ZEP rose to a maximum level and subsequently decreased. The expression levels of upstream carotenogenic genes (PSY, PDS, and CRTISO) between ‘Hong Anliu’ and WT were almost the same. However, the gene expression of LCYb and LCYe was much higher in ‘Hong Anliu’ than in WT.
Carotenoid concentration and biosynthetic gene expression profile in the segment membrane
At the green stage (120–150 DAF), ‘Hong Anliu’ accumulated predominantly lycopene (<1.8 µg g–1 FW) in the segment membrane, which accounted for 70.3% of the total identified carotenoids (Fig. 8). Yet no lycopene was detected in WT. The concentration of lutein and violaxanthin increased, while -carotene decreased in both ‘Anliu’ and ‘Hong Anliu’. The concentration of β-carotene increased in ‘Hong Anliu’, yet decreased in ‘Anliu’. Clearly, the gene expression of PSY, CRTISO, LCYb, HYb, and ZEP increased in both genotypes. The expression levels of these genes were also higher in WT than in ‘Hong Anliu’ at the time point of 150 DAF. However, the expression of LCYe was higher in ‘Hong Anliu’ than in WT.
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After the green stage, phytoene, lycopene, β-cryptoxanthin, and violaxanthin accumulated noticeably, although the concentrations of β-cryptoxanthin and violaxanthin were very low compared with that of lycopene. In December, ‘Hong Anliu’ accumulated predominantly lycopene and phytoene (29.2 µg g–1 and 12.4 µg g–1), which accounted for 66.3% and 28.2% of the total identified carotenoids, respectively. However, only a trace amount of lycopene and phytoene was detected in WT. The expression profiles of PSY, PDS, ZDS, and CRTISO were very similar between ‘Hong Anliu’ and WT. Although the gene expression of LCYb increased in both ‘Hong Anliu’ and WT, the increased level was much higher in ‘Hong Anliu’. The expression of the LCYe gene remained constantly high in ‘Hong Anliu’ compared with WT.
Carotenoid concentration and biosynthetic gene expression profile in the juice sac
During the green stage (120–150 DAF), the concentration of total carotenoids was low in the juice sacs, compared with those of albedo and membrane, in both ‘Hong Anliu’ and WT (Fig. 9), but the concentration of total carotenoids in ‘Hong Anliu’ was still higher than that in WT. Violaxanthin and lutein made up the major carotenoids identified in both ‘Hong Anliu’ and WT. Lycopene accumulated noticeably in ‘Hong Anliu’, although the concentration was low. No lycopene was detected in WT. The gene expression of upstream genes leading to the synthesis of lycopene, PSY, PDS, ZDS, and CRTISO, was higher in ‘Hong Anliu’ than in WT, whereas the expression of LCYb was lower in ‘Hong Anliu’ than in WT.
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After the green stage, the concentration of total carotenoids increased rapidly in both ‘Hong Anliu’ and WT. The increase in ‘Hong Anliu’ was caused by the accumulation of lycopene (from 0.09 µg g–1 to 2.35 µg g–1), whereas in WT, it was caused by the accumulation of β-cryptoxanthin and violaxanthin (from an undetectable level to 2.28 µg g–1 and from 0.99 µg g–1 to 4.63 µg g–1, respectively). The concentration of lutein remained at a relatively high level in both ‘Hong Anliu’ and WT. Thus, lycopene, lutein, β-cryptoxanthin, and violaxanthin, which account for 31.6, 31.5, 11.7, and 25.3% of the identified carotenoids, respectively, made up the major carotenoids in ‘Hong Anliu’. In the WT, however, lutein (8%), β-cryptoxanthin (30%), and violaxanthin (60%) were the main carotenoids. The expression of the set of genes needed to produce β, β-xanthophylls (PSY, PDS, ZDS, LCYb, and ZEP) increased, reaching a maximum level in December in both ‘Hong Anliu’ and WT. Moreover, the expression levels of PSY, PDS, and ZDS were much higher in ‘Hong Anliu’ than in WT. At the same time, the expression of LCYe decreased rapidly in ‘Hong Anliu’.
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Discussion |
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‘Hong Anliu’, which exerts a profound effect on temporal and spatial patterns of carotenoid accumulation, is a spontaneous mutation isolated from ‘Anliu’ sweet orange. The mutation confers a phenotype that is regulated in a fruit-specific pattern, with albedo, segment membrane, and juice sac exhibiting obvious red colour in the mutant. The distinctive red colour in fruit has clearly been shown to be due to the massive accumulation of lycopene. The fruits of other red-fleshed citrus mutants, e.g. Cara Cara navel orange (C. sinensis L. Osbeck), ‘Fengdu’ pummelo (C. grandis Osbeck and C. maxima Merr.), ‘Guanxi’ sweet pummelo, Chuzhou Early Red pummelo, Ruby Red grapefruit, and Star Ruby grapefruit were also previously reported to accumulate lycopene (Lee, 2001; CJ Xu et al., 2006; J Xu et al., 2006). However, expression of carotenoid biosynthetic genes was not comparatively examined in these mutants and their WTs. Moreover, according to the present examination, the albedo and segment membrane of Cara Cara fruit are not red (data not shown), which is different from the case of ‘Hong Anliu’ sweet orange.
Carotenoid accumulation is the net result of several processes, including those that determined the level of GGPP available to the pathway, the catalytic activity of the pathway, and the storage of the synthesized carotenoids (Li et al., 2001). Since multiple pathways are directly or indirectly associated with each of these processes, it provides many hypothetical regulation points for the mutation. Previous research has identified certain steps in carotenoid biosynthesis which appear to be important rate-limiting steps (Fraser et al., 1994; Lois et al., 2000). Based on a low content of total carotenoids in the fruit of WT, the lycopene accumulation caused by the mutation, and the increased total carotenoids in the ‘Hong Anliu’, it was speculated that the mutation might be up-regulating expression of a gene for one of the previously identified flux control points. The expression profile of carotenoid biosynthetic genes in the juice sacs has proved this hypothesis. The upstream genes in the carotenoid biosynthetic pathway (PSY, PDS, ZDS, and CTRISO) exhibited an obvious increase in expression in the juice sacs of ‘Hong Anliu’ compared with WT, indicating that the mutation exerted a major effect on carotenoid accumulation via modification of the level of transcription. The cyclization of lycopene is a key branch point in the pathway of carotenoid biosynthesis in higher plants, in which LCYe and LCYb are key enzymes for the lycopene cyclases (Cunningham et al., 1996). The expression of LCYe decreased rapidly in the juice sac of ‘Hong Anliu’ during fruit ripening, which leads to the lycopene accumulation in the juice sacs. In tomato fruit, Li et al. (2001) concluded that the induction of lycopene accumulation coincided with increased expression of upstream carotenogenic genes and reduced expression of genes downstream of lycopene synthesis. Thus, the mechanism regulating the lycopene accumulation in this report seems to be consistent with that of tomato.
However, in albedo and segment membrane, our analyses did not reveal that the expression of upstream carotenogenic genes exhibited a dramatic increase in ‘Hong Anliu’ compared with WT, indicating that the mechanisms regulating lycopene accumulation in albedo and segment membrane are different from that in the juice sac. This result implies that there must be at least two different modes regulating lycopene accumulation in ‘Hong Anliu’ fruit. The carotenoid synthesis pathway may also be regulated by feedback inhibition by end-products (Bejarano and Cerdaolmedo, 1989; Bramley, 2002). In the albedo and segment membrane, the expression of upstream carotenogenic genes did not increase, yet the expression of downstream carotenogenic genes increased in ‘Hong Anliu’ compared with WT. This cannot lead to the accumulation of lycopene in ‘Hong Anliu’, which is in contrast to the fact that lycopene is the dominant carotenoid in the albedo and segment membrane of ‘Hong Anliu’. As carotenoids are formed in plastids, it is likely that exchanges of cytoplasmic and plastidic metabolites occur (McCaskill and Croteau, 1998). Thus, the lycopene that accumulated in the albedo and segment membrane might be transported from another part of the fruit instead of being synthesized by these tissues.
It is hypothesized that the lycopene is transported from juice sac to albedo and segment membrane, which would accommodate three of our observations. The first is the lack of congruence between carotenogenic gene expression and carotenoid accumulation in albedo and segment membrane of WT. The second is the higher sucrose content in the juice sac of ‘Hong Anliu’ compared with WT as sufficient sucrose can promote lycopene accumulation (Telef et al., 2006). The third is that the basal part of the juice sac of ‘Hong Anliu’ where it connects to the segment membrane is red, but the remainder of the juice sac is yellow (Fig. 2C). Taken together, these observations suggest that the large amount of lycopene that accumulated in albedo and segment membrane is synthesized in juice sacs and then transported to albedo and segment membrane, leading to the feedback regulation of carotenogenic genes there. However, conclusive proof of this hypothesis requires further characterization of the mutation.
This study is the first in-depth examination of the biochemical and molecular alterations associated with a red-fleshed fruit mutation in citrus. Additional details on this mutation's actions are expected to provide new insight into the regulation of lycopene accumulation.
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Acknowledgements |
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We would like to thank Professors Carol Lovatt and Jihong Liu for critical reading of this manuscript, and Mrs Hongyan Zhang for technical support. This work was supported by the National Science Foundation of China (NSFC Grant nos 30471201 and 30570973), the Ministry of Science and Technology of China (863 Project and 973 Project, 2006CB708202), and the Ministry of Education of China (IRT0548).
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