JXB Advance Access originally published online on August 28, 2007
Journal of Experimental Botany 2007 58(12):3135-3144; doi:10.1093/jxb/erm132
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RESEARCH PAPER |
A comparison of the carotenoid accumulation in Capsicum varieties that show different ripening colours: deletion of the capsanthin-capsorubin synthase gene is not a prerequisite for the formation of a yellow pepper
1National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Republic of Korea
2Department of Crops Biotechnology, Jinju National University, Jinju 660-758, Republic of Korea
* To whom correspondence should be addressed. E-mail: shha{at}rda.go.kr
Received 16 April 2007; Revised 21 May 2007 Accepted 23 May 2007
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Abstract |
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Ripe pepper (Capsicum sp.) fruits can display a range of colours from white to deep red. To understand better the regulatory mechanisms of the carotenoid biosynthetic pathways that underlie these ripening colours, Capsicum varieties that show seven different fully ripe colour types were analysed. The levels and composition of the carotenoid accumulation in these samples at different stages of ripening were measured, and the resulting data were analysed in conjunction with the expression patterns of the carotenoid biosynthetic genes. It was found that red peppers accumulate increasing levels of total carotenoids during ripening, whereas non-red peppers accumulate lower levels of total carotenoids of varying composition. The expression levels of the phytoene synthase, phytoene desaturase, and capsanthin-capsorubin synthase (Ccs) genes are high in peppers with high levels of total carotenoid, whereas one or two of these genes are not expressed in peppers with lower levels of total carotenoid. Surprisingly, it was found that the Ccs gene is present in two Capsicum varieties whose ripe colour is yellow. This gene has never previously been shown to be present in yellow peppers. Sequence analyses of the Ccs gene further revealed two structural mutations in yellow peppers that may result in either a premature stop-codon or a frame-shift. Taken together with the fact that the Ccs transcript is not detectable in yellow peppers, our current results suggest that nonsense-mediated transcriptional gene silencing of Ccs and not the deletion of this gene is responsible for yellow ripening in Capsicum.
Key words: Capsanthin-capsorubin synthase, capsanthin, carotenoid, pepper (Capsicum sp.), promoter
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Among the different species of crops, the pepper (Capsicum sp.) harbours the most evolved carotenoid biosynthetic pathway (Fig. 1). Although capsanthin and capsorubin are the key components that cause the typical red colour during the ripening process in the pepper, there are many other types of carotenoids that can accumulate in pepper fruits (Deli et al., 2001; Maoka et al., 2001). In addition to fruit colour changes during ripening, which are due to alternations in carotenoid composition, diverse ripe colours from white to deep red can also be found across the different pepper varieties. Pepper fruits are therefore a good model system to study the regulatory mechanisms underlying carotenoid biosynthesis.
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-carotene desaturase; β-LCY, lycopene-β-cyclase; In plants, carotenoids play indispensable roles in light harvesting, photo-protection, and as precursors for abscisic acid (ABA) synthesis. Plants attract insects and other animals to act as pollinators and vehicles of seed dispersion by using stimuli generated by carotenoid pigments, including red, orange, and yellow, which are present in fruits and flowers (Bartley and Scolnik, 1995). Carotenoids have also been used for many years as natural colorants in both animal and human foodstuffs, and some of the β-carotenoids are essential components of the human diet as pro-vitamin A. Moreover, some carotenoid components have been shown to have important biological functions as antioxidants and free-radical scavengers, which can reduce the risk of cancer (Hornero-Méndez et al., 2000; Hirschberg, 2001; Maoka et al., 2001).
Many of the genes that are involved in carotenoid biosynthesis have now been cloned from several plants including Arabidopsis, tomato, pepper, daffodil, and marigold (DellaPenna and Pogson, 2006). These carotenogenic genes are developmentally regulated and also directly connected with the colour phenotype of these plants, as shown in tomato fruits and merigold petals (Ronen et al., 1999, 2000; Moehs et al., 2001). These studies have demonstrated that carotenoid accumulation is mainly controlled by the transcriptional regulation of carotenoid biosynthetic genes.
Pepper fruit is one of the oldest and most widely used natural food additives. The typical red colour found in Capsicum is derived from the capsanthin and capsorubin components that are exclusively synthesized and accumulated during fruit ripening in this genus (Mínguez-Mosquera and Hornero-Méndez, 1994). Hence, red cultivars of Capsicum have been the focus of a number of studies over many years on the composition of carotenoid pigments (Cholnoky et al., 1955; Deli et al., 2001).
The quantitative and qualitative changes in the carotenoid profile in red paprika have previously been compared with both yellow and black paprika varieties (Matus et al., 1991; Deli et al., 1992). In addition, early efforts to identify the factors that determine the colour of ripening have also been made in Capsicum using genetic approaches (Kormos and Kormos, 1960; Hurtado-Hernandez and Smith, 1985). Since diverse colour phenotypes in the F2 segregation from a cross between white and red pepper fruits were observed, three independent loci, y, c1, and c2, were proposed as putative genes that determine fruit pigmentation. Among these candidates in the pepper, a single dominant gene corresponding to the y locus was subsequently determined to be the capsanthin-capsorubin synthase (Ccs) gene, the deletion of which results in a yellow ripening phenotype (Lefebvre et al., 1998). More recently, the orange fruit colour in Capsicum was also found to result from the absence of the Ccs gene (Popovsky and Paran, 2000; Lang et al., 2004). Popovsky and Paran further demonstrated that this orange colour might originate from two possible genotypes that are either dominant or recessive for Ccs (Popovsky and Paran, 2000).
To delineate the relationship between carotenoid accumulation and the expression of carotenoid biosynthetic genes in the pepper further, Capsicum varieties were selected that show different ripening fruit colours to analyse these properties. These results show that the number of genes that are active during ripening appears to correlate with the total carotenoid levels. It is also reported here for the first time that the Ccs gene is present in two Capsicum varieties whose ripening colour is yellow, which differs from previous reports about other yellow peppers where the Ccs gene was found to be deleted.
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Materials and methods |
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Plant materials
Capsicum varieties were selected from the Genebank of the National Institute of Agricultural Biotechnology (Rural Development Administration, Suwon, Korea) on the basis of their different colour types in the fully ripe fruit. Their introduction numbers (IT) are as follows: 158770 (C. baccatum var. pendulum, LR2), 158773 (C. chinense, R2), 158782 (C. annuum, W), 158806 (C. baccatum, LR1), 163499 (C. annuum, DR), 164918 (C. chinense, Y3), 191655 (C. chinense, PYO), 203499 (C. chinense, O), 709424 (C. pubescens, R1), 800065 (C. chinense, Y2), and 800070 (C. baccatum, Y1) (see Supplementary Fig. 1 at JXB online for phenotypic features and general Capsicum information). A commercial cultivar (C. annuum cv. Nockwang, R3) was also included in these analyses, which was purchased from HungNong Seeds Company (Seoul, Korea). All 12 pepper varieties chosen for this study were grown in the field and the fruit samples were harvested at fully expanded stages where differences in their respective colours during ripening were evident. At this time, the fruit colours were also visually recorded.
Extraction of carotenoid pigments and quantification by HPLC
Pepper fruits that had been harvested at different ripening stages for each pepper variety were homogenized under liquid N2, weighed after freeze-drying, and subjected to carotenoid extraction as described previously (Mínguez-Mosquera and Hornero-Méndez, 1993). After saponification, the organic layer of the extracts was vacuum-dried and then analysed by HPLC. The HPLC system consisted of a HP 1050 auto-sampler, a gradient pump, a column thermo-regulator (Mistral, Spark, The Netherlands), and a C-30 carotenoid column (5 µm, 25x4.6 mm, YMC Europe, Schermbeck, Germany) equipped with a pre-column (Nucleosil 5 µm-C18, 10x4.6 mm, Bischoff, Leonberg, Germany). The data were acquired using HP3D-chemstation software (Hewlett-Packard). As authentic standards for the qualitative and quantitative analyses of carotenoids, 
-carotene (β, 
-carotene) and β-carotene (β, β-carotene) were purchased from Sigma-Aldrich Company (St Louis, Missouri, USA). Lutein [(3R, 3'R, 6'R)-β, -carotene-3, 3'-diol], zeaxanthin [(3R, 3'R)-β, β-carotene-3, 3'-diol], β-cryptoxanthin [(3R)-β, β-caroten-3-ol], and capsanthin [(3R, 3'S, 5'R)-3, 3'-dihydroxy-β, 
-carotene-6'-one] were purchased from Extrasynthese SA (Genay, Cedex, France). Total carotenoid levels were calculated as the sum of the six carotenoids -carotene, lutein, β-carotene, β-cryptoxanthin, zeaxanthin, and capsanthin.
Northern blotting
Total RNAs were extracted from the same fruit samples used in the HPLC analysis. The cDNAs for phytoene synthase (Psy), phytoene desaturase (Pds), β-carotene hydroxylase (Bch), and the Ccs genes were amplified from Korean red pepper (C. annuum cv. Nockwang) for use as probes in northern blot analysis (Ha et al., 1999). Hybridization was then carried out overnight at 65 °C in 0.5 M Na2PO4 (pH 7.2) buffer, 1% bovine serum albumin fraction V and 7% SDS with the 32P-labelled probes. After membranes were rinsed in 2x SSC solution, and then washed once in 1x SSC solution at 65 °C for 15 min, and finally in 1x SSC and 0.1% (w/v) SDS at 65 °C, they were exposed on the X-ray film to develop the signals.
PCR and sequencing of genomic DNA
Genomic DNA extracts were prepared from the ripe fruits of each of the pepper varieties under study using a modified cetyltriammonium bromide (CTAB) method. PCR was then performed using Super taq PLUS (SuperBio, Suwon, Korea) with 100 ng of each DNA template and 10 pmol of gene-specific primers for geranylgeranyl pyrophosphate synthase (Ggps), Psy, and Ccs (Ha et al., 1999), and also the promoter region of the Ccs genes (forward primer/reverse primer; 5'-TTGAACCTCCTTGATAAAA-3'/3'-AGGTAGAGGAAATGAAAGG-5'). The PCR conditions were: 95 °C for 10 min, and then 30 cycles of 95 °C for 15 s, 58 °C for 15 s, and 72 °C for 1 min. PCR fragments were then subcloned into a reading frame A cassette of the Gateway vector conversion system through pDONA201 by BP and LR recombination (Invitrogen, Carlsbad, CA) and further sequenced with a Big Dye terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA). Analyses of DNA and protein sequences were performed using the Lasergene (DNASTAR, Madison, WI) and BioEdit programs (v7.0.5, Carlsbad, CA).
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Results |
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Assessment of the accumulation of carotenoids at the fully ripe stage in Capsicum fruits
The Capsicum fruit colour phenotype is determined by both the amount and composition of the carotenoids that have accumulated at the chromoplasts. Fruits of different Capsicum varieties showing a diversity of mature colours that includes deep red (DR), red (R), light-red (LR), orange (O), pale-yellow orange (PYO), yellow (Y), and white (W), were analysed by HPLC to compare the total levels and composition of their carotenoids at the fully ripe stage.
As shown in Fig. 2, in pepper varieties whose ripening colour is red, the DR pepper accumulates the highest level of total carotenoids (1043 mg kg–1 dry weight). Three R peppers (R1–3) accumulate total carotenoids at lower levels compared with the DR pepper, but at higher levels than the two LR peppers of LR1 and LR2. Interestingly, in Fig. 3, it was found that for pepper varieties whose ripe colour is non-red, both O and PYO peppers accumulate total carotenoids at lower levels than the three Y peppers (Y1–3). No carotenoids are detectable in ripe W pepper.
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All of the ripe pepper fruits, with the exception of the W and Y (Y1-3) peppers, were found to accumulate capsanthin at various levels. These capsanthin levels are decreased in ripened fruits with a lighter red pigmentation in the order DR, R, LR, O, and PYO, suggesting that they directly correlate with the red colour-saturation in these pepper varieties. Capsanthin was found to be the major component (80%) of the total carotenoids in the ripe fruits of red peppers. The levels of other β-carotenoids including β-carotene, β-cryptoxanthin, and zeaxanthin varied from 1.5% to 16%, and that of lutein was never detected above 0.3% of the total carotenoid content in most of the red peppers. In terms of the main carotenoid content, the O pepper was a surprising exception among pepper varieties as it displayed capsanthin and lutein levels of 39% and 48%, respectively. Similarly high lutein levels were detected in the Y1, Y2, and Y3 varieties with levels of 40.5%, 49.8%, and 67%, respectively, whereas capsanthin in undetectable in yellow peppers. Alpha-carotene was detectable only in the Y1–3 samples showing a mature colour and was found to be present at levels ranging from 10.6% to 13.3% of the total carotenoids. These results thus indicate that the non-red peppers, O and Y, accumulate
-carotenoids such as lutein and -carotene at higher levels and maintain total carotenoids at lower levels than red peppers (see Supplementary Fig. 1 at JXB online for the detailed carotenoid composition of each pepper variety).
Changes to the carotenoid composition during the ripening of Capsicum fruits
To examine whether changes in fruit colour in the pepper can be correlated with changes in the carotenoid composition of this fruit, the samples were harvested at different stages of ripening and their carotenoid contents were analysed by HPLC. In the fully expanded non-ripe pepper fruits (commonly S1 stage in all varieties), the total carotenoid profile mainly comprises the chlorophyll-dependent carotenoids such as lutein and β-carotene. These compounds were detectable at their highest levels of (11.8 mg kg–1 and 7.3 mg kg–1 dry weight, respectively) in the Y2 pepper that showed the deepest green colour at maturation. By contrast, the W and LR1 varieties that do not show any green colour resulting from chlorophyll contain much lower levels of lutein (1.28 mg kg–1and 0.35 mg kg–1 dry weight, respectively).
As shown in Fig. 2, the total carotenoid levels increase with ripening in the red Capsicum varieties and were found to increase by 11–92-fold in the order LR1, LR2, R1, R2, R3, and DR during this process. These increases in the total carotenoid levels are principally due to the increased capsanthin levels during ripening. In the four red pepper samples LR1, LR2, R2, and R3, the levels of all of the β-carotenoids tested, including capsanthin, steadily increase until the final stages of ripening. The accumulation of capsanthin in R1 and DR is also similar to these other four red varieties, reaching maximum levels of 180.7 mg kg–1 and 1013.2 mg kg–1 dry weight, respectively, at their fully ripe stages. The accumulation of other β-carotenoids in R1 and DR differs, however, as it reaches a maximum level just before the fully ripe stage and then decreases from 12.7 mg kg–1 to 5.0 mg kg–1 dry weight for R1, and from 49.4 mg kg–1 to 27.9 mg kg–1 dry weight for DR.
By contrast with the red peppers, the total carotenoid levels in the W, Y, PYO, and O peppers do not increase significantly during ripening (Fig. 3). In the W pepper, the lutein and β-carotene levels that are present at the early stages of ripening were found to have completely disappeared at the fully ripe stage. In the three Y peppers, the levels of lutein that are present at relatively high levels before ripening are maintained during ripening. In addition, -carotene was found to be produced de novo and β-carotene was observed to be depleted in order to synthesize other β-carotenoids, without affecting the total carotenoid levels during ripening in the Y samples.
Of the two non-red varieties that accumulate capsanthin (O and PYO) the PYO pepper showed a small increase in its total carotenoid content (1.4-fold) with evidence of capsanthin accumulation de novo. In the O pepper, the total carotenoid levels had slightly decreased in parallel with the reduction of β-carotene during ripening. Beta-carotene appears to be mainly converted into capsanthin and small amounts of zeaxanthin at the fully ripe stage in the O pepper (see Supplementary Fig. 1 at JXB online for a detailed description of the carotenoid composition of each pepper variety).
The carotenogenic gene expression profile in ripening peppers of different colours
To investigate whether the observed differences in carotenoid accumulation in our pepper sample's cohort correlated with the expression profile of the carotenoid biosynthetic genes, northern blotting of the same samples used in the HPLC analysis was performed (Fig. 4). Transcripts for Psy, Pds, Bch, and Ccs were barely detectable at the fully expanded non-ripe stage (S1) in any of these varieties. The expression of these genes begins to be induced at the onset of ripening at different levels in each variety, except for the W pepper that shows no expression of any of these genes at the ripening stage as expected.
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At least two, and sometimes three of these four genes are expressed in the Y, PYO, and LR1 peppers that accumulate low levels of total carotenoids (below 28 mg kg–1 dry weight). By contrast, all four genes are expressed in the five R pepper samples (LR2, R1, R2, R3, and DR) that accumulate high total carotenoid levels (greater than 163 mg kg–1 dry weight). Interestingly, the expression patterns of these four genes are similar in LR2, R1, and R2, which accumulate similar levels of total carotenoid (163, 186, and 226 mg kg–1 dry weight), with a strong induction of Psy evident during ripening. The expression levels of these genes are higher in R3 and DR, which is consistent with their higher levels of total carotenoids (400 mg kg–1 and 1043 mg kg–1 dry weight), and a more dramatic induction of Ccs expression is observed in these varieties compared with the other genes. Hence, these results reveal that the activity of each of the four carotenoid biosynthetic genes tested is required to achieve high levels of carotenoid accumulation. Moreover, the strong induction of the Psy and Ccs genes appears to be required during ripening in red peppers.
Structural variations in the carotenogenic genes among peppers of different ripening colours
Any alteration in either the coding and/or promoter region of a gene can result in a reduction or even a failure in expression. Since the expression levels of the carotenoid biosynthetic genes were found to correlate closely with the total carotenoid accumulation, the genomic sequences of several of these genes were examined to determine whether defects in the gene structure could account for different ripe-colours in Capsicum. As shown in Fig. 5, genomic PCR analysis of the different pepper samples reveals that the Ggps gene is equivalently sized in all of the varieties tested in this study. This is also the case for the Psy gene with the exception of the W pepper. Both the coding and promoter regions of Ccs are absent from the genomes of the W and Y1 peppers, but are present in the remaining varieties.
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The coding region of the Ccs gene has previously been shown to have the same 1.5 kb size in the bell pepper (Bouvier et al., 1994) that was observed in this study's samples, but the promoter regions of this gene differ between Capsicum varieties and can be grouped into three patterns; 920/922 bp, 998/999 bp, and 1175 bp. Interestingly, both the coding and promoter regions of the Ccs gene are present in the genomes of two of the Y varieties in the sample cohort, Y2 and Y3 (Fig. 5), both of which share a yellow ripe-colour and a similar carotenoid composition with the Y1 pepper, that is lacking in capsanthin. To our knowledge, this is the first evidence of a yellow ripe-colour pepper that harbours the Ccs coding region.
As shown in Fig. 6A, multiple alignments of the nucleotide sequences of the Ccs promoter regions among the different pepper varieties in this study's samples have identified three kinds of insertions/deletions, in addition to several minor variations, when compared with the previously reported sequence for the bell pepper (Bouvier et al., 1998). The 11 bp-deletion and 88 bp-insertion that were identified in these analyses occur in seven of the varieties tested, but not in three Capsicum annuum species. A 176 bp-insertion is observed in all four C. chinense species except for the O pepper, which in fact shows a higher Ccs promoter sequence homology with the C. baccatum species and is quite closely related to this genus in the phylogenic tree (Fig. 6B). It is concluded from these data, therefore, that differences in the sequence of the Ccs gene promoter region reflect species-specific variations in Capsicum (see Supplementary Fig. 2 at JXB online for the detailed sequence comparison data).
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Multiple alignments of the nucleotide sequences from the Ccs coding regions were also performed and the presence of two structural mutations in the Y2 and Y3 peppers was found (Fig. 7; see Supplementary Fig. 3 at JXB online for the detailed sequence alignment data). One of these mutations results in a frame-shift, thus causing early translation-termination, via an 8 bp-insertion at position 1431 of the gene in Y2. The Ccs mutation in Y3 causes a premature stop-codon via a single base change at position 599. It is interesting that both of these mutations occur in peppers where the Ccs gene is present but which have a yellow ripe-colour.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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In general, carotenoid accumulation does not appear to increase until a particular fruit is fully expanded. In the fruits of the red Capsicum, the concentration of lutein, a major chloroplast pigment, decreases during the ripening process from its highest level during the non-ripe mature stage, whereas the levels of β-carotene gradually increase and other β-carotenoids including β-cryptoxanthin, zeaxanthin, and capsanthin begin to be synthesized de novo. As a result of this, the total carotenoid levels in red pepper varieties increase as they ripen. In the present study, it was observed that the increases in the total carotenoid content during ripening in red pepper varieties are highly variable (from 11–92-fold) and are dependent on the ripe colours (Fig. 1). Such a large variation is not unexpected because the red varieties sampled for this study were selected on the basis of seven different ripe colours.
In non-red pepper fruits, in contrast, the total carotenoid levels do not increase and remain low during ripening. Moreover, the composition of the total carotenoids in non-red pepper fruits significantly differs from red pepper fruits where more than 80% of this total is capsanthin. In particular, the lutein content of the Y peppers is far higher than any of the R peppers at the non-ripe stages and remains high, at 41–67% of the total carotenoids, in fully ripe Y peppers. Based on these results, this study's pepper varieties could be categorized into two groups: one showing an accumulation of increasing levels of total carotenoids, and one that maintains low levels of carotenoids that vary in composition. The fact that all red peppers seem to accumulate increasing amounts of total carotenoids with a large proportion of capsanthin suggests that capsanthin production is mainly responsible for this increase. Accordingly, the structure and expression levels of the Ccs gene could be considered to be the most important determinant of whether high levels of total carotenoids are produced in the pepper.
All non-red peppers were found to accumulate limited amounts of total carotenoids regardless of whether capsanthin accumulation occurred (Fig. 3). In the case of the O pepper, which shows low amounts of total carotenoid, it is possible that an alteration in the expression of the Psy gene causes the orange-ripening phenotype in spite of the capsanthin accumulation, as described previously (Huh et al., 2001). Huh et al. (2001) reported here that the orange pepper (C. chinense cv. Habanero) produces aberrant Psy transcripts by abnormal splicing, resulting in a reduced activity of PSY.
The present results are consistent with those of Bouvier et al. (1994) in showing that all red peppers accumulate high levels of capsanthin and express the Ccs gene at high levels, which accounts for the high levels of total carotenoid in these fruits. In addition, it was also observed that high levels of total carotenoid in Capsicum required the induction of the Psy, Pds, and Bch genes in addition to the Ccs gene during ripening, even though these patterns vary between the different red pepper varieties (Fig. 4). Minimal levels of expression of other genes such as β-lycopene cyclase (β-Lcy) and violaxanthin de-epoxidase (Vde) also appear to be required for the accumulation of high levels of carotenoid during ripening in red peppers (data not shown). Our current data indicating that the expression levels of the carotenoid biosynthetic genes are the key regulators of the high levels of total carotenoid accumulation in peppers are in good agreement with previous results obtained in tomato fruits (Pecker et al., 1996; Ronen et al., 1999, 2000), marigold petals (Moehs et al., 2001), and red peppers (Romer et al., 1993; Hugueney et al., 1996; Ha et al., 1999).
This relationship between the presence of structural genes for carotenoid biosynthesis and the phenotypic variability in ripe colours has been studied for several decades using genetic approaches in diverse Capsicum species (Kormos and Kormos, 1960; Hurtado-Hernandez and Smith, 1985; Lefebvre et al., 1998; Popovsky and Paran, 2000; Thorup et al., 2000; Lang et al., 2004). In particular, several studies have reported that the Ccs gene is either deleted or absent in yellow and orange colour pepper fruits (Bouvier et al., 1994; Lefebvre et al., 1998; Popovsky and Paran, 2000; Lang et al., 2004). Bouvier et al. (1994) have also demonstrated that the Ccs gene is not present in two yellow pepper cultivars, Jaune de Pignerolle and Golden Summer. Interestingly, Lang et al. (2004) and Popovsky and Paran (2000) reported similar results, showing that the Ccs gene is deleted in yellow fruits at 211 bp and 220 bp from the 3'-end, respectively. Nucleotide sequences of Ccs genes were analysed in the pepper varieties in the present study and it was revealed that the coding sequence of this gene and its promoter region are present in two of the pepper varieties whose ripe-colour is yellow (Fig. 5). The Ccs gene, however, is not expressed in the corresponding yellow peppers (Fig. 4), probably due to the presence of mutations in the coding region that generate a premature stop-codon (Y2) and a frame-shift (Y3) (Fig. 7).
Neither unusual cis-acting regulatory sequences nor critical mutations are evident that would impair the promoter activity of the Ccs genes in the two yellow pepper samples, Y2 and Y3. The Y2 pepper can produce a truncated CCS protein of 495 amino acids that contains a novel 18 amino acid stretch at its C-terminus caused by an 8 bp insertion in the coding region of the gene. The premature translation-termination codon (PTC) caused by the single base change in the Ccs gene of the Y3 pepper can generate a truncated CCS protein of 199 amino acids. Such PTCs or nonsense mutations can result in two kinds of yellow ripe colours. Such a phenomenon can be explained by a mechanism called ‘nonsense-mediated mRNA decay’, in which aberrant mRNAs harbouring premature translation-termination codons decay rapidly because the resulting C-terminally truncated proteins can function as dominant negative inhibitors of the full-length protein (Buhler et al., 2005). Recently, a 1.9-fold higher activity of PSY has been reported in the high pigment-1 (hp-1) tomato mutant without any increase of gene expression compared with the wild-type tomato, indicating translational or post-transcriptional control of carotenoid gene expression (Cookson et al., 2003). A post-transcriptional regulation also appears to play a role in carotenoid metabolism. Our elucidation of structural mutations in the Ccs gene in yellow peppers is likely to be another such example of this regulation and may be useful in increasing our understanding of carotenoid metabolism in the future.
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Supplementary data |
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Supplementary data in the form of three figures can be found at JXB online. Figure 1: Phenotypic features, carotenoid compositions and general information for the Capsicum varieties used in this study. Figure 2: Sequence comparisons in the region of the Ccs promoter among the indicated Capsicum varieties. Figure 3: Multiple alignments of the nucleotide and predicted amino acid sequences of the Ccs coding genes among the indicated Capsicum varieties.
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Acknowledgements |
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This work was supported by research funds of the National Institute of Agricultural Biotechnology (NIAB 06-2-11-10-2 and NIAB 06-2-12-6-1 to S-H Ha) and the Ministry of Science and Technology through the Crop Functional Genomics Center (grant to S-H Ha) in Korea.
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