JXB Advance Access originally published online on May 23, 2005
Journal of Experimental Botany 2005 56(417):1877-1886; doi:10.1093/jxb/eri177
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Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.
RESEARCH PAPER |
Ethylene regulation of carotenoid accumulation and carotenogenic gene expression in colour-contrasted apricot varieties (Prunus armeniaca)
1INRA-UMR Sécurité et Qualité des Produits d'Origine Végétale, Domaine Saint Paul, Site Agroparc, F-84914 Avignon cedex 9, France
2INRA-UGAFL Arboriculture, Domaine Saint Paul, Site Agroparc, F-84914 Avignon cedex 9, France
* To whom correspondence should be addressed. Fax: +33 4 32 72 24 92. E-mail: marty{at}avignon.inra.fr
Received 1 December 2004; Accepted 31 March 2005
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Abstract |
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In order to elucidate the regulation mechanisms of carotenoid biosynthesis in apricot fruit (Prunus armeniaca), carotenoid content and carotenogenic gene expression were analysed as a function of ethylene production in two colour-contrasted apricot varieties. Fruits from Goldrich (GO) were orange, while Moniqui (MO) fruits were white. Biochemical analysis showed that GO accumulated precursors of the uncoloured carotenoids, phytoene and phytofluene, and the coloured carotenoid, ß-carotene, while Moniqui (MO) fruits only accumulated phytoene and phytofluene but no ß-carotene. Physiological analysis showed that ethylene production was clearly weaker in GO than in MO. Carotenogenic gene expression (Psy-1, Pds, and Zds) and carotenoid accumulation were measured with respect to ethylene production which is initiated in mature green fruits at the onset of the climacteric stage or following exo-ethylene or ethylene-receptor inhibitor (1-MCP) treatments. Results showed (i) systematically stronger expression of carotenogenic genes in white than in orange fruits, even for the Zds gene involved in ß-carotene synthesis that is undetectable in MO fruits, (ii) ethylene-induction of Psy-1 and Pds gene expression and the corresponding product accumulation, (iii) Zds gene expression and ß-carotene production independent of ethylene. The different results obtained at physiological, biochemical, and molecular levels revealed the complex regulation of carotenoid biosynthesis in apricots and led to suggestions regarding some possible ways to regulate it.
Key words: Apricot, carotenoid, ethylene, fruit, 1-MCP, Prunus armeniaca, ripening-related genes
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Carotenoid pigments are synthesized by all higher plants, algae, and some bacteria and fungi (Goodwin, 1980
). In plants, they are involved in multiple functions related to (i) accessory pigments and photo-protectants in the chloroplasts of photosynthetic tissues, (ii) attracting pollinating insects and animals that contribute to seed dispersal in fruits and flowers, and (iii) a precursor of the abscisic acid hormone. In animals and human, they are essential health compounds that are not synthesized de novo in the body and must be acquired from the diet (Parker, 1996). Some carotenoids serve as precursors for vitamin A and as antioxidants that may prevent degenerative diseases and reduce the risk of certain forms of cancer (Giovannucci, 1999). Fruits and vegetables are a natural source of such compounds. The accumulation of carotenoids in fruits occurs during the ripening stage concomitantly with changes in pigmentation, firmness, sweetness, acidity, and aroma. The distribution of carotenoids in fruits is subject to considerable variation from one species to another, such as from the accumulation of an intermediate in the pathway as is the case of red lycopene in ripe tomato, orange ß-carotene in orange, and species-specific carotenoids in pepper fruits.
Carotenoid biosynthesis and sequestration take place within the plastids of higher plants (reviewed by Cunningham and Gantt, 1998). Carotenoids are C40 isoprenoids that derive from the common C20 precursor, geranygeranyldiphosphate (GGDP). The first step (Fig. 1) is catalysed by the enzyme phytoene synthase (Psy) which condenses two GGDP (C20) molecules into the colourless compound phytoene (C40). The next steps include four sequential desaturations of phytoene catalysed by two enzymes, phytoene desaturase (Pds) and 
-carotene desaturase (Zds). Phytoene desaturase catalyses the conversion of phytoene into the colourless phytofluene and the yellow -carotene compounds. -Carotene desaturase catalyses the conversion of -carotene into orange neurosporene and red lycopene. Subsequent cyclization of lycopene by ß-cyclase leads to the formation of ß-carotene and its derivative xanthophylls: zeaxanthin, violaxanthin, and neoxanthin. The last two compounds are precursors for the synthesis of the hormone abscisic acid.
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Carotenoid biosynthesis has been studied in various plant species, such as Arabidopsis (Park et al., 2002
), tomato (Isaacson et al., 2002), pepper (Bouvier et al., 1998), tobacco (Busch et al., 2002), and an alga (Steinbrenner and Linden, 2001). Bramley (2002) reviewed carotenoid biosynthesis and regulation during ripening and development in tomato fruit. Tomato mutant analysis has provided a wealth of information on genes involved in carotenoid biosynthesis and sequestration. In tomato fruits, the induction of lycopene accumulation coincides with the increased expression of upstream genes of lycopene synthesis (Psy and Pds genes) and reduced expression of downstream genes (cyclase genes) (Giuliano et al., 1993; Pecker et al., 1996; Ronen et al., 1999). In a similar way in pepper fruits, carotenoid accumulation coincides with an increase in mRNA in several carotenoid biosynthetic genes (Hugueney et al., 1996). Recent investigations performed on carotenoid biosynthesis in citrus fruits showed an increase in Psy mRNA with the onset of colouring (Ikoma et al., 2001), whereas the levels of Pds mRNA remained constant once fruit was fully developed (Kita et al., 2001). Although significant advances have been made in understanding the molecular biology of carotenogenesis, the regulatory mechanisms that control carotenoid biosynthesis are still poorly understood. In another connection, it has been demonstrated that ethylene, produced at the onset of ripening in climacteric fruits (Lelièvre et al., 1997), controlled ripening processes including colour changes. The objective of this study was thus to investigate the regulatory effects of ethylene on carotenogenesis in colour-contrasted apricots, at the biochemical and molecular levels.
In this study, two colour-contrasted apricots, the orange Goldrich (GO) and the white Moniqui (MO), were analysed for their carotenoid content and carotenogenic gene expression as a function of ethylene production and treatments. The major compounds found in apricots, colourless phytoene and phytofluene, and coloured ß-carotene, were measured. The genes involved in their biosynthesis, encoding phytoene synthase (Psy), phytoene desaturase (Pds), and -carotene desaturase (Zds), were isolated. Carotenogenic gene expression and carotenoid accumulation were quantified with respect to ethylene production which is initiated in mature green fruits at the onset of the climacteric stage and was also measured with respect to exo-ethylene or ethylene-receptor inhibitor (1-MCP) treatments. As far as the authors are aware, this is the first study to provide information on the regulation of carotenogenesis in apricots. On the basis of the comparison of biochemical, molecular, and physiological data, the mechanisms causing the difference in apricot colour, carotenoid accumulation and regulation are discussed.
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Materials and methods |
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Plant material, fruit treatments and storage
Experiments were performed over two years on fruits of two contrasting varieties: Goldrich, an American variety with orange fruits, high acidity, and slow development, and Moniqui, a Spanish variety with white fruits, good taste, and rapid development. They were obtained from INRA experimental orchards. Fruits were picked on full-grown trees that were goblet-trained and received routine horticultural care. Fruits were harvested once or twice a week during the pre-climacteric and ripening periods in 1998 and 2000 for Moniqui and in 1999 and 2000 for Goldrich. Different physiological stages of fruits were defined according to their ability to produce ethylene. Stage A: no ethylene had been produced at picking day and production was not inducible; stage B: no ethylene had been produced at picking day but was induced by ethylene treatment; stage C: no ethylene had been produced at picking day but was induced by air; stage D: fruit had initiated its own production of ethylene at picking day; stage E: half-ripe fruits; stage F: ripe fruits; stage G: over-ripe fruits. For stages A, B and C, fruits were divided into four homogeneous groups for treatments: (i) fruits were frozen immediately after picking, (ii) continuous treatment with 1-methylcyclopropene (MCP) at approximately 1 ppm, (iii) treatment with ethylene for 48 h at 20 ppm, and (iv) untreated control in the same conditions.
Physiological measurements
The physical measurements, i.e. weight, external ground colour, and firmness, were individually determined on the fruits. Fruit colour was estimated daily through the CIE L*a*b* system using a Minolta chromameter CM-1000R (Minolta, Ramsey, NJ) and expressed by the Hue value (H°ab=tg–1 (b*/a*). Ethylene rate was measured daily by gas chromatography (IGC 121 FL) after 1.5 h of confinement in a jar (Chambroy et al., 1995). It was expressed in nmol kg–1 h–1. Fruits from every sample were frozen in liquid nitrogen without stones, ground to a fine powder and stored at –80 °C for biochemical and molecular experiments. At the end of the 5 d treatments, fruits were frozen in liquid nitrogen without stones, ground to a fine powder and stored at –80 °C for biochemical and molecular analysis.
Carotenoid determination
Carotenoids were extracted from 10 g of apricot powder with 50 ml acetone. ß-apo-8'-carotenal was added as an internal standard. Two successive acetone extractions were performed to remove carotenoids from the powder. The compounds were isolated with 50 ml diethyl ether that was washed three times and concentrated to dryness. The extract was dissolved in 5 ml acetonitrile/methanol (60/40; v/v) and 10 µl were injected in HPLC. Carotenoids were analysed using a HPLC with a diode array detector (HP1100) equipped with a RP C18 5 µm, 250x4.6 mm Vydac column. The system was operated at 30 °C with a flow rate of 1 ml min–1 of isocratic mobile phase (acetonitrile/methanol/methylene chloride (60/38/2; by vol.). Carotenoid levels were expressed in mg kg–1 fresh weight.
Isolation of carotenogenic cDNA clones by RT-PCR and RACE-PCR
A method of reverse-transcription polymerase chain reaction (RT-PCR) was used to isolate cDNA clones of Psy, Pds, and Zds using total RNA from Goldrich apricot fruits. Total RNA was extracted from the fruits according to Manning's protocol (Manning, 1991). RT-PCR was performed on DNase-treated RNAs (Sambrook et al., 1989). Degenerate primers (Table 1) were designed for Psy, Pds, and Zds genes on the basis of conserved peptides as shown by aligning several plant homologues. PCR reactions were performed in 50 µl with 1 µg of RNA, 200 µM dNTP, 5 mM of DTT, 0.3 µM of combined primers, 1.5 mM MgCl2, 1x RT-PCR buffer, 1x Titan enzyme mix (Boehringer Mannheim GmbH) using a ‘DNA Thermal Cycler’ (Perkin Elmer, USA). The RT-PCR program was run as follows: once at 94 °C for 2 min, 35 times at 94 °C for 30 s, specific annealing temperature (Table 1) for 30 s and at 68 °C for 45 s, and once at 68 °C for 7 min. The PCR products were cloned in the pGEM-T vector (pGEM-T Vector System, Promega) at the EcoRI sites and submitted to nucleotide sequencing using SP6 and T7 as universal primers (Eurogentec). Partial cDNA sequences obtained for each gene were compared to EMBL and GenBank databases using the BLAST (Basic Local Alignment Search Tool) program.
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The complete sequence of Pds cDNA was carried out by RACE-PCR (Rapid Amplification of cDNA Ends). The 5' end of Pds cDNA was obtained by three successive 5' RACE-PCR (Boehringer Mannheim) according to the manufacturer's recommendations. Poly(A)+ RNA was obtained from total RNA of Goldrich apricot fruits (Oligotex mRNA mini kit, Qiagen). Each 5' RACE-PCR was performed on first-strand cDNA synthesized using cDNA-specific primer SP1. After degradation of the mRNA template, a homopolymeric A-tail was added to the 3' end of the cDNA. PCR-amplification of this template was then performed with an oligo dT-anchor primer (5'-GACCACGCGTATCGATGTCGAC(T)16-3') and another cDNA-specific primer SP2. The resulting cDNA was further amplified by another PCR using a nested specific primer SP3 and a PCR anchor primer (5'-GACCACGCGTATCGATGTCGAC-3'). The PCR products were cloned in the pGEM-T vector (pGEM-T Vector System, Promega) at the EcoRI sites and submitted to nucleotide sequencing (Eurogentec). The first 5' RACE-PCR was performed with primers SP1 (5'-CAGCGCCTTCCATTGAAGCC-3'), SP2 (5'-GGGAGATCTTGCAAGGGACGG-3'), and SP3 (5'-GGGACGGCAAGGTTCACAATCTGG-3') and led to a PCR product of 835 bp. The second 5' RACE-PCR was performed using primers designed on the 5' sequence of the 800 bp cDNA, SP1 (5'-GGACTTCACCGCCAATGACTG-3'), SP2 (5'-TGGTGCACAGAGTCTCTCAGG-3'), and SP3 (5'-GAACCGTGTTTCTCCTGAAGG-3') leading to a PCR product of 1256 bp. The third 5' RACE-PCR was performed with primers designed on the 5' end of the 1250 bp PCR product: SP1 (5'-GCCTGCCCACCAAGAATTGCTG-3'), SP2 (5'-CTCCTGGTTTGTTTGGCATTGC-3'), and SP3 (5'-TCATAGAATGCTCCTTCCAC-3') leading to the complete 5' Pds cDNA. The 3' end of Pds cDNA was obtained by 3' RACE-PCR (Boehringer Mannheim) according to the manufacturer's recommendations. The PCR was performed on first-strand cDNA using cDNA-specific primer 3pds (5'-ATTGTGAACCTTGCCGTCCCTTGC-3') and the oligo dT-anchor primer leading to a 457 PCR product. The complete sequence of Pds cDNA was 2155 bp.
Gene expression by Real-Time-quantitative PCR
cDNA pools were synthesized from each RNA sample. Samples of total RNA (2 µg) were incubated at 65 °C for 10 min with 1.5 µM of random primer P(dN)6 (Boerhinger Mannheim), then cooled for 10 min in ice to allow random primers to anneal to the poly(A) tail of mRNA. Four µl of 5x Expand Reverse Transcriptase Buffer (Roche Diagnostics), 10 mM DTT, 1 mM each dNTP, 20 units of RNase Inhibitor, and 50 units of Expand Reverse Transcriptase (Roche) were added to the RNAs which were then incubated at 30 °C for 10 min and at 42 °C for 45 min. To inactivate reverse transcriptase the reaction was incubated at 70 °C for 5 min. The cDNA was then ready for Real-Time-PCR performed with the LightCycler system (Roche Diagnostics, Mannheim, Germany), using SYBR Green I double strand DNA binding dye. The primers (Table 2) were based on the Prunus armeniaca sequences previously characterized by RT-PCR (see above). The amplification of a target was carried out in a total volume of 20 µl containing 500 nM of the sense and anti-sense primers, 5 mM MgCl2, 10 µl LightCycler DNA Master SYBR Green I (containing 1.25 units of Taq polymerase, 10x Taq buffer (500 mM KCl, 100 mM TRIS–HCl, pH 8.3), dNTPs each at 2 mM, 10x SYBR Green I; Roche Diagnostics) and 150 ng of cDNA prepared as described above. The following cycling conditions were chosen: melting of the DNA at 95 °C for 6 min, amplification with 45 cycles with a desaturation step at 95 °C for 15 s, annealing at 60 °C for 15 s, and elongation at 72 °C for 5 s. Fluorescence data were collected after each extension step. Fluorescence was analysed using LightCycler Analysis Software. The crossing point for each reaction was determined using the Second Derivative Maximum algorithm and manual baseline adjustment. Amplification efficiency (75–95% with slopes of 3.31 to 3.5) was determined for each gene by amplification of the target from a dilution series (1/5, 1/10, 1/20, 1/40, 1/80) of a pool of RNA (data not shown). The transcript level of the target gene was measured relative to ribosomal RNA. Each qRT-PCR reaction was duplicated at least once. As the error bar values were extremely low (less than 2.5% of the average value) they were not shown on the graphs.
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Results |
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Cloning of carotenogenic cDNAs and determination of the number of gene copies in the apricot genome
The partial Psy, Pds, and Zds cDNAs were isolated by classical Reverse Transcriptase PCR and degenerate primers (Table 1) that were designed by back-translating a conserved region of deduced amino acid sequences among plant orthologue genes. Supplementary RACE-PCR were carried out for Psy and Pds cDNAs in order to isolate longer and/or entire cDNAs, respectively.
The partial Psy cDNA obtained by classical RT-PCR was 237 bp in length. The nucleic acid sequence presented 85% of homology to known Psy-1 genes. Since two different Psy genes, Psy-1 and Psy-2, have been identified in fruits (Fraser et al., 1999) as well as in apricot (data not shown), a longer and more specific fragment was isolated by RACE-PCR. The 3' end of the 237 bp cDNA was isolated by one 3' RACE-PCR leading to a fragment of 750 bp in length (AprPsy-1: accession no. AY822067). The cDNA presented 633 bp of coding sequence and 117 bp of 3' untranslated region. The 633 nucleic acid and deduced amino acid sequences of AprPsy-1 showed high homology to genes encoding the Psy-1 isozyme of citrus, 82% and 88%, respectively (accession nos AF220218 and AAT33237), melon, 82% and 88%, respectively (accession nos Z37543 and CAA85775), and pepper, 80% and 87%, respectively (accession nos X68017 and CAA48155). According to the results of these comparisons, AprPsy-1 encoded the Psy-1 isozyme. In order to follow Psy-1 gene expression in apricot, two specific primers were designed in the 3' untranslated region of AprPsy-1 (Table 2).
The Pds cDNA isolated from classical RT-PCR and degenerate primers (Table 1) was 348 bp in length. The nucleic and amino acid sequences showed high homology to known Pds genes (around 85% and 90%, respectively). Using sequence data of this 348 bp fragment, the complete cDNA was isolated by successive 3' and 5' RACE-PCR leading to a fragment of 2155 bp in length (AprPds: accession no. AY822065). The complete cDNA contained 174 bp of 5' leader, 1719 bp of coding region, and 260 bp of the 3' untranslated region including 16 bp of polyA tract. AprPds encoded a predicted protein of 573 amino acid residues with a predicted molecular mass of 64 kDa. The nucleic and deduced amino acid sequences of AprPds showed high homology to Pds genes of citrus, 84% and 85%, respectively (accession nos AF36451 and AAK51545), soybean, 83% and 85%, respectively (accession nos M64704 and AAA34001), and pepper, 81% and 87%, respectively (accession nos X68058 and CAA48195). In order to analyse Pds gene expression in apricot, two specific primers were designed in the 3' untranslated region (Table 2).
The isolated partial cDNA clone of Zds, AprZds (accession no. AY822066) was 229 bp in length. The nucleic and amino acid sequences deduced from AprZds showed high homology to Zds genes of citrus, 85% and 92%, respectively (accession nos AJ319762 and CAC85667), pepper, 82% and 96%, respectively (accession nos X89897 and CAA61985), and tomato, 81% and 93%, respectively (accession nos AF195507 and AAF13698). The DNA gel blot performed with the AprZds fragment as a probe suggested that there was only one copy of this gene per haploid apricot genome (data not shown). Since only one gene was found in apricot (data not shown) as well as in other fruits, two primers were designed on this small AprZds sequence (Table 2) in order to analyse Zds gene expression.
Physiological characterization of mature green fruits at the pre-climacteric period
Fruits were collected around the climacteric period and were characterized for their ethylene production and their colour. At picking day, the A, B, and C stages of GO and MO fruits did not produce ethylene while the D stage had already initiated ethylene production (Fig. 2). Ethylene production increased rapidly in the more mature stages (E, F, and G), with a 3-fold higher maximum for MO compared with GO fruits. The colour of GO and MO fruits remained green (Hue value=105–110) until the B and C stage, respectively; in GO it began to change to orange (Hue value=65) at stage C and in MO there was a very slight change to ivory (Hue value=90) at stage D.
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Exo-ethylene treatment of green fruits enabled characterization of the A, B, and C stages (Fig. 3) during post-harvest development at 23 °C. Fruits at stage A can be classified as immature green fruit because they produced very low ethylene (GO) or no ethylene (MO) after ethylene treatment. Fruits at stage B are mature green fruits since ethylene production was induced 2 d after ethylene treatment, while stage C fruits are induced mature green fruits with an increase in ethylene production after 3 d in air. At this pre-climacteric stage, 1-MCP treatment inhibited ethylene autocatalysis while, by contrast, ethylene treatment stimulated it, in agreement with the results given in Chahine et al. (1999)
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Carotenoid accumulation and carotenogenic gene expression as a function of the physiological stage of development
Carotenoid content and carotenogenic gene expression were analysed at the fourth initial stage (picking day) for GO and MO apricots (Fig. 4). The qualitative accumulation profiles of the colourless carotenes, phytoene and phytofluene, were quite similar in the two varieties (Fig. 4A, B). A basal and constant level was found at stages A and B with an increase in accumulation from stage B. This accumulation gradually increased in GO until stage D whereas in MO it reached the maximum at stage C. However, the carotene content was always higher in GO than in MO whatever the developmental stage. Similarly, the expression profiles of Psy-1 and Pds genes were strictly identical in the two varieties. No Psy-1 transcript and basal amounts of Pds transcripts were detected at stages A, B, and C and were strongly accumulated at stage D. The qualitative behaviour of product accumulation and gene expression was quite well correlated even if phytoene and phytofluene were accumulated earlier at stage C. However, a marked quantitative difference was found since Psy-1 transcripts were 50 times more accumulated in MO than in GO at stage D. This tendency was not detected or was inverted at the compound level since MO accumulated 2 times less phytoene than GO at stage D. A similar, but less drastic, difference was also detected in Pds gene expression and phytofluene accumulation of which transcripts accumulated 2 times more in MO than GO, while phytoene accumulation was 2 times lower.
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There was a significant difference in the accumulation profiles of ß-carotene (Fig. 4C) between the two varieties. The compound was not detectable in the white MO fruits while its accumulation increased gradually, but considerably, in the orange GO fruits from stage B to D. Zds gene expression profiles were rather similar in the two varieties but differed significantly from Psy-1 and Pds. A significant expression was already detectable at stage A that gradually increased throughout the physiological stages whatever the variety. As for Psy-1 gene expression, the relative amount of Zds transcripts was higher in MO than in GO (up to 20-fold).
Carotenoid accumulation and carotenogenic gene expression under exo-ethylene or 1-MCP treatments
Treatments with exogenous ethylene and also with 1-MCP, an inhibitor of ethylene perception, were conducted on immature (A) and mature green fruits (stages B and C) that did not produce ethylene at picking, but were able to produce it a few days after ethylene (B) or air (C) treatments (see Fig. 3). Carotenoid contents and carotenogenic gene expression were performed on these fruits 5 d post-harvest at 23 °C.
Ethylene-treated fruits accumulated a large amount of phytoene compared with control fruits (2 times more at stages B and C: Fig. 5A). The behaviour of fruits treated with 1-MCP was quite similar to controls. The Psy-1 expression profiles were similar in the two varieties. Induction of Psy-1 gene expression by ethylene was clearly visible at stage C. The transcript level was significantly higher in ethylene treated fruits (25-fold in GO and 10-fold in MO) than in control or 1-MCP-treated fruits. A similar but slighter induction was detected as early as stage B (10-fold in GO and 5-fold in MO) while no significant induction was detected in fruits at stage A. In a similar way in both varieties, treatment with the ethylene-receptor inhibitor (1-MCP) did not significantly modulate Psy-1 gene expression compared with controls. This ethylene up-regulation was found for Psy-1 gene expression and for its product accumulation, phytoene.
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In the two varieties, phytofluene accumulated considerably in ethylene-treated fruits from stages A to C (Fig. 5B) while no significant difference was detected in compound content between air and 1-MCP-treated fruits. Similarly, in both varieties, the Pds transcript level was significantly higher (around 5-fold) in ethylene-treated fruits at stages B and C compared with the basal level in controls. Strong induction was already detectable at stage A in ethylene-treated GO. However, fruits treated with 1-MCP also showed a consequent induction of Pds expression from stage B (up to 3-fold in GO and 2-fold in MO at stage C).
The behaviour of ß-carotene accumulation (Fig. 5C) was clearly different in GO and MO. Its accumulation in GO was constant whatever the physiological stage or treatment. In Moniqui, no accumulation was found in control and ethylene-treated fruits, but a slight increase was detected in 1-MCP-treated fruits from stage B. By contrast, the behaviour of Zds gene expression was similar in the two varieties and no significant induction by ethylene was detected compared with controls. However, Zds transcripts accumulated significantly in fruits treated with 1-MCP. In GO, this up-regulation by 1-MCP was constant whatever the physiological stage (around 5-fold compared with the air-control) while it decreased from stage B in MO.
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Discussion |
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The remarkable contrast in colour between MO and GO apricots suggested an alteration of the mechanisms regulating the accumulation of carotenoids that might provide new insights into the regulation of carotenogenesis in fruits. Analysis of carotenoid content showed that the orange GO fruits accumulated a high level of coloured ß-carotene as well as colourless precursors, phytoene and phytofluene. By contrast, MO white fruits only accumulated the two colourless compounds but no coloured compound. This quantitative and qualitative variation in carotenoids presumably yielded the colour-contrast between MO and GO varieties. Such a variation could originate from a regulation at the genomic level. Studies that were mainly performed on tomato and pepper have suggested a correlation between carotenoid accumulation and an increased level of mRNA for a few carotenoid biosynthetic genes (Hugueney et al., 1996
). In the case of tomato, the availability of a large number of carotenoid genes and efficient genetic transformation resulted in genetic modifications and changes in the level of carotenoids in fruits. Transgenic tomato plants transformed with an antisense Psy-1 cDNA were impaired for carotenoid accumulation in ripe fruit (Bramley et al., 1992), a phenotype also characteristic of the yellow-flesh r-mutant, mutated in the Psy-1 gene (Fray and Grierson, 1993). Similarly, a rise in ß-carotene content was obtained in transgenic tomatoes that constitutively expressed an Erwinia phytoene desaturase (crtI) gene (Romer et al., 2000). These results showed that Psy-1 and Pds genes played key roles in carotenoid synthesis and in the colour of tomato fruits. To understand the relationship between carotenoid content and gene expression better, key carotenogenic cDNAs, encoding phytoene synthase 1 (Psy-1), phytoene desaturase (Pds) and -carotene desaturase (Zds), were isolated in apricot. The three corresponding cDNAs presented high and significant homologies to their plant orthologues (up to 85% and 90% at the nucleic and amino acid levels, respectively). While two Psy genes (Psy-1 and Psy-2) were found in tomato, only the orthologue Psy-1 cDNA was isolated in apricot. It has been postulated that Psy-1 is responsible for carotenoid formation in ripening fruit while the chloroplastic Psy-2 is related to foliar carotenoid formation (Fraser et al., 1999). In order to analyse the impact of ethylene on this process, gene expression and carotenoid content were measured on mature green fruits at the pre-climacteric stage and after ethylene treatments. Regardless of the experimental conditions and apricot varieties, the accumulation of colourless carotenes, phytoene and phytofluene, correlated well with the expression of their corresponding genes, Psy-1 and Pds. Subsequent accumulation and expression were found in fruits that produced ethylene at picking (stage D in Fig. 4) or after ethylene treatments (stages B and C in Fig. 5A, B) while basal accumulation and gene expression were detected in immature fruits that did not produce ethylene (stage A in Figs 4 and 5). These results showed that phytoene and phytofluene synthesis was similarly regulated in the two varieties and was up-regulated by ethylene that controlled the expression of the corresponding genes, Psy-1 and Pds. By contrast, the accumulation of ß-carotene in MO and GO was clearly different, since MO did not accumulate this compound, while Zds transcripts accumulated in both varieties and in greater quantity in MO. In addition, the behaviour of ß-carotene synthesis and Zds gene expression towards ethylene was also different, since no significant regulation was detected (Figs 4C, 5C). One hypothesis concerning the uncorrelated ß-carotene and Zds transcript accumulation in MO would be a post-transcriptional modification of the Zds enzyme leading to an inactive form. This type of regulation has already been found for carotenogenesis in Narcissus where both Psy and Pds were detected in inactive forms in the soluble fraction, although they were active when membrane-bound (Al-Babili et al., 1996). The in situ and/or ex situ measurement of Zds activity could provide an answer to this hypothesis. An alternative explanation is that the MO Zds gene was mutated and resulted in an inactive form of the enzyme. When comparing MO and GO cDNA fragments isolated by RT-PCR (229 bp), no mutations were found, but it cannot be excluded that mutations were localized elsewhere on the gene. The isolation of the entire cDNA in MO and GO could provide information about this hypothesis.
The carotenoid end-products could be potential regulators of the carotenoid pathways. The idea of feedback regulation by end-products is in agreement with some tomato studies. Boosted expression of both Pds and Psy-1 genes was observed in tomato leaves where lycopene cyclase was inhibited (Corona et al., 1996). Similarly, a reduction in total carotenoid content of tomato transformants expressing the constitutive Pds gene (Erwinia crtI) was thought to be a consequence of feedback regulation from ß-carotene or one of its metabolites (Giuliano et al., 2000). This study's results suggest feedback up-regulation of genes upstream of ß-carotene synthesis (Psy-1, Pds, and Zds) by one of the ß-carotene metabolites. In practice, no indication of the nature of the active compound was obtained.
These regulations might also be controlled by the ethylene that was produced at the onset of ripening in climacteric fruits and thus controlled some ripening-related processes. In addition, ethylene production in MO and GO was drastically different (around 6000 and 2000 nmol h–1 kg–1, respectively). Results showed qualitatively similar behaviour in both varieties where product (phytoene and phytofluene) accumulation and upstream gene (Psy-1 and Pds) expression of phytofluene were ethylene up-regulated while downstream ones (ß-carotene and Zds) were ethylene-independent. Nevertheless, a different quantitative behaviour was observed that might be regulated by ethylene.
In conclusion, this study on apricot provided a new insight on the regulation of carotenogenesis in fruits. Although the major regulatory control of carotenogenesis was found at the transcriptional level in tomato, this study showed other possible ways of regulation, including post-transcriptional or metabolic regulation. Complementary studies are necessary in order to understand the regulatory mechanisms of carotenogenesis in fruits and to improve their nutritional and commercial quality.
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Acknowledgements |
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We are grateful to Linda Hagen for her analysis of gene copy and Line Tichit for her technical help. This work was supported by post-doctoral INRA grant to Gaiané Sarkissian.We wish to thank Mathilde Causse for her critical reading of this paper.
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Footnotes |
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Abbreviations: Psy, phytoene synthase; Pds, phytoene desaturase; Zds, zeta-carotene desaturase; GGDP, geranylgeranyl diphosphate; 1-MCP, 1-methylcyclopropene; GO, Goldrich; MO, Moniqui; ABA, abscisic acid; Real-Time-PCR, Real-Time-Polymerase Chain Reaction; RACE-PCR, Rapid Amplification of cDNA Ends by Polymerase Chain Reaction; RT-PCR, Reverse-Transcription-Polymerase Chain Reaction.
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