BACKGROUND
among rosaceae, peach  is an appealing model crop, because of its economical value, small genome, rapid generation time and several mendelian traits  still to be functionally characterized  <cit> .
peaches are appreciated for their visual, nutritional and organoleptic features, partially contributed by carotenoids, sugars, acids and volatile organic compounds , which vary as a function of genetic, developmental and post-harvest factors .
in particular, carotenoid accumulation in the mesocarp determines the difference between yellow- and white-fleshed genotypes, the latter being generally characterized by a peculiar and more intense aroma.
flesh color is a mendelian trait , associated with the y locus that has been mapped on the linkage group  <dig> of the prunus map  <cit>  but which has not been yet functionally characterized from the molecular or enzymatic point of view.
natural mutations, originating flesh color chimera with irregular yellow and white distribution, have long been observed in peach  <cit> .
carotenoids are a widespread class of compounds having important functions across living organisms, whose accumulation shows striking phylum- and genotype-specific regulation  <cit> .
following the formation of the first carotenoid phytoene from the general isoprenoid pathway, the pathway bifurcates after lycopene with respect to the ring type, giving rise to carotenes and xanthophylls with either β-β or β-ε rings .
in addition to their roles in plants as photosynthetic accessory pigments and colorants, carotenoids are also precursors to norisoprenoids .
norisoprenoids are commonly found in flowers, fruits, and leaves of many plants  <cit>  and possess aromatic properties together with low odor thresholds , thus having a strong impact on fruit and flower aroma even at low levels  <cit> .
an increasing number of dioxygenase enzymes that specifically cleave carotenoid compounds to form volatile norisoprenoids, abscisic acid  and regulators of plant growth and development has been characterized.
these enzymes have been referred to as carotenoid cleavage dioxygenases  and 9-cis-epoxycarotenoid dioxygenases   <cit>  and represent a plant multienzyme family: arabidopsis has nine ccd/nced members, of which four have been classified as ccds  and the remaining as aba-related nceds  <cit> .
functional analysis of ccd enzymes determined that ccd <dig> and ccd <dig> are mostly related to the synthesis of norisoprenoid  plant hormones, while ccd <dig> and ccd <dig> are preferentially involved in volatile production, by using different carotenoid substrates with variable specificity and cleavage site, which probably contributes to the diversity of norisoprenoids found in nature .
the synthesis of β-ionone, geranylacetone and 6-methyl-5-hepten-2-one in tomato fruits increases 10- <dig> fold during fruit ripening and these compounds were produced by the activity of ccd products  <cit> .
silencing of ccd genes resulted in a significant decrease of the β-ionone content of tomato ripe fruits and petunia flowers  <cit> , and increased pigmentation of potato tubers and chrysanthemum flowers  <cit> .
ccds were also implied to be involved in the formation of important apocarotenoid aroma compounds in melon fruit  <cit> .
furthermore, comparative genetics studies have indicated that carotenoid pigmentation patterns have profound effects on the norisoprenoid and monoterpene aroma volatile compositions of tomato and watermelon fruits  <cit> .
many norisoprenoids strongly contribute to peach fruit aroma, and their levels increase during fruit ripening  <cit> .
the partial purification and biochemical characterization of β-carotene degrading enzyme from nectarine skin extracts was associated with the formation of c <dig> norisoprenoids  <cit> .
the study of two nced-encoding genes from peach showed their differential expression, suggesting a functional relation to aba formation during fruit ripening  <cit> .
the recent synthesis of specific carotenoid dioxygenase inhibitors  <cit>  enables to assess the role of such enzymes in vivo, not only in aba biosynthesis but also in fruit voc metabolism.
in order to improve our knowledge of carotenoid and voc biosynthesis in peach fruit and determine the factor controlling carotenoid accumulation in peach flesh, the two cultivars 'redhaven'  and its white-fleshed bud sport mutant 'redhaven bianca'   <cit>  were investigated.
carotenoid accumulation, voc content and transcript levels of relevant carotenoid biosynthetic genes were studied at five stages of fruit development .
the effect of the carotenoid dioxygenase inhibitor, abamine sg, on fruit voc composition at full ripening was also studied.
RESULTS
fruit phenotype during ripening
at s1-s <dig> stages, whole fruits of rhb  and rh  look similar, while the final ripening stages br and s <dig>  carotenogenesis is well established in rh fruits, and differences in flesh color between yellow-fleshed rh and white-fleshed rhb fruits become dramatic.
the yellow pigmentation visible along the suture at full ripening stage  indicates that rhb is a bud sport chimera mutant, in which the mutation does not involve the l-i apical cell layer, that originates the epidermis and several cells layers at the suture of the ovary wall  <cit> .
the flesh color phenotype has remained stable throughout several cycles of clonal propagation, pointing out the stability of the chimera in rhb .
the mutation is transmitted to the progeny in a mendelian fashion, and is associated to the y locus controlling fruit flesh color.
rhb is heterozygous for this locus , originating either 1: <dig> of yellow- to white-fleshed seedlings when selfed or 1: <dig> of yellow- to white-fleshed progenies when crossed with yellow  peach accessions .
the yellow strip is not observed in white-fleshed rhb progenies , because the gametes does not originate from the l <dig> cell layer but from the fully-mutated l <dig> layer.
the major fruit quality traits and skin color parameters of the two cultivars were also measured, showing statistically significant differences only for soluble solids content .
carotenoid composition of rhb and rh fruits
in carotenoid-containing fruits, the massive biosynthesis of such compounds is generally associated with late ripening stages and plastid transition from chloroplasts into chromoplasts.
at early ripening stages s <dig> and s <dig>  fruits of rhb and rh had similar total carotenoid levels and accumulated only a few carotenoid compounds, dominated by the presence of lutein and β-carotene .
from the s <dig> stage, rh mesocarp accumulated increasing amounts of carotenoids that peaked at the s <dig> stage to provide the solid yellow flesh color, while carotenoid content in rhb flesh remained low .
detailed hplc analysis revealed the presence of specific carotenoid compounds in the two genotypes , some of which rather uncommon and present in rh only, whose main chemical structures are illustrated .
until stage s <dig>  and br , lutein and its -isomers were the major carotenoids in fruits of both genotypes, accounting for over 50% of the total carotenoid pool.
other major carotenoids at early stages were β-carotene, relatively abundant in fruits of both cultivars, and neochrome epimers, which accumulated only in rh fruits.
from stage s <dig>  not only did rh fruits have higher carotenoid levels, but also a range of carotenoid compounds much wider than rhb .
at full ripening s <dig> stage, xanthophylls made up the majority of carotenoids - zeaxanthin was the main carotenoid in rhb, while antheraxanthin, luteoxanthin and zeaxanthin were the most abundant compounds in rh fruits .
average values are listed in descending order with respect to rh composition and expressed in ng/g fresh weight.
pda λ:  <dig> nm.
tent.
: tentative identification.
n.d.: not detectable.
gene expression analyses
relative transcript levels of three genes involved in isoprenoid metabolism  and twelve genes involved in carotenoid biosynthesis and cleavage   were measured in rh and rhb mesocarp at s <dig>  s <dig>  s <dig>  br and, s <dig> stages by reverse transcription quantitative real-time pcr .
the isoprenoid pathway genes, dxs and cmk, had very low transcript levels throughout fruit development in both cultivars.
hdr showed a sharp peak of expression at the s <dig> stage which declined at later stages in the yellow-fleshed rh, while its expression remained high in rhb .
similarly, early carotenoid pathway genes psy and zds showed a peak at the s <dig> stage in rh, while in rhb these transcripts showed a constant increase until the s <dig> stage .
among later pathway genes , only chy-b was strongly up-regulated in rhb , while, ccd and nced expression was generally low in both genotypes, with the exception of ccd <dig>  which was significantly up-regulated in rhb at late ripening stages, its transcript level being 13-fold higher than that in rh at the s <dig> stage .
hierarchical clustering analysis  of gene expression data clustered the ripening stages consistently with their chronological order in joint analysis of both genotypes  and in rh alone .
in rh, a clear co-regulation of genes encoding enzymes closely positioned in the pathway  was observed .
instead, in the rhb mutant the majority of these co-regulation clusters was broken, with the exception of ccd <dig> and chy-b genes which remained co-regulated .
voc analyses
levels of different vocs were studied in rhb and rh during fruit ripening by gc-ms. in total,  <dig> vocs were detected, assigned to aromatic and branched chain amino acid-related, fatty acid-related, furan-related, lactone, monoterpene and norisoprenoid classes, quantified and underwent further analyses .
average data of  <dig> to  <dig> replicates.
values are in ng/g fresh weight.
id.
: identification method .
positive compound identification was obtained by matching both ms and ki or standard compound data.
otherwise, putative  best compound is listed.
ri: retention index.
aa: amino acid.
n.d.: not detectable.
the two genotypes had a similar, ripening-associated accumulation of total vocs starting from the s <dig> stage, while early s <dig> and s <dig> stages were characterized by very low volatile content .
detailed analysis pointed out differences in the accumulation of the distinct voc pools in the two genotypes .
furan-related compounds accumulated at the highest levels in both genotypes, followed by norisoprenoids and fatty acid-related compounds, whose maximum levels were about 5-fold lower than those of the furans .
the other classes accumulated at lower absolute levels, with maxima in the range of hundreds of μg/g fresh weight.
the two genotypes displayed similar ripening-associated patterns for aromatic and branched chain amino acid-, fatty acid-, and furan-related classes, with a peak at the s3/br stages and a more or less pronounced decline at later ripening stage .
total lactone and monoterpene contents displayed a different pattern, with a strong up-regulation only at final s <dig> ripening stage in the two cultivars .
at the s <dig> stage, all the six above-mentioned voc classes had higher levels in rh fruits .
a remarkable exception was the norisoprenoid pool, which accumulated in rhb fruits at levels higher than those of rh from s <dig> stage on.
norisoprenoid pattern in rhb fruits peaked at br and was constant through s <dig> stage, while in rh fruits it displayed a linear increase from s <dig> stage .
in particular, the three identified norisoprenoids 3-hydroxy- <dig> -epoxide-β-ionone, 3-hydroxy-β-damascone and 4-hydroxy- <dig> ,6-trimethyl-4--2-cyclo-hexen-1-one were responsible for the higher total norisoprenoid levels in fruits of rhb, with an almost 3-fold difference at the s <dig> stage , when the typical floral scent of white-fleshed peach fruits reaches its maximum.
at any ripening stage, the level of each identified norisoprenoid compound was always higher in the white-fleshed rhb than in rh .
instead, the less prominent unidentified norisoprenoids had a similar accumulation pattern in the two genotypes, with higher levels in rh fruits .
pca was performed on the whole gc-ms dataset  to provide a more intuitive visualization of data and to discriminate the different ripening stages in the two varieties with respect to voc composition.
a preliminary pca analysis was carried out including all five stages, and resulted in a poor separation of most samples , with the exception of rhb-s <dig>  rh-s <dig>  rh-s <dig> and rh-br.
principal components  <dig> and  <dig> explained 67% and 21% of the total variability, respectively .
a narrower analysis was then carried out by excluding the s <dig> and s <dig> samples, which allowed the complete discrimination of the six late ripening samples of both genotypes .
in this closer analysis, the new calculated principal components  <dig> and  <dig> accounted for 76% and 13% of the total variability, respectively .
effect of a carotenoid dioxygenase inhibitor on voc production
the effect of abamine sg treatment was assessed in rhb ripe fruits, injected once a week from the s <dig> stage.
as expected, abamine sg injection resulted in a drastic reduction of the levels of both identified and unknown norisoprenoids .
unexpectedly, this treatment also down-regulated the content of the other vocs.
the strongest reduction was observed for furan-related, monoterpene and lactone pools, while the total content of aromatic amino acid- and fatty acid-related compounds was the least affected by abamine sg treatment .
discussion
peach fruits contain carotenoid compounds with significant antioxidant capacity and claimed beneficial health effects.
the enzymatic cleavage of these compounds results in the production of volatile norisoprenoids .
our study investigated the expression of carotenoid genes, the carotenoid content and the volatile composition in the wild type yellow-fleshed rh and its white-fleshed rhb during fruit ripening.
key regulatory steps and regulation mechanisms controlling isoprenoid and carotenoid flux in many species, also through biotechnology-based approaches to carotenoid manipulation, have been extensively reviewed  <cit> .
differential accumulation of carotenoids in rh and rhb became evident at stage s <dig>  and reached its maximum at stage s <dig>  when rh fruits accumulated approximately 10-fold more carotenoids than rhb , composed mainly of β-ring carotenoids .
accordingly, the two cultivars showed strikingly different developmental patterns of expression of several isoprenoid and carotenoid genes .
however, several differential regulation events appeared to be the effect, rather than the cause, of the differential carotenoid accumulation.
for instance, in rhb the up-regulation of hdr, psy, and zds genes at late  stages appears to be the result of a feed-back repression by either the lower carotenoid levels in this genotype or their cleavage products, acting on the transcript levels of these genes.
examples of feed-back regulation of carotenoid gene transcripts have been described in plants where carotenoid biosynthesis has been altered through the use of inhibitors or metabolic engineering  <cit> .
a similar higher expression of early carotenoid genes was found in a white-fleshed apricot cultivar with respect to an orange-fleshed genotype  <cit> , possibly suggesting common regulation mechanisms of early carotenoid gene expression based on feedback regulation mediated by carotenoid end products.
the generally low expression at late br/s <dig> stages of the studied carotenoid genes in the yellow-fleshed rh might point out a control of metabolite flux based on steady-state gene/enzyme expression rather than up- or down regulation of transcription.
on the other hand, in rhb fruits the strong up-regulation at late stages of chy-b and ccd <dig> genes is negatively correlated with the accumulation of β-ring carotenoids, and positively correlated with that of identified β-ring norisoprenoids.
the biochemical function of the chy-b and ccd <dig> gene products is compatible with the observed phenotype of rhb: chy-b funnels carotenoids into the β-xanthophyll branch, producing substrates cleaved by carotenoid dioxygenases, including ccd <dig>  furthermore, chy-b is a negative regulatory step in several plant systems: inhibition of its expression through transgenosis or natural genetic variation results in higher β-carotenoid levels in potato tubers and maize endosperm  <cit> .
similarly, the levels of ccd <dig> transcript negatively correlate with carotenoid levels .
aubert et al.
<cit>  described the presence of several c <dig> norisoprenoids, mainly derived from the cleavage of β-ring xanthophylls like in the present study, in yellow- white-fleshed nectarines.
unlike rh and rhb, the two cultivars were not isogenic, but similarly to the present case, the white-fleshed nectarine 'vermeil' had higher norisoprenoid contents than the yellow-fleshed 'springbright'.
however, in two other studies, the β-ionone levels detected in a number of white-fleshed genotypes were lower than those of several unrelated yellow-fleshed accessions  <cit> .
such examples show that flesh color per se is not sufficient to determine the levels of norisoprenoid vocs, confirming that volatile composition strongly depends on genetic factors other than carotenoid levels.
in our study, rhb ripe fruits had higher norisoprenoid content than that of rh, derived from carotenoid cleavage.
as a likely consequence of the redirection of metabolite flux towards the synthesis of nor/isoprenoid compounds in rhb fruits, the levels of all other voc pools were lower than those in rh fruits.
among aroma-related carotenoid dioxygenases, both ccd <dig> and ccd <dig> enzymes cleave carotenoids at the  <dig>  and 9',10' positions and have a major role in the formation of β-ionone and other fruit and flower norisoprenoids  <cit> : the β-carotene degradation displayed by yellow nectarine skin extracts  <cit>  most likely corresponds to ccd <dig> and/or ccd <dig> activity.
compared with ccd1s, the ccd <dig> enzymes were characterized more recently from several crops  <cit> , and shown to have a major impact on organ pigmentation: ccd <dig> expression was higher in white-fleshed potato and in white-flowered chrysanthemum genotypes than in their respective yellow-pigmented counterparts, and rnai-mediated knockout boosted carotenoid accumulation  <cit> .
the small ccd <dig> family contains at least two forms of genes with different structure, expression patterns and genome position, and their encoded enzymes show different substrate specificity  <cit> .
the existence of plastid target peptides and the demonstrated plastid localization of ccd <dig> enzymes  <cit>  allow these enzymes direct access to the carotenoid substrates, suggesting that they start the carotenoid degradation and norisoprenoid synthesis pathway.
on the other hand, the lack of correlation between the patterns of ccd <dig> expression  and norisoprenoid content  in the flesh of both cultivars reflects a situation common to other fruits like grape, melon, and tomato  <cit> .
this is a likely consequence of the different localization of ccd1s and their substrates, since carotenoids are accumulated in plastids but known ccd1s lack plastid transit peptides  <cit> , and/or substrate preference, since each carotenoid dioxygenase can accept different carotenoids.
our data suggest that because of their different subcellular localization, ccd1s only contribute to volatile production, while ccd4s are likely to control also carotenoid degradation.
as to other dioxygenase-encoding genes studied, nced <dig> and nced <dig> expression patterns were up-regulated during ripening , in agreement with the reported physiological increase of aba content at late fruit ripening stages  <cit> , with a profile similar to that of ethylene-related accs and acco genes .
the transcriptional control of peach norisoprenoid content by specific dioxygenase genes is similar to that of fatty acid pathway-related genes and enzymes, whose expression patterns generally show a strong positive correlation with those of the corresponding volatiles  <cit> .
the strong down-regulation of norisoprenoids in ripe rhb fruits treated with abamine sg, a carotenoid dioxygenase inhibitor applied at late steps of fruit development, confirmed the hypothesis that their production is mediated by carotenoid cleavage dioxygenases.
although abamine sg was reported to be a specific carotenoid cleavage dioxygenase and aba biosynthesis inhibitor, more potent than the previously synthesized abamine  <cit> , the authors did not investigate pathways other than that leading to aba formation under stress conditions.
therefore, the observed unexpected reduction of other voc classes could be due to the non-specific inhibition of other dioxygenase enzymes active in different aroma pathways, suggesting that abamine sg is a useful tool to investigate voc metabolism.
if ccd <dig> is confirmed to be the major factor responsible for carotenoid degradation, then the mutation that generated the rhb cultivar is likely to be a gain-of-function mutation, restoring ccd <dig> function.
this is compatible with the dominant nature of the white flesh phenotype over the yellow one.
since the mutation originating rhb is associated with the y locus , a crucial step of future research is to determine whether the y locus, mapped on lg <dig>  is linked to the ccd <dig> gene.
the recently released draft of the peach genome sequence  will enable future comprehensive research towards the functional characterization of the peach ccd/nced family, required to confirm the presented data on ccd <dig> and to elucidate the function of each member in different peach genotypes.
CONCLUSIONS
this study presented a comprehensive molecular and biochemical research of the carotenoid and voc metabolism in peach fruit, driven by developmental and genetic cues, and pointed out the central role of carotenoid cleavage dioxygenases, namely the product of ccd <dig>  in flesh color and peach aroma formation.
by taking advantage of a wild type-mutant system contrasted in fruit flesh color, we provided new information for understanding the mechanisms controlling carotenoid biosynthesis, flesh color and volatile content in peach, which could be useful for the optimization of these important fruit quality traits.
the completion of peach genome will improve existing databases  and boost genetic and molecular research to confirm the role and interactions of carotenoid cleavage dioxygenases with other factors controlling fruit pigmentation and aroma, stimulating new comprehensive research on peach fruit quality traits  <cit> .

 among rosaceae, peach is an appealing model crop, because of its economical value, small genome, rapid generation time and several mendelian traits still to be functionally characterized. peaches are appreciated for their visual, nutritional and organoleptic features, partially contributed by carotenoids, sugars, acids and volatile organic compounds, which vary as a function of genetic, developmental and post-harvest factors. in particular, carotenoid accumulation in the mesocarp determines the difference between yellow- and white-fleshed genotypes, the latter being generally characterized by a peculiar and more intense aroma. flesh color is a mendelian trait, associated with the y locus that has been mapped on the linkage group of the prunus map but which has not been yet functionally characterized from the molecular or enzymatic point of view. natural mutations, originating flesh color chimera with irregular yellow and white distribution, have long been observed in peach. carotenoids are a widespread class of compounds having important functions across living organisms, whose accumulation shows striking phylum- and genotype-specific regulation. following the formation of the first carotenoid phytoene from the general isoprenoid pathway, the pathway bifurcates after lycopene with respect to the ring type, giving rise to carotenes and xanthophylls with either β-β or β-ε rings. in addition to their roles in plants as photosynthetic accessory pigments and colorants, carotenoids are also precursors to norisoprenoids. norisoprenoids are commonly found in flowers, fruits, and leaves of many plants and possess aromatic properties together with low odor thresholds, thus having a strong impact on fruit and flower aroma even at low levels. an increasing number of dioxygenase enzymes that specifically cleave carotenoid compounds to form volatile norisoprenoids, abscisic acid and regulators of plant growth and development has been characterized. these enzymes have been referred to as carotenoid cleavage dioxygenases and 9-cis-epoxycarotenoid dioxygenases and represent a plant multienzyme family: arabidopsis has nine ccd/nced members, of which four have been classified as ccds and the remaining as aba-related nceds. functional analysis of ccd enzymes determined that ccd and ccd are mostly related to the synthesis of norisoprenoid plant hormones, while ccd and ccd are preferentially involved in volatile production, by using different carotenoid substrates with variable specificity and cleavage site, which probably contributes to the diversity of norisoprenoids found in nature. the synthesis of β-ionone, geranylacetone and 6-methyl-5-hepten-2-one in tomato fruits increases 10- fold during fruit ripening and these compounds were produced by the activity of ccd products. silencing of ccd genes resulted in a significant decrease of the β-ionone content of tomato ripe fruits and petunia flowers, and increased pigmentation of potato tubers and chrysanthemum flowers. ccds were also implied to be involved in the formation of important apocarotenoid aroma compounds in melon fruit. furthermore, comparative genetics studies have indicated that carotenoid pigmentation patterns have profound effects on the norisoprenoid and monoterpene aroma volatile compositions of tomato and watermelon fruits. many norisoprenoids strongly contribute to peach fruit aroma, and their levels increase during fruit ripening. the partial purification and biochemical characterization of β-carotene degrading enzyme from nectarine skin extracts was associated with the formation of c norisoprenoids. the study of two nced-encoding genes from peach showed their differential expression, suggesting a functional relation to aba formation during fruit ripening. the recent synthesis of specific carotenoid dioxygenase inhibitors enables to assess the role of such enzymes in vivo, not only in aba biosynthesis but also in fruit voc metabolism. in order to improve our knowledge of carotenoid and voc biosynthesis in peach fruit and determine the factor controlling carotenoid accumulation in peach flesh, the two cultivars'redhaven' and its white-fleshed bud sport mutant'redhaven bianca' were investigated. carotenoid accumulation, voc content and transcript levels of relevant carotenoid biosynthetic genes were studied at five stages of fruit development. the effect of the carotenoid dioxygenase inhibitor, abamine sg, on fruit voc composition at full ripening was also studied.  fruit phenotype during ripening at s1-s stages, whole fruits of rhb and rh look similar, while the final ripening stages br and s carotenogenesis is well established in rh fruits, and differences in flesh color between yellow-fleshed rh and white-fleshed rhb fruits become dramatic. the yellow pigmentation visible along the suture at full ripening stage indicates that rhb is a bud sport chimera mutant, in which the mutation does not involve the l-i apical cell layer, that originates the epidermis and several cells layers at the suture of the ovary wall. the flesh color phenotype has remained stable throughout several cycles of clonal propagation, pointing out the stability of the chimera in rhb. the mutation is transmitted to the progeny in a mendelian fashion, and is associated to the y locus controlling fruit flesh color. rhb is heterozygous for this locus, originating either 1: of yellow- to white-fleshed seedlings when selfed or 1: of yellow- to white-fleshed progenies when crossed with yellow peach accessions. the yellow strip is not observed in white-fleshed rhb progenies, because the gametes does not originate from the l cell layer but from the fully-mutated l layer. the major fruit quality traits and skin color parameters of the two cultivars were also measured, showing statistically significant differences only for soluble solids content. carotenoid composition of rhb and rh fruits in carotenoid-containing fruits, the massive biosynthesis of such compounds is generally associated with late ripening stages and plastid transition from chloroplasts into chromoplasts. at early ripening stages s and s fruits of rhb and rh had similar total carotenoid levels and accumulated only a few carotenoid compounds, dominated by the presence of lutein and β-carotene. from the s stage, rh mesocarp accumulated increasing amounts of carotenoids that peaked at the s stage to provide the solid yellow flesh color, while carotenoid content in rhb flesh remained low. detailed hplc analysis revealed the presence of specific carotenoid compounds in the two genotypes, some of which rather uncommon and present in rh only, whose main chemical structures are illustrated. until stage s and br, lutein and its -isomers were the major carotenoids in fruits of both genotypes, accounting for over 50% of the total carotenoid pool. other major carotenoids at early stages were β-carotene, relatively abundant in fruits of both cultivars, and neochrome epimers, which accumulated only in rh fruits. from stage s not only did rh fruits have higher carotenoid levels, but also a range of carotenoid compounds much wider than rhb. at full ripening s stage, xanthophylls made up the majority of carotenoids - zeaxanthin was the main carotenoid in rhb, while antheraxanthin, luteoxanthin and zeaxanthin were the most abundant compounds in rh fruits. average values are listed in descending order with respect to rh composition and expressed in ng/g fresh weight. pda λ: nm. tent.: tentative identification. n.d.: not detectable. gene expression analyses relative transcript levels of three genes involved in isoprenoid metabolism and twelve genes involved in carotenoid biosynthesis and cleavage were measured in rh and rhb mesocarp at s s s br and, s stages by reverse transcription quantitative real-time pcr. the isoprenoid pathway genes, dxs and cmk, had very low transcript levels throughout fruit development in both cultivars. hdr showed a sharp peak of expression at the s stage which declined at later stages in the yellow-fleshed rh, while its expression remained high in rhb. similarly, early carotenoid pathway genes psy and zds showed a peak at the s stage in rh, while in rhb these transcripts showed a constant increase until the s stage. among later pathway genes, only chy-b was strongly up-regulated in rhb, while, ccd and nced expression was generally low in both genotypes, with the exception of ccd which was significantly up-regulated in rhb at late ripening stages, its transcript level being 13-fold higher than that in rh at the s stage. hierarchical clustering analysis of gene expression data clustered the ripening stages consistently with their chronological order in joint analysis of both genotypes and in rh alone. in rh, a clear co-regulation of genes encoding enzymes closely positioned in the pathway was observed. instead, in the rhb mutant the majority of these co-regulation clusters was broken, with the exception of ccd and chy-b genes which remained co-regulated. voc analyses levels of different vocs were studied in rhb and rh during fruit ripening by gc-ms. in total, vocs were detected, assigned to aromatic and branched chain amino acid-related, fatty acid-related, furan-related, lactone, monoterpene and norisoprenoid classes, quantified and underwent further analyses. average data of to replicates. values are in ng/g fresh weight. id.: identification method. positive compound identification was obtained by matching both ms and ki or standard compound data. otherwise, putative best compound is listed. ri: retention index. aa: amino acid. n.d.: not detectable. the two genotypes had a similar, ripening-associated accumulation of total vocs starting from the s stage, while early s and s stages were characterized by very low volatile content. detailed analysis pointed out differences in the accumulation of the distinct voc pools in the two genotypes. furan-related compounds accumulated at the highest levels in both genotypes, followed by norisoprenoids and fatty acid-related compounds, whose maximum levels were about 5-fold lower than those of the furans. the other classes accumulated at lower absolute levels, with maxima in the range of hundreds of μg/g fresh weight. the two genotypes displayed similar ripening-associated patterns for aromatic and branched chain amino acid-, fatty acid-, and furan-related classes, with a peak at the s3/br stages and a more or less pronounced decline at later ripening stage. total lactone and monoterpene contents displayed a different pattern, with a strong up-regulation only at final s ripening stage in the two cultivars. at the s stage, all the six above-mentioned voc classes had higher levels in rh fruits. a remarkable exception was the norisoprenoid pool, which accumulated in rhb fruits at levels higher than those of rh from s stage on. norisoprenoid pattern in rhb fruits peaked at br and was constant through s stage, while in rh fruits it displayed a linear increase from s stage. in particular, the three identified norisoprenoids 3-hydroxy- -epoxide-β-ionone, 3-hydroxy-β-damascone and 4-hydroxy-,6-trimethyl-4--2-cyclo-hexen-1-one were responsible for the higher total norisoprenoid levels in fruits of rhb, with an almost 3-fold difference at the s stage, when the typical floral scent of white-fleshed peach fruits reaches its maximum. at any ripening stage, the level of each identified norisoprenoid compound was always higher in the white-fleshed rhb than in rh. instead, the less prominent unidentified norisoprenoids had a similar accumulation pattern in the two genotypes, with higher levels in rh fruits. pca was performed on the whole gc-ms dataset to provide a more intuitive visualization of data and to discriminate the different ripening stages in the two varieties with respect to voc composition. a preliminary pca analysis was carried out including all five stages, and resulted in a poor separation of most samples, with the exception of rhb-s rh-s rh-s and rh-br. principal components and explained 67% and 21% of the total variability, respectively. a narrower analysis was then carried out by excluding the s and s samples, which allowed the complete discrimination of the six late ripening samples of both genotypes. in this closer analysis, the new calculated principal components and accounted for 76% and 13% of the total variability, respectively. effect of a carotenoid dioxygenase inhibitor on voc production the effect of abamine sg treatment was assessed in rhb ripe fruits, injected once a week from the s stage. as expected, abamine sg injection resulted in a drastic reduction of the levels of both identified and unknown norisoprenoids. unexpectedly, this treatment also down-regulated the content of the other vocs. the strongest reduction was observed for furan-related, monoterpene and lactone pools, while the total content of aromatic amino acid- and fatty acid-related compounds was the least affected by abamine sg treatment. discussion peach fruits contain carotenoid compounds with significant antioxidant capacity and claimed beneficial health effects. the enzymatic cleavage of these compounds results in the production of volatile norisoprenoids. our study investigated the expression of carotenoid genes, the carotenoid content and the volatile composition in the wild type yellow-fleshed rh and its white-fleshed rhb during fruit ripening. key regulatory steps and regulation mechanisms controlling isoprenoid and carotenoid flux in many species, also through biotechnology-based approaches to carotenoid manipulation, have been extensively reviewed. differential accumulation of carotenoids in rh and rhb became evident at stage s and reached its maximum at stage s when rh fruits accumulated approximately 10-fold more carotenoids than rhb, composed mainly of β-ring carotenoids. accordingly, the two cultivars showed strikingly different developmental patterns of expression of several isoprenoid and carotenoid genes. however, several differential regulation events appeared to be the effect, rather than the cause, of the differential carotenoid accumulation. for instance, in rhb the up-regulation of hdr, psy, and zds genes at late stages appears to be the result of a feed-back repression by either the lower carotenoid levels in this genotype or their cleavage products, acting on the transcript levels of these genes. examples of feed-back regulation of carotenoid gene transcripts have been described in plants where carotenoid biosynthesis has been altered through the use of inhibitors or metabolic engineering. a similar higher expression of early carotenoid genes was found in a white-fleshed apricot cultivar with respect to an orange-fleshed genotype, possibly suggesting common regulation mechanisms of early carotenoid gene expression based on feedback regulation mediated by carotenoid end products. the generally low expression at late br/s stages of the studied carotenoid genes in the yellow-fleshed rh might point out a control of metabolite flux based on steady-state gene/enzyme expression rather than up- or down regulation of transcription. on the other hand, in rhb fruits the strong up-regulation at late stages of chy-b and ccd genes is negatively correlated with the accumulation of β-ring carotenoids, and positively correlated with that of identified β-ring norisoprenoids. the biochemical function of the chy-b and ccd gene products is compatible with the observed phenotype of rhb: chy-b funnels carotenoids into the β-xanthophyll branch, producing substrates cleaved by carotenoid dioxygenases, including ccd furthermore, chy-b is a negative regulatory step in several plant systems: inhibition of its expression through transgenosis or natural genetic variation results in higher β-carotenoid levels in potato tubers and maize endosperm. similarly, the levels of ccd transcript negatively correlate with carotenoid levels. aubert et al. described the presence of several c norisoprenoids, mainly derived from the cleavage of β-ring xanthophylls like in the present study, in yellow- white-fleshed nectarines. unlike rh and rhb, the two cultivars were not isogenic, but similarly to the present case, the white-fleshed nectarine'vermeil' had higher norisoprenoid contents than the yellow-fleshed'springbright'. however, in two other studies, the β-ionone levels detected in a number of white-fleshed genotypes were lower than those of several unrelated yellow-fleshed accessions. such examples show that flesh color per se is not sufficient to determine the levels of norisoprenoid vocs, confirming that volatile composition strongly depends on genetic factors other than carotenoid levels. in our study, rhb ripe fruits had higher norisoprenoid content than that of rh, derived from carotenoid cleavage. as a likely consequence of the redirection of metabolite flux towards the synthesis of nor/isoprenoid compounds in rhb fruits, the levels of all other voc pools were lower than those in rh fruits. among aroma-related carotenoid dioxygenases, both ccd and ccd enzymes cleave carotenoids at the and 9',10' positions and have a major role in the formation of β-ionone and other fruit and flower norisoprenoids: the β-carotene degradation displayed by yellow nectarine skin extracts most likely corresponds to ccd and/or ccd activity. compared with ccd1s, the ccd enzymes were characterized more recently from several crops, and shown to have a major impact on organ pigmentation: ccd expression was higher in white-fleshed potato and in white-flowered chrysanthemum genotypes than in their respective yellow-pigmented counterparts, and rnai-mediated knockout boosted carotenoid accumulation. the small ccd family contains at least two forms of genes with different structure, expression patterns and genome position, and their encoded enzymes show different substrate specificity. the existence of plastid target peptides and the demonstrated plastid localization of ccd enzymes allow these enzymes direct access to the carotenoid substrates, suggesting that they start the carotenoid degradation and norisoprenoid synthesis pathway. on the other hand, the lack of correlation between the patterns of ccd expression and norisoprenoid content in the flesh of both cultivars reflects a situation common to other fruits like grape, melon, and tomato. this is a likely consequence of the different localization of ccd1s and their substrates, since carotenoids are accumulated in plastids but known ccd1s lack plastid transit peptides, and/or substrate preference, since each carotenoid dioxygenase can accept different carotenoids. our data suggest that because of their different subcellular localization, ccd1s only contribute to volatile production, while ccd4s are likely to control also carotenoid degradation. as to other dioxygenase-encoding genes studied, nced and nced expression patterns were up-regulated during ripening, in agreement with the reported physiological increase of aba content at late fruit ripening stages, with a profile similar to that of ethylene-related accs and acco genes. the transcriptional control of peach norisoprenoid content by specific dioxygenase genes is similar to that of fatty acid pathway-related genes and enzymes, whose expression patterns generally show a strong positive correlation with those of the corresponding volatiles. the strong down-regulation of norisoprenoids in ripe rhb fruits treated with abamine sg, a carotenoid dioxygenase inhibitor applied at late steps of fruit development, confirmed the hypothesis that their production is mediated by carotenoid cleavage dioxygenases. although abamine sg was reported to be a specific carotenoid cleavage dioxygenase and aba biosynthesis inhibitor, more potent than the previously synthesized abamine, the authors did not investigate pathways other than that leading to aba formation under stress conditions. therefore, the observed unexpected reduction of other voc classes could be due to the non-specific inhibition of other dioxygenase enzymes active in different aroma pathways, suggesting that abamine sg is a useful tool to investigate voc metabolism. if ccd is confirmed to be the major factor responsible for carotenoid degradation, then the mutation that generated the rhb cultivar is likely to be a gain-of-function mutation, restoring ccd function. this is compatible with the dominant nature of the white flesh phenotype over the yellow one. since the mutation originating rhb is associated with the y locus, a crucial step of future research is to determine whether the y locus, mapped on lg is linked to the ccd gene. the recently released draft of the peach genome sequence will enable future comprehensive research towards the functional characterization of the peach ccd/nced family, required to confirm the presented data on ccd and to elucidate the function of each member in different peach genotypes.  this study presented a comprehensive molecular and biochemical research of the carotenoid and voc metabolism in peach fruit, driven by developmental and genetic cues, and pointed out the central role of carotenoid cleavage dioxygenases, namely the product of ccd in flesh color and peach aroma formation. by taking advantage of a wild type-mutant system contrasted in fruit flesh color, we provided new information for understanding the mechanisms controlling carotenoid biosynthesis, flesh color and volatile content in peach, which could be useful for the optimization of these important fruit quality traits. the completion of peach genome will improve existing databases and boost genetic and molecular research to confirm the role and interactions of carotenoid cleavage dioxygenases with other factors controlling fruit pigmentation and aroma, stimulating new comprehensive research on peach fruit quality traits. 
abamine sg - an inhibitor of ccd enzymes - decreased the levels of both isoprenoid and non-isoprenoid volatiles in rhb fruits, indicating a complex regulation of volatile production. to investigate the regulation of carotenoid metabolism, as related to norisoprenoids and other volatile compounds in peach, and the role of carotenoid dioxygenases in determining differences in flesh color phenotype and volatile composition, the expression patterns of relevant carotenoid genes and metabolites were studied during fruit development along with volatile compound content. more in general, dioxygenases appear to be key factors controlling volatile composition in peach fruit, since abamine sg-treated'redhaven bianca' fruits had strongly reduced levels of norisoprenoids and other volatile classes. lower carotenoid levels and higher levels of norisoprenoid volatiles were observed in rhb, which might be explained by differential activity of carotenoid cleavage dioxygenase enzymes. differential expression of ccd is likely to be the major determinant in the accumulation of carotenoids and carotenoid-derived volatiles in peach fruit flesh. comparative functional studies of peach carotenoid cleavage enzymes are required to fully elucidate their role in peach fruit pigmentation and aroma. the two genotypes also showed differences in the expression patterns of several carotenoid and isoprenoid transcripts, compatible with a feed-back regulation of these transcripts.
 carotenoids are plant metabolites which are not only essential in photosynthesis but also important quality factors in determining the pigmentation and aroma of flowers and fruits. to investigate the regulation of carotenoid metabolism, as related to norisoprenoids and other volatile compounds in peach, and the role of carotenoid dioxygenases in determining differences in flesh color phenotype and volatile composition, the expression patterns of relevant carotenoid genes and metabolites were studied during fruit development along with volatile compound content. two contrasted cultivars, the yellow-fleshed'redhaven' and its white-fleshed mutant'redhaven bianca' were examined.  the two genotypes displayed marked differences in the accumulation of carotenoid pigments in mesocarp tissues. lower carotenoid levels and higher levels of norisoprenoid volatiles were observed in rhb, which might be explained by differential activity of carotenoid cleavage dioxygenase enzymes. in fact, the ccd transcript levels were dramatically higher at late ripening stages in rhb with respect to rh. the two genotypes also showed differences in the expression patterns of several carotenoid and isoprenoid transcripts, compatible with a feed-back regulation of these transcripts. abamine sg - an inhibitor of ccd enzymes - decreased the levels of both isoprenoid and non-isoprenoid volatiles in rhb fruits, indicating a complex regulation of volatile production.  differential expression of ccd is likely to be the major determinant in the accumulation of carotenoids and carotenoid-derived volatiles in peach fruit flesh. more in general, dioxygenases appear to be key factors controlling volatile composition in peach fruit, since abamine sg-treated'redhaven bianca' fruits had strongly reduced levels of norisoprenoids and other volatile classes. comparative functional studies of peach carotenoid cleavage enzymes are required to fully elucidate their role in peach fruit pigmentation and aroma. 