Fruit phenotype during ripening

At S1-S3 stages, whole fruits of RHB (Figure 2A) and RH (Figure 2B) look similar, while the final ripening stages Br and S4, 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 (Figure 2A, S4) 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 [28]. The flesh color phenotype has remained stable throughout several cycles of clonal propagation, pointing out the stability of the chimera in RHB (Liverani A., unpublished data). 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 (Yy), originating either 1:3 of yellow- to white-fleshed seedlings when selfed or 1:1 of yellow- to white-fleshed progenies when crossed with yellow (yy) peach accessions (Liverani A., unpublished data). The yellow strip is not observed in white-fleshed RHB progenies (Liverani A., unpublished data), because the gametes does not originate from the L1 cell layer but from the fully-mutated L2 layer.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 (Additional File 2).

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 S1 and S2, 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 (Figure 3; Table 1). From the S3 stage, RH mesocarp accumulated increasing amounts of carotenoids that peaked at the S4 stage to provide the solid yellow flesh color, while carotenoid content in RHB flesh remained low (Figure 3). Detailed HPLC analysis revealed the presence of specific carotenoid compounds in the two genotypes (Table 1), some of which rather uncommon and present in RH only, whose main chemical structures are illustrated (Additional File 1). Until stage S3 (RH) and Br (RHB), lutein and its (Z)-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 S3, not only did RH fruits have higher carotenoid levels, but also a range of carotenoid compounds much wider than RHB (Table 1). At full ripening S4 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 (Table 1).

Gene expression analyses

Relative transcript levels of three genes involved in isoprenoid metabolism [1-deoxy-d-xylulose 5-phosphate synthase (dxs), 4-(cytidine 5'-diphospho)-2-C-methyl-d-erythritol kinase (cmk) and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (hdr)] and twelve genes involved in carotenoid biosynthesis and cleavage [phytoene synthase (psy), phytoene desaturase (pds), ζ-carotene desaturase (zds), lycopene β-cyclase (lcy-b), lycopene ε-cyclase (lcy-e), carotene β-hydroxylase (chy-b), carotene ε-hydroxylase (chy-e), zeaxanthin epoxidase (zep), two carotenoid cleavage dioxygenases (ccd1 and ccd4), and two 9-cis-epoxycarotenoid dioxygenases (nced1 and nced2)] (Figure 1) were measured in RH and RHB mesocarp at S1, S2, S3, Br and, S4 stages by reverse transcription quantitative real-time PCR (RT-qPCR).

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 S2 stage which declined at later stages in the yellow-fleshed RH, while its expression remained high in RHB (Figure 4A). Similarly, early carotenoid pathway genes psy and zds showed a peak at the S3 stage in RH, while in RHB these transcripts showed a constant increase until the S4 stage (Figure 4B). Among later pathway genes (lcy-b , lcy-e, chy-b, chy-e and zep), only chy-b was strongly up-regulated in RHB (Figure 4C), while, ccd and nced expression was generally low in both genotypes, with the exception of ccd4, which was significantly up-regulated in RHB at late ripening stages, its transcript level being 13-fold higher than that in RH at the S4 stage (Figure 4D).

Hierarchical clustering analysis (HCA) of gene expression data clustered the ripening stages consistently with their chronological order in joint analysis of both genotypes (Additional File 3A) and in RH alone (Additional File 3B). In RH, a clear co-regulation of genes encoding enzymes closely positioned in the pathway (dxs and cmk; psy, pds and zds; lcy-b and lcy-e; nced1, ccd1, ccd4 and, surprisingly, chy-b) was observed (Additional File 3C). Instead, in the RHB mutant the majority of these co-regulation clusters was broken, with the exception of ccd4 and chy-b genes which remained co-regulated (Additional File 3C).

VOC analyses

The two genotypes had a similar, ripening-associated accumulation of total VOCs starting from the S3 stage, while early S1 and S2 stages were characterized by very low volatile content (Additional File 4). Detailed analysis pointed out differences in the accumulation of the distinct VOC pools in the two genotypes (Figure 5). 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 (compare Figures 5D, C and 5H). 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(s) (Figures 5A-D). Total lactone and monoterpene contents displayed a different pattern, with a strong up-regulation only at final S4 ripening stage in the two cultivars (Figure 5E-F). At the S4 stage, all the six above-mentioned VOC classes had higher levels in RH fruits (Figure 5A-F).

A remarkable exception was the norisoprenoid pool, which accumulated in RHB fruits at levels higher than those of RH from S3 stage on. Norisoprenoid pattern in RHB fruits peaked at Br and was constant through S4 stage, while in RH fruits it displayed a linear increase from S3 stage (Figure 5H). In particular, the three identified norisoprenoids 3-hydroxy-5,6-epoxide-β-ionone, 3-hydroxy-β-damascone and 4-hydroxy-3,5,6-trimethyl-4-(3-oxo-1-butenyl)-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 S4 stage (Figure 5 H1), 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 (Additional File 5). Instead, the less prominent unidentified norisoprenoids had a similar accumulation pattern in the two genotypes, with higher levels in RH fruits (Figure 5 H2).

PCA was performed on the whole GC-MS dataset (41 major VOCs) 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 (Figure 6A), with the exception of RHB-S1, RH-S2, RH-S3 and RH-Br. Principal components 1 and 2 explained 67% and 21% of the total variability, respectively (Figure 6A). A narrower analysis was then carried out by excluding the S1 and S2 samples, which allowed the complete discrimination of the six late ripening samples of both genotypes (Figure 6B). In this closer analysis, the new calculated principal components 1 and 2 accounted for 76% and 13% of the total variability, respectively (Figure 6B).

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 S3 stage. As expected, abamine SG injection resulted in a drastic reduction of the levels of both identified and unknown norisoprenoids (Figure 7). 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 (Figure 7).

Plant material and sampling

Fruits of cv RH and RHB were harvested from trees grown in an experimental field located near Forlì (Po Valley, Italy; 44.161° N, 12.088° E) at five different ripening stages according with the growth curve (Figure 2): S1 (about 35 days after pollination, dap), S2 (about 50 dap), S3 (about 90 dap), Breaker (Br; about 115 dap), and S4 (about 122 dap) (Figure 2). For molecular and biochemical analyses, two replicates of four representative fruits of each stage were sampled, peeled, cut into 0.5-cm slices and the mesocarp was immediately frozen in liquid nitrogen and stored at -80 °C. In RHB, the tissues near the suture were discarded since were not affected by the mutation originating the white-fleshed phenotype. For visual and organoleptic quality scoring (Additional File 2), S4 fruits underwent the following measurements immediately after harvest: flesh firmness (FTA penetrometer, Turroni, Italy, equipped with an 8-mm plunger tip), soluble solid content (Smart1 automatic refractometer; Atago Co., Tokyo, Japan), titratable acidity (titration of 10 ml peach juice with NaOH 0.2 M until pH 8.2), skin color (Chroma Meter CR-200 reflectance colorimeter; Minolta Camera Co., Osaka, Japan).

For abamine SG treatments, 100 μl of an abamine SG solution (0,1 mM in DMSO) were injected with a hypodermical needle in different points of RHB fruits once a week from S3 stage until S4. DMSO-injected fruits were taken as controls. Mesocarp samples were collected at S4 stage as previously described, and used for GC-MS analyses.

Molecular procedures

Experiments complied with the MIQE guidelines [49], and relevant information is contained in the manuscript. Total RNA (two independent replicates for each sample) was isolated from frozen fruit tissue as reported [50]. First strand cDNA was synthesized from 1 μg total RNA in 30 μL with oligo-d(T)17 and Superscript III (Invitrogen, Milan, Italy), according to manufacturer's instructions. cDNA concentration in the RT mix was quantified using a ND-1000 UV spectrophotometer (Nanodrop, Wilmington, USA). First strand cDNA (10 ng) was used as template for RT-qPCR assays, carried out with primers for rps28 and carotenoid genes. The rps28 gene was chosen as reference gene, based on preliminary tests (data not shown). All reactions were performed using the Applied Biosystems 7900HT Real Time PCR system and Platinum^® SYBR^® Green RT-qPCR SuperMix-UDG plus ROX (Applera Italia, Monza, Italy), following the manufacturer's procedures. No template and no amplification controls were included in each experiment.

Three RT-qPCR runs were carried out for each cDNA and gene to serve as technical replicates. PCR conditions were: 5 min at 95°C, followed by 45 cycle at 95°C for 15 s and at 58°C for 60 s. At the end of the PCR, melting curve analysis was carried out to check for the presence of the correct amplicons only. Relative transcript abundance was quantified using the relative standard curve method described in the ABI PRISM 7900 HT manual, and the data was normalized against the quantity of the reference rps28 transcript. Serial 10-fold dilution of each gene fragment were used to calculate the standard curve and measure the amplification efficiency for each target and reference gene. Sequences of peach dxs, cmk, hdr, psy, pds, zds, lcy-b, lcy-e, chy-b, chy-e, zep, ccd1, nced1 and nced2 genes, and the reference gene rps28, were obtained from NCBI database and [51]. A 1.84-kb putative peach ccd4 gene was retrieved from the recently published peach draft genome [52] (Phytozome v6.0 search tool at http://www.phytozome.org/peach) by searching with a Malus ccd4 sequence (EU327777). The identified peach ccd4 coding sequence share 80% identity with its Malus ortholog. All RT-qPCR primers for studied genes were designed with Primer Express (Applera Italia, Monza, Italy) software (Additional File 6).

Analysis of VOCs

Frozen fruit tissue (15 g) was ground in liquid N₂ into a fine powder. The volatile components were extracted with 100 ml methyl tert-butyl ether (MTBE) by vigorous shaking on a shaker apparatus overnight. Iso-butyl benzene (5 μg) was added as internal standard. The upper MTBE layer was separated, dried with sodium sulfate, and concentrated to a volume of 0.5 ml under a N₂ stream. Samples were kept at 4°C until analysis. A 1-μl aliquot of the concentrated MTBE extract was injected into a GC-MSD (splitless mode). Results were an average of 6-8 replicate measurements.

GC-MS analysis was carried out using an Agilent GC-MSD system (CA, USA) EI Scan, equipped with a RTx-5sil MS column (injector temperature: 250°C; detector temperature: 280°C, 70 eV). Oven temperature profile was: 50 °C (1 min), 5° C/min to 280° C, 280° (5 min). Gas flow: 0.8 ml/min. Mass range: 41 to 500 m/z. Compound identification was performed by comparing their relative retention indices and mass spectra with those of authentic standards or with those found in the literature and supplemented with NIST 98 and QuadLib 1607 GC-MS libraries. A mixture of straight-chain alkanes (C7-C23) was injected into the column under the above-mentioned conditions for retention index calculation [53].

Isolation and HPLC analysis of carotenoids

Plant material was homogenized in the presence of methanol and extracted three-times with MeOH. The methanolic extracts were combined and the carotenoid content of this solutions was transferred in a separatory funnel into toluene:hexane (1:1) mixture; evaporated and dissolved in diethyl-ether. After methanolic extractions, plant materials were then extracted once with diethyl-ether. The resulting extracts (ethereal solution of methanolic extracts + ethereal extract) were combined and saponified in a heterogeneous phase with 30% KOH/MeOH overnight (the 30% KOH/MeOH solution was layered on the lower part of the ethereal total extracts). After this process the reaction mixture was washed to alkali-free in a separatory funnel, dried over anhydrous Na₂SO₄, evaporated and the residues were dissolved in EtOH preparing the corresponding stock solutions for quantitative analysis. All of these procedures were carried out in semi-darkness and in N₂-atmosphere [54–56].

The quantitative UV/VIS spectra of the corresponding stock solutions were recorded by a Jasco V-530 spectrophotometer. The total carotenoid content of the samples was calculated according to the Lambert-Beer's law (average molar extinction coefficient: 100,000; average molar mass of carotenoids: 600). The HPLC separation was performed with a Dionex Softron instrument (Germering, Germany) equipped with a Dionex P680 gradient pump, a Dionex PDA-100 detector and an end-capped C18 column (250 × 4.6 mm internal diameter; Merck LiChrospher 100 RP-18; 5 μm) thermostated at 22 ˚C. Elution was performed using 12% H₂O/MeOH (A), MeOH (B), 50% Acetone/MeOH (C) at a flow rate of 1.25 ml min^-1. The gradient program was: 100% A (0-2 min); 80% A and 20% B (2-10 min); 50% A and 50% B (10-18 min); 100% B (18-27 min); 100% C (27-34 min); 100% B (34-43 min); 100% A (43-56 min). Data acquisition was performed at 450 nm detection wavelength by Chromeleon 6.70 software.

Statistical analyses

GC-MS data were submitted to principal component analysis (PCA) using Systat 11 software (http://www.systat.com). The data set was made up of data from eight repetitions of each ripening stage of RH and RHB. The variable set was made of the major 41 volatile aroma compounds. PCA involves a mathematical procedure that transforms a number of possibly correlated variables into a smaller number of uncorrelated variables called principal components. The first principal component accounts for as much of the variability in the data as possible, and each succeeding component accounts for as much of the remaining variability as possible. Gene expression data were submitted to hierarchical clustering analysis (HCA) using Systat 11. The HCA data set was made up from the means from independent experiments, for every stage of fruit development, of both genotypes. Means from independent RT-qPCR experiments were subjected to one-way ANOVA and Tukey's pairwise comparisons, and fruit quality data to t test, carried out using PAST (http://folk.uio.no/ohammer/past/).