TYPE Original Research PUBLISHED 01 August 2022 DOI 10.3389/fpls.2022.913433 Environmentally driven OPEN ACCESS transcriptomic and metabolic changes leading to color EDITED BY Claudio Bonghi, University of Padua, Italy REVIEWED BY Jinwook Lee, differences in “Golden Reinders” apples Chung-Ang University, South Korea Joanna Lado, National Institute for Agricultural Research (INIA), Uruguay *CORRESPONDENCE Pablo Fernández-Cancelo1, Ariadna Iglesias-Sanchez2,3, Manuel Rodriguez-Concepcion manuelrc@ibmcp.upv.es Salvador Torres-Montilla2,3, Albert Ribas-Agustí4, Neus Teixidó1, Jordi Giné-Bordonaba Manuel Rodriguez-Concepcion2,3* and Jordi Giné-Bordonaba1* jordi.gine@irta.cat 1 Postharvest Programme, Institute of Agrifood Research and Technology (IRTA), Lleida, Spain, SPECIALTY SECTION 2 Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Barcelona, Spain, This article was submitted to 3 Institute for Plant Molecular and Cell Biology (IBMCP), CSIC-Universitat Politècnica de València, Crop and Product Physiology, Valencia, Spain, 4 Food Safety and Functionality Programme, IRTA, Monells, Spain a section of the journal Frontiers in Plant Science RECEIVED 05 April 2022 Apple is characterized by its high adaptation to diverse growing environments. ACCEPTED 13 July 2022 PUBLISHED 01 August 2022 However, little is still known about how different environments can regulate at the metabolic or molecular level specific apple quality traits such as the CITATION Fernández-Cancelo P, Iglesias-Sanchez A, yellow fruit peel color. In this study, changes in carotenoids and chlorophylls, Torres-Montilla S, Ribas-Agustí A, Teixidó N, antioxidants as well as differences in the transcriptome were investigated by Rodriguez-Concepcion M and Giné-Bordonaba J (2022) Environmentally comparing the peel of “Golden Reinders” apples grown at different valley driven transcriptomic and metabolic and mountain orchards. Mountain environment favored the development changes leading to color differences in of yellow color, which was not caused by an enhanced accumulation of “Golden Reinders” apples. Front. Plant Sci. 13:913433. carotenoids but rather by a decrease in the chlorophyll content. The yellow doi: 10.3389/fpls.2022.913433 phenotype was also associated to higher expression of genes related to COPYRIGHT chloroplast functions and oxidative stress. Time-course analysis over the © 2022 Fernández-Cancelo, last stages of apple development and ripening, in fruit from both locations, Iglesias-Sanchez, Torres-Montilla, Ribas- Agustí, Teixidó, Rodriguez-Concepcion and further revealed that the environment differentially modulated isoprenoids Giné-Bordonaba. This is an open-access and phenylpropanoid metabolism and pointed out a key role for H2O2 in article distributed under the terms of the triggering apple peel degreening. Overall, the results presented herein provide Creative Commons Attribution License (CC BY). The use, distribution or new insights into how different environmental conditions regulate pigment reproduction in other forums is permitted, and antioxidant metabolism in apple leading to noticeable differences in the provided the original author(s) and the copyright owner(s) are credited and that apple peel color. the original publication in this journal is cited, in accordance with accepted KEYWORDS academic practice. No use, distribution or reproduction is permitted which does not antioxidant metabolism, color, environment, isoprenoids, oxidative stress, comply with these terms. phenylpropanoids, Malus domestica (Bork.) Introduction Fruit peel color is an attribute highly appreciated by consumers (Iglesias et al., 2008) which is controlled by the relative concentrations of pigments, including isoprenoids (chlorophylls and carotenoids) and phenylpropanoids (anthocyanins; Merzlyak et al., 2003). Chlorophylls and carotenoids are both photosynthetic pigments associated with Frontiers in Plant Science 01 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 light-harvesting proteins which are involved in light absorption 2013). For instance, a recent study demonstrated that “Golden processes within the thylakoids (Polívka and Frank, 2010). While Reinders” apples grown in the Ebro valley never attained the chlorophylls are responsible for the green color in unripe apples yellow phenotype as observed in fruit grown in mountain regions, (Reay et al., 1998), carotenoids are the major pigments in yellow even if the fruit was left on the tree far beyond the commercial peel apples such as “Golden Delicious” (Delgado-Pelayo et al., harvest date (Fernández-Cancelo et al., 2022). This said, no 2014). During ripening, but also as a response to stress, chlorophyll information is currently available on how the environment levels decrease accompanying the perceived fruit color changes influences, at the metabolic and transcriptomic level, yellow peel (Yin et al., 2016). Carotenoids are synthetized and stored in color in “Golden” apples, despite being one of the most widely plastids, including chloroplasts, where they are required for the cultivated and consumed varieties worldwide (Bonany et al., 2013). assembly of the photosynthetic apparatus and are essential for Accordingly, our study aimed to decipher the biochemical and photoprotection by scavenging reactive oxygen species (ROS) and transcriptomic shifts leading to color differences in the peel of by dissipating excess light energy as heat (Rodriguez-Concepcion “Golden Reinders” apples grown in different environments et al., 2018). Carotenoids also act together with tocopherols (mountain vs. valley) as well as to elucidate if color changes were (another group of plastidial isoprenoids) as thylakoid antioxidants only caused by differences in pigments content or rather to other (Ramel et al., 2012). In addition, carotenoids are the precursors of metabolic shifts driven by environmental signals able to alter peel abscisic acid (ABA), a hormone involved in the regulation of fruit ripening. Special emphasis was also given to clarify how pigment ripening (Leng et al., 2013) and in the fruit or plant adaptation to content and antioxidant metabolism of the apple peel change environmental conditions (Wang et al., 2020). As an example, during the last stages of fruit development and ripening in ABA is able to orchestrate the fruit tolerance to abiotic stresses by response to diverse growing environments. regulating the accumulation of ROS (Zhang et al., 2019) and the expression of antioxidant enzymes (Mou et al., 2015), such as superoxide dismutase (SOD), catalase (CAT), and peroxidases Materials and methods (APX and POX; Meitha et al., 2020). Indeed, antioxidant enzymes in combination with non-enzymatic antioxidants, such as phenolic Plant material and experimental design compounds, tocopherols, carotenoids, ascorbic acid, and glutathione (Shao et al., 2008), are able to limit the accumulation In 2018, four different orchards located in the province of of damaging ROS produced either during aerobic metabolism Lleida (Catalonia, North-East Spain) were used to assess the (Venditti et al., 2013) or under stress conditions (Choudhury et al., influence of the environment on apple color at the commercial 2017). ROS (i.e., H2O2) not only act as damaging but also as harvest date (CHD): two orchards from valley areas, Alcanó signaling molecules participating in transduction cascades (214 meters above sea level, masl) and Vilanova (195 masl), and (Saxena et al., 2016), interacting with plant hormones (Chen et al., two from mountain areas, Llesp (989 masl) and Gotarta (1,191 2016), and regulating physiological processes such as fruit masl). “Golden Reinders” apples were harvested at the CHD development (Khandaker et al., 2012). Some studies suggest that based on flesh firmness (71 ± 2.5 N) and starch index values H2O2 may regulate, for instance, the chloroplast-to-chromoplast (6 ± 0.8) from trees of similar age and grown under the same transition in carotenoid-rich fruit during ripening (Bouvier et al., rootstock (M9) as well as similar agronomical practices. Further 1998), favoring carotenoids accumulation and color changes characterization of the fruit maturity at the time of harvest (Sadali et al., 2019). However, in “Golden” apples, carotenoid (focused on ethylene biosynthesis) is available from Fernández- levels do not appear to increase but to decrease during fruit Cancelo et al. (2021). A set of 45 fruit were harvested at the growth (Gross et al., 1978), suggesting that other mechanisms are same canopy height from 10 random trees in each location at likely involved in color development within this particular CHD. Thirty fruit were used for quality evaluations (color, apple cultivar. firmness, and starch index), whereas the peel from 15 fruit (5 The fruit pigment content is strongly influenced by the apples per replicate and 3 replicates per location) were grinded environment, mainly temperature (Lin-Wang et al., 2011) and and frozen in liquid nitrogen and kept at −80°C until further light (Llorente et al., 2016). For example, it has been observed that biochemical determinations. growing “Fuji” apples at high altitudes, where temperatures are Based on the color differences found in 2018, the next year low and sunlight incidence is high, promotes the accumulation of fruit development and ripening were monitored in Vilanova anthocyanins (Karagiannis et al., 2020). Anthocyanins own (valley) and Gotarta (mountain) orchards at 4 sampling different roles within the fruit, including the protection against points, including the CHD (165 and 167 days after full bloom photo-oxidative stress induced by high light incidence (Zheng [DAFB] in valley and mountain orchards, respectively), based et al., 2020) and contributing, as non-photosynthetic pigments, to on the percentage of final fruit size (S1: 60% final fruit size, the red fruit color. Nowadays, and as a result of climate change, S2: 80% final fruit size, S3: 90% final fruit size, and S4: 100% the environmental conditions in certain production areas are no final fruit size). Samplings started at 89 DAFB (corresponding longer suitable for developing fruit with optimal peel color (Lin- with S1) and were performed approximately every 25–30 days Wang et al., 2011), or other optimum quality traits (Sugiura et al., differing less than 5 days between locations for each sampling Frontiers in Plant Science 02 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 point. The final fruit size (equivalent to CHD) also from Rosacea (GDR2). Differentially expressed genes (DEGs) were corresponded to 1,388 and 848 growing degree days (GDD) identified with the DESeq2 statistical method in the AIR platform. for apples from Vilanova and Gotarta, respectively, within the The resulting lists were filtered using cut-offs of false discovery studied period. At each harvest, 50 fruit per location and rate < 0.05 and log2-transformed fold change (log2FC) >0.585 for sampling point were harvested at the same canopy height from upregulated genes and < −0.599 for downregulated genes. GO 10 random trees and immediately transported to the enrichment analyses were performed using default parameters laboratory for quality evaluations (30 fruit per location or with AppleMDO3 automatically followed by AgriGO4. Enriched sampling point). In parallel, peel from 20 fruit (5 apples per GO terms and p-values were then loaded into REVIGO5 to replicate and 4 replicates per location and sampling point) identify and visualize non-redundant GO terms using a cutoff were ground and frozen in liquid nitrogen and kept at −80°C value of 0.5. until further biochemical analysis. Meteorological data including temperature, relative humidity (HR), evapotranspiration, and solar radiation were recorded at cDNA and qPCR analysis Vilanova and Gotarta orchards by portable meteorological stations (Decagon Devices, Pullman, WA, United States) equipped with a The cDNA synthesis was performed on 1 μg of RNA using pyranometer (Apogee Instruments, Logan, UT, United States). the SuperScript IV First-Strand Synthesis System (Invitrogen, Carlsbad, CA, United States). The gene expression analysis was ® performed by mixing KAPA SYBR Fast qPCR Master Mix Fruit quality evaluation (Kapa Biosystems, Inc., Wilmington, MA, United States), 100 nM of each primer, and the corresponding diluted On arrival to the laboratory, fruit weight, diameter, color, cDNA. The reaction was performed on a 7,500 Real-Time PCR firmness, and starch index were measured on 30 individual fruit System (Applied Biosystems, Waltham, MA, United States) per orchard and sampling point. In the particular case of color and under the following conditions: 10 s at 95°C followed by firmness determinations, measurements were made on two 40 cycles of 95°C during 15 s and 60°C for 1 min. A non-template opposite sides of each examined fruit using a portable colorimeter control (NTC) was included by using DNA-free water instead (CM-2600d; Konica Minolta Sensing, Osaka, Japan) and a GÜSS of cDNA. A melt curve analysis was performed to check primer FTA penetrometer (FR Turoni, Forlì, Italy), respectively. Starch specific by including a final amplification cycle at 95°C for 15 s, index was evaluated by dipping equatorial fruit slices in an iodine 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s. Primers used solution (I2-KI) for 10 min. The starch index was assigned to each in this study were retrieved from the literature or designed de fruit using the starch scale from 1 to 10 developed by the Centre novo when indicated (Supplementary Table S1). Relative gene Technique Interprofessionnel des Fruits et Légumes (CTIFL, Paris, expression was expressed as Mean Normalized Expression France). Further quality measurements were earlier reported by (MNE) according to previous studies (Muller et al., 2002) using Fernández-Cancelo et al. (2021). Md8283 as a reference gene. RNA extraction and sequencing Photosynthetic pigments and tocopherols analysis RNA was extracted from lyophilized peel samples, using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St Louis, MO, Carotenoids, chlorophylls, and tocopherols were extracted United States) following the manufacturer’s recommendation. from lyophilized apple peel tissue as described for tomato leaves Electrophoresis on an agarose gel by staining with GelRed™ (Barja et al., 2021). An Agilent 1,200 series HPLC system (Agilent Nucleic Acid Gel Stain (Biotium, Hayward, CA, United States) was Technologies, Santa Clara, CA, United States) was used for the used for the evaluation of RNA integrity and the absence of separation and detection of individual species with a photodiode contaminant DNA. RNA-seq service was performed by Sequentia array for (chlorophylls and carotenoids) or a fluorescence detector Biotech SL (Barcelona, Spain). Assessment of RNA quality and (for tocopherols). Quantification was performed by comparison indexed library preparation was carried out as described (Llorente of peak areas with commercial standards. et al., 2020). Sequencing was performed using the NovaSeq6000 System (Illumina) 2×150 bp, 30 M paired reads per sample. Data analysis was performed with the online platform AIR1 using the Malus × domestica “Golden Delicious” GDDH13 v1.1 reference genome (Daccord et al., 2017) deposited in the Genome Database 2 https://www.rosaceae.org/ 3 https://www.rosaceae.org/ 4 https://systemsbiology.cau.edu.cn/agriGOv2 1 https://www.transcriptomics.cloud 5 https://revigo.irb.hr Frontiers in Plant Science 03 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 ABA quantification HCl in Milli-Q water. Sample extraction was held at 20°C with constant shaking for 2 h and mixing the samples every 30 min. The ABA was extracted from lyophilized apple peel tissue as extract was centrifuged at 24,000 × g for 30 min at 20°C. From the described elsewhere (Torres et al., 2017). The extract (5 μl) was same extract, total phenolic compounds were measured by means injected on a Waters ACQUITY UPLC chromatograph (Waters of the Folin–Ciocalteu method and total antioxidant capacity was Corporation, Milford, MA, United States) fitted with a Waters measured by the Ferric Reducing Antioxidant Power (FRAP) assay ACQUITY UPLC HSS T3 1.8 μm 100×2.1 mm column (Waters as described in previous works (Giné-Bordonaba et al., 2016). Corporation, Milford, MA, United States). Separation was carried Individual phenolic compounds from peel tissue were out at a flow rate of 0.40 ml/min using a mobile phase consisting in quantified from the same extracts used for antioxidant capacity a mixture of 0.1% formic acid and 2% of acetonitrile in water (A) determination. The methanolic extract was filtered through a and 0.1% formic acid in acetonitrile (B). The gradient program was 0.22 μm filter and injected (5 μl) on an Agilent 1,260 Infinity II held for the first 1 min at 5% B, increased linearly to 100% B in liquid chromatograph UHPLC (Agilent Technologies, Santa Clara, 1.5 min, and then maintained at 100% B for 1 min. The temperature CA, United States) fitted with a Waters XSelect HSS T3 (4.6×100 of the column was kept at 35°C and the sample compartment was mm, 2.5 μm; Waters Corporation, Milford, MA, United States) refrigerated at 10°C. Quantification was performed by comparison and a guard column Waters XSelect HSS T3 VanGuard (3.9×5 μm, of peak areas with the commercial standard. 3.5 μm; Waters Corporation, Milford, MA, United States). Separation was carried out at a flow rate of 0.75 ml/min using a mobile phase consisting of 0.1% acetic acid in water (A) and 0.1% Quantification of hydrogen peroxide and acetic acid in acetonitrile (B). The gradient program was held for malondialdehyde the first 2 min at 6% B, increased linearly to 12% B in 3 min, then raised to 30% B in 29 min, and finally elevated to 100% B in 6 min Hydrogen peroxide concentration expressed in mmol kg−1 and maintained at 100% B for 5 min. The temperature of the was determined using the PeroxiDetect Kit (Sigma-Aldrich, Saint column was kept at 40°C and the sample compartment was Louis, MO, United States) after the extraction of 2.5 g of frozen refrigerated at 10°C. Detection was performed at 280, 320, and peel in 10 ml of 5% trichloroacetic acid (TCA) based on the 520 nm, yet the online spectra were acquired in the 220–650 nm protocol described elsewhere (Giné-Bordonaba et al., 2019). wavelength range with a resolution of 0.5 nm. Phenolic Malondialdehyde (MDA) was analyzed as an index of lipid compounds were identified according to their retention time, peroxidation using the thiobarbituric acid reactive substrates spectral features, and ratios of maximum absorption peaks (λ). (TBARS) based on the protocol previously described (Martínez- When no commercial standard was available, compounds were Solano et al., 2005) using 0.5 g of frozen tissue mixed with 4 ml of tentatively identified in an Acquity UPLC system equipped with 0.1% trichloroacetic acid (TCA) solution. diode array (DAD) and triple quadrupole mass spectrometer (MS) detectors using electrospray ionization interface (Waters, Milford, MA, USA). MS scan mode was used to obtain parent molecular Ascorbic acid and dehydroascorbic acid ions and MS/MS daughters mode was used to obtain their content fragmentation patterns, with argon as collision gas and 20–30 V as collision energies. Quantification was performed using Based on previous protocols (Rassam and Laing, 2005), the calibration curves prepared with standard stock solutions of extraction of ascorbic acid was carried out mixing 2.5 g of frozen chlorogenic acid, catechin, and quercetin-3-O-glucoside, and their peel tissue with 5 ml of metaphosphoric acid suspension (3% concentrations were expressed as g kg−1 FW. Detected phenolic metaphosphoric acid, 8% acetic acid) and centrifuging at acids and quercetin-derivatives were quantified using chlorogenic 24,000 × g for 22 min at 4°C. Quantification of ascorbic acid (AsA) acid and quercetin-3-O-glucoside standard curves. and total ascorbic acid was performed as previously described (Fernández-Cancelo et al., 2021). Dehydroascorbic acid (DHA) was calculated by subtracting the ascorbic acid content from that Antioxidant enzymes activity of total ascorbic acid. Peroxidase (POX, EC 1.11.1.7) was extracted by mixing 5 g of frozen peel with 10 ml of phosphate buffer (100 mM, pH 6) with Determination of phenolic compounds 0.5 mM cysteine and 0.5 g of PVPP and centrifuged at 20,000 g for and the peel antioxidant capacity 15 min at 4°C. A 2.5 ml of supernatant was loaded into a Sephadex G-25 column (PD 10; Pharmacia, Madrid, Spain) that had Total phenolic compounds (TPC) and antioxidant capacity of previously been equilibrated with 10 ml phosphate buffer the apple peel were determined using frozen tissue as previously (100 mM, pH 6), and the enzyme was eluted with 3.5 ml of the described (Giné Bordonaba and Terry, 2008) by mixing 3 g of apple same buffer. POX activity was determined as described elsewhere frozen peel tissue with 10 ml of 79.5% (v/v) methanol and 0.5% (v/v) (Giné-Bordonaba et al., 2017). Frontiers in Plant Science 04 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 Ascorbate peroxidase (APX; EC 1.11.1.11) extraction, 5 g of differences in peel color (determined both visually and as changes frozen peel was mixed with 15 ml of 100 mM base phosphate in Hue angle, H°) among locations, but only apples harvested in buffer (pH 7.5) containing 0.8 mM ascorbic acid and 1 mM Gotarta mountain orchard exhibited an optimal yellow peel color EDTA. The mixture was centrifuged at 10000 g for 15 min at 4°C (H° = 96 ± 0.8) if compared with fruit from valley locations—Alcanó (Collazo et al., 2018). APX activity was determined at 290 nm for (H° = 107 ± 0.5) and Vilanova (H° = 108 ± 0.4)—and even with 10 min by monitoring the H2O2-dependent decomposition of apples harvested in the mountain orchard of Llesp (H° = 104 ± 0.7). ascorbate in a mixture containing 20 μl of supernatant and 280 μl A principal component analysis (PCA; Figure 1A) using individual of a reaction solution containing 0.22 mM ascorbic acid, 1 mM isoprenoids (chlorophylls, carotenoids, and tocopherols), H°, MDA, EDTA, and 1 mM H2O2 (Nakano and Asada, 1981). TPC, and antioxidant capacity as variables was used to differentiate Extraction of superoxide dismutase (SOD, EC 1.15.1.1) and the apple peel from fruit grown at the four different locations (two catalase (CAT, EC 1.11.1.6) was carried out at the same time by valley vs. two mountain orchards). The first two principal homogenizing 5 g of peel tissue in 15 ml potassium phosphate buffer components captured 90.2% of the total variability and were (100 mM, pH 7.8) containing 2 mM DTT, 5% (w/v) PVPP, 0.1 mM sufficient to clearly differentiate the samples. The bidimensional plot EDTA, and 1.25 mM polyethylene glycol (Chiriboga et al., 2013). The illustrated that greenish peel apples were clustered together in the homogenate was centrifuged at 20000 g for 15 min at 4°C and then a PC1-positive part of the plot, while yellow apples were plotted in the 2.5 ml aliquot was loaded into a Sephadex G-25 column (PD 10; negative part. Enhanced yellow coloration of Gotarta apples was Pharmacia, Madrid, Spain) equilibrated with 10 ml 100 mM accompanied by a distinct pigment and antioxidant profile as phosphate buffer (pH 7.8). The enzyme was eluted with 3.5 ml of the depicted in Figure 1A. Interestingly, no statistically significant same buffer. The resulting supernatant was used as an enzyme extract differences on carotenoid content were found between the four to determine SOD and CAT activity. SOD activity was assayed by orchards studied, being its levels ca. 0.03 ± 0.003 mg kg−1 DW measuring its ability to inhibit the photochemical reduction of (Figure 1B). However, chlorophyll levels on apples from Gotarta nitroblue tetrazolium (NBT) using the method described elsewhere were around 2.5-fold lower if compared to fruit from the other (Giannopolitis and Ries, 1977). One unit of SOD (Unit of activity; orchards (Figure 1C), hence owning a higher carotenoid/chlorophyll UA) was considered the amount of enzyme required to inhibit NBT ratio (ca. 1.8 in Gotarta vs. 1.2 in the green apples). Gotarta apples reduction by 50%. CAT activity was measured by the Claiborne also presented higher levels of oxidative stress markers such as method (Claiborne, 1986) following the disappearance of H2O2 at MDA (20 ± 1.7 μmol kg−1) if compared with valley apples 240 nm. Except for SOD, enzymatic activity was expressed in UA per (17 ± 2.3 μmol kg−1 and 16 ± 1.0 μmol kg−1 for Alcanó and Vilanova milligram of protein, with one UA representing the quantity of apples, respectively) and higher content of antioxidants, specifically enzyme responsible for a change in 1 absorbance unit per minute. γ-tocopherol (Figures 1D,E). Protein was quantified by the method of Bradford (Bradford, 1976). Transcriptome reprograming in response Statistical data analysis to orchard location Data were subjected to analysis of variance (ANOVA) tests To study whether the expression of genes for enzymes in using JMP 13.1.0 SAS Institute. When the analysis was statistically metabolic pathways involved in plastidial isoprenoid (chlorophyll, significant, Tukey’s HSD test at the level p ≤ 0.05 was performed for carotenoid, and tocopherol) biosynthesis and degradation was comparison of means for the interaction location and development altered by the orchard location, a RNA-seq analysis of the peel stage. A principal component analysis (PCA) was also performed from apples grown at the four different locations was performed to characterize the samples harvested at CHD in the four orchards (Figure 2; Supplementary Figure S1). Comparison of Gotarta studied. Data were centered and weighted using the inverse of the (yellow phenotype) with the other three locations (green standard deviation of each variable in order to avoid the influence phenotype) showed that most genes of the carotenoid of the different scales used for the variables. Spearman’s rank biosynthetic pathway were downregulated in Gotarta, whereas correlation matrix (p ≤ 0.05) was done using the R corplot package. those involved in carotenoid degradation were upregulated, consistent with the observed trend toward lower levels of carotenoids in Gotarta apples (Supplementary Figure S1). By Results contrast, gene-encoding enzymes involved in chlorophyll degradation and tocopherol biosynthesis were strongly Orchard location influences peel color upregulated in “Gotarta” apples (Supplementary Figure S1). and composition To further identify other potential processes associated to color differences among orchards, a more detailed analysis of the “Golden Reinders” apples grown in four different locations (two RNA-seq data was performed (Figure 2). Comparison of Gotarta valleys – Alcanó and Vilanova and two mountains - Llesp and with the other three locations identified a common set of 797 Gotarta - orchards) and picked at the CHD showed significant genes that were upregulated in Gotarta and 693 genes that were Frontiers in Plant Science 05 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 A B C D E FIGURE 1 Principal component analysis (PCA) characterization of the apple peel from four different locations (two orchards from the valley: Alcanó and Vilanova; and two orchards from the mountain: Llesp and Gotarta) using 16 variables including Hue angle (H°) individual pigments and tocopherols, antioxidant capacity, TPC and MDA (A). Total carotenoids (B), total chlorophylls (C), α-tocopherol (D), and γ-tocopherol (E) in the peel of apples grown in different locations. Error bars represent the standard error (n=4). Letters, whenever available, indicate significant differences between locations. downregulated if compared to all three locations leading to green S4, the latter being the CHD). Differences in carotenoid levels were apples (Figure 2A). GO term analysis of the whole set of observed between locations and developmental stages, yet the differentially expressed genes (1,490 genes) showed an enrichment carotenoid profile, understood as the individual carotenoids in processes related to photosynthesis, metabolism (including identified among samples, remains relatively unchanged (Figure 3). pigment and isoprenoid biosynthesis), plastid organization, and Lutein was the main carotenoid found in the apple peel, accounting response to environmental cues (Figures 2B,C). Many terms for 60% of total carotenoids. β-carotene and violaxanthin each within metabolism and cellular responses were actually related to contributed to 15% of the total carotenoids blend, while photosynthesis, carbon fixation, and carbohydrate metabolic neoxanthin accounted only for the 8% of total carotenoids. In both processes (Figure 2C), whereas another important group of GO locations, carotenoids content decreased as the fruit ripened terms was linked to response to oxidative stress, including genes on-tree, yet the reduction rate was 2-fold higher in mountain than involved in the regulation of ROS and H2O2 metabolism in valley fruit. In consequence, although carotenoid levels were ca. (Figure 2C). We also observed that pathways regulating nitrogen 30% higher in Gotarta at the two initial samplings (S1 and S2), and phosphorus metabolic process as well as general organic carotenoid content in mountain apples reached similar levels to substance metabolism were overrepresented among the set of those observed in valley apples in the last two samplings (S3 and differentially expressed genes. In terms of cellular responses, the S4) (Supplementary Figure S2A). The global decrease in carotenoid regulation of biological quality, protein metabolic process, and content during fruit development in both locations only translated molecular function was the most represented (Figure 2C). into an increase of ABA from 27 ± 2.9 to 59 ± 2.5 μg kg−1 in valley fruit from S1 to S3 (Figure 3). In contrast, ABA levels in mountain fruit remained around 23 ± 2.2 μg kg−1 DW until S4, when it Pigment metabolism during fruit reached similar levels to those found in valley apples development is differentially affected by (49 ± 0.4 μg kg−1 DW). Chlorophyll levels followed the same pattern the environment as carotenoids in both locations (Supplementary Figure S2B). Mountain apples accumulated a 40% more chlorophyll in the first Based on the major differences on peel color, pigment two stages of development (S1 and S2) and then its levels matched composition, and gene expression, Gotarta (mountain) and with those from the valley at S3 and S4 (0.02 ± 0.001 and Vilanova (valley) orchards were selected to further monitor major 0.01 ± 0.001 mg kg−1 DW, respectively). metabolic and transcriptomic shifts associated to fruit color Carotenogenic gene expression (MdPSY, MdVDE, and changes along fruit development (four developmental stages: S1 to MdZEP) progressively increased during fruit development for Frontiers in Plant Science 06 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 A B C FIGURE 2 Number of genes upregulated and down-regulated (A) in Gotarta apples (yellow phenotype) compared with the other 3 orchards (green phenotypes). The 20 most frequent GO upregulated in Gotarta were displayed as % Total GO counted (B). Gene functional categories enriched in Gotarta apples (C) provided by REVIGO’s “Scatterplot view” (http://revigo.irb.hr/). Disc color indicates enrichment value of p (the softer the color the highest the value) and size represents the frequency of the category. Thickness of grey lines represent semantic similarity between categories while spatial arrangement of discs corresponds to grouping of categories by semantic similarity. Those related to photosynthesis are circled in green, and terms associated with oxidative stress are circled in blue. Frontiers in Plant Science 07 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 FIGURE 3 Comparison of carotenoid metabolism in “Golden Reinders” apples grown in mountain (Gotarta- ) or valley (Vilanova- ) orchards during fruit development/ripening. Dashed lines indicate that some steps have been omitted. Quantified carotenoid compounds are represented in bar plots, including minor carotenoid isomers marked with the superscript “iso.” Error bars represent the standard error (n=4). Letters, whenever available, indicate significant differences for the interaction location*developmental stage according to Tukey test (p≤0.05). The corresponding transcript levels of the main genes are represented as heatmaps. Letters within the heatmaps indicate significant differences (p≤0.05) between development stages, while asterisk denote differences among locations at each specific development stage. both studied locations (Supplementary Figures S3A–C). In the last acid), dihydrochalcones (phloretin glycosides), flavanols stage, MdPSY expression was 1.4-fold higher in Vilanova whereas (catechin, epicatechin, and procyanidin B2), and flavonols MdZEP and MdVDE showed a 30% greater expression in Gotarta. (quercetin glycosides; Figure 4). In general, the peel from Regarding gene-encoding apocarotenoid production, MdCCD4 mountain apples showed higher levels of phenolic compounds in expression remained constant along fruit development in the the two first development stages (S1 and S2), except for phloretin- valley, while it increased from S1 to S2 and significantly declined 2’-O-glucoside and procyanidin-B2 whose levels were not affected at S4 in mountain apples (Supplementary Figure S3D). The by the orchard location. All phenolic compounds identified expression of MdNCED1 and MdNCED2 was similar between followed a downward trend in both location as the fruit developed, locations and remained unchanged during fruit development being the decrease rate between 1.2 and 2.0-fold higher in except at the first stage (S1), where mountain apples showed 3-fold mountains than in valley apples yet depending on the phenolic higher expression of both genes in comparison to fruit from the compound (Figure 4). At the CHD, despite most differences valley (Supplementary Figures S3E,F). between locations levelled off, the peel from mountain apples showed generally higher levels of phenolic compounds than those from the valley. The levels of tocopherols and phenolic Analysis of MdPAL1 and MdPAL2 showed expression compounds are greater in mountain differences between locations mainly at S4, being significantly apples higher and lower the expression of MdPAL1 and MdPAL2 in mountain apples than in fruit from the valley. Mountain apples showed 10-fold higher levels of γ-tocopherol at S4 if compared with valley apples (Supplementary Figure S2C), whereas during earlier stages (S1–S3), γ-tocopherol content The environment strongly influences remained below 0.004 mg kg−1 DW and constant in both locations. ROS-scavenging enzymes in the apple In contrast, α-tocopherol remained unchanged peel (0.03 ± 0.003 mg kg−1 DW) in both locations during fruit development and ripening on-tree (Supplementary Figure S2D). A completely different pattern of H2O2 accumulation was Eleven phenolic compounds were identified in both locations observed when comparing apples grown in mountain or valley during fruit development, including phenolic acids (chlorogenic orchards. Levels of H2O2 remained unchanged in valley apples Frontiers in Plant Science 08 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 FIGURE 4 Comparison of phenolic compounds metabolism in “Golden Reinders” apples grown in (Gotarta- ) or valley (Vilanova- ) orchards during fruit development/ripening. Dashed lines indicate that some steps have been omitted. Phenolic compounds are represented in plots. Error bars represent the standard error (n=4). Letters, whenever available, indicate significant differences for the interaction location*developmental stage according to Tukey’s test (p≤0.05). The corresponding transcript levels of the main genes are represented as heatmaps. Letters within the heatmaps indicate significant differences (p≤0.05) between development stages, while asterisk denote differences among locations at each specific development stage. from S2 to S4 (around 54 ± 4.2 mmol kg−1), whereas in valley (134 ± 4.5 UA mg−1 protein). In contrast, no differences mountain apples, H2O2 raised from 77 ± 2.0 to in CAT activity were observed between valley and mountain 110 ± 8.1 mmol kg−1 within the same period (Figure 5). apples except at S4, when CAT activity in fruit from the valley Enhanced H2O2 accumulation in the peel from mountain increased until reaching values of 1.7 ± 0.07 UA mg−1 protein. apples was not associated to higher levels of SOD, the enzyme Significant differences were found for the expression of all responsible for the dismutation of O2·- into H2O2, since the genes encoding for antioxidant enzymes along the different activity of this enzyme remained relatively unchanged fruit development stages and locations except for MdDHAR3 throughout fruit development/ripening in both locations. In and MdMDHAR2 that showed no changing pattern along fruit contrast, the activities of enzymes involved in the development/ripening (Supplementary Figure S5). Copper decomposition of H2O2 into water and oxygen were location superoxide dismutase encoding genes (MdCSD1 and MdCSD2) and developmental stage dependent. The activity of APX, the showed the same downward pattern in both locations along enzyme responsible to catalyze the H2O2-dependent oxidation fruit development/ripening. However, the expression of of ascorbate was nearly 2-fold greater in mountain apples at MdCSD2, which encodes for chloroplastic SOD, was around S1, but it decreased in the rest of developmental stages to 40-fold higher than its cytosolic counterpart MdCSD1 13 ± 1.5 UA mg−1 protein, matching then the activity levels (Supplementary Figures S5A,B). In turn, MdFSD2 showed the found in valley apples. Ascorbic acid levels in the valley opposite trend, reaching maximum expression levels at S4 for remained unchanged with values below 75 mg kg−1, whereas both locations (Supplementary Figure S5C). In the case of AsA levels massively increased from 92 ± 8.0 to MdAPX1, encoding for cytosolic APX, only mountain apples 252 ± 30.0 mg kg−1 in mountain apple peel during fruit showed an increase of gene expression from 30 to 60 MNE development and on-tree ripening. A similar pattern was during development/ripening (Supplementary Figure S5D). observed for POX, whose activity was significantly higher in The expression of MdAPX6 and MdMDHAR1 increased mountain apples at S1 and S2 (Figure 5). In the last two during fruit development/ripening and significant differences samplings (S3 and S4), POX activity from mountain apples were found for the expression of these genes at the CHD decreased and matched the levels observed in fruit from the (Supplementary Figures S5F,G). Frontiers in Plant Science 09 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 FIGURE 5 Comparative overview of the ROS-scavenging metabolism and ascorbate recycling pathway of “Golden Reinders” apples grown in mountain (Gotarta- ) or valley (Vilanova- ) orchards during fruit development/ripening. The activity of the main antioxidant enzymes, as well as the content of ascorbic acid and H2O2, is represented in bar plots. Error bars represent the standard error (n=4). Letters, whenever available, indicate significant differences for the interaction location*stage according to Tukey test (p≤0.05). The corresponding transcript levels of the main genes are represented as heatmaps. Letters indicate significant differences (p≤0.05) between development stages, while asterisk denote differences among locations at each specific development stage. Discussion color, as desired by consumers, but also higher antioxidant capacity and elevated levels of MDA, a marker of lipid Optimal yellow peel color in “Golden peroxidation (Hodges et al., 1999). Since the apples were Reinders” apples is associated to harvested at a similar maturity stage (Fernández-Cancelo et al., oxidative stress 2021), climatic differences between orchards may have influenced peel metabolism (McTavish et al., 2020) leading to different color “Golden Reinders” apples were grown under similar phenotypes. Indeed, in our previous study, we demonstrated that agronomical practices in four orchards of Lleida Province (two optimal fruit color in “‘Golden Reinders” apples was not from the valley and two from the mountains) and harvested at associated to the fruit maturity stage and that fruit grown in the the same ripening status in terms of fruit size (75 ± 0.4 mm), Ebro valley never attain the optimal yellow color desired by the firmness (71 ± 0.5 N) and other ripening-related traits consumer, even if left to ripen on-tree far beyond the CHD (Fernández-Cancelo et al., 2021) showed different peel color (Fernández-Cancelo et al., 2021). Among the wide variety of phenotype (Figure 1). Notably, fruit from mountain orchards, environmental factors that can influence fruit color (temperature, and especially those from Gotarta, showed enhanced yellow solar radiation, hours of sun, precipitations, and relative Frontiers in Plant Science 10 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 humidity), the low temperatures and solar radiation reached days oxidative stress responses (Figure 2), strongly suggest that the yellow before harvest in Gotarta (Supplementary Figures S8, S9) in peel of Gotarta apples may be the result of higher exposure to combination with a probable higher accumulation of UV oxidative damage. radiation due to the high altitude (data not measured; Blumthaler et al., 1997) may have favored chlorophyll degradation (León- Chan et al., 2017) and color changes in Gotarta apples. Pigments and phenolic compounds Given the difficulties to evaluate the specific environmental decrease during fruit development and factors affecting apple color, we decided to focus our study in ripening investigating the metabolic or transcriptomic shifts capable to explain the color phenotype differences observed between growing Based on the intense color differences observed between locations, with a special emphasis on pigment and antioxidant Vilanova and Gotarta apples, both locations were chosen as the most metabolism. The pigment profiling presented herein revealed that suitable to better understand the metabolic and transcriptomic yellow apples had similar levels of carotenoids but significant lower differences underlying different apple phenotypes. To elucidate the levels of chlorophylls than apples grown at other locations and point when color changes and pigment differences occur, apple showing greenish coloration (Figure 1). These observations strongly development was followed at four different stages (S1 to S4) based suggest that higher chlorophylls levels in greenish “Golden Reinders” on fruit size in both valley (Vilanova) and mountain (Gotarta) apples may be shielding the carotenoid contribution to peel color as orchards. The optimal yellow color was only perceived in Gotarta it was described in pepper (Filyushin et al., 2020). Hence, it may apples at the last developmental stage (S4; equivalent to the CHD), be that chlorophyll levels in apple peel must fall below a certain while in Vilanova, the apple peel remained green (Table 1). Color threshold for achieving an optimal yellow color as previously changes in fruit are generally associated to the transition from observed during olive ripening (Mínguez-Mosquera and Gallardo- chloroplast to the chromoplast and the accumulation of carotenoids Guerrero, 1995). Besides, our data demonstrated that enhanced (Sadali et al., 2019). However, the observed differences in “Golden yellow color was not due to enhanced content of carotenoids, as it Reinders” peel color were again not due to enhanced accumulation may be deduced from available literature (Delgado-Pelayo et al., of carotenoids but rather to a more intense degradation of 2014) but rather associated to higher carotenoids/chlorophyll ratios chlorophylls (Supplementary Figure S2A). The characteristic triggered by environmental cues. Yellow peel apples showed also decrease of chlorophyll and carotenoids in yellow peel apples significantly higher levels of tocopherols which are antioxidant (Solovchenko et al., 2005) was observed in both locations, yet the molecules involved in the protection of thylakoid membranes against final carotenoid/chlorophyll ratio was higher in the mountain lipid peroxidation (Piller et al., 2014). These observations, together (Gotarta) location (Table 1). This accelerated degradation of with the results of the RNA-seq analyses showing an chlorophylls not only explains the more yellowish color of these overrepresentation of gene categories related to photosynthesis and apples but also the increased levels of tocopherols (Figure 1), which TABLE 1 Peel color evolution during the four developmental stages studied in Vilanova (valley) and Gotarta (mountain) orchards. S1 (60% final fruit size) S2 (80% final fruit size) S3 (90% final fruit size) S4 (100%final fruit size) Vilanova (Valley Orchard) Ho 104.5 ± 0.14 ab 104.2 ± 0.16 bc 103.6 ± 0.27 bc 100.5 ± 0.32 e Carotenoids/ 1.05 ± 0.023 a 0.95 ± 0.024 bc 0.98 ± 0.021 abc 0.94 ± 0.016 c Chlorophylls Gotarta (Mountain Orchard) Ho 105.1 ± 0.12 a 103.3 ± 0.21 c 102.2 ± 0.24 d 95.8 ± 0.38 f Carotenoids/ 1.03 ± 0.005 ab 1.03 ± 0.017 abc 0.96 ± 0.027 abc 1.03 ± 0.018 abc Chlorophylls Hue angles (H°) were determined by measuring both sides of 30 fruits (n = 60), while the ratio carotenoids/chlorophylls were determined from four replicates (n = 4). Data are represented as the means of replicates ± standard error. Letters indicate significant differences for the interaction location*developmental stage according to Tukey’s test (p ≤ 0.05). Frontiers in Plant Science 11 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 in many fruits are made from phytol recycled from the chlorophylls Besides carotenoids, tocopherols play an important role in degraded during ripening (Muñoz and Munné-Bosch, 2019). maintaining chloroplast integrity. In our study, α-tocopherol levels Differences in the chlorophyll and tocopherol levels may be driven, in the apple peel remained stable during fruit ripening in both in turn, by differences in temperature and solar radiation among the locations, whereas a burst in γ-tocopherol was observed in studied locations (Karagiannis et al., 2020). In fact, a higher exposure mountain apples at the latest stage (S4). Given that tocopherol to sunlight is known to favor the accumulation of chlorophyll and synthesis utilizes phytyl-PP released during chlorophyll carotenoids in apple (Jing et al., 2020), while an excess of it can also breakdown, it is plausible to hypothesize that the drop of night induce apple ripening and accelerate chlorophyll loss (Solovchenko temperatures observed in high-altitude orchards days before et al., 2006). Higher solar radiation combined with milder harvest (Supplementary Figure S8) may strongly activate phytol temperatures during the early development stages may have favored recycling mechanisms and promote the accumulation of the accumulation of photosynthetic pigments (chlorophylls and γ-tocopherol (Vom Dorp et al., 2015), thereby enhancing stress carotenoids) in mountain locations at S1 and S2 tolerance (Abbasi et al., 2007). The decrease in chlorophyll content (Supplementary Figures S2, S8, S9). As summer progressed, the and the accumulation of oxidative stress markers observed in pronounced decrease in solar radiation and temperatures in Gotarta apples (Figures 3, 5) are signs compatible with senescence mountains possibly favored, to some extent, the inhibition of (Muñoz and Munné-Bosch, 2018). Senescence can be activated by pigment biosynthesis (Nagata et al., 2007) and, hence, a more environmental cues such as the low temperatures and low solar pronounced decline in the chlorophyll and carotenoids content in radiation reached in Gotarta just before harvest mountain apples if compared with those from the valley (Supplementary Figures S8, S9), or also by an accumulation of ROS (Supplementary Figures S2, S8, S9). (Figure 5) able to cause a potential oxidative damage in the peel of The differences in the temporal changes of carotenoids along Gotarta apples (Thakur et al., 2016). Hence, the development of fruit development between locations were not a consequence of yellow color in mountain apples may be interpreted as the result of differences on the expression of MdPSY, the main regulator of an activation of a peel senescence-like process driven by carotenoid synthesis (Cazzonelli and Pogson, 2010). The upward environmental stresses. However, the RNA-seq analysis did not trend in MdPSY expression in both locations did not lead to an show a differential expression of senescence-associated genes accumulation of carotenoids, suggesting the possibility that (SAGs) between locations able to support this hypothesis, and degradation pathways were more active and rather responsible for therefore further studies are warranted. the final levels of carotenoids observed in apples at the time of Although carotenoids and tocopherols play a key role as harvest (Figure 3). Nonetheless, carotenoid catabolism seemed to antioxidants in chloroplasts, the total antioxidant capacity in apple be strongly influenced by the orchard location. The upregulation mainly depends on phenolic compounds (Lee et al., 2003). of MdCCD4 in S2 and S3 combined with the absence of correlation Phenolic compounds can be subdivided into different classes between carotenoids and ABA observed in mountain apples, including hydroxycinnamic acid derivatives, chalcones, flavanols, suggest that high-altitude environments may favor alternative flavonols, and anthocyanins (Haminiuk et al., 2012). The main degradation pathways than those mediated by NCED, hence in phenolic compounds detected in “Golden Reinders” peel were agreement with available literature for other organisms (Liang chlorogenic acid and quercetin-3-O-glycosides (Figure 4), which et al., 2021). Indeed, the accumulation of ROS in mountain apples followed the same trend as carotenoids, decreasing as the fruit (Figure 5) may favor the synthesis of stress response developed and ripened, in accordance with previous studies made apocarotenoids through the CCD4 pathway (Felemban et al., on apple (Wojdyło and Oszmiański, 2020). The higher levels of 2019). The pattern of ABA accumulation may be, in turn, flavanols, flavonols, and hydroxycinnamic acid derivatives found influenced by temperature differences between locations since at early development stages in mountain fruit may also be a ABA is considered a stress response hormone involved in consequence of the higher solar radiation received by apple peel at thermotolerance (Iqbal et al., 2021). In this regard, apple is a crop high altitudes in the first months of fruit growth (Awad et al., 2000). adapted to moderate climates (Funes et al., 2016); hence, the high The first step of the phenolic compound synthesis pathway is temperatures reached in Vilanova during fruit development may catalyzed by Phenylalanine Ammonia-Lyase (PAL), an enzyme activate the accumulation of ABA for better coping with heat encoded by several gene isoforms regulated by the environment stress (Torres et al., 2017). Likewise, the moderate temperatures in (Huang et al., 2010). Extensive evidence has shown that PAL gene mountain during the three first stages (Supplementary Figure S8) expression in model species (i.e., Arabidopsis thaliana) is likely kept ABA levels low until the last stage, when ABA may responsive to a variety of environmental stimuli, including be induced not by warm but rather by cold temperatures (Lee pathogen infection, wounding, nutrient depletion, UV et al., 2021). Indeed, evidence suggesting that ABA accumulates irradiation, and extreme temperatures, among others (Huang in response to high- or low-temperature stress is abundant for et al., 2010). Accordingly, the PAL gene isoforms analyzed in our other species (Daie and Campbell, 1981), yet no data are currently study (MdPAL1 and MdPAL2) showed different expression available for apples. Future studies are encouraged to better patterns (Supplementary Figure S4) not only along fruit growth understand the actual relationship between ABA content and but also in response to the growing location, suggesting that each environmental temperatures during apple growth. isoform may be differentially regulated by environmental stimuli. Frontiers in Plant Science 12 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 In this sense, the observed MdPAL1 gene expression pattern and MdMDHAR and MdDHAR (Supplementary Figure S7), it is the high correlation with γ-tocopherol (Supplementary Figure S7) possible to speculate that the differential accumulation pattern suggest that both MdPAL1 gene expression and γ-tocopherol may of ascorbic acid found between locations may be a mere be regulated by similar environmental cues, being in this consequence of a higher biosynthetic activity driven by particular case, likely low temperatures prior to the time of environmental cues (Zhang et al., 2011). Furthermore, the high harvest (Maeda et al., 2006; Olsen et al., 2008). MdPAL2 gene correlation between AsA and H2O2 (Supplementary Figure S7) expression, in contrast, seemed to mainly change along fruit suggests that AsA may play an important role in modulating the developmental, showing an upward trend as the fruit developed protection against ROS (Akram et al., 2017). In this sense, the and ripened yet with more pronounced and significant higher increase of H2O2 levels observed in the peel of mountain-grown expression levels in fruit grown in the valley at S4. In this apples may be favored by the reduction of peroxidase activities particular case, and based on our metereological data, enhanced during fruit development combined with the low catalase activity UV or solar radiation at this particular phenological stage may (Figure 5). Since during the early development stages (90 days account for the higher MdPAL2 gene expression in valley fruit. before harvest) climatic differences were not appreciable between Overall, our results are in agreement with previous studies valley and mountain orchards, we would like to point out that showing a differential expression of PbPAL genes among different other environmental stresses such as higher UV radiation tissues, phenological stages or even in response to exogenous incidence on mountain apples may induce the accumulation of hormonal treatments in other pome fruit species (i.e., pears; Li H2O2 (Nawkar et al., 2013). Given that fruit ripening is et al., 2019). Nonetheless, further studies are encouraged to considered an oxidative process by itself (Steelheart et al., 2019), elucidate the possible specialization or environmentally driven it is reasonable to hypothesize that the accumulation of H2O2 in regulation of MdPAL genes in apple fruit. mountain apples may trigger peel ripening associated processes such as color changes (Kumar et al., 2016) without affecting pulp ripening parameters such as firmness or starch index The accumulation of H2O2 favored by the (Fernández-Cancelo et al., 2021). These results would further mountain environment may trigger apple reinforce the results from recent studies suggesting that apple peel degreening peel and flesh ripening are independent processes (Karagiannis et al., 2020). In addition to the antioxidant molecules described above, The phenotypic differences observed between locations fruit has enzymatic mechanisms able to neutralize toxic ROS may be regulated epigenetically through DNA methylation species generated by aerobic metabolism and stress (Apel and and specific histone modifications driven by the environment Hirt, 2004). The RNA-seq data showed that a large number of (Baulcombe and Dean, 2014). Moreover, recent studies have genes related to oxidative stress were differentially expressed shown that the DNA methylation can regulate pigment when comparing yellow and green peel “Golden Reinders” accumulation in apple (Jiang et al., 2019) but little is known apples (Figure 2C). In this sense, we further investigated changes about the involvement of histone modifications in apple color. in the activity of enzymes involved in ROS-homeostasis during Given that some deacetylases seem to have a key role in the the last stages of apple development/ripening in both locations modulation of color development in tomato (Guo et al., 2017) (mountain vs. valley; Figure 5). The enzyme SOD is present in and since environmental stress may also alter the epigenetic the cytoplasm, mitochondrion, and the chloroplast (Feng et al., regulation (Baulcombe and Dean, 2014), we decided to 2016), and is able to convert oxygen radicals produced in the perform a gene expression analysis of a class of histone processes of photosynthesis and respiration into a less reactive deacetylases known as sirtuins (MdSRT1 and MdSRT2). compound such as H2O2 (Meitha et al., 2020), which can Sirtuins are suggested to be involved in the epigenetic be reduced by peroxidases present in cytosol and chloroplast regulation of fruit ripening in other Rosaceae spp. (Vall-llaura (Maruta et al., 2016) and by catalase in peroxisome (Corpas, et al., 2021) and well known for their association to several 2015). Although orchard location and developmental stages antioxidant and oxidative stress-related processes and influenced SOD gene expression levels, SOD activity remained functions (Singh et al., 2018). Indeed, the fact that sirtuins unchanged regardless of the environment. In contrast, peroxidase deacetylase activity is dependent on NAD+, a key redox activities tended to be higher in the peel of mountain apples for signaling molecule, sustains the idea that these molecules POX in S1 and S2 and for ascorbic acid-dependent peroxidase may be integral players in regulating cellular antioxidant and (APX) in S1 (Figure 5). In general, the expression of genes redox signaling pathways (Singh et al., 2018). In our study, involved in ascorbate metabolism or the antioxidant enzymes the high correlation between the expression of MdSRT2 and detailed above was poorly correlated with the equivalent ripening associated traits such as Hue angle (H°), H2O2, and enzymatic activity but well correlated with the concentration of AsA levels (Supplementary Figure S7) suggests that fruit the corresponding metabolite (Supplementary Figure S7). Based ripening and color changes may be induced epigenetically on the absence of correlation between APX activity, ascorbic acid through the action of histone deacetylases as described in (AsA), and the gene expression of ascorbate recycling enzymes tomato (Guo et al., 2017). On the other hand, the analysis of Frontiers in Plant Science 13 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 MdSRT1 gene expression, a sirtuin involved in abiotic stress and writing—review and editing. MR-C and JG-B: responses in A. thaliana (Liu et al., 2017), showed a conceptualization, methodology, supervision, funding consistently higher expression in mountain apples at all acquisition, project administration, and writing—review and ripening stages (Supplementary Figure S6). Overall, the editing. All authors contributed to the article and approved the expression analysis of sirtuins may point out that mountain submitted version. apples suffer an additional stress during fruit growth able to induce epigenetic changes that lead to an acceleration of ripening processes including color changes. However, further Funding studies should be performed to analyze which specific epigenetic modifications regulate peel color in “Golden This work has been financially supported by the Spanish Reinders” apples. Agencia Estatal de Investigación (AEI) and European Regional Development Fund (ERDF) through the national projects RTA2015-00037-CO2-01, BIO2017-84041-P, BIO2017-90877- Conclusion REDT and PID2020-115810GB-I00. Funding was also provided by the internal CRAG project “Green2Gold” in the context of The results from this study demonstrate that differences in the Severo Ochoa Programme for Centres of Excellence in R&D apple peel color among valley and mountain orchards at the 2016–2019 (SEV-2015-0533). This work has been also supported time of harvest are explained by the influence of environmental by the CERCA Programme from the “Generalitat de Catalunya.” conditions on antioxidant and pigment metabolism occurring Thanks are also given to AEI and ERDF for the predoctoral earlier during fruit development. Optimal yellow coloration in fellowships awarded to PF-C (BES-2017-080741) and AI-S “Golden Reinders” apples, as exclusively observed in mountain (PRE2018-083610). ST-M is supported by a PhD fellowship environments, was mainly a consequence of enhanced from the Spanish Ministry of Education, Culture and Sports chlorophyll degradation instead of enhanced carotenoid levels (FPU16/04054). as well as alterations in the expression of genes involved in chloroplasts functions. Likewise, the yellow phenotype observed in mountain apples seems also to be regulated through Acknowledgments epigenetic modifications driven by the environment and may be related, to some extent, to oxidative stress and to the specific We are grateful to Dolors Ubach and Mª Rosa Rodriguez for action of H2O2 during fruit growth/ripening. In this sense, H2O2 their technical support and to Dr. Núria Vall-Llaura for her help seemed to play a key role triggering some peel ripening events, with RT-qPCR analysis. including those involved in peel degreening in fruit grown in the mountains. Further studies may be required to dissect how specific environmental cues (solar radiation, temperature and Conflict of interest so on) can trigger or inhibit pigment and antioxidant metabolism in different apple varieties hence leading to fruit The authors declare that the research was conducted in the with different phenotypes. Besides, the results from this study absence of any commercial or financial relationships that could may assist in selecting specific compounds that when applied be construed as a potential conflict of interest. exogenously are capable to trigger color changes in fruit grown under specific environments. Publisher’s note Data availability statement All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated RNA-seq raw data are freely available in SRA database with organizations, or those of the publisher, the editors and the the Bioproject ID PRJNA839574. Other data supporting the reviewers. Any product that may be evaluated in this article, or findings of this study are available within the paper and within its claim that may be made by its manufacturer, is not guaranteed or Supplementary material published online. endorsed by the publisher. Author contributions Supplementary material PF-C: methodology, writing—original draft preparation, The Supplementary Material for this article can be found online and writing—review and editing. AI-S, AR-A and ST-M: at: https://www.frontiersin.org/articles/10.3389/fpls.2022.913433/ methodology and writing—review and editing. NT: supervision full#supplementary-material Frontiers in Plant Science 14 frontiersin.org Fernández-Cancelo et al. 10.3389/fpls.2022.913433 References Abbasi, A.-R., Hajirezaei, M., Hofius, D., Sonnewald, U., and Voll, L. M. (2007). 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