Apple fruit undergoes a broad metabolic adaption and unreversed specialization during the fruit ripening progress, which is known to involve internal signals refined by environmental cues13. Essentially all of what is known about apple fruit development pertains to the changes of mesocarp or of the whole fruit, and little is known about the function of the peel tissue, as an isolated component, during apple ripening. In addition to this, our current knowledge about the influence of altitude on apple peel is entirely derived from research on fruit coloration-related aspects of ripening3,4,6,11. Therefore, an overall molecular framework is needed for better understanding of the altitude-associated changes in metabolism during peel ripening.
In this study, we characterized the impact of discrete environments (altitudes) on apple peel metabolism. It is notable that apples sampled at the two different altitudes exhibited a similar pattern of key ripening traits, including fresh weight, starch content, fruit diameter, firmness, soluble solids content, and dry matter (Fig. 1b). This evidenced that the sampled fruit at both environmental conditions displayed the same ripening status; therefore, the observed phenotypes (Figs. 1a, 2) cannot be due to an advance/delay in fruit ripening time but rather has to be related to differences in altitude-driven regulation of ripening. It is, however, worth noting that the different growth conditions do influence several peel color parameters, namely the hue angle (ho), lightness (L), redness (a) yellowness (b) as well as the total anthocyanin level and the percentage (%) of peel color index (Fig. 2), verifying previous findings that the red coloration in apple and other fruit is stimulated by altitude6,7. These differences between apples grown at the two distinct altitude cultivation regions could be a result of specific metabolic and biochemical adjustments to the environmental factors, such as day length, ambient light, temperature, or even an interplay between these parameters collectively affecting apple peel biology. For example, the sensitivity and responsiveness of anthocyanin biosynthesis to temperature was demonstrated in an on-tree experiment with ‘Mondial Gala’ and ‘Royal Gala’ apples where a single night of lower temperature was sufficient to elicit a large increase in transcription of MYB10 and consequently also in anthocyanin abundance10.
It was previously shown that numerous environmental signals induced major metabolic changes during fruit ripening17. Data from our current study indicates that apples grown at high altitude provoke the accumulation of specific carbohydrates, such as arabinose, xylose, sucrose, turanose, and talose at distinct developmental stages (Fig. 3). Our results, when taken together with previous ones18, suggest a crucial roles of carbohydrate biosynthesis in response to high altitude. In this regard, an interesting observation was the greater accumulation of SQD (UDP–sulfoquinovose synthase), that is related to sucrose accumulation19, in apple fruit grown at high altitude (Figs. 3, 6). This observation suggests that cell-wall loosening across apple peel development would provide arabinose and xylose, as the major neutral sugars that contribute carbon to the enlarging cells towards environmental stimuli14,20. This observation is supported by present data showing that xyloglucan–xyloglucosyl transferase (XTH), a protein which catalyzes fundamental cell-wall properties21, and is subsequently involved in the biosynthesis of xylose/arabinose in peel cells was induced by high altitude.
Schematic representation of a metabolite-protein network that particularly induced in each altitude. The ‘—’ and ‘—’ arrows indicate the metabolite and protein connection to the network, respectively. ICDH isocitrate dehydrogenase, GR glutathione reductase, CAT catalase, MTHFR methylenetetrahydrofolate reductase, GGAT glutamate–glyoxylate aminotransferase, MAN, mannan endo-1,4-beta-mannosidase, SQD UDP–sulfoquinovose synthase, SDR (+)-neomenthol dehydrogenase, SHM glycine hydroxymethyltransferase, S6PDH aldose-6-phosphate reductase, GRXS monothiol glutaredoxin, RPL13B large subunit ribosomal protein L13e, PSBO1 photosystem II oxygen-evolving enhancer protein 1, PSBO2 photosystem II oxygen-evolving enhancer protein 2, PSBO3 photosystem II oxygen-evolving enhancer protein 3, TROL thylakoid rhodanese, PSAE photosystem I subunit IV, Cy-3-O-gal cyanidin-3-O-galactoside, Cyan-3-O-samb cyanidin-3-O-sambubioside
Another interesting observation made in our study was the decrease in fucose and especially rhamnose at high altitude, with the simultaneous accumulation of (+)-neomenthol dehydrogenase (SDR), an enzyme active in reduction-oxidation- processes of the plant cell22 (Figs. 3, 6; Supplementary Table 1). The reduction of a rhamnose biosynthetic enzyme at high altitude indicates that the apple peel exhibits a loss in its capacity to convert the newly synthesized polyuronides to a more tightly bound form compared to low-altitude environments. The accumulation of these compounds at high altitude might play a key role in the enrichment of the outer cell membrane due to the higher temperatures during fruit development, as already proposed in peach peel7. However, further experiments are required in order to substantiate this hypothesis.
The present study also showed that both the citrate level and isocitrate dehydrogenase (ICDH) abundance were increased in fruit exposed to high altitude (Figs. 3, 5c; Supplementary Table 4). Given that the citrate and isocitrate conversions play important roles in TCA metabolism23, these findings might be suggestive of a general increase of TCA cycle activity, and, consequently, a likely induction of the mitochondrial levels of NADH and NADPH leading to alterations in apple fruit pigment content (Fig. 2). Evidence for such a compensatory mechanism has been provided by expressing a fragment of the mitochondrial NAD-dependent isocitrate dehydrogenase gene (SlIDH1) in tomato24. The transgenic lines exhibited considerably altered metabolism, being characterized by altered TCA flux, NAD(P)H levels, and leaf pigmentation24.
In addition to primary metabolism, we also focused on secondary metabolites that are known to be important for apple fruit biology and quality25. Data revealed a widespread accumulation of various polyphenolic compounds, including quercetin-3-O-rhamnoside, quercetin-3-O-glucoside+galactoside (SUM), quercetin-3-O-rutinoside, and chlorogenic acid in apple peel at high-altitude environment (Fig. 4, Supplementary Table 2). In addition, chlorogenic acid and epicatechin, as major phenolic compounds in several apple cultivars, including ‘Fuji’26,27, were also stimulated by high altitude (Fig. 4, Supplementary Table 2), indicating an environment-based regulation of these phenolic metabolites. Previous studies in apple peel demonstrated that the accumulation of anthocyanin cyanidin-3-O-galactoside was induced by low temperatures, and thus promoted fruit coloration by up-regulating the expression of genes involved in anthocyanin biosynthesis28. In the present study, the anthocyanin Cy-3-gal and cyanidin-3-O-sambubioside were increased in apple grown at high altitude (Fig. 4, Supplementary Table 2). It is interesting that cyanidin-3-O-sambubioside is one of the major anthocyanin compounds in black peanut skin29; however, till now, no information is available to the impact of cyanidin-3-O-sambubioside on apple peel metabolism. The greater accumulation of this anthocyanin at high-altitude grown apples, might therefore, be related to the higher abundance of the total anthocyanin levels in apple peel (Fig. 2). Apple fruits, and especially peel tissues, at high altitude can counteract severe environmental conditions such as high UV radiation, low temperature, large variations in the day–night temperature, etc. as compared to the low-altitude grown fruit. Thus, fruit growing at high-altitude environment may develop strategies in order to adapt to these conditions. For example, UV light has a sustained effect on anthocyanin and flavonol production via regulation of HY5 and MYB10/MYB22 in ‘Royal Gala’ apple fruit30.
Another major target of interest in this study was the changes of peel protein profiles in response to altitude. In particular, the high-altitude-dependent induction of linoleate 9S-lipoxygenase reported here (Fig. 5c, Supplementary Table 4) is indicative of the involvement of this protein in regulating the synthesis of flavor compounds. Consistent with this, the strong accumulation of isoleucine at high altitude (Fig. 3) could provide precursors to branched-chain esters and may contribute to the formation of short-chain fatty acids31, which are linked to the lipoxygenase pathway and thereby to apple aroma composition. As such these results seem to imply a diverse role of the lipoxygenase pathway in apple fruit across different environmental conditions.
Apple fruit undergo a physiological transition on the differentiation of photosynthetically active chloroplasts into chromoplasts during ripening, and this transition would appear to be coupled to a decline in enzymatic activities associated with carbon assimilation32. In this work, the abundances of several photosynthetic proteins, including photosystem II oxygen-evolving enhancer protein 1, photosystem II oxygen-evolving enhancer protein 2, photosystem II oxygen-evolving enhancer protein 3, thylakoid rhodanese, and photosystem I subunit IV were repressed by high altitude (Fig. 5c). However, this photosynthetic-related repression had remarkably little effect on either fruit growth dynamics or sugar accumulation (Fig. 1a), which suggests that the vast majority of photoassimilates are supplied by the leaves rather than produced de novo in the apple fruit. This is in-line with the results of a previous study33, where it was shown that transgenic tomato plants exhibiting decreased expression of glutamate 1-semialdehyde aminotransferase were characterized by a reduced photosynthetic activity, as determined by the levels of intermediates of the Calvin–Benson cycle, but without effects on fruit growth and ripening. The authors suggested that under certain environmental conditions (i.e. higher light irradiance), the repression of fruit photosynthetic capacity could probably be compensated by an enhanced import of photoassimilates from source tissues33. The observed reduction of these photosynthetic proteins (Fig. 5c) is coincident with the loss of green color during chlorophyll degradation and the red coloration in apple fruit during maturity transition (Figs. 1a, 2). Indeed, both are potentially linked to the increased anthocyanin content of the high-altitude fruit (Figs. 2, 4), suggesting the presence of tight regulatory control between photosynthetic proteins and anthocyanin biosynthesis. The link between photosynthesis/anthocyanin homeostasis and fruit development is by no means unprecedented. Previous data proposed that anthocyanins protect photosynthetic tissues by shading chloroplasts from blue–green light34, while anthocyanins have been shown to reduce photobleaching and to decrease photoinhibition in the apple peel exposed low temperature35.
Oxidative stress in the fruit is known to increase markedly during the last stages of normal fruit development; and this has been proposed to be part of the ripening process with the aim of facilitating metabolic changes associated with this phenomenon17. During fruit ripening, chloroplasts develop into chromoplasts, accumulating high amounts of antioxidant compounds, which presumably will protect the chromoplast and fruit cells32. In this sense, the massive peel coloration at high altitude (Figs. 1a, 2) was accompanied with a strong increase in the abundance of several oxidative stress-related proteins, including catalase, ascorbate peroxidase, glutathione reductase, and monodehydroascorbate reductase as well as of various defense-related proteins such as the heat shock 70 kDa protein 1/2/6/8 and pathogenesis-related thaumatin superfamily proteins (Fig. 5c, Supplementary Table 4). Thus, it is possible that the high-altitude environment may alter defense metabolism, thereby enabling apple peel to withstand these conditions.
An important result revealed by our study was that the high-altitude peel tissues had substantially lower fumarate, malate and glutamic-acid contents than those grown at low altitude (Figs. 3, 6). Using protein-metabolite correlation network analysis (Supplementary Fig. 3), we also found that the reduction of these metabolites was accompanied with the decrease of several glutamic-acid-related proteins such as glycine hydroxymethyltransferase (SHM), methylenetetrahydrofolate reductase (MTHFR), monothiol glutaredoxin (GRXS), glutamate–glyoxylate aminotransferase (GGAT), aldose-6-phosphate reductase (S6PDH), and the large subunit ribosomal protein L13e (RPL13B) (Figs. 5c, 6; Supplementary Table 4). Although very little is known about glutamate fluxes and the dynamics of apple peel tissues, it seems possible that glutamate could participate in intercellular signaling36. There is preliminary evidence that environmental factors trigger glutamate efflux into the apoplast to activate glutamate-gated channels, causing a transient change in [Ca2+]cyt and a cascade of downstream responses in Arabidopsis37. Consistent with this possibility, plant tissues have evolved the capacity to respond to variations in glutamate concentration in the external environment38. Moreover, the observation that isocitrate dehydrogenase (ICDH) is present at higher levels in high-altitude fruit (Fig. 5 and Fig. 6) along with the established role of this enzyme in glutamate biosynthesis36, prompts the question as to whether this metabolic route has an undescribed function in apple peel tissues that is associated with the altitudinal gradient. However, more work is needed to characterize the biological function of the proposed network (Fig. 6). For example, functional genomics analysis alongside the application of artificial methods to change specific climatic characteristic (e.g., light, temperature), would likely allow one to verify how altitude environmental factors may influence the whole-peel metabolism during apple ripening transition.