Global characteristics of the ‘Yellow’ non-climacteric melon ripening
Fruit ripening and development is a genetically programmed and irreversible process that involves physiological, biochemical and organoleptic changes influencing the fruit quality such as flavour, texture, colour and aroma [27]. However, the study of the metabolic networks is complex and the central signal of genic cascade is not completely understood. In our study, we used an important commercial non-climacteric ‘Yellow’ melon fruit (Cucumis melo, inodorus group) as experimental material to comprehend the main metabolic processes that involve maturation in this phenotype, focusing on the sugar pathway study that is a main quality attribute in melon fruits.
RNA-seq technology was used to analyze the transcriptomic differences between young (10 DAP) and mature (40 DAP) non-climacteric melon fruit. A total of 895 DE genes are down-regulated and 909 are up-regulated during melon ripening. GO enrichment analysis showed that the DE genes in young fruit were more related to molecular transport and metabolic processes including the ‘carbohydrate metabolism’; while in ripe fruit the most DE genes are required for peptide metabolism and protein biosynthesis. In addition, the integrative KEGG analysis conducted for metabolic pathways demonstrated that ‘carbon fixation in photosynthetic organisms’ and ‘carbon metabolism’ pathways were enriched in both fruit development stages; however different genes or isoforms are DE. At the beginning of fruit development there is high anabolism and catabolism of sugar that is the metabolic process required for carbon skeleton construction and energy supply in plants. In strawberry fruit, an important role of oxidative phosphorylation in ripening was demonstrated [28]. The DE genes enriched in 40 DAP melon fruits are related to the sucrose accumulation function [6]. The protein processes in the endoplasmic reticulum, spliceosome mechanism and ribosome biogenesis were also significantly enriched in KEGG analysis in the late development of melon indicating high transcription and translation rate. Moreover, the high splicing process is reflected in the production of different proteins that can act and control a specific metabolic route. This characteristic associated with the activation of different protein isoforms can also explain the KEGG enrichment of the same pathways in both maturation stages as previously mentioned. Studies have reported the presence of paralogous copies acting in diverse metabolic pathways in plants, including in sugar metabolism, that in melon have definite functionalization concerning both development stages and tissue specificity [9]. The high activity of photosynthesis in young melon when compared to full-ripe fruit has also been described for grape and other melon varieties [6, 29].
The ‘plant hormone signal transduction’ is an important process in fruit ripening [30, 31], and this pathway was significantly enriched in the early melon fruit development. Ethylene (ETH), abscisic acid (ABA) and brassinosteroids (BRs) have been suggested to promote ripening through complex interactions; while auxin (IAA), cytokinins (CYT), gibberellin (GA) and jasmonic acid (JA) are putative inhibitors of ripening [28, 32]. In our study, the DE genes present in IAA, JA, GA and CYT signal transduction decreased during maturation which also occurs in other non-climacteric fruits [28, 33, 34]. In the ABA pathway, only the ‘protein phosphatase 2C 77’ gene (repressor of the abscisic acid signalling pathway [35]) was DE in the 10 DAP fruit. Studies have been suggested that ABA plays an important role in the regulation of non-climacteric fruits [36, 37] and the key gene for its biosynthesis is 9-cis-epoxycarotenoid dioxygenase (CmNCED) that was significantly more expressed in full-ripe fruit (Additional file 7: Table S6). This can indicate that ABA might be involved in the regulation of melon maturation and senescence. Interestingly, there are intimate connections between sugar and ABA signalling [38]. The BR burst production generally occurs in the colour change stage in late fruit development [28, 32, 39]. In our study, the genes related to BR signal transduction are more expressed in young ‘Yellow’ melon fruit; however the colour change occurs from 20 DAP to 30 DAP fruits. Thus further studies should be conducted to understand the transcriptome profile of these stages. The expression of some genes present in the ethylene and salicylic acid metabolism were highest in mature fruit (Additional file 7: Table S6). One of these genes is the ethylene receptor 1 that has been shown to negatively regulate ethylene signal transduction and suppress ethylene responses [40]. Thus, it can be a candidate gene in non-climacteric and climacteric melon comparative study.
In the subnetwork protein-protein interaction (PPI), the results of the 10 DAP fruits showed the interaction of 6 metabolic pathways: ‘Starch and sucrose metabolism’; ‘Amino and nucleotide sugar metabolism’; ‘Galactose metabolism’; ‘Pentose and glucuronate interconversions’; ‘Cynoamino acid metabolism’; and ‘Pentose phosphate pathway’. Furthermore, enzymes related to cell wall degradation were identified such as pectinesterase and polygalacturonase that are mainly responsible for the pectin changes. The up-regulation of these genes and those associated with sucrose synthesis in the early stage of development are involved with progressive fruit softening and sucrose accumulation. In flesh watermelon, some isoforms of pectinesterase and polygalacturonase also show an increase in the first development stages, decreasing in the full-ripe fruit [41]. Another cell wall enzyme was alpha-L-arabinofuranosidase that catalyzes the breaks in the arabinoxylan (major component of cell wall plant hemicellulose) [42]. Saladié et al. (2015) demonstrated that several genes related to cell wall degradation were more strongly up-regulated in climacteric melon (cv. Védrantais) than non-climacteric (cv. Piel de Sapo) [6]. The sugar metabolism is an important process in fruit ripening and development and sucrose accumulation is the major determinant of melon sweetness [6, 43]. One enzyme of this pathway is the acid invertase (CmAIN2) that had the second highest number of interactions in the young melon fruit subnetwork (Fig. 5; Additional file 9: Table S9). This reinforces the idea of its key function in the catabolism of sucrose [6]. Two beta-glucosidases (CmGL18, CmGL24) have an interaction with CmAIN2, these enzymes have the function of hydrolyzing the terminal, non-reducing beta-D-glucosyl residues (final reaction in cellulose hydrolysis) with the release of beta-D-glucose (primary energy source in plants) [44] that suggest a high sugar conversion to energy in the early fruit development stage. Another important enzyme in the subnetwork is sucrose synthase 2 (CmSUS2) that has a strong interaction with alpha-trehalose phosphate synthase 9 (CmTPS9) followed by trehalose phosphate phosphatase (CmTPP1). These enzymes and others of sugar metabolism will be discussed in more detail below in the next topic.
Although the majority of DE genes of auxin and sugar metabolism are up-regulated in 10 DAP melons, some isoforms or different genes from these pathways are more expressed in 40 DAP. The subnetwork generated for mature fruit is represented by a different sucrose synthase (CmSUS1) which also has high interaction with two alpha-trehalose phosphate synthase isoenzymes (CmTPS7, CmTPS5). The trehalose phosphate synthases (TPS) convert glucose-6-phosphate and uridine diphosphate (UDP) glucose into trehalose-6phosphate (T6P) and the subsequent dephosphorization of T6P is catalyzed by trehalose-phosphate phosphatases. A recent study reported that threalose-6-phosphate inhibited sucrose synthase and consequently the sucrose cleavage in castor bean [45]. The T6P may be undergoing a higher conversion into trehalose in young melon due to the greater trehalose-phosphate phosphatase gene expression. Thus T6P accumulation is expected in full-ripe fruit, once that TPP is down-regulated, contributing to the increase of sucrose content [46]. In addition, genes involved with the auxin pathway such as auxin response factor (CmAUXRF1, CmAUXRF2) and responsive auxin protein (CmAUXRS) are present in this subnetwork and have interaction through hexosyltransferase and argonaute proteins with the CmSUS1, CmTPS7 and CmTPS5 (Fig. 5). In that respect, previous studies reported that auxin reduces the sugar content in fruits [47]. However, the precise association of the genes CmAUXRF1, CmAUXRF2 and CmAUXRS with the sugar pathway requires further studies. Regarding the argonaute proteins, they bind to micro RNAs (miRNA) and act in transcript cleavage [48]. Plant miRNAs typically target transcription factors including the auxin-response factor [48]. A weak interaction was detected between hexosyltransferases, unknown proteins and argonaute proteins and further studies should be conducted to better understand this association. It is also noteworthy that the chromatin structure-remodelling complex protein SYD (CmCSREM) gene present in this subnetwork is related to a promotor regulation of several genes downstream of the jasmonate and ethylene signalling pathways [49].
Sucrose metabolism
Sugar metabolism is an important pathway related to the sweetness of fruits and it is the most attractive characteristic for consumers [50]. Furthermore, studies have reported that sugars may serve as important signals that modulate a wide range of processes in plant physiology including fruit maturation [38, 51, 52]. In our study, a total of 17 genes were DEs in the sucrose; amino and nucleotide sugar; and galactosidase pathways and 8 were evaluated in two additional development stages (20 and 30 DAP). Sucrose is the main sugar component that gives the sweet taste in melon and its high content at the mature stage could be used as a marker [6]. Only sucrose synthases and invertases are known enzymes responsible for sucrose cleavage [10]. The sucrose synthases convert sucrose to fructose and UDP glucose that is a reversible reaction [10]. In our study, two isoforms were DEs by RNA-seq analysis, the CmSUS1 that was up-regulated in full-ripe fruit while CmSUS2 had a burst of gene expression in 10 DAP fruit. In fruit maturation, there is a gradual expression increase of CmSUS1 and decrease of CmSUS2 in the ‘Yellow’ melon. In non-climacteric melons, ‘Hami’ [50] and ‘Piel del Sapo’ [6], the same expression profile was observed. In ‘Dulce’ climacteric melon, the CmSUS1 was more expressed in young fruit, followed by near-silencing in mature fruit. CmSUS2 showed low levels of expression throughout fruit development and the third sucrose synthase (CmSUS3) was DE being weakly expressed in the young fruit and increased in the maturing fruit [9]. Thus, it may be suggested that in non-climacteric melons, CmSUS1 is mainly responsible for the synthesis of sucrose for storage in the vacuole, contributing to ripe fruit taste, while CmSUS2 acts in an opposite way providing the substrate for energy production by sucrose catabolism during early development (Fig. 7). Also, the TPS and TPP have an important function in the sucrose synthase activities contributing to sucrose content in the fruit as previously described (Fig. 7).
Invertases produce glucose instead of UDP-glucose and fructose in a non-reversible reaction. Acid invertases have been attributed to vacuole localization while neutral invertases have generally been located in the cytosol, consistent with the optimal neutral pH activity and absence of glycosylation [9]. In the RNA-seq analysis, only the acid invertase (CmAIN2) was DE in non-climacteric ‘Yellow’ melon. Previous studies with ‘Piel del Sapo’ [6] and ‘Hami’ non-climacteric melon fruit [50] also showed only transcriptional activity of acid invertase 2 (CmAIN2). In ‘Dulce’ climacteric melons, four neutral invertase (CmNIN1, CmNIN2, CmNIN3 and CmNIN4) were DE, as well as the acid invertase 2 (CmAIN2) [9]. The peak of CmAIN2 expression occurs in the 20 DAP ‘Yellow’ melon fruits and consistently decreased in the following developmental stages (Fig. 7). Studies have demonstrated that acid and neutral invertase genes are highly expressed in young developing fruit, and subsequently declined substantially at the sucrose accumulation stage [6, 9, 18, 50]. This reduction of soluble acid invertase activity signals the metabolic transition from fruit growth to sucrose accumulation [3, 17]. The higher expression of neutral invertases in climacteric melon fruit suggests that cytoplasmatic sugar catabolism might be an additional source of energy, supporting the hypothesis that climacteric melon fruit spends more energy during fruit development, due to respiration, than non-climacteric ones. In the non-climacteric and climacteric melon comparison, studies demonstrated that the acid invertase gene (CmAIN2) was almost 10-fold higher in ‘Védrantais’ (climacteric) than in ‘Piel del Sapo’ (non-climacteric). The high activity of soluble acid invertase (CmAIN2) might limit the accumulation of sucrose during climacteric ripening and increase organic acids, such as malate, that impart a stale flavour to the fruit [3, 6, 9, 17]. Thus, the inhibition of CmAIN2 can be a key process in the differences of sugar content and melon quality.
Invertase inhibitors are responsible for decreasing the activity of soluble acid invertases through post-translational regulation, reducing sugar consumed in respiration and regulating the accumulation of sucrose during melon development and ripening [6, 17]. Two invertase inhibitors (CmINH2; CmINH-LIKE3) were DE by RNA-seq analysis and are characterized by the presence of plant invertase/pectin methyltransferase inhibitor domain (Additional file 12: Figure S11). The invertase inhibitor 2 presented higher transcription level in full-ripe (40 DAP) than in youngest melon (10 DAP) (Fig. 7). On the other hand the putative invertase inhibitor 3 (CmINH-LIKE3) had high activity in the beginning of development and low expression in the following stages (Fig. 7). Hence, in the 20 DAP stage the inactivation of CmAIN2 by CmINH-LIKE3 protein interaction may be occurring. Subsequently, the CmAIN2 expression rapidly decreases and the CmINH-LIKE3 transcription is not necessary anymore. The CmINH2 can have affinity with other invertases that have not been evaluated on the intermediate stages (20 DAP and 30 DAP). The putative CmINH-LIKE3 expression was not reported in previous studies. The DE of CmINH2 was also detected in climacteric ‘Dulce’ melon; however, it is more strongly expressed in the first stages of development decreasing during ripening [9]. One other DE isoform detected for this same variety was CmINH1 that was expressed at high levels in the 30 DAP stage [9]. The results demonstrated different expression profiles for non-climacteric ‘Yellow’ melon and climacteric ‘Dulce’ melon, indicating recruitment of distinct INH isoforms in the regulation of the invertases or its activation in different stages of ripening. Moreover, two invertase inhibitors (cCL2226Contig1 and c15d_02-B02-M13R_c) are about 30 times higher in non-climacteric ‘Piel del Sapo’ melon than in climacteric ‘Védrantais’ melon [6]. These characteristics are in accordance with invertase expression differences.
The raffinose oligosaccharides (RFOs) synthesized by photosynthesis can be converted into sucrose and galactose by α-galactosidases in fruit tissues. In melon, α-galactosidase includes acid and neutral isoenzymes and in young fruit, both can be used to provide energy for the growth metabolism. In mature fruits, sucrose can be stored in the vacuole while galactose can be metabolized to sucrose [3, 6, 9]. In our study, three neutral α-galactosidases (CmNAG2, CmNAGLIKE2, CmNAG3) were DEs (Fig. 7). In the early development stages of ‘Yellow’ melon, there is an increase of the CmNAG2 expression, that has the highest level in the 20 DAP stage and decreases in 30 and 40 DAP. Though the RNA-seq analyses demonstrated that in the 40 DAP stage the expression of this enzyme was higher than in 10 DAP, the log2 fold change was low (1.07). These differences might be due to individual variations. The CmNAGLIKE2 also has high activity in the beginning of fruit development, while CmNAG3 peaks in the 40 DAP stage. Previous studies showed no DE of neutral α-galactosidases in non-climacteric melon. In climacteric ‘Dulce’ melon the high activity transcriptional of CmNAG2 was also detected in early stages and low activity of acid α-galactosidases in the maturation process [9]. We suggest that the sucrose produced in the catabolic reaction of CmNAG2 and CmNAGLIKE2 in young fruit is recruited in respiration by its conversion to hexoses, evidenced by the high activity of CmAIN2 and CmSUS2 in the same stages. The CmNAG3 has a function in the sucrose accumulation in full-ripe melon.
Another important route of sucrose synthesis is by conversion of galactose-1P into glucose-1P by UDP- glucose epimerase (UGE) [3, 6, 9, 16]. In the present study, we observed the increase of CmUGE3 expression in the initial development, followed by subsequent decrease until the full-ripe stage, implying a concomitant gene expression with CmNAG2 (Fig. 7). The activity of both enzymes denotes high sucrose production in young fruits. In climacteric ‘Dulce’ melon, three UGEs were DE throughout fruit development (CmUGE1, CmUGE2 and CmUGE3), but CmUGE3 expression increased significantly during fruit maturation [9]. The CmUGE3 gene expression or its enzyme activity were not reported in other non-climacteric melon in previous studies [6, 50]. Antagonistic profile of CmUGE3 is observed in the two melon phenotypes that can be associated with differences in sugar accumulation.
Sucrose-P synthase (SPS) is considered the key gene for sucrose accumulation in fruit ripening [9, 17, 18]. This enzyme catalyzes the reversible transfer of a hexosyl group from UDP-glucose to D-fructose 6-phosphate to form UDP and D-sucrose-6-phosphate [53]. Only CmSPS2 was DE in our study, which has an increase during fruit maturation, reaching the highest levels in colour change melons (30 DAP) (Fig. 7). This is in agreement with gene expression observed in ‘Hami’ non-climacteric melon [50]. In climacteric ‘Dulce’ melon, CmSPS2 is weakly expressed during fruit development, but CmSPS1 rapidly increased from 20 DAP, peaking at late developmental stages [9]. The gene expression of SPS was similar when comparing climacteric and non-climacteric melon, however different isoenzymes are responsible for syntheses D-sucrose-6-phosphate. The sucrose phosphate phosphatase (SPP) has complementary activity with SPS by conversion of sucrose-6-phosphate to sucrose [3, 9, 17, 18, 50]. In our study only CmSPP1 was DE, having a moderate expression without pronounced differences in the first two stages but with evident decrease in the full-ripe fruit. In non-climacteric ‘Hami’ melon, the DE of sucrose-P phosphatase was not observed [50]. In climacteric ‘Dulce’ melon, the CmSPP1 gene was weakly expressed with a slight increase in the 40 DAP stage [9].