Global characteristics of the ‘Yellow’ non-climacteric melon ripening
The regulation of fruit ripening and development is an important key process in fruticulture. Several metabolic and physiological changes that occur in fruit ripening reflect in its quality such as taste, color, aroma, and texture. 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 mel, 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.
The RNA-seq technology was used to analyze the transcriptomic differences between young (10 DAP) and mature (40 DAP) non-climacteric melon fruit. The DE genes were annotated with diverse GO functional descriptions related to energetic metabolism including carbohydrate metabolic process (BP), photosynthesis (BP), integral component of membrane (CC), ATP binding (MF), metal ion binding (MF). Based on KEGG analysis, carbon fixation in photosynthetic organisms and carbon metabolism pathways were enriched in both fruit development stages; however different genes or isoforms are DE. Studies demonstrated that in the beginning of fruit development there are a high anabolism and catabolism of sugar; and in strawberry fruit was demonstrated an important role of oxidative phosphorylation in ripening [26]. In mature melon fruits, the enrichment of these same pathways can be explaining by accumulation of sucrose in this stage [6]. The pathways of protein processes in the endoplasmic reticulum, spliceosome and ribosome biogenesis are also represented in the late development of melon indicating high transcription and translation rate. Besides that, the high splicing process reflects on 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 demonstrated the presence of paralogous copies acting in diverse metabolic pathways in plants including in sugar that in melon have definite functionalization concerning both development stages and tissue specificity [9]. Besides, the high activity of photosynthesis in young melon when compared to full-ripe fruit has also been reported in grape and other melon varieties [6, 27].
The ‘plant hormone signal transduction’ is an important pathway in the ripening process [28, 29]. 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 [26, 30]. In the early melon fruit development, the KEGG enrichment analysis was significant for this pathway. The majority of DE genes present in IAA, JA, GA and CYT pathways decreased in the ripening process as well as occurs in other non-climacteric fruits [26, 31, 32]. The ABA and BR burst production generally occurs in the color change stage in late fruit development [26, 30, 33], however, in our study, the genes related to both hormones pathways are more expressed in young ‘Yellow’ melon fruit. The expression of some genes presents in the downstream of ethylene and acid salicylic pathways increased along fruit development of non-climacteric melon (Additional file 4: Table S2). One of these genes is the ethylene receptor 1 that has been shown to negatively regulate ethylene signal transduction and suppress ethylene responses [34]. 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 10 DAP fruits demonstrated the interaction of 6 metabolic pathways: ‘Starch and sucrose metabolism’; ‘Amino and nucleotide sugar metabolism’; ‘Galactose metabolism’; ‘Pentose and glucuronate interconversions’; ‘Cynoamino acid metabolism’; ‘Pentose phosphate pathway’. Furthermore, enzymes related to cell wall degradation were identified like 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 probably occur to prepare tissue for maturation and for the development process, which involves progressive fruit softening and sucrose accumulation. In flesh watermelon, some isoforms of pectinesterase and polygalacturonase also have an increase in the first development stages decreasing in the full-ripe fruit [35]. Also, the alpha-L-arabinofuranosidase had a higher interaction number and it is involved in the degradation of arabinoxylan (the major component of cell wall plant hemicellulose) [36]. Saladié et al. (2015) demonstrated that several genes related to cell wall degradation were 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 having the sucrose accumulation as the major determinant of melon sweetness [6, 37]. One enzyme of this pathway is the acid invertase (CmAIN2) that had the second-highest number of interactions in the young melon fruits subnetwork (Fig. 5; Additional file 5 Table S3). This reinforces the idea of its key function in the catabolism of sucrose [6]. Two beta-glucosidases (CmGL18, CmGL24) have 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) [38] that reinforce the idea of high sugar conversion to energy in early fruit development stage. Another important enzyme in the subnetwork is sucrose synthase 2 (CmSUS2) that has 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 further 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 synthases isoenzymes (CmTPS7, CmTPS5). The trehalose phosphate synthases 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 demonstrated that threalose-6-phosphate inhibited sucrose synthase and consequently the sucrose cleavage in castor bean [39]. The TP6 may be having a higher conversion into trehalose in young melon because there is the highest gene expression of trehalose-phosphate phosphatase. Thus a higher TP6 accumulation is expected in full-ripe fruit, once that TPP is down-regulated, contributing to the increase of sucrose content [40]. Besides, some genes involved with the auxin pathway are present in this subnetwork and have interaction through hexosyltransferase and argonaute proteins with the CmSUS1, CmTPS7, CmTPS5. Previous studies reported that auxin reduced the sugar content in fruits [41]. Two genes of this subnetwork are auxin response factor genes (CmAUXRF1, CmAUXRF2) and one is a responsive auxin protein (CmAUXRS). Responsive auxin proteins are transcriptional factors that have repression function through interaction with auxin response factors [42, 43]. This potentiates the sucrose accumulation in 40 DAP fruit. Regarding the argonaute proteins, they bind to micro RNAs (miRNA) and act in transcript cleavage [44]. Plant miRNAs typically target transcription factors including auxin-response factor [44]. The weak interaction is detected into hexosyltransferases, unknown proteins and argonaute proteins and deeper studies should be conducted to better understand this association. It is noteworthy that the chromatin structure-remodeling complex protein SYD (CmCSREM) gene presents in this subnetwork is related to a promotor regulation of several genes downstream of the jasmonate and ethylene signaling pathways [45].
Sucrose metabolism
Sugar metabolism is an important pathway related to the sweetness of melon fruits and it is the most attractive characteristic for consumers [46]. 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). The sucrose is the main sugar component that gives sweet taste in melon and its high content in 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 are a gradual expression increase of CmSUS1 and decrease of CmSUS2 in the ‘Yellow’ melon. In non-climacteric melons ‘Hami’ [46] and ‘Piel del Sapo’ [6] were observed the same expression profile. 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. 8). Because CmSUS1 and CmSUS2 are located in different chromosomes (chr1 and chr9 respectively) they may be subject to different epigenetic regulatory mechanisms. 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. 8).
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 [46], also showed just 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’ melons and consistently decreased in the following developmental stages (Fig. 8). Studies have demonstrated that acid and neutral invertases genes are highly expressed in young developing fruit, and subsequently declined substantially at the sucrose accumulation stage [6, 9, 18, 46]. 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 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 flavor 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]. The RNA-seq results showed two DE genes that encoding invertase inhibitors (CmINH2; CmINH-LIKE3) that are characterized by the presence of plant invertase/pectin methyltransferase inhibitor domain (Additional file 8: Figure S9). The putative invertase inhibitor 3 (CmINH-LIKE3) had high activity in the beginning of development and low expression in the following stages (Fig. 8). On the other hand, invertase inhibitor 2 (CmINH2) presented the higher transcription level in full-ripe (40 DAP) than in youngest melon (10 DAP) (Fig. 8). Hence, in the 20 DAP stage may be occurring the inactivation of CmAIN2 by CmINH-LIKE3 protein interaction. 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 invertase or its activation in different stages of ripening. Besides, two invertase inhibitors (cCL2226Contig1 and c15d_02-B02-M13R_c) are about the 30 times higher in non-climacteric ‘Piel sapo’ melon than in climacteric climacteric ‘Védrantais’ melon [6]. These characteristics are in accordance with invertases expression differences.
The raffinose oligosaccharides (RFOs) synthesized by photosynthesis can be converted into sucrose and galactose by α-galactosidases on 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. 8). 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 can be explaining by individual variations that do not invalidate the profile of gene expression detected by RT-qPCR. The CmNAGLIKE2 also has high activity in the beginning of fruit development, while CmNAG3 peaking in the 40 DAP stage. Previous studies no showed 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 the respiration by its conversion to hexoses, evidenced by the high activity of CmAIN2 and CmSUS2 in same stages. The CmNAG3 has 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 the subsequent decrease until the full-ripe stage, implying a concomitant gene expression with CmNAG2 (Fig. 8). 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, 46]. 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 to 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 [47]. Only CmSPS2 was DE in our study, which has an increase during fruit maturation, reaching the highest levels in color change melons (30 DAP) (Fig. 8). This is in agreement with gene expression observed in ‘Hami’ non-climacteric melon [46]. In climacteric ‘Dulce’ melon CmSPS2 is weekly 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, 46]. In our study only CmSPP1 was DE, having a moderate expression without pronounced differences in the first two stages but with evident decreased in the full-ripe fruit. In non-climacteric ‘Hami’ melon the DE of Sucrose-P Phosphatase was not observed [46]. In climacteric ‘Dulce’ melon CmSPP1 gene was weakly expressed and slightly increase in the 40 DAP stage [9].