An overview on metabolites involved in durian fruit ripening
The changes in the underlying mechanisms during the ripening process contribute to durian fruit characteristics. To study the alterations of primary metabolites in these related mechanisms, we performed metabolome analysis in durian cv. Monthong pulp at the unripe and ripe stages. CE-TOF/MS and GC-TOF/MS techniques were used for comprehensive analysis of primary metabolites, which identified 167 and 56 metabolites, respectively. Among these metabolites, 28 were annotated using both techniques (Supplementary Table S1, Supplementary Fig. S1). In total, 195 annotated metabolites were obtained and used for further analysis. Our annotation showed that results from each technique validated those from other techniques because the intensities of the same metabolites exhibited similar changes during the ripening period (Supplementary Table S1). It is known that both CE-TOF/MS and GC-TOF/MS annotate primary metabolites of slightly different kinds. CE-TOF/MS can separate a wider range of charged metabolites, including amino acids and organic acids, while GC-TOF/MS is more dependent on volatility and can identify uncharged metabolites, such as sugar and sugar derivatives.
Metabolites were further analyzed using the web-based tool MetaboAnalyst 4.0. From the PCA plot, the first two components on the x- and y-axes represented the total variance of the data at 42.5% and 33.2%, respectively. Unripe durian samples were clearly separated from the ripe groups by principal component 1 (Fig. 1A). As expected, the results showed that ripening greatly influences the metabolic change at the biochemical level and leads to physiological changes, such as the expression of flavor and softening of the edible ripe fruit. This phenomenon is likewise observed in other fruit species, such as peach, including strawberry, tomato, and grape5,11, constituted the different fruit models. To obtain the relevant biological pathways, Metabolomic Pathway Analysis (MetPA) was used. Of 66 enriched KEGG biological pathways, 30 metabolic pathways were perturbed (Supplementary Table S2). Some of the significant altered metabolic pathways, such as the TCA cycle, amino acid metabolism, and starch and sucrose metabolism, are primary metabolic pathways and are possibly involved in flavor precursor biosynthesis (Fig. 1B).
Ripening-associated metabolites contributing to durian flavor
Out of 195 annotated metabolites, 94 metabolites in durian pulp were significantly altered between the unripe and ripe stages. The ripening-associated metabolites were classified into six groups, including proteinogenic amino acids, non-proteinogenic amino acids, sugar and sugar derivatives, polyamine, organic acid, and nucleotide and nucleotide derivatives. All others were placed in a miscellaneous group (Fig. 2a, 2b, Supplementary Table S3).
Sugar is an important metabolite group responsible for sweetness in fruits. Previously, sucrose, fructose, glucose, and maltose were found to be increased in ripe durian pulp samples1. Using GC-TOF/MS, we confirmed that these four major sugars and a newly annotated sugar, raffinose, were significantly increased during ripening with the log2 FC ranging between 1.16. and 4.84 (Fig. 2), while a newly annotated 1-kestose was unchanged. Since sugars were only annotated from GC-TOF/MS in this work, we applied HPAEC-PAD analysis to validate and confirm the result (Fig. 3). According to the results, the contents of all four major sugars agreed with GC-TOF/MS. However, the content of 1-kestose was significantly increased, while the content of raffinose remained unchanged during the ripening period (Fig. 3). Disagreements between the two methods could be obtained when the concentrations in the samples were very low, as raffinose and 1-kestose were in this case. When using GC-MS, it is suggested that optimum derivatization and separation are needed before measuring sugars in each individual plant extract12. Therefore, in our analysis, the quantification from targeted HPAEC-PAD analysis is more reliable than the untargeted analysis, which can lead to the discovery of novel metabolites. Fruits can accumulate different types of sugars, but sucrose is the primary storage sugar in many fruits, such as melon, banana, peach, and strawberry13. Notably, durian pulp contains over 50% sucrose by dry weight (Fig. 3), which makes it the highest sucrose-accumulating fruit among those reported. Our study showed that sucrose is an important flavor-providing metabolite in ripe durian pulp.
In addition, we identified two ripening-associated sugar alcohols found at low concentration related to raffinose, galactinol and myo-inositol (Fig. 2). Galactinol content decreased during ripening, which was in agreement with the two analytical methods (Fig. 3), whereas myo-inositol content remained unchanged, as confirmed by HPAEC-PAD. Raffinose and myo-inositol were synthesized from galactinol. The decrease in galactinol during the ripening period suggested that it might be stored before ripening and then converted to the downstream sugars later. The accumulation of raffinose-family sugars is quite different between various fruit14, and their role in fruits has not been studied much to date; however, they might be involved in stress tolerance during ripening. Furthermore, we found an increase in sugar phosphates, including glucosamine-6-phosphate, N-acetyl-D-galactosamine-6-phosphate, lactose-1-phosphate, sucrose-6-phosphate, and trehalose-6-phosphate in ripe durian pulp with log2 FC ranging between 1.34 and 2.09 (Fig. 2). Sugar phosphates are intermediates in different biological pathways. In addition, they also act as signaling molecules. One interesting metabolite is trehalose-6-phosphate, which is an intermediate for trehalose biosynthesis. It is an important signaling molecule for sucrose consumption and a negative feedback molecule for sucrose biosynthesis in plants15. Therefore, the increase in trehalose-6-phosphate in durian pulp possibly helps to control the sucrose content and supports trehalose content at the same time.
We detected 21 ripening-related organic acids in the durian pulp. Of these, the levels of 19 organic acids significantly increased during durian ripening, with log2 FC ranging between 1.38 - 10.33, while those of 2-isopropylmalate and threonate decreased (Log2 FC = -1.25 and -2.56, respectively) (Fig. 2). There were five ripening-related organic acids, namely cis-aconitate, fumarate, malate, pyruvate, and succinate, that participated in central carbon pathways. Malate and succinate were the highest among others (Supplementary Table S1), which was consistent with the results in the ripe durian pulps from other cultivars16. Another abundant organic acid was citrate, and its content was not significantly changed during the ripening period (Supplementary Table S1). Therefore, malate and succinate probably contribute to sour taste in the ripe fruit, even if the sweet taste is dominant in the pulp, due to high sugar composition. In addition, we also detected a significant change in 4-methyl-2-oxovalerate and 2-isopropylmalate (Fig. 2), which are two important intermediates in branched-chain amino acid metabolism that will be elaborated in the section below.
Amino acids serve as important flavor precursors in many fruits, including durian. We detected 31 ripening-associated amino acids, three amino acids that were unchanged: asparagine, glutamate, and threonine. Levels of 15 proteinogenic and 14 non-proteinogenic amino acids increased during durian ripening with log2 FC ranging between 1.34 - 5.65 and between 1.4 - 9.15, respectively, while only aspartate and ornithine decreased (Log2 FC = -1.36 and -5.64, respectively) (Fig. 2). Durian pulp releases sulfuryl odor during ripening. The odor is associated with amino acids in sulfur-related pathways. Notably, cysteine is the most modulated amino acid in the pathway (Fig. 2, Supplementary Table S3) and is an important precursor for the biosynthesis of methionine, another sulfur-containing amino acid. We found that methionine levels slightly increased in ripening durian pulp (Fig. 2, Supplementary Table S3). Methionine is a central metabolite of the sulfur pathway and ethylene biosynthesis, in which ethylene is an important compound for climacteric ripening. Therefore, this may be the reason for the slight changes in methionine content during ripening. In addition, we also found the accumulation of serine and homoserine (Fig. 2), which are intermediates in the sulfur pathway, supporting the involvement of the pathway during the ripening process.
In addition to sulfuryl odor, durian pulp also exhibits fruity aroma during ripening. Branched-chain amino acids, including isoleucine, leucine, and valine, are precursors for alcohols, esters, aldehydes, and lipid-derived volatiles17, which are associated with the fruity smells. We found a significant increase in the levels of these three amino acids in ripe durian pulp (Fig. 2). Interestingly, the increase in isoleucine enhances the biosynthesis of 3-methylbutyl alcohols, 2-methylbutyl alcohols, and their ester derivatives, while the increase in valine enhances 2-methylpropyl alcohol derivatives18. These volatiles can also be detected in the durian and provide the fruit with a fruity odor19. Therefore, it implies that the accumulation of branched-chain amino acids during ripening is important for the biosynthesis of volatile compounds in durian pulp. Moreover, the altered composition of branched-chain amino acids may lead to differences in proportions of volatile compounds and may cause different aroma among durian cultivars.
For the aromatic amino acid pathway, phenylalanine, tryptophan, and tyrosine are ripening-related amino acids in durian fruit (Fig. 2). These amino acids are precursors for the biosynthesis of volatile compounds. For example, phenylalanine increases the production of aldehyde- and ester- type aromatic volatiles4, and some phenyl volatiles are also found in durians19. Furthermore, aromatic amino acids are precursors for secondary metabolites such as flavonoids, alkaloids, indoles, etc. Interestingly, durian pulp contains a high amount of flavonoids at the ripe stage, which positively correlates with the high antioxidant activity of the pulp20. Accumulation of these amino acids may be important for durian pulp to synthesize secondary metabolites during ripening.
Aspartate was the only proteinogenic amino acid whose levels decreased during ripening (Fig. 2). It can be directly used for the biosynthesis of glutamate and asparagine. Moreover, it is involved in the biosynthesis of lysine, methionine, isoleucine, leucine, and valine, whose contents increased during ripening. Therefore, the decrease in aspartate levels during ripening could result from its usage for biosynthesis of other amino acids. Glutamate is a highly abundant amino acid that provides umami taste in durian pulp7. We found that the glutamate content remained constant during the ripening period (Supplementary Table S1). Glutamate can be converted to other metabolites, such as glutamine, arginine, proline, and GABA21. In fact, we observed increased levels of proline and hydroxyproline during ripening. Until now, the roles of these two metabolites in fruits have not been well understood. Previous studies have shown that accumulation of proline is involved in the nitrogen sink mechanism, and it helps to increase the fruit quality traits and yield22. Regarding the precursors for polyamine biosynthesis, we found that the arginine content was unchanged (Supplementary Table S1), while ornithine content decreased during the ripening period (Fig. 2). Both amino acids can be converted to putrescine, a central metabolite of the polyamine pathway.
For polyamine, we found significant increases in N8-acetylspermidine and putrescine, while spermidine and spermine were unchanged and decreased, respectively (Fig. 2, Supplementary Table S1). Polyamines are associated with abiotic stress responses in fruits23. Furthermore, previous studies have shown that they are also involved in the modulation of fruit ripening by intricate crosstalk with the ethylene pathway24. The role of polyamines in durian pulp still requires further investigation, and their accumulation might affect ripening behavior among various durian cultivars. Polyamines are also precursors for GABA biosynthesis. Interestingly, GABA was highly enriched in ripe durian pulp (Supplementary Table S3, Fig. 2). GABA can provide a bitter taste to the fruit. Although the role of GABA accumulation in fruit has not been clearly ascertained, previous studies have shown that it is associated with abiotic stress responses in strawberry25, tomato26, and apple27. It has been shown that these fruits accumulate high GABA content under elevated CO2 and low O2. Therefore, the role of GABA in durian might also be involved in stress response. Further studies in durian may help to promote its market value by developing appropriate storage conditions for higher GABA content in the future.
The levels of nine nucleotide derivatives increased during ripening, with log2 FC ranging between 1.32 - 8.28 (Fig. 2), while those of cytidine diphosphate (CDP) and deoxyguanosine decreased (Log2 FC = -2.06 and -2.18, respectively). To our knowledge, 4 of 11 nucleotide derivatives are known to be involved in fruit flavor. AMP, IMP, and GMP accumulation is associated with enhancing of the umami taste in tomato28. In addition, we also detected additional 17 miscellaneous metabolites with significantly altered levels during durian ripening with log2 FC ranging between -2.18 and 8.45 (Fig. 2). Some of these metabolites are food supplements in the durian pulp, such as gluconate and pyridoxamine, so called vitamin B group7.
Transcriptome analysis of durian cv. Monthong during ripening
The metabolome data provided us only the ripening-associated metabolites and the related pathways associated with durian flavor. Therefore, to deepen and more comprehensively understand the modification of the pathways at the biochemical level, we integrated transcriptome analysis to study the expression of candidate genes in associated pathways.
Approximately 10 - 16 million clean reads from unripe and ripe Monthong were generated. The clean reads were then used to perform de novo assembly, which generated 164,618 unigenes with an average length of 1,082.68 bp, and total reads of each library were mapped to the de novo assembled transcriptome (Supplementary Table S4). The annotation results of unigenes are shown in Supplementary Table S5. Differentially expressed gene analysis identified a total of 18,616 DEGs (Supplementary Table S6) with 8,145 up-regulated genes including the key ethylene biosynthetic genes (1-aminocyclopropane-1-carboxylate synthase and 1-aminocyclopropane-1-carboxylate oxidase) (Supplementary Table S7), and 10,471 down-regulated genes. DEGs were then used to perform GO analysis, and most of the GO terms were assigned to biological processes. Within biological processes, the largest proportion was in protein modification by small protein conjugation and ubiquitin-dependent protein catabolic process categories, respectively. Within molecular function, the largest proportion was in the ATPase activity category. Lastly, within the cellular compartment, the largest proportion was in the host cell nucleus category (Supplementary Fig. S3). To reveal the biological pathways associated with DEGs data, DEGs were assigned to KEGG. The results showed that the DEGs participated in 144 biological pathways. The top five pathways are purine metabolism, thiamine metabolism, starch and sucrose metabolism, glycolysis/gluconeogenesis, and pyruvate metabolism (Supplementary Fig. S4). This result is consistent to the metabolic pathway analysis where the change of nucleotide metabolism, sugar metabolism, and central carbon metabolism was highly associated to durian ripening process.
To validate the DEGs identified from RNA-seq results, we randomly selected 11 key genes (ALDH (LOC111284558), ADC (LOC111308806), CM (LOC111286253), GABA-TK (LOC111305652), GAD (LOC111281776), BCAT (LOC111305652), MDH (LOC111293205), NADP-MEs (LOC111286064), OAT (LOC111278083), PFK (LOC111285266), and TrpAB (LOC111287933)) from various metabolic pathways possibly related to durian flavor, and their expression levels were analyzed by RT-qPCR using Monthong cDNA at the unripe and ripe stages. According to the RT-qPCR analysis, the expression results of all selected genes agreed with results obtained from RNA-seq analysis showing the reliability of the RNA-seq method for expression quantitation (Fig. 4).
Identification of candidate genes in metabolic pathways contributing to durian flavor
Our results demonstrated that sugar and central carbon pathways are important pathways during the ripening process (Supplementary Fig. S4). High sugar accumulation promotes sweet taste in ripe durian pulp. Alternatively, sugars are metabolized into the central carbon pathway, which not only produces cellular energy, but also provides organic acids for fruit acidic taste. In this section, we focused on the modulation of the central carbon pathway and its connection to flavor-related pathways. Therefore, a metabolic network was constructed to elucidate the relationship between changes in metabolite levels and candidate genes (Fig. 5). Information related to candidate genes, such as locus number and expression level, are provided in Supplementary Table S7.
Central carbon pathway
We identified genes in the glycolytic pathway and found the upregulation of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) (Fig. 5). These three genes control the rate-limiting steps of the glycolytic pathway in fruit29. The gene expression is consistent with the accumulation of pyruvate, which is the final product of the pathway (Fig. 5), showing the activation of glycolysis during durian ripening. Afterward, pyruvate is converted to acetyl-CoA and enters the TCA cycle by pyruvate dehydrogenase (PDH), which was also found to be upregulated in the ripe durian pulp (Fig. 5). The first step of the TCA cycle is to produce citrate, which is controlled by citrate synthase (CS). Although CS was upregulated, the content of citrate remained constant during ripening (Fig. 5), suggesting that the rate of citrate biosynthesis is probably equal to citrate consumption. We also observed the upregulation of other genes in the TCA cycle, including isocitrate dehydrogenase (IDH), α-ketoglutarate dehydrogenase (2-OGDH), fumarase (FUM), and malate dehydrogenase (MDH), together with the upregulated metabolites, isocitrate, succinate, fumarate, and malate (Fig. 5), showing the activation of the TCA cycle, supporting citrate biosynthesis during ripening. Citrate usage probably goes through glutamate metabolism through α-ketoglutarate. We found that the content of α-ketoglutarate was unchanged during the ripening period. Notably, the gene for its biosynthesis, IDH, and the gene for its degradation, 2-OGDH, and glutamate dehydrogenase (GDH), were upregulated (Fig. 5). This result suggests that α-ketoglutarate content remains constant because it is converted to the TCA cycle by 2-OGDH and bypassed to glutamate metabolism by GDH. Similar observations were found in other fruits, such as tomato30, showing that the change in citrate is correlated to α-ketoglutarate content during ripening and salt-stress treatment. Although some metabolites in this pathway do not provide flavor to ripe durian, they serve as important precursors for the biosynthesis of other flavor-related compounds, which will be discussed later.
Glutamate and glutamate-derived metabolic pathways
Glutamate, an amino acid providing umami taste, is linked to the TCA cycle via α-ketoglutarate, which is controlled by GDH, and we found that the gene was upregulated during the period of ripening (Fig. 5, Supplementary Table S7). Additionally, there are three routes for glutamate biosynthesis, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glutamate synthase (GOGAT). We found that only AST and GOGAT were upregulated during the period of ripening (Fig. 5). Notably, the route from AST (LOC111284590) contained the highest log2 FC (4.83) and RPKM expression value among the other routes (Supplementary Table S7). Therefore, such a route is highly activated for glutamate biosynthesis during ripening. Glutamate interconverts with aspartate, and this step is controlled by AST. We found that the decrease in aspartate content during durian ripening correlated with the upregulation of AST (Fig. 5), suggesting a transfer of amino group from aspartate toward glutamate.
Although glutamate biosynthesis was highly activated, its content remained unchanged during the period of ripening (Fig. 5), implying an equal rate of biosynthesis and consumption. Interestingly, we found upregulation of several genes related to glutamate conversion. GS was correlated with an increase in glutamine content (Fig. 5). In addition, we also found the upregulation of nitrate reductase (NR) (Supplementary Table S7). Based on the upregulation of these genes, the activation of nitrogen assimilation, leading to enhanced fruit flavor by modulating levels of amino acid and soluble sugars can be suggested. Glutamate can also be sequentially converted to Δ1-pyrroline-5-carboxylate and proline by enzymes encoded from Δ1-pyrroline-5-carboxylate synthetase (P5CS) and Δ1-pyrroline-5-carboxylate reductase (P5CR), respectively. Only one isoform of P5CS and P5CR were found in our transcriptome data, and they were both downregulated during durian ripening, which was not consistent with increased proline content (Fig. 5). Previous report showed that proline content may not be correlated to the expression of P5CR in developing tissues of Arabidopsis31. Therefore, these durian P5CS and P5CR could still be the only candidate genes of proline metabolism in the pulp. We assumed that although both genes were downregulated during ripening, they might still encode enough enzymes that catalyze the reactions and produce proline. In addition, the up-accumulation of proline might be partially caused by a lower rate in proline degradation since we observed the downregulation of a gene encoding proline dehydrogenase, an enzyme involved in proline catabolism (Supplementary Table S7).
Furthermore, glutamate can be used for the biosynthesis of ornithine and arginine, which are precursors for polyamine biosynthesis. These two amino acids can later be converted to putrescine, a precursor for further polyamine production. We propose that the primary route for polyamine biosynthesis of durian pulp is arginine because the key gene arginine decarboxylase (ADC) was upregulated and highly expressed during durian ripening (Fig. 5), while the expression level of ornithine decarboxylase (ODC), controlling the reaction from ornithine to putrescine, was not significantly changed. The arginine route has been found to be a major pathway for polyamine biosynthesis in other fruits such as apple and tomato32. For the polyamine pathway, spermidine synthase (SPDS) and spermine synthase (SPMS) control steps that convert putrescine to spermidine and spermine, respectively. We found upregulation of SPDS and SPMS during durian ripening (Fig. 5), which were not correlated with spermidine and spermine content. Spermidine is either converted to 4-aminobutyraldehyde or is used for spermine biosynthesis. Moreover, spermine can be degraded into β-alanine metabolism. Therefore, these probably are the reasons for the unchanged spermidine content and the decreased spermine content during the ripening period.
GABA is a bioactive compound that its biosynthetic pathway bypasses from α-ketoglutarate. Interestingly, we found a significant increase in GABA levels in the ripe durian pulp (Fig. 2). There are two main GABA biosynthetic pathways in fruits, which are GABA shunts and alternative pathways from polyamines. For the GABA shunt, we found the upregulation of glutamate decarboxylase (GAD) in ripening durian pulp, which positively correlated with GABA content (Fig. 5). GABA shunts are the major pathway in many fruits. GAD is the key gene that controls the conversion from glutamate to GABA in this pathway. In addition, we found the upregulation of several genes in polyamine-derived-GABA biosynthesis, including polyamine oxidase (PAO) and 4-aminobutanal dehydrogenase (ALDH10). Therefore, our gene expression results supported that GABA production during durian ripening was contributed by the two pathways.
Branched-chain amino acid pathway
An overpowering smell is a well-known characteristic of ripe durian. Besides the sulfuryl aroma, ripe durian also contains a sweet, fruity aroma, and the volatiles providing such aroma are associated with branched-chain amino acid metabolism. Interestingly, we observed high upregulation of threonine ammonia-lyase (TAL) (Log2FC = 5.52) (Supplementary Table S7), which converts threonine to 2-oxobutanoate, an important intermediate for BCAA biosynthesis, during durian ripening (Fig. 5). Moreover, we also observed high upregulation of genes in the BCAA pathway, such as branched-chain amino acid aminotransferase (BCAT) (Log2FC = 6.32) (Supplementary Table S7), which is a key regulatory gene controlling branched-chain amino acid biosynthesis and degradation18,33, supporting the activation of this pathway. In addition, branched-chain amino acids are precursors for volatile ester production. Notably, the upregulation of 3-methyl-2-oxobutanoate dehydrogenase (BCKDHA) and long-chain-enoyl-CoA hydratase (EHHADH), key genes in pathways connected to branched-chain amino acid degradation34, was observed during durian ripening (Fig. 5, Supplementary Table S7). Our results support the hypothesis that branched-chain amino acid biosynthesis is upregulated during durian ripening, and these amino acids are possibly used for volatile ester production, providing aroma to the ripe durian.
In conclusion, our findings provide fundamental knowledge for future durian molecular studies that will allow comparison of the candidate gene expression and their related metabolites among different durian cultivars, thereby assisting in developing molecular markers for durian breeding in the future.