The ripening process may be completed to varying extents in mature soft and dry types of dates causing major variation in fruit composition
In this study, two collections of date fruits were measured with metabolomics techniques and multivariate statistical analysis applied to both extract and characterize the principal components that explain most variability in their metabolomics data. The two date collections present fundamental differences: Dates from the first sample collection originated mostly from the Gulf region and the subset that was obtained from shops is likely to have been through a grading and drying process to meet market criteria. In contrast, dates from the second collection were mostly North African varieties collected fresh from the trees and local markets. Despite these differences, PC1 separately derived for each dataset was found in both cases significantly associated with the fruit country of production with dates from the East Gulf region and the West North African side being observed, in broad terms, at opposite ends of the PC1 scale. Another factor that proved to be strongly associated with PC1 is the extent of metabolic conversion during the ripening process. The moisture analysis indicated that this is partly explained by the incidental occurrence of immature moist fruits which possibly had not yet completed their ripening. This was rather anticipated and was the reason for the inclusion of a development stage dataset as part of the second cohort. However, low moisture dates which should not incur any further changes in their phenotype and are by definition mature, were found to span the entire range of PC1 displaying varying extents of ripening metabolic turnover. In other words, some date samples reach maturity after a rigorous ripening activity (negative range of PC1) whilst others become mature and dry out having carried out a lower extent of metabolic turnover (positive range of PC1). Further analysis suggested that the former dates are generally from the soft type whilst the latter are mostly from the dry type. The soft/dry phenotype is variety specific and was, in this study, collected from the literature. It is important to note that the phenotype in question does not refer to a development or maturity stage per-se but to a collection of physiochemical properties that distinguish the fresh naturally ripened fruit from the two types: Dates from the soft class are characterized by higher moisture, softer texture and higher levels of sucrose to reducing sugars in the ripe fruit (refer to background for more details). Due to their high moisture level, dates from the soft class often necessitate additional drying to become mature unlike the dry type which matures naturally on the trees.
Importantly, the observed variation in metabolic turnover in dates from the soft and dry types can explain their known phenotypic differences. Low metabolic turnover in the dry type may limit the synthesis of color substances and efficient degradation of fibrous structures. This could explain fruit discoloration and the hard texture typical to this type of date . On the other hand the high metabolic turnover with the soft type is likely to be accompanied by optimal degradation of fibers and accumulation of color molecules, which justifies the soft texture and intensified color typical to this type of dates. Also, the high sucrose versus high reducing sugar levels in dry versus soft types of dates can be attributed to the development effect since early accumulating sucrose is readily broken down into reducing sugars as ripening progresses. Interestingly, hydrolysis of sucrose by invertase during ripening was found to display faster kinetics in soft than in the dry type of dates . Slower degradation of sucrose could be a limiting factor for the ripening process in dry varieties as it would impact a significant proportion of downstream ripening reactions. Whether the activity of invertase is alone causative of the soft/dry classification of dates remains to be investigated. Also, underlying factors whether of genetic nature or simply consisting of low water activity or both need to be addressed; as although evidence in the literature suggests genetic diversity between the soft and dry types , a direct link to the fruit ripening process remains to be established. One venue for investigating the role of water activity is via experimental modification of water content in developing soft and dry dates through altering irrigation amount and frequency [36, 37]. This combined with global metabolome characterization of a larger cohort of dry and soft dates may provide important clues on the relevance of water activity to the soft and dry phenotypes in dates. In summary, the ripening effect captured in PC1 in this study is not exclusive to fresh immature dates with ongoing ripening activity but also mature dates from the dry/soft classes that display varying extent of metabolic turnover during ripening.
Importantly, the enrichment of dry and soft type of dates at opposite ends of PC1 provides an explanation for observed association between PC1 scores and geography. In the Arab world, different types of date palm cultivation areas with varying climates tend to be more suitable for either type of dates: Oasis sites typical to North African countries including Tunisia, Morocco, Algeria, Libya and Egypt are famous for the semi-dry and dry types of dates whilst offshore dry areas found in Egypt, Sudan, Libya, Saudi Arabia and Oman are mainly suitable for dry varieties. Finally, the humid nature of coastal areas typical to Bahrain, United Arab Emirates and Qatar are more suitable for soft varieties of dates [15, 16]. Importantly, the established genetic variation in dates between the North African and Arabian Gulf regions  could be linked to varying climatic conditions imparting a bias in the type of cultivar between the two regions.
In comparison to PC2, 3 and 4, PC1 captures a higher proportion of the variance in the data. Also, in this work, unlike PC2, 3 and 4, PC1 is significantly associated with available phenotypic characteristics of the dates including the country of production, soft/dry type and color intensity. This justifies its being at the focus of this study. Nevertheless, the metabolic signatures of PC2, 3 and 4 will be later discussed in some details.
Multivariate techniques are useful exploratory and integrative tools of single and multi-measured metabolomics datasets
In this study, a range of multivariate techniques were used to reach a comprehensive understanding of determinants of metabolic variation in date samples. Initially, non-supervised PCA was used to extract this variation. In order to assess the relationship with the ripening process, an OPLS-DA classifier was trained on the DS2-immature dataset to model the ripening process in dates. However, a prerequisite for the OPLS-DA model is class segregation of samples, which was clearly missing with this dataset. This is because the fruits in DS2-immature were not collected at pre-set time intervals during the ripening process and therefore could not be aligned across samples to create the required classes. Instead, a PCA analysis revealed a dominant PC1 that essentially captured the ripening process in DS2-immature and organized the constituent fruits accordingly into three broad clusters. Clusters 2 and 3 served as the training set for the OPLS-DA model leaving out cluster 1. This is rationalized by the anticipation that the prediction set, consisting of DS2-mature, would lay between clusters 2 and 3 as cluster 1 featured green dates from the early phase of ripening that was not represented in the prediction set. Mapping the development effect onto DS1-bolon from the first date collection was important for the sake of replicating the association between PC1 and the biochemistry of fruit ripening in a yet independent dataset. The model used was based on the O2PLS-DA procedure which is able to extract systematic variation from batch 1 and 2 measurements of the training set that consistently differentiate the designated sample classes. It follows that the O2PLS-DA procedure was used in this study to consolidate separate batch measurements of the same samples as although the measuring technique was essentially the same, slight operational changes may have been introduced between the two batch measurements which were well separated in time. This is, in principal, similar to the way the technique has been traditionally applied to bring together measurements of the same biological samples by different analytical methods .
Comprehensive characterization of temporal aspects of ripening metabolism in dates
At the metabolic level, PC1 has a ripening signature and is the reason why it is able to differentiate between the dry and soft phenotypes that undergo varying ripening kinetics. Soft and dry types of dates have different climatic requirements which could explain the association between PC1 and geography. We now focus on the metabolic signature of PC1 and dedicate the remaining part of the discussion section to contrasting observed enrichment in classes of metabolites along PC1 with the known biochemistry of fruit ripening (though, we will occasionally refer to other PCs when discussing metabolic classes that are relevant to them). We would implicitly refer to the positive and negative ends of PC1 by their corresponding ripening profiles as early ripening and late ripening based on the results on Fig. 5. We will frequently refer to Additional file 6 which shows scatter plots of all metabolite abundance profiles along PC1 organized within their respective biological classes.
Amino acids and related metabolites
Enrichment in free amino acids was observed in this study in dates with an early ripening profile similar to other fruit [8, 39] (Fig. 5). Amongst all detected amino acids, the levels of alanine, glutamate and aspartate declined least in dates with late ripening profile (Additional file 6), consistent with previous work looking at ripening in tomato . In general, amino acids serve as building blocks for synthesis of key intermediates and end-products of the ripening process in fruits . In particular the aromatic amino acids, also measured in this study, give rise to a myriad of secondary metabolites, notably color and flavor-conferring phenylpropanoids. The observed enrichment in dipeptides in date fruits with an early ripening profile in this study (Fig. 5) may be linked to protein degradation activity recruited by hormones to eliminate pre-ripening enzymes at the onset of ripening . Another potential source for the dipeptides is the targeting peptide sequence, attached to pre-folded nuclear proteins, which is digested upon protein entry into organelle structures including chromoplast . Chromoplasts, which are differentiated forms of chloroplasts lacking chlorophyll, act as metabolic hubbs at early ripening, necessicating constant in-flow of effector proteins from the nucleus . Targeting peptide sequences contain mostly hydrophobic amino acid residues , consistent with the high proportion (70 %) of hydrophobic valine, leucine, isoleucine and phenylalanine amongst amino acid constituents of the dipeptides observed in this study. Interestingly, N-acetylation of chromoplast-targeted pre-folded proteins was suggested as a mechanism of organelle specificity . This may account for the enrichment of N-acetylated amino acids in date samples with an early ripening profile in this study (Fig. 5); although acetylation can sometimes be a necessary intermediate reaction during metabolism of amino acids. Finally, enrichment in glutathione activity in dates with an early ripening profile (Fig. 5) is consistent with increased antioxidant activity in fruits at early ripening .
Primary amines and polyamines
In this study, ethanolamine, GABA, serotonin, tyramine, tryptamine and phenethylamine from the decarboxylation of serine, glutamate, 5-hydroxy tryptamine, tyrosine, tryptophan and phenylalanine were all found enriched in dates with early ripening profiles (Additional file 6). This is consistent with early ripening expression of amino acid decarboxylases leading to amine synthesis in a number of fruits [44, 45]. Tyramine and tryptamine serve as precursors for the synthesis of defense-mediating alkaloids previously detected in dates  and a turnover of phenethylamine into antipathogen volatiles phenylacetaldehyde and phenylethanol serves the same purpose [8, 45]. The role of serotonin in fruit ripening has not been fully investigated; however, melatonin that derives from N-acetylserotonin (a derivative of serotonin also observed in our data), has recently been found to promote various physiological aspects of ripening when given exogenously to green tomato . Recently, the decrease in GABA with ripening was linked to maintaining high levels of essential glutamate and aspartate during tomato ripening . Enrichment in the polyamine putrescine in dates with an early ripening profile (at the positive end of PC1) is consistent with previously reported expression of a mouse ornithine decarboxylase conjugated to a ripening specific promotor at the onset of ripening in transgenic tomato . Previous work suggested a synergy between the ripening hormone ethylene and the polyamines spermine and spermidine, derivatives of putrescine [39, 50]. The potential regulatory role of putrescine may justify its co-occurrence with products from its degradation pathway in dates with an early ripening profile (Additional file 6).
The earliest sign of secondary metabolites from the phenylpropanoid pathway in our data consisted of tannins procyanidin B1 and procyanidin B2 and catechin monomers all showing maximal level in dates with an early ripening profile (Additional file 6), in accordance with the literature [8, 51]. Astringent tannin oligomers are abundant in green fruit and only lose their astringency when undergoing structural changes as ripening progresses . In addition to tannins, a wide range of color and flavor flavonoids and hydroxycinnamates were observed to peak at different ranges of PC1 and some showed no correlation with PC1. In general, variance from these metabolites was poorly explained by PC1 (Additional file 6). This could be due to a much stronger influence by genetic background , which may contribute to unique taste and color characteristics of individual varieties. Interestingly, PC4 from DS2-mature revealed an opposing trend between classes phenylpropanoids and TCA on one hand and the accumulation of phosphorylated sugars, captured under the class glycolysis, on the other hand. This effect can be explained by energy requirement for the synthesis of phenylpropanoids through initial degradation of phosphorylated sugars during glycolysis and downstream TCA activity. A third class of secondary metabolites consisted of volatiles, major contributors to aroma in fruits. In this study, an increase in branched chain amino acid derived volatiles and hydroxycinnamate derived volatiles was observed in dates with a late ripening profile (Fig. 5 and Additional file 6), consistent with the literature . Volatiles are strong attractants of seed dispersers and their sharp increase in overripe fruit could constitute a mechanism to maximize the chance of consumption before onset of senescence.
Changes to cell wall and cell membrane
Alterations in cell membrane composition have long been known to occur during ripening [53–55], but have been given little attention by the more recent literature. Key changes to membrane phospholipids during ripening include an increased desaturation level of fatty acyl chains facilitating their peroxidation. Induction of expression of a handful of desaturase isomers was found to be strongly associated with a continuous flux of linoleate and linolenate substrates of the lipoxygenase (LOX) pathway during peach ripening . This pathway is known for being the mechanism of synthesis of a myriad of C6 volatile aldehydes and alcohols that contribute significantly to fruit aroma . In this study, a range of mono and poly-unsaturated fatty acids including the LOX substrates and their oxidized derivative oxylipins were observed to generally plummet in dates with a late ripening profile at the negative range of PC1.
Initial excision of fatty acyl chains resulted in accumulation of lysophospholipids in dates with an early ripening profile and late accumulation of lysophospholipid degradation products in dates with a late ripening profile (Fig. 5). These included monoacylglycerols and phosphorylated head groups, free head groups and remaining lysophosphatidic acids  as well N-acylethanolamines, derivatives of lysophospholipids via N-acylphosphatidylethanolamine intermediates  (Additional file 6). These degradation products, in addition to sphingoid bases, are likely to have a signaling role in fruits and some are already known to be downstream mediators of abscisate signaling in other plant organs [58–60]. The opposing trend in lysophospholipids versus sphingoids by PC2 in both datasets is interesting and may suggest a change in signaling patterns during ripening of certain date varieties or as a response to certain external stimuli. For instance, sphingolipid signaling is known to be induced under drought conditions in plants .
Fruit softening during ripening is known to be associated with an increased activity of fiber degrading enzymes, many of which targeting cell wall structures . Pectin is a major constituent of the plant cell wall and its hydrolysis seems to make way for synergistic disassembly of cellulose and hemicellulose polysaccharide matrices . In this study, the abundance of keto-deoxyoctulosonic acid, an acidic monosaccharide located in the side chain of the rhamnogalacturonan II class of pectin peaked in dates with middle range PC1 (Fig. 6b). This served to establish a link to the Rutab phase which is characterized by maximal fruit softness (refer to results).
Sugar metabolism, energy and gene expression activity
In this study, sucrose and related non-reducing sugars kestose and melezitose (from DS1-bolon) were most abundant in fruits with an early ripening profile at the positive range of PC1 (Fig. 5, Additional file 6). In fruits, an increase in sucrose levels is observed at the late mature green stage and sucrose is broken down by the enzyme invertase following the onset of ripening . Metabolism of released sugar monomers proceeds via the early pentose phosphate pathway, which serves to provide carbon precursor molecules for the synthesis of aromatic amino acids, secondary metabolites, vitamins and purine/pyrimidines. In parallel, flux through glycolysis and the downstream TCA cycle serves to sustain energy levels required for gene expression and anabolic reactions during ripening. The relationship between precursor non-reducing sugars and product TCA intermediates is captured by PC3 from DS1-bolon and may reflect varying glycolysis/TCA kinetics across the date cohort.
In this study, the abundance levels of energy molecules NAD+ and AMP were lowest in dates with a late ripening profile (Fig. 5, Additional file 6). This together with accumulation of the TCA cycle intermediate fumarate, members of the glycolysis pathway and ribosomal nucleosides possibly originating from ribosome degradation (Additional file 6) may indicate a diminished metabolic activity at this late stage of ripening. Interestingly, metabolites from the TCA/rRNA nucleosides classes appeared to contrast the unsaturated fatty acids and oxylipins in PC4/PC3 from DS1-bolon/DS2-mature respectively. Late synthesis of oxylipins in some species of dates may require residual TCA activity and late synthesis of key enzymes.
The accumulation of reducing sugars in dates with a late ripening profile in this study (Fig. 5) confirmed similar reports in dates at the late Tamr stage . Amongst the sugars observed (also shown in Additional file 6) xylose, fucose, arabinose and glucose may derive from cell wall hydrolysis activity during ripening. Ribulose and xylulose could derive from their phosphorylated forms from the pentose phosphate pathway whilst sugar alcohols, sugar lactones and derivative acids may result from upregulation of aldose/ketose oxidoreductases in ripe dates, previously shown using a proteomics approach . It is important to note that besides contributing to fruit flavor, sugar alcohols are a type of polyol osmoprotectant that may act to alleviate the impact of fruit dryness at this stage.
Vitamins and hormones
In this study, a range of vitamins has been detected including riboflavin, niacin, pyridoxine and nicotinate (Additional file 6). The enrichment in pyridoxine in dates with an early ripening profile may be attributed to its essential role in amino acid synthesis and metabolism by amino acid decarboxylases at the early phase of ripening. In contrast, the accumulation of threonate (Additional file 6), a degradation product of vitamin C, in dates with a late ripening profile is consistent with the previously described decrease in vitamin C at the Tamr stage .
Dates behave like climacteric fruits implying a leading regulatory role by ethylene although interplay with abscisate may be operating on certain ripening events . In this study, the ripening hormone ethylene was not measured (below the mass cut-off imposed on the instrumentation) but two related metabolites were observed (Additional file 6). One is cyanoalanine, which is a conjugate of cysteine and cyanide (cyanide being a toxic byproduct of ethylene synthesis ) and 5-methylthioadenosine, an intermediate in the Yang cycle that replenishes the ethylene precursor SAM. Both molecules showed maximum levels in dates with an early ripening profile at the positive range of PC1 (Additional file 6). This range was previously mapped to the Khalal stage (refer to results), which follows the climacteric ethylene peak in dates . A similar pattern by isopentenyl adenosine, the precursor of the cytokinin hormone zeatin, is concordant with the reported abundance profile of zeatin during tomato ripening . Abscisate is an early regulator that precedes ethylene in the climacteric fruit model tomato [65, 66]. Following the peak in ethylene, abscisate was shown to increase in abundance slightly , which could explain the small peak in abscisate observed in this study in dates with middle range PC1 values. In summary, key hormones and related metabolites were measured as part of this work motivating future analysis of partial correlations with other measured metabolites in order to uncover regulatory mechanisms operating on distinct aspects of the ripening process in date fruit.