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Metabolite profiling of mangosteen seed germination highlights metabolic changes related to carbon utilization and seed protection
Scientia Horticulturae 243 (2019) 226–234
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Metabolite profiling of mangosteen seed germination highlights metabolic changes related to carbon utilization and seed protection
T
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Othman Mazlan, Wan Mohd Aizat , Nor Shahida Aziz Zuddin, Syarul Nataqain Baharum, ⁎ Normah Mohd Noor Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia (UKM), 43600 Bangi, Selangor, Malaysia
A R T I C LE I N FO
A B S T R A C T
Keywords: Garcinia mangostana L. LC–MS GC–MS Metabolomics Recalcitrant seed Germination
Mangosteen seed is categorized as recalcitrant, becoming inviable if exposed to desiccation and low temperature. However, the molecular mechanism of mangosteen seed germination has not been fully understood. This study profiled the metabolites that were present in germinating mangosteen seeds at different stages (zero, one, three, five, seven and nine days after sowing) using liquid chromatography-mass spectrometry (LC–MS) and gas chromatography-mass spectrometry (GC–MS). A total of 38 tentative metabolites were profiled and classified into amino acid, organic acid, sugar, polyol, alkaloid and others. Metabolites identified implied a timed regulation for germination. Sugars as well as amino acids exhibited a declining trend throughout the germination period as they are likely to be utilized for development and growth of seedling. Furthermore, secondary metabolites such as alkaloids, flavonoids, and xanthone presence displayed an increasing trend at the middle of germination period which may provide protection for mangosteen seed against herbivory and stress. In brief, carbon utilization and seed protection associated with the modulation of primary and secondary metabolites, respectively are important metabolic signatures of mangosteen seed germination.
1. Introduction Mangosteen (Garcinia mangostana L.), a tropical fruit widely known for its sweet unique taste can be found in Southeast Asian countries such as Malaysia, Philippines and Thailand (Lim, 1984; Palapol et al., 2009). Additionally, mangosteen is well known in traditional medicine as some part of it, either from trees or fruit, has been used to treat wounds, diarrhea, dysentery, skin infections and more (Lu et al., 1998; Nakatani et al., 2002; Pedraza-Chaverri et al., 2008; Obolskiy et al., 2009). Mangosteen extracts have also been shown to possess various medicinal properties such as antifungal (Obolskiy et al., 2009), antioxidant (Yoshikawa et al., 1994), antibacterial (Iikubo et al., 2002), anti-HIV (Chen et al., 1996; Vlietinck et al., 1998) and anticancer agents (Matsumoto et al., 2003; Moongkarndi et al., 2004; Han et al., 2009; Shan et al., 2011). These health benefits are mainly contributed by xanthones, a class of polyphenolic compounds present in only a few plant species including mangosteen (Jinsart et al., 1992; Jung et al., 2006; Obolskiy et al., 2009). The two major xanthones that can be found in mangosteen include α-mangostin and γ-mangostin (Obolskiy et al., 2009). Despite its medicinal potential, mangosteen tree growth is slow and
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only matures in about five to seven years before it starts bearing fruit (Osman and Milan, 2006). Mangosteen is mainly propagated through seeds which are categorized as recalcitrant. These seeds are sensitive to desiccation and low temperature (Normah et al., 1997) as they maintain active metabolism when shed (Berjak and Pammenter, 2013). Also, at later stages of development, mangosteen seed has an elevated level of soluble sugars and secondary metabolites (Mazlan et al., 2018). The seed will not be able to germinate if its moisture content drops to 30% or lower (Normah et al., 1997, 2016). It can only be stored for a short period before becoming inviable for germination (Normah et al., 1997), making it difficult for a long-term seed preservation and maintaining planting materials throughout the year. Mangosteen seed shows Garcinia-type germination where radicle protrusion is followed by plumule emergence on the opposite side (Lim, 1984; Normah et al., 2016). Studies on mangosteen seed are still limited particularly during the seed germination. Seed germination is dependent upon multitude of endogenous and exogenous factors such as temperature and pH level (Kulkarni et al., 2014) and is strongly supported by various types of reserve compounds synthesized and stored during seed development such as carbohydrates and proteins (Pritchard et al., 2002; Galili et al., 2014). Germination are mainly divided into three phases – imbibition
Corresponding authors. E-mail addresses: [email protected] (W.M. Aizat), [email protected] (N.M. Noor).
https://doi.org/10.1016/j.scienta.2018.08.022 Received 17 April 2018; Received in revised form 10 August 2018; Accepted 11 August 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
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before 0.1 min increase to 100% B. At 100% B, the mixture was held for 4 min prior drop to 5% B in 2 min. Finally, column reconditioning was performed for 7 min with the initial gradient. Mass spectrometry analysis was using a MicroTOF-QIII (Bruker, Germany) system with electrospray ionization (ESI) source in a positive mode of ionization. The drying gas (nitrogen) was set at 45 psi with flow at 8 L min−1 and gas temperature of 200 °C. The voltage of ESI spray and the fragmentor were fixed at 4.5 kV and 200 V respectively. Range of the scan was between 50 and 1000 Da. Subsequently, the data were logged in a centroid mode.
(Phase 1), lag interval (Phase 2) and radicle protrusion (Phase 3) (Nonogaki et al., 2010; Rajjou et al., 2012). Rapid water uptake by seeds occurs during Phase 1 followed by Phase 2 which is characterised by an active seed metabolism. Lastly, seeds germinate at Phase 3 with radicle protrusion. Seed germination has been investigated in many plant species such as in Arabidopsis (Rajjou et al., 2006), barley (Yang et al., 2011), tea (Chen et al., 2011), maize (Huang et al., 2012), rice (Kim et al., 2008) and soybean (Cheng et al., 2010). For instance, it was revealed that key compounds such as sugars and amino acids were temporally regulated during Arabidopsis seed germination (Fait et al., 2006). Still, investigation on recalcitrant seeds such as mangosteen are relatively scarce and hence demands further analysis. Previous studies by Normah et al. (2016) and Lim (1984) have highlighted the physical properties and morphology of germinating mangosteen seed in effort to understand its biology. However, metabolic changes during the germination of this recalcitrant seed have not been reported. Hence, the aim of this study is to analyze mangosteen seed germination using metabolomics approach via liquid chromatography-mass spectrometry (LC–MS) and gas chromatography-mass spectrometry (GC–MS). Information gained would provide better comprehension on the metabolism of recalcitrant mangosteen seed germination.
2.4. LC–MS metabolite identification The raw data for LC–MS were first analyzed using Compass Data Analysis (Bruker, Germany) to detect peaks and deconvolute the total ion current chromatogram. This was to generate the list of peaks for retention time to mass per charge ratio (Rt-m/z) that corresponds to putative compounds detected and intensity data sets. The data were then binned and tabulated. MetaboAnalyst (http://www. metaboanalyst.ca/) for data normalization (by sum) and transformation, scaling and statistical analysis. Rt-m/z values that were detected in all three biological replicates, in at least three stages of germination were selected for putative identification. The m/z values were compared with three online metabolite databases namely Massbank (http:// www.massbank.jp), Metlin (https://metlin.scripps.edu) and Metfrag (http://msbi.ipb-halle.de/MetFrag/). For each m/z values, top three compounds that are found in plants were selected and narrowed down to one, based on thorough literature review. Calculation of relative intensity (in percentage, %) was done by using the Formula (1). Finalized identified metabolites were then tabulated and graphed.
2. Materials and methods 2.1. Seed sample preparation Mature mangosteen fruit were obtained from trees at mangosteen plots in Universiti Kebangsaan Malaysia (UKM), Selangor, Malaysia (GPS coordinate: 2.922662 °E, 101.786690 °N). Two batches of sample were made; one for metabolomics analysis and the other for representative pictures. Aril was removed from seeds to avoid fungal infection during germination. Clean seeds were planted in autoclaved sand and were grown in a greenhouse (24–26 °C) with daily watering. Mangosteen seed takes nine days of sowing before both radicle and plumule emerge (Osman and Milan, 2006; Normah et al., 2016). Hence, samples were taken every two days at one (D1), three (D3), five (D5), seven (D7) and nine (D9) day(s) after sowing with day zero (D0) as control. At each timepoint, three germinating seeds, acted as biological replicates, were cleaned, blotted dry and flash frozen in liquid nitrogen prior storage at −80 °C.
Relative intensity, reI =
Compound peak intensity × 100 Total intensity in all metabolite across all stages
(1) 2.5. Gas chromatography-mass spectrometry (GC–MS) protocol The dried supernatant was dissolved and derivatized in 80 μL of methoxyamine hydrochloride (20 mg mL−1) in pyridine at 30 °C for 90 min. Then, it was incubated for 30 min with temperature of 37 °C after the addition of 80 μL of N-Methyl-N-(trimethylsilyl)-trifluoracetamide (MSTFA). Both chemicals were obtained from SigmaAldrich (St. Louis, MO, USA). GC–MS and its analysis was done as per Chen et al. (2014) with revision. For each replicate, 1.0 μL of derivatized sample was injected into GC–MS with a split ratio of 25:1. The GC–MS system consisted of a GC Agilent 5975 gas chromatograph (Agilent, China), an autosampler and a single quadruple mass spectrometer (Agilent, USA). Using tris-(perfluorobutyl)-amine (CF43), adjustment to the mass spectrometer was made according to recommendations by the manufacturer. The AB-5MS 30 m column used has 0.25 mm for both inner diameter and film thickness (Abel Industries, Canada). The parameters were set for injection temperature (230 °C), interface (250 °C) and ion source (200 °C). Helium, the carrier gas used was set to flow at the rate of 1 mL min−1. The temperature setup started with isothermal heating (70 °C, 5 min) followed by a 5 °C min−1 rising of oven temperature (up to 310 °C) and a final heating (310 °C, 1 min).
2.2. Metabolite extraction Seeds were ground to fine powder and 0.1 g from each replicate was homogenized in 1.4 mL of methanol (Merck, Germany) and ribitol (50 μL, 2 mg mL−1) as an internal standard. For 15 min at 70 °C, the mixtures were incubated for extraction. Extracts obtained were added with one volume of water, mixed briskly and subsequently centrifuged at 2200×g. Supernatant acquired was vacuum-dried for four to six hours to eliminate the extraction solvent and stored in −80 °C until used. 2.3. Liquid chromatography-mass spectrometry (LC–MS) protocol Methanol were used to resolubilised dried samples. This study follows Glauser et al. (2013) method with modifications (Mamat et al., 2018; Mazlan et al., 2018). In extract compound separation, liquid chromatography system (Ultimate 3000 UHPLC+, Thermo Scientific, USA) equipped with a C18 column was utilized. LC parameters were set for injection volume (1.0 μL), column temperature (60 °C) and flow rate (0.3 mL min−1). The mobile phases consisted of 0.1% formic acid in water (mobile phase A) and acetonitrile (mobile phase B). Elution was performed with a 35-min gradient starting with increase from 0 to 5% B in the first 2 min, then further to 40% in the following 2 min and up to 95% B within the next 16 min. At 95% B, the mixture was held for 2 min
2.6. GC–MS metabolite identification Metabolites were identified using NIST11 library. Then, metabolites with match score of below 700 were discarded. Compounds that were found to be redundant in the data were further trimmed. The metabolites that were detected in all three replicates for at least three stages were selected as the final list of metabolites. The data were imported into MetaboAnalyst where they were subjected to normalization using 227
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3.2. LC–MS analysis
ribitol, transformation, scaling and statistical analysis. These compounds were classified into amino acid, organic acid, sugar, polyol and glycoside. Then, semi quantitative concentration of compounds was calculated using the formula below based on the known concentration of internal standard (ribitol, 2 mg mL−1) as outlined in Roessner et al. (2000) and Roessner et al. (2006). Semi quantitative concentration Area percent of a replicate = × 2 mg mL-1 Average area percent of ribitol in a particular stage
From the LC–MS analysis, 68 Rt-m/z values were obtained and subjected for MVA. Both PCA and PLS-DA show no distinct separation (Supplementary Fig. 1a and b). Further analysis using OPLS-DA shows distinction between D0 and other germination stages with a total variance of 27% (Fig. 2a). The loading plot shows Rt-m/z pairs responsible for the separation (Fig. 2b), and 21 Rt-m/z pairs has Variable Importance in Projection (VIP) scores of more than 1 (Supplementary Table 1). Seventeen identified compounds were classified into alkaloid, glycoside, flavonoid, organic acid, xanthone-related compound, amino acid and ‘other’ (Fig. 3). Alkaloids (Fig. 3a) include alpinine, anopterine, 1,2-dimethoxy-13-methyl-[1,3]benzodioxolo[5,6-c]phenanthridine (DMMBP) and indoleacrylic acid. Meanwhile, cyanidin 3-O-(6O-p-coumaroyl)glucoside (cyanidin coumaroylglucoside), cyclo-Dopaglucuronylglucoside, isopropyl apiosylglucoside and lusitanicoside are in the glycoside group (Fig. 3b). Furthermore, 4-(2,4-dihydroxyphenyl)8-[(Z)-2-(2,4-dihydroxyphenyl)ethenyl]-10,18-dihydroxy-14-methyl3,5,15-trioxahexacyclo[12.7.1.0²,⁴.0²,¹².0⁶,¹¹.0¹⁶,²¹]docosa6,8,10,16(21),17,19-hexaen-13-one (kuwanon Z) and epicathechin(4β→8)-epicathechin (procyanidin B2) are flavonoids (Fig. 3c) while chlorogenic acid and pantothenic acid are organic acids (Fig. 3d). Xanthone-related compounds (Fig. 3e) are 2,4,6-triHB and garcimangosone D. Amino acid group (Fig. 3f) consists of phenylalanine while terpenoid (Fig. 3g) is (all-E)-6′-Apo-y-caroten-6′-al (psi-carotenal). Lastly, methyl phenyl disulfide is grouped as ‘other’ (Fig. 3h).
(2)
2.7. Statistical analysis MetaboAnalyst was used for data normalization, log transformation and pareto scaling as well as ANOVA (p ≤ 0.05) for statistical analysis and heatmap generation. Heatmap generated was added to simple metabolic pathways made by referring to literatures (Aizat et al., 2014). Multivariate analysis performed was principal component analysis (PCA), partial least squares to latent structure-discriminant analysis (PLS-DA) and orthogonal partial least squares to latent structure-discriminant analysis (OPLS-DA) using SIMCA-P 14.1 (Umetrics, Sweden). Tabulation and graph were made using Microsoft Excel 2016 and GraphPad Prism 7 (GraphPad Software). Graphs display mean for each stage with standard error of the mean (SEM). 3. Results
3.3. GC–MS analysis 3.1. Mangosteen seeds physical appearance A total of 21 compounds were identified using NIST library of GC–MS. PCA and PLS-DA score plot do not show distinction between sample groups (Supplementary Fig. 1c and d). However, a clear separation between the sample groups and metabolites was observed particularly between D0 and the rest of seed germination stages via OPLS-DA (total variance of 35.2%) (Fig. 2c) with compounds that explain the separation seen in the loading plot (Fig. 2d). There are eight compounds with VIP > 1 (Supplementary Table 2). The metabolites
Mangosteen seed at D0 (Fig. 1) contains undifferentiated embryo with a procambium ring which extends outward at one side at D1. Eventually, the procambium ring grows towards the seed coat and at D3 a small protuberance (radicle) occurs on the surface of the seed. At D5 the seed grew a second protuberance (plumule) at the opposite end of the seed. The radicle and plumule then grew up to 1–2 mm at D7 and 2–4 mm by D9.
Fig. 1. Mangosteen seeds at six different germination stages: zero (D0), one (D1), three (D3), five (D5), seven (D7) and nine (D9) days after sowing. The arrows indicate procambium ring that eventually grows towards radicle (r) protrusion and plumule (p) emergence. All seeds were obtained from ripe mangosteen prior sowing. Scale bar, 1 cm.
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Fig. 2. Orthogonal partial least squares to latent structure-discriminant analysis (OPLS-DA) of Rt-m/z pairs and metabolites identified by liquid chromatographymass spectrometry (LC–MS) and gas chromatography-mass spectrometry (GC–MS) during mangosteen seed germination at zero (D0), one (D1), three (D3), five (D5), seven (D7) and nine (D9) day(s) after sowing. Circles are labelled with respect to the germination stages. Bigger darker diamond dots correspond to Rt-m/z pairs and metabolites with Variable Importance of Projection (VIP) larger than 1. Compounds chosen were found in all replicates for at least three seed germination stages. LC–MS score plot (a) and loading plot (b) of the OPLS-DA (27% total variance). GC–MS score plot (c) and loading plot (d) of the OPLS-DA (35.2% total variance).
can be classified into five groups (Fig. 4). Firstly, the amino acids which comprised of alanine, aspartate, glutamate, glycine, isoleucine, serine, threonine and valine (Fig. 4a). Secondly, the organic acids; citrate, fumarate, glycerate, malate, mucic acid and succinate (Fig. 4b). There were also sugars (fructose, galactose and sucrose) (Fig. 4c) as well as polyols (mannitol, myo-inositol and scyllo-inositol) (Fig. 4d). Lastly, glyceryl-glycoside is classified as glycoside (Fig. 4e).
instance, anopterine (Fig. 3a), kuwanon Z (Fig. 3c), 2,4,6-triHB (Fig. 3e), methyl phenyl disulfide (Fig. 3h) and glycerate (Fig. 4b) levels were increasing during germination. DMMBP, lusitanicoside, garcimangosone D and pantothenic acid (Fig. 3) as well as glyceryl-glycoside (Fig. 4e) levels were also increased particularly at the early germination and radicle protrusion phase (D0–D3) but decrease at later stages. Other metabolites fluctuated with increase levels at the middle timepoints of the germination stages, for examples alpinine and cyclo-Dopaglucuronylglucoside (D5), isopropyl apiosylglucoside (D1, D5), chlorogenic acid (D1, D5–D7) as well as psi-carotenal (D1, D7) (Fig. 3). The third major trend is indicated by indoleacrylic acid, cyanidin coumaroylglucoside and procyanidin B2 (Fig. 3), as well as mannitol and scyllo-inositol (Fig. 4) where their levels were mostly constant throughout germination. Briefly, the germination of mangosteen seed involved modulation of both primary and secondary metabolites.
3.4. Metabolite trends Metabolite levels throughout the stages showed three major trends of regulation. First is the general decrease of most primary metabolites during the germination period, either consistently dropping or increasing slightly towards the end of the process (Fig. 4, Table 1). For examples, phenylalanine (Fig. 3f), alanine, glutamate, threonine, valine (Fig. 4a) and myo-inositol (Fig. 4d) levels were decreasing during early germination stages (D0–D3) and remained low throughout the stages. Similar downward trend but rising slightly at a later germination stage was also observed for other amino acids (e.g. aspartate, glycine, isoleucine, serine) (Fig. 4a), organic acids (e.g. citrate, fumarate, malate, mucic acid, succinate) (Fig. 4b) and sugars (e.g., fructose, galactose) (Fig. 4c). The second major trend illustrates an increasing pattern during mangosteen germination, mainly by secondary metabolites. For
3.5. Metabolic pathways Fig. 5 illustrates major metabolic pathways associated with mangosteen seed germination. Metabolites putatively identified in this study are positioned within relevant pathways and their levels are represented with heatmaps. Soluble sugars such as fructose, galactose and sucrose were mainly fed into glycolysis to be used for subsequent pathways such as TCA cycle and amino acid biosynthesis. Moreover,
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Fig. 3. Tentative metabolites identified by liquid chromatography-mass spectrometry (LC–MS) presented in relative intensity (in percentage, %) across all stages of mangosteen seed germination at zero (D0), one (D1), three (D3), five (D5), seven (D7) and nine (D9) day(s) after sowing. The metabolites are categorized into alkaloid (a), glycoside (b), flavonoid (c), organic acid (d), xanthone-related compound (e), amino acid (f), terpenoid (g) and ‘other’ (h). Each stage was comprised of three biological replicates ± standard error of the mean. DMMBP, 1,2-Dimethoxy-13-methyl-[1,3]benzodioxolo[5,6-c]phenanthridine; cyanidin coumaroylglucoside, cyanidin 3-O-(6-O-p-coumaroyl)glucoside; kuwanon Z, 4-(2,4-dihydroxyphenyl)-8-[(Z)-2-(2,4-dihydroxyphenyl)ethenyl]-10,18-dihydroxy-14-methyl-3,5,15trioxahexacyclo[12.7.1.0²,⁴.0²,¹².0⁶,¹¹.0¹⁶,²¹]docosa-6,8,10,16(21),17,19-hexaen-13-one; procyanidin B2, epicathechin-(4β→8)-epicathechin; psi-carotenal, (all-E)6′-Apo-y-caroten-6′-al; 2,4,6-triHB, 2,4,6-trihydroxybenzophenone.
The metabolite content of these mangosteen stages was investigated using metabolomics (LC–MS and GC–MS) approaches, which revealed the regulation of both primary and secondary metabolism important for the germination of this recalcitrant seed.
TCA cycle may also consume metabolites such as from fatty acid metabolism as well as amino acids and via malate, succinate, fumarate, and acetyl-CoA conversion. Meanwhile, the secondary metabolic pathway through shikimate splits into two biosynthesis pathways; the anthranilate path for alkaloids and terpenoids, and the prephenate path for flavonoid and xanthones.
4.1. Modulation of sugar, amino acid and TCA cycle intermediates during seed germination
4. Discussion Primary metabolites such as sugars are important sources of carbon in supporting seed germination (Eveland and Jackson, 2011; Sami et al., 2016). During seed development, mangosteen seed continues to accumulate soluble sugars until late maturation (Mazlan et al., 2018). In this study, we found that the level of soluble sugars (fructose, galactose, sucrose) dropped during the initial germination stages (imbibition-lag phase, D0–D3) and remained low until their increase at
In this study, the germination of mangosteen seed was investigated at D0, D1, D3, D5, D7 and D9. The seed radicle started to appear by D3 and hence regarded as the radicle protrusion stage (Fig. 1). The stages prior to the radicle protrusion (D0–D3) are regarded as the imbibitionlag phase and the stages after D3 are considered as the seedling establishment phase (growth of radicle and plumule protrusion, Table 1). 230
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Fig. 4. Putative metabolites detected using gas chromatography-mass spectrometry (GC–MS) and presented as semi quantitative (mg mL−1) across all stages of mangosteen seed germination; zero (D0), one (D1), three (D3), five (D5), seven (D7) and nine (D9) day(s) after sowing. The metabolites are categorized into amino acid (a), organic acid (b), sugar (c), polyol (d) and glycoside (e). Each stage was comprised of three biological replicates ± standard error of the mean.
phase (D0–D1, Fig. 4 and Table 1). Interestingly, the levels of malate, succinate and citrate were increasing when the orthodox seeds of Arabidopsis germinate sensu stricto (Fait et al., 2006) implying the different method for carbon metabolism compared to mangosteen as stated previously. Among the four TCA cycle intermediates identified, citrate level is relatively higher than others by the end of germination (Fig. 4b). It could be that the germinating seeds may employ a noncyclic TCA cycle flux, bypassing malate, fumarate and succinate through an alternative pathway such as via pyruvate-oxaloacetate conversion (Fig. 5) to increase the citrate level for nitrogen assimilation (Krömer et al., 1996; Sweetlove et al., 2010; Eastmond et al., 2015). Moreover, the non-cyclic bypass (Fig. 5) may have affected aspartate level to increase (Fig. 4a). Aspartate might be used to synthesize other amino acids such as phenylalanine, isoleucine and threonine (Galili, 2011; Aizat et al., 2014; de la Torre et al., 2014). Their levels were decreasing throughout the germination stages especially from D0 to D1 (Figs. 3f and 4 a), implying some involvement in biosynthetic pathways or cellular processes predominantly for procambium expansion during imbibition-lag phase at D1. For instance, phenylalanine is a precursor towards secondary metabolic pathways, in particular flavonoid biosynthesis pathway (Tohge et al., 2013), can contribute majorly to their formation and may assist in oxidative stress suppression (Teixeira et al., 2017) and ultraviolet protection (Sullivan et al., 2014) during seedling establishment. Meanwhile, glutamate (from α-ketoglutarate conversion (Fig. 5) was also decreasing, inferring a role in germination initiation particularly for the procambium expansion at D1 (Fig. 1). In Cola acuminate (Onomo et al., 2010) and Punica granatum
later stages by which is similarly observed in germinating recalcitrant Guilfoylia monostylis seed (Nkang, 2002). These soluble sugars are thought to be consumed for a quick source of energy as they are readily available by the end of reserve accumulation phase (Mazlan et al., 2018). The energy generation might have been directed towards seed cellular growth including procambium ring expansion and radicle protrusion at D1–D3 (Fig. 1). In orthodox seeds such as Arabidopsis, fructose, galactose and sucrose were reduced up to the end of its vernalization period (Table 1), limiting their availability towards its germination phase (Fait et al., 2006). The orthodox seed may need to opt for alternative pathways to fuel its germination for example through fermentation and amino acid consumption (Fait et al., 2006; Rosental et al., 2014). While mangosteen seed may have utilized the freely available sugars during early germination for energy supply, their low levels in the following stages of germination, late imbibition-lag phase (D1–D3) as well as radicle and plumule protrusion (D3–D9) (Fig. 4, Table 1) might prompt energy outsourcing to other carbon reserve such as lipid. Storage lipid was found to be a prominent reserve in mangosteen (Normah et al., 2016) as well as other recalcitrant species such as Landolphia kirkii Dyer (Berjak et al., 1992), Guilfoylia monostylis (Nkang, 2002) and Telfairia occidentalis (Nkang et al., 2003), and is probably used for germination metabolism. As soluble sugars were consumed and limited during late imbibition-lag phase (D1–D3) and the rest of mangosteen seed germination, biochemical processes such as glycolysis and TCA cycle might have been affected. Evidently, metabolic intermediates such as malate, citrate, succinate and fumarate decreased during the early imbibition-lag
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Table 1 Metabolite comparison between mangosteen (this study) and Arabidopsis thaliana (Fait et al., 2006) at different stages of development and germination for metabolites found in both species. Timepoints for these studies are as follow: mangosteen seed germination at zero (D0), one (D1), three (D3), five (D5), seven (D7) and nine (D9) day(s) after germination (this study); Arabidopsis at 18, 21 and 22 days after flowering (DAF). Germination in Arabidopsis is germination sensu stricto where it precedes radicle protrusion. Up arrow (↑) represents increasing level, down arrow (↓) represents decreasing level, ‘no change’ (n.c.) represents no change in level from previous stage and ‘negligible’ represents negligible presence or not detected. Metabolite
Alanine Aspartate Glutamate Glycine Isoleucine Phenylalanine Serine Threonine Valine Citrate Fumarate Glycerate Glycerate 3P Malate Succinate Myo-inositol Fructose Fructose 6P Galactose Sucrose
Mangosteen seed germination (this study)
Arabidopsis vernalization and germination (Fait et al., 2006)
D0–D1 D1–D3 Imbibition-lag phase
D3–D5 D5–D7 Radicle and plumule protrusion
D7–D9
↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↑ Negligible ↓ ↓ ↓ ↓ Negligible ↓ ↓
n.c. ↑ n.c. n.c. ↑ ↓ ↑ n.c. n.c. ↑ n.c. ↑ Negligible n.c. n.c. n.c. n.c. Negligible n.c. n.c.
n.c. ↑ n.c. n.c. ↑ n.c. ↑ n.c. n.c. n.c. ↑ ↑ Negligible n.c. ↑ n.c. n.c. Negligible ↑ n.c.
n.c. ↑ n.c. n.c. ↑ n.c. ↑ n.c. ↓ ↑ ↓ ↓ Negligible n.c. n.c. n.c. ↓ Negligible n.c. n.c.
n.c. ↓ n.c. n.c. ↓ ↓ ↑ n.c. n.c. n.c. n.c. ↑ Negligible n.c. n.c. n.c. n.c. Negligible n.c. n.c.
18–21 DAF Vernalization ↑ ↑ ↑ ↓ ↓ ↓ ↑ ↑ ↓ ↓ ↓ ↓ ↑ ↑ ↑ ↓ ↓ ↑ ↓ ↓
21–22 DAF Germination ↑ ↑ ↑ ↓ ↓ ↑ ↑ ↑ n.c. ↑ n.c. ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓
cell metabolism for germination (Araujo et al., 2011). The declining levels of almost all identified amino acids (Fig. 4a) during early imbibition-lag phase suggest that they were exploited for energy generation (Hildebrandt et al., 2015). Alanine, glycine, serine, threonine and aspartate (Fig. 4a) for instance may be converted into pyruvate and metabolized in TCA cycle to yield some energy or consumed for nitrogen metabolism (Hildebrandt et al., 2015), and utilized for seedling growth as seen in recalcitrant seed of Ocotea catharinensis (Dias et al., 2009).
seed (Alhadi et al., 2012), glutamate level decrease during germination and is thought to have novel signalling function during amino acid interconversion. Both C. acuminata and P. granatum prefer arginine conversion and this may suggest the same for mangosteen, although further investigation to target and identify this amino acid in mangosteen seed is needed in future study. Amino acid metabolism is imperative in germinating seeds as they, in coherence with stored carbohydrates and sugars are necessary to drive
Fig. 5. Simplified depiction of metabolic pathways for mangosteen seed germination. Accumulation of metabolites is denoted by heatmap with scale of between −2 (green) to 2 (red) that indicates low and high-level buildup respectively. Consecutive arrows indicate multiple reactions. Dashed arrow indicates alternative pathway (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 232
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Acknowledgements
Additionally, amino acids might have some importance during seedling establishment. The general decrease of amino acids was inferred to support seedling growth in recalcitrant Cedrela fissilis (Aragão et al., 2015) after germination while developing to be an autotroph (Bove et al., 2002). In mangosteen, amino acids most probably assist in the expansion of procambium ring towards radicle and plumule protrusion (D3–D9, Fig. 1 and Table 1). Comparatively, Arabidopsis seed showed the opposite trend for most amino acids (increasing during germination sensu stricto) (Fait et al., 2006). This allude that recalcitrant seed such as mangosteen may utilize amino acids for growth and development differently during germination, in contrast to the orthodox seeds. Even so, it should be noted that the increase in some amino acid levels might be due to the need for alternative energy metabolism (Rosental et al., 2014). In short, the recalcitrant mangosteen seed might employ a unique mechanism for energy supply and amino acid metabolism during seed germination compared to orthodox Arabidopsis seed.
The project was supportedby University Kebangsaan Malaysia Research University grants (DLP-2013-011 and GUP-2015-051). OM was sponsored by Skim Zamalah Universiti Kebangsaan Malaysia. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2018.08.022. References Aizat, W.M., Dias, D.A., Stangoulis, J.C.R., Able, J.A., Roessner, U., Able, A.J., 2014. Metabolomics of capsicum ripening reveals modification of the ethylene relatedpathway and carbon metabolism. Postharvest Biol. Technol. 89, 19–31. Alhadi, F.A., AS, A.-A.A., SA, A.A., AA, A.Q., 2012. The effects of free amino acids profiles on seeds germination/dormancy and seedlings development of two genetically different cultivars of Yemeni Pomegranates. J. Stress Physiol. Biochem. 8. Aragão, V.P.M., Navarro, B.V., Passamani, L.Z., Macedo, A.F., Floh, E.I.S., Silveira, V., Santa-Catarina, C., 2015. Free amino acids, polyamines, soluble sugars and proteins during seed germination and early seedling growth of Cedrela fissilis Vellozo (Meliaceae), an endangered hardwood species from the Atlantic Forest in Brazil. Theor. Exp. Plant Phys. 27, 157–169. Araujo, W.L., Tohge, T., Ishizaki, K., Leaver, C.J., Fernie, A.R., 2011. Protein degradation–an alternative respiratory substrate for stressed plants. Trends Plant Sci. 16, 489–498. Berjak, P., Pammenter, N.W., 2013. Implications of the lack of desiccation tolerance in recalcitrant seeds. Front. Plant Sci. 4, 478. Berjak, P., Pammenter, N.W., Vertucci, C., 1992. Homoiohydrous (recalcitrant) seeds: developmental status, desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer. Planta 186, 249–261. Bove, J., Jullien, M., Grappin, P., 2002. Functional genomics in the study of seed germination. Genome Biol. 3, 1002.1–1002.5. Chen, S.X., Wan, M., Loh, B.N., 1996. Active constituents against HIV-1 protease from Garcinia mangostana. Planta Med. 62, 381–382. Chen, Q., Yang, L., Ahmad, P., Wan, X., Hu, X., 2011. Proteomic profiling and redox status alteration of recalcitrant tea (Camellia sinensis) seed in response to desiccation. Planta 233, 583–592. Chen, M., Rao, R.S.P., Zhang, Y., Zhong, C.X., Thelen, J.J., 2014. A modified data normalization method for GC–MS-based metabolomics to minimize batch variation. SpringerPlus 3, 1–7. Cheng, L., Gao, X., Li, S., Shi, M., Javeed, H., Jing, X., Yang, G., He, G., 2010. Proteomic analysis of soybean [Glycine max (L.) Meer.] seeds during imbibition at chilling temperature. Mol. Breed. 26, 1–17. Cook, D., Green, B.T., Welch, K.D., Gardner, D.R., Pfister, J.A., Panter, K.E., 2011. Comparison of the toxic effects of two duncecap larkspur (Delphinium occidentale) chemotypes in mice and cattle. Am. J. Vet. Res. 72, 706–714. de la Torre, F., Cañas, R.A., Pascual, M.B., Avila, C., Cánovas, F.M., 2014. 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The role of photosynthesis and amino acid metabolism in the energy status during seed development. Front. Plant Sci. 5 (447), 1–6. Gardner, D.R., Pfister, J.A., 2009. HPLC-MS analysis of toxic norditerpenoid alkaloids: refinement of toxicity assessment of low larkspurs (Delphinium spp.). Phytochem. Anal. 20, 104–113. Glauser, G., Veyrat, N., Rochat, B., Wolfender, J.L., Turlings, T.C., 2013. Ultra-high
4.2. Secondary metabolites in seed defense during germination Germination period may pose seeds vulnerable to stresses such as pathogen infection and herbivory. Hence, secondary metabolites may be utilized for seed defense (Fuller et al., 2012; Li et al., 2013; Ramegowda and Senthil-Kumar, 2015). The antibacterial and antimicrobial properties of alkaloids and flavonoids are vital for seed to fend off against herbivores and pathogen-borne diseases (Wink, 1988; Shirley, 1998; Mierziak et al., 2014). In this study, the increase of both alkaloids (anopterine and DMMBP, Fig. 3a) and flavonoid (kuwanon Z, Fig. 3c) were observed particularly during radicle protrusion (D3) and onwards, suggesting their importance during mangosteen seed germination. Furthermore, the transient increase of alpinine, an alkaloid throughout germination stages (Fig. 3a) might also support seedlings defense against herbivory by insects and animals as the alkaloid can be irritant to them as seen in Delphinium (Gardner and Pfister, 2009; Cook et al., 2011) and Lupinus (Frick et al., 2017) plants. During early seed germination, free radicals can be produced as the by-products of metabolism and respiration, and the levels may elevate in response to stress (Fernie et al., 2004; van Dongen et al., 2011). As free radicals can damage cells and impede germination if their levels are high, compounds with antioxidant activity such as xanthones can be elevated to neutralize and limit their deleterious effect. In this study, a xanthone called garcimangosone D was found highly increased in the germinating mangosteen seed particularly during D1–D5 (Fig. 3e). Previous reports have also shown its presence in mangosteen fruit hull or pericarp (Huang et al., 2001; Yoshimura et al., 2015) and may confer protection against free radicals. Furthermore, garcimangosone D may be part of a synthesis pathway that involved 2,4,6-triHB as a precursor for synthesizing other xanthones such as mangostin and mangiferin (Schmidt and Beerhues, 1997; El-Awaad et al., 2016). The increase in both garcimangosone D and 2,4,6-triHB as well as other secondary metabolites during mangosteen seed germination may imply a seed defense strategy against metabolic stress. Nonetheless, further analysis that specifically targets xanthones and other putatively identified metabolites are needed to clarify their functions in mangosteen seed germination.
5. Conclusion This study has elucidated the metabolic profiles of mangosteen seed germination. Primary metabolites such as sugars and amino acids were mostly reduced during the initial phase of mangosteen seed germination, suggesting high carbon utilization to drive the process. Subsequently, secondary metabolites such as alkaloids and xanthones generally increased throughout the later phases of seed germination, highlighting the importance of seed defense during this period.
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Metabolite profiling of mangosteen seed germination highlights metabolic changes related to carbon utilization and seed protection
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Othman Mazlan, Wan Mohd Aizat , Nor Shahida Aziz Zuddin, Syarul Nataqain Baharum, ⁎ Normah Mohd Noor Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia (UKM), 43600 Bangi, Selangor, Malaysia
A R T I C LE I N FO
A B S T R A C T
Keywords: Garcinia mangostana L. LC–MS GC–MS Metabolomics Recalcitrant seed Germination
Mangosteen seed is categorized as recalcitrant, becoming inviable if exposed to desiccation and low temperature. However, the molecular mechanism of mangosteen seed germination has not been fully understood. This study profiled the metabolites that were present in germinating mangosteen seeds at different stages (zero, one, three, five, seven and nine days after sowing) using liquid chromatography-mass spectrometry (LC–MS) and gas chromatography-mass spectrometry (GC–MS). A total of 38 tentative metabolites were profiled and classified into amino acid, organic acid, sugar, polyol, alkaloid and others. Metabolites identified implied a timed regulation for germination. Sugars as well as amino acids exhibited a declining trend throughout the germination period as they are likely to be utilized for development and growth of seedling. Furthermore, secondary metabolites such as alkaloids, flavonoids, and xanthone presence displayed an increasing trend at the middle of germination period which may provide protection for mangosteen seed against herbivory and stress. In brief, carbon utilization and seed protection associated with the modulation of primary and secondary metabolites, respectively are important metabolic signatures of mangosteen seed germination.
1. Introduction Mangosteen (Garcinia mangostana L.), a tropical fruit widely known for its sweet unique taste can be found in Southeast Asian countries such as Malaysia, Philippines and Thailand (Lim, 1984; Palapol et al., 2009). Additionally, mangosteen is well known in traditional medicine as some part of it, either from trees or fruit, has been used to treat wounds, diarrhea, dysentery, skin infections and more (Lu et al., 1998; Nakatani et al., 2002; Pedraza-Chaverri et al., 2008; Obolskiy et al., 2009). Mangosteen extracts have also been shown to possess various medicinal properties such as antifungal (Obolskiy et al., 2009), antioxidant (Yoshikawa et al., 1994), antibacterial (Iikubo et al., 2002), anti-HIV (Chen et al., 1996; Vlietinck et al., 1998) and anticancer agents (Matsumoto et al., 2003; Moongkarndi et al., 2004; Han et al., 2009; Shan et al., 2011). These health benefits are mainly contributed by xanthones, a class of polyphenolic compounds present in only a few plant species including mangosteen (Jinsart et al., 1992; Jung et al., 2006; Obolskiy et al., 2009). The two major xanthones that can be found in mangosteen include α-mangostin and γ-mangostin (Obolskiy et al., 2009). Despite its medicinal potential, mangosteen tree growth is slow and
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only matures in about five to seven years before it starts bearing fruit (Osman and Milan, 2006). Mangosteen is mainly propagated through seeds which are categorized as recalcitrant. These seeds are sensitive to desiccation and low temperature (Normah et al., 1997) as they maintain active metabolism when shed (Berjak and Pammenter, 2013). Also, at later stages of development, mangosteen seed has an elevated level of soluble sugars and secondary metabolites (Mazlan et al., 2018). The seed will not be able to germinate if its moisture content drops to 30% or lower (Normah et al., 1997, 2016). It can only be stored for a short period before becoming inviable for germination (Normah et al., 1997), making it difficult for a long-term seed preservation and maintaining planting materials throughout the year. Mangosteen seed shows Garcinia-type germination where radicle protrusion is followed by plumule emergence on the opposite side (Lim, 1984; Normah et al., 2016). Studies on mangosteen seed are still limited particularly during the seed germination. Seed germination is dependent upon multitude of endogenous and exogenous factors such as temperature and pH level (Kulkarni et al., 2014) and is strongly supported by various types of reserve compounds synthesized and stored during seed development such as carbohydrates and proteins (Pritchard et al., 2002; Galili et al., 2014). Germination are mainly divided into three phases – imbibition
Corresponding authors. E-mail addresses: [email protected] (W.M. Aizat), [email protected] (N.M. Noor).
https://doi.org/10.1016/j.scienta.2018.08.022 Received 17 April 2018; Received in revised form 10 August 2018; Accepted 11 August 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
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before 0.1 min increase to 100% B. At 100% B, the mixture was held for 4 min prior drop to 5% B in 2 min. Finally, column reconditioning was performed for 7 min with the initial gradient. Mass spectrometry analysis was using a MicroTOF-QIII (Bruker, Germany) system with electrospray ionization (ESI) source in a positive mode of ionization. The drying gas (nitrogen) was set at 45 psi with flow at 8 L min−1 and gas temperature of 200 °C. The voltage of ESI spray and the fragmentor were fixed at 4.5 kV and 200 V respectively. Range of the scan was between 50 and 1000 Da. Subsequently, the data were logged in a centroid mode.
(Phase 1), lag interval (Phase 2) and radicle protrusion (Phase 3) (Nonogaki et al., 2010; Rajjou et al., 2012). Rapid water uptake by seeds occurs during Phase 1 followed by Phase 2 which is characterised by an active seed metabolism. Lastly, seeds germinate at Phase 3 with radicle protrusion. Seed germination has been investigated in many plant species such as in Arabidopsis (Rajjou et al., 2006), barley (Yang et al., 2011), tea (Chen et al., 2011), maize (Huang et al., 2012), rice (Kim et al., 2008) and soybean (Cheng et al., 2010). For instance, it was revealed that key compounds such as sugars and amino acids were temporally regulated during Arabidopsis seed germination (Fait et al., 2006). Still, investigation on recalcitrant seeds such as mangosteen are relatively scarce and hence demands further analysis. Previous studies by Normah et al. (2016) and Lim (1984) have highlighted the physical properties and morphology of germinating mangosteen seed in effort to understand its biology. However, metabolic changes during the germination of this recalcitrant seed have not been reported. Hence, the aim of this study is to analyze mangosteen seed germination using metabolomics approach via liquid chromatography-mass spectrometry (LC–MS) and gas chromatography-mass spectrometry (GC–MS). Information gained would provide better comprehension on the metabolism of recalcitrant mangosteen seed germination.
2.4. LC–MS metabolite identification The raw data for LC–MS were first analyzed using Compass Data Analysis (Bruker, Germany) to detect peaks and deconvolute the total ion current chromatogram. This was to generate the list of peaks for retention time to mass per charge ratio (Rt-m/z) that corresponds to putative compounds detected and intensity data sets. The data were then binned and tabulated. MetaboAnalyst (http://www. metaboanalyst.ca/) for data normalization (by sum) and transformation, scaling and statistical analysis. Rt-m/z values that were detected in all three biological replicates, in at least three stages of germination were selected for putative identification. The m/z values were compared with three online metabolite databases namely Massbank (http:// www.massbank.jp), Metlin (https://metlin.scripps.edu) and Metfrag (http://msbi.ipb-halle.de/MetFrag/). For each m/z values, top three compounds that are found in plants were selected and narrowed down to one, based on thorough literature review. Calculation of relative intensity (in percentage, %) was done by using the Formula (1). Finalized identified metabolites were then tabulated and graphed.
2. Materials and methods 2.1. Seed sample preparation Mature mangosteen fruit were obtained from trees at mangosteen plots in Universiti Kebangsaan Malaysia (UKM), Selangor, Malaysia (GPS coordinate: 2.922662 °E, 101.786690 °N). Two batches of sample were made; one for metabolomics analysis and the other for representative pictures. Aril was removed from seeds to avoid fungal infection during germination. Clean seeds were planted in autoclaved sand and were grown in a greenhouse (24–26 °C) with daily watering. Mangosteen seed takes nine days of sowing before both radicle and plumule emerge (Osman and Milan, 2006; Normah et al., 2016). Hence, samples were taken every two days at one (D1), three (D3), five (D5), seven (D7) and nine (D9) day(s) after sowing with day zero (D0) as control. At each timepoint, three germinating seeds, acted as biological replicates, were cleaned, blotted dry and flash frozen in liquid nitrogen prior storage at −80 °C.
Relative intensity, reI =
Compound peak intensity × 100 Total intensity in all metabolite across all stages
(1) 2.5. Gas chromatography-mass spectrometry (GC–MS) protocol The dried supernatant was dissolved and derivatized in 80 μL of methoxyamine hydrochloride (20 mg mL−1) in pyridine at 30 °C for 90 min. Then, it was incubated for 30 min with temperature of 37 °C after the addition of 80 μL of N-Methyl-N-(trimethylsilyl)-trifluoracetamide (MSTFA). Both chemicals were obtained from SigmaAldrich (St. Louis, MO, USA). GC–MS and its analysis was done as per Chen et al. (2014) with revision. For each replicate, 1.0 μL of derivatized sample was injected into GC–MS with a split ratio of 25:1. The GC–MS system consisted of a GC Agilent 5975 gas chromatograph (Agilent, China), an autosampler and a single quadruple mass spectrometer (Agilent, USA). Using tris-(perfluorobutyl)-amine (CF43), adjustment to the mass spectrometer was made according to recommendations by the manufacturer. The AB-5MS 30 m column used has 0.25 mm for both inner diameter and film thickness (Abel Industries, Canada). The parameters were set for injection temperature (230 °C), interface (250 °C) and ion source (200 °C). Helium, the carrier gas used was set to flow at the rate of 1 mL min−1. The temperature setup started with isothermal heating (70 °C, 5 min) followed by a 5 °C min−1 rising of oven temperature (up to 310 °C) and a final heating (310 °C, 1 min).
2.2. Metabolite extraction Seeds were ground to fine powder and 0.1 g from each replicate was homogenized in 1.4 mL of methanol (Merck, Germany) and ribitol (50 μL, 2 mg mL−1) as an internal standard. For 15 min at 70 °C, the mixtures were incubated for extraction. Extracts obtained were added with one volume of water, mixed briskly and subsequently centrifuged at 2200×g. Supernatant acquired was vacuum-dried for four to six hours to eliminate the extraction solvent and stored in −80 °C until used. 2.3. Liquid chromatography-mass spectrometry (LC–MS) protocol Methanol were used to resolubilised dried samples. This study follows Glauser et al. (2013) method with modifications (Mamat et al., 2018; Mazlan et al., 2018). In extract compound separation, liquid chromatography system (Ultimate 3000 UHPLC+, Thermo Scientific, USA) equipped with a C18 column was utilized. LC parameters were set for injection volume (1.0 μL), column temperature (60 °C) and flow rate (0.3 mL min−1). The mobile phases consisted of 0.1% formic acid in water (mobile phase A) and acetonitrile (mobile phase B). Elution was performed with a 35-min gradient starting with increase from 0 to 5% B in the first 2 min, then further to 40% in the following 2 min and up to 95% B within the next 16 min. At 95% B, the mixture was held for 2 min
2.6. GC–MS metabolite identification Metabolites were identified using NIST11 library. Then, metabolites with match score of below 700 were discarded. Compounds that were found to be redundant in the data were further trimmed. The metabolites that were detected in all three replicates for at least three stages were selected as the final list of metabolites. The data were imported into MetaboAnalyst where they were subjected to normalization using 227
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3.2. LC–MS analysis
ribitol, transformation, scaling and statistical analysis. These compounds were classified into amino acid, organic acid, sugar, polyol and glycoside. Then, semi quantitative concentration of compounds was calculated using the formula below based on the known concentration of internal standard (ribitol, 2 mg mL−1) as outlined in Roessner et al. (2000) and Roessner et al. (2006). Semi quantitative concentration Area percent of a replicate = × 2 mg mL-1 Average area percent of ribitol in a particular stage
From the LC–MS analysis, 68 Rt-m/z values were obtained and subjected for MVA. Both PCA and PLS-DA show no distinct separation (Supplementary Fig. 1a and b). Further analysis using OPLS-DA shows distinction between D0 and other germination stages with a total variance of 27% (Fig. 2a). The loading plot shows Rt-m/z pairs responsible for the separation (Fig. 2b), and 21 Rt-m/z pairs has Variable Importance in Projection (VIP) scores of more than 1 (Supplementary Table 1). Seventeen identified compounds were classified into alkaloid, glycoside, flavonoid, organic acid, xanthone-related compound, amino acid and ‘other’ (Fig. 3). Alkaloids (Fig. 3a) include alpinine, anopterine, 1,2-dimethoxy-13-methyl-[1,3]benzodioxolo[5,6-c]phenanthridine (DMMBP) and indoleacrylic acid. Meanwhile, cyanidin 3-O-(6O-p-coumaroyl)glucoside (cyanidin coumaroylglucoside), cyclo-Dopaglucuronylglucoside, isopropyl apiosylglucoside and lusitanicoside are in the glycoside group (Fig. 3b). Furthermore, 4-(2,4-dihydroxyphenyl)8-[(Z)-2-(2,4-dihydroxyphenyl)ethenyl]-10,18-dihydroxy-14-methyl3,5,15-trioxahexacyclo[12.7.1.0²,⁴.0²,¹².0⁶,¹¹.0¹⁶,²¹]docosa6,8,10,16(21),17,19-hexaen-13-one (kuwanon Z) and epicathechin(4β→8)-epicathechin (procyanidin B2) are flavonoids (Fig. 3c) while chlorogenic acid and pantothenic acid are organic acids (Fig. 3d). Xanthone-related compounds (Fig. 3e) are 2,4,6-triHB and garcimangosone D. Amino acid group (Fig. 3f) consists of phenylalanine while terpenoid (Fig. 3g) is (all-E)-6′-Apo-y-caroten-6′-al (psi-carotenal). Lastly, methyl phenyl disulfide is grouped as ‘other’ (Fig. 3h).
(2)
2.7. Statistical analysis MetaboAnalyst was used for data normalization, log transformation and pareto scaling as well as ANOVA (p ≤ 0.05) for statistical analysis and heatmap generation. Heatmap generated was added to simple metabolic pathways made by referring to literatures (Aizat et al., 2014). Multivariate analysis performed was principal component analysis (PCA), partial least squares to latent structure-discriminant analysis (PLS-DA) and orthogonal partial least squares to latent structure-discriminant analysis (OPLS-DA) using SIMCA-P 14.1 (Umetrics, Sweden). Tabulation and graph were made using Microsoft Excel 2016 and GraphPad Prism 7 (GraphPad Software). Graphs display mean for each stage with standard error of the mean (SEM). 3. Results
3.3. GC–MS analysis 3.1. Mangosteen seeds physical appearance A total of 21 compounds were identified using NIST library of GC–MS. PCA and PLS-DA score plot do not show distinction between sample groups (Supplementary Fig. 1c and d). However, a clear separation between the sample groups and metabolites was observed particularly between D0 and the rest of seed germination stages via OPLS-DA (total variance of 35.2%) (Fig. 2c) with compounds that explain the separation seen in the loading plot (Fig. 2d). There are eight compounds with VIP > 1 (Supplementary Table 2). The metabolites
Mangosteen seed at D0 (Fig. 1) contains undifferentiated embryo with a procambium ring which extends outward at one side at D1. Eventually, the procambium ring grows towards the seed coat and at D3 a small protuberance (radicle) occurs on the surface of the seed. At D5 the seed grew a second protuberance (plumule) at the opposite end of the seed. The radicle and plumule then grew up to 1–2 mm at D7 and 2–4 mm by D9.
Fig. 1. Mangosteen seeds at six different germination stages: zero (D0), one (D1), three (D3), five (D5), seven (D7) and nine (D9) days after sowing. The arrows indicate procambium ring that eventually grows towards radicle (r) protrusion and plumule (p) emergence. All seeds were obtained from ripe mangosteen prior sowing. Scale bar, 1 cm.
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Fig. 2. Orthogonal partial least squares to latent structure-discriminant analysis (OPLS-DA) of Rt-m/z pairs and metabolites identified by liquid chromatographymass spectrometry (LC–MS) and gas chromatography-mass spectrometry (GC–MS) during mangosteen seed germination at zero (D0), one (D1), three (D3), five (D5), seven (D7) and nine (D9) day(s) after sowing. Circles are labelled with respect to the germination stages. Bigger darker diamond dots correspond to Rt-m/z pairs and metabolites with Variable Importance of Projection (VIP) larger than 1. Compounds chosen were found in all replicates for at least three seed germination stages. LC–MS score plot (a) and loading plot (b) of the OPLS-DA (27% total variance). GC–MS score plot (c) and loading plot (d) of the OPLS-DA (35.2% total variance).
can be classified into five groups (Fig. 4). Firstly, the amino acids which comprised of alanine, aspartate, glutamate, glycine, isoleucine, serine, threonine and valine (Fig. 4a). Secondly, the organic acids; citrate, fumarate, glycerate, malate, mucic acid and succinate (Fig. 4b). There were also sugars (fructose, galactose and sucrose) (Fig. 4c) as well as polyols (mannitol, myo-inositol and scyllo-inositol) (Fig. 4d). Lastly, glyceryl-glycoside is classified as glycoside (Fig. 4e).
instance, anopterine (Fig. 3a), kuwanon Z (Fig. 3c), 2,4,6-triHB (Fig. 3e), methyl phenyl disulfide (Fig. 3h) and glycerate (Fig. 4b) levels were increasing during germination. DMMBP, lusitanicoside, garcimangosone D and pantothenic acid (Fig. 3) as well as glyceryl-glycoside (Fig. 4e) levels were also increased particularly at the early germination and radicle protrusion phase (D0–D3) but decrease at later stages. Other metabolites fluctuated with increase levels at the middle timepoints of the germination stages, for examples alpinine and cyclo-Dopaglucuronylglucoside (D5), isopropyl apiosylglucoside (D1, D5), chlorogenic acid (D1, D5–D7) as well as psi-carotenal (D1, D7) (Fig. 3). The third major trend is indicated by indoleacrylic acid, cyanidin coumaroylglucoside and procyanidin B2 (Fig. 3), as well as mannitol and scyllo-inositol (Fig. 4) where their levels were mostly constant throughout germination. Briefly, the germination of mangosteen seed involved modulation of both primary and secondary metabolites.
3.4. Metabolite trends Metabolite levels throughout the stages showed three major trends of regulation. First is the general decrease of most primary metabolites during the germination period, either consistently dropping or increasing slightly towards the end of the process (Fig. 4, Table 1). For examples, phenylalanine (Fig. 3f), alanine, glutamate, threonine, valine (Fig. 4a) and myo-inositol (Fig. 4d) levels were decreasing during early germination stages (D0–D3) and remained low throughout the stages. Similar downward trend but rising slightly at a later germination stage was also observed for other amino acids (e.g. aspartate, glycine, isoleucine, serine) (Fig. 4a), organic acids (e.g. citrate, fumarate, malate, mucic acid, succinate) (Fig. 4b) and sugars (e.g., fructose, galactose) (Fig. 4c). The second major trend illustrates an increasing pattern during mangosteen germination, mainly by secondary metabolites. For
3.5. Metabolic pathways Fig. 5 illustrates major metabolic pathways associated with mangosteen seed germination. Metabolites putatively identified in this study are positioned within relevant pathways and their levels are represented with heatmaps. Soluble sugars such as fructose, galactose and sucrose were mainly fed into glycolysis to be used for subsequent pathways such as TCA cycle and amino acid biosynthesis. Moreover,
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Fig. 3. Tentative metabolites identified by liquid chromatography-mass spectrometry (LC–MS) presented in relative intensity (in percentage, %) across all stages of mangosteen seed germination at zero (D0), one (D1), three (D3), five (D5), seven (D7) and nine (D9) day(s) after sowing. The metabolites are categorized into alkaloid (a), glycoside (b), flavonoid (c), organic acid (d), xanthone-related compound (e), amino acid (f), terpenoid (g) and ‘other’ (h). Each stage was comprised of three biological replicates ± standard error of the mean. DMMBP, 1,2-Dimethoxy-13-methyl-[1,3]benzodioxolo[5,6-c]phenanthridine; cyanidin coumaroylglucoside, cyanidin 3-O-(6-O-p-coumaroyl)glucoside; kuwanon Z, 4-(2,4-dihydroxyphenyl)-8-[(Z)-2-(2,4-dihydroxyphenyl)ethenyl]-10,18-dihydroxy-14-methyl-3,5,15trioxahexacyclo[12.7.1.0²,⁴.0²,¹².0⁶,¹¹.0¹⁶,²¹]docosa-6,8,10,16(21),17,19-hexaen-13-one; procyanidin B2, epicathechin-(4β→8)-epicathechin; psi-carotenal, (all-E)6′-Apo-y-caroten-6′-al; 2,4,6-triHB, 2,4,6-trihydroxybenzophenone.
The metabolite content of these mangosteen stages was investigated using metabolomics (LC–MS and GC–MS) approaches, which revealed the regulation of both primary and secondary metabolism important for the germination of this recalcitrant seed.
TCA cycle may also consume metabolites such as from fatty acid metabolism as well as amino acids and via malate, succinate, fumarate, and acetyl-CoA conversion. Meanwhile, the secondary metabolic pathway through shikimate splits into two biosynthesis pathways; the anthranilate path for alkaloids and terpenoids, and the prephenate path for flavonoid and xanthones.
4.1. Modulation of sugar, amino acid and TCA cycle intermediates during seed germination
4. Discussion Primary metabolites such as sugars are important sources of carbon in supporting seed germination (Eveland and Jackson, 2011; Sami et al., 2016). During seed development, mangosteen seed continues to accumulate soluble sugars until late maturation (Mazlan et al., 2018). In this study, we found that the level of soluble sugars (fructose, galactose, sucrose) dropped during the initial germination stages (imbibition-lag phase, D0–D3) and remained low until their increase at
In this study, the germination of mangosteen seed was investigated at D0, D1, D3, D5, D7 and D9. The seed radicle started to appear by D3 and hence regarded as the radicle protrusion stage (Fig. 1). The stages prior to the radicle protrusion (D0–D3) are regarded as the imbibitionlag phase and the stages after D3 are considered as the seedling establishment phase (growth of radicle and plumule protrusion, Table 1). 230
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Fig. 4. Putative metabolites detected using gas chromatography-mass spectrometry (GC–MS) and presented as semi quantitative (mg mL−1) across all stages of mangosteen seed germination; zero (D0), one (D1), three (D3), five (D5), seven (D7) and nine (D9) day(s) after sowing. The metabolites are categorized into amino acid (a), organic acid (b), sugar (c), polyol (d) and glycoside (e). Each stage was comprised of three biological replicates ± standard error of the mean.
phase (D0–D1, Fig. 4 and Table 1). Interestingly, the levels of malate, succinate and citrate were increasing when the orthodox seeds of Arabidopsis germinate sensu stricto (Fait et al., 2006) implying the different method for carbon metabolism compared to mangosteen as stated previously. Among the four TCA cycle intermediates identified, citrate level is relatively higher than others by the end of germination (Fig. 4b). It could be that the germinating seeds may employ a noncyclic TCA cycle flux, bypassing malate, fumarate and succinate through an alternative pathway such as via pyruvate-oxaloacetate conversion (Fig. 5) to increase the citrate level for nitrogen assimilation (Krömer et al., 1996; Sweetlove et al., 2010; Eastmond et al., 2015). Moreover, the non-cyclic bypass (Fig. 5) may have affected aspartate level to increase (Fig. 4a). Aspartate might be used to synthesize other amino acids such as phenylalanine, isoleucine and threonine (Galili, 2011; Aizat et al., 2014; de la Torre et al., 2014). Their levels were decreasing throughout the germination stages especially from D0 to D1 (Figs. 3f and 4 a), implying some involvement in biosynthetic pathways or cellular processes predominantly for procambium expansion during imbibition-lag phase at D1. For instance, phenylalanine is a precursor towards secondary metabolic pathways, in particular flavonoid biosynthesis pathway (Tohge et al., 2013), can contribute majorly to their formation and may assist in oxidative stress suppression (Teixeira et al., 2017) and ultraviolet protection (Sullivan et al., 2014) during seedling establishment. Meanwhile, glutamate (from α-ketoglutarate conversion (Fig. 5) was also decreasing, inferring a role in germination initiation particularly for the procambium expansion at D1 (Fig. 1). In Cola acuminate (Onomo et al., 2010) and Punica granatum
later stages by which is similarly observed in germinating recalcitrant Guilfoylia monostylis seed (Nkang, 2002). These soluble sugars are thought to be consumed for a quick source of energy as they are readily available by the end of reserve accumulation phase (Mazlan et al., 2018). The energy generation might have been directed towards seed cellular growth including procambium ring expansion and radicle protrusion at D1–D3 (Fig. 1). In orthodox seeds such as Arabidopsis, fructose, galactose and sucrose were reduced up to the end of its vernalization period (Table 1), limiting their availability towards its germination phase (Fait et al., 2006). The orthodox seed may need to opt for alternative pathways to fuel its germination for example through fermentation and amino acid consumption (Fait et al., 2006; Rosental et al., 2014). While mangosteen seed may have utilized the freely available sugars during early germination for energy supply, their low levels in the following stages of germination, late imbibition-lag phase (D1–D3) as well as radicle and plumule protrusion (D3–D9) (Fig. 4, Table 1) might prompt energy outsourcing to other carbon reserve such as lipid. Storage lipid was found to be a prominent reserve in mangosteen (Normah et al., 2016) as well as other recalcitrant species such as Landolphia kirkii Dyer (Berjak et al., 1992), Guilfoylia monostylis (Nkang, 2002) and Telfairia occidentalis (Nkang et al., 2003), and is probably used for germination metabolism. As soluble sugars were consumed and limited during late imbibition-lag phase (D1–D3) and the rest of mangosteen seed germination, biochemical processes such as glycolysis and TCA cycle might have been affected. Evidently, metabolic intermediates such as malate, citrate, succinate and fumarate decreased during the early imbibition-lag
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Table 1 Metabolite comparison between mangosteen (this study) and Arabidopsis thaliana (Fait et al., 2006) at different stages of development and germination for metabolites found in both species. Timepoints for these studies are as follow: mangosteen seed germination at zero (D0), one (D1), three (D3), five (D5), seven (D7) and nine (D9) day(s) after germination (this study); Arabidopsis at 18, 21 and 22 days after flowering (DAF). Germination in Arabidopsis is germination sensu stricto where it precedes radicle protrusion. Up arrow (↑) represents increasing level, down arrow (↓) represents decreasing level, ‘no change’ (n.c.) represents no change in level from previous stage and ‘negligible’ represents negligible presence or not detected. Metabolite
Alanine Aspartate Glutamate Glycine Isoleucine Phenylalanine Serine Threonine Valine Citrate Fumarate Glycerate Glycerate 3P Malate Succinate Myo-inositol Fructose Fructose 6P Galactose Sucrose
Mangosteen seed germination (this study)
Arabidopsis vernalization and germination (Fait et al., 2006)
D0–D1 D1–D3 Imbibition-lag phase
D3–D5 D5–D7 Radicle and plumule protrusion
D7–D9
↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↑ Negligible ↓ ↓ ↓ ↓ Negligible ↓ ↓
n.c. ↑ n.c. n.c. ↑ ↓ ↑ n.c. n.c. ↑ n.c. ↑ Negligible n.c. n.c. n.c. n.c. Negligible n.c. n.c.
n.c. ↑ n.c. n.c. ↑ n.c. ↑ n.c. n.c. n.c. ↑ ↑ Negligible n.c. ↑ n.c. n.c. Negligible ↑ n.c.
n.c. ↑ n.c. n.c. ↑ n.c. ↑ n.c. ↓ ↑ ↓ ↓ Negligible n.c. n.c. n.c. ↓ Negligible n.c. n.c.
n.c. ↓ n.c. n.c. ↓ ↓ ↑ n.c. n.c. n.c. n.c. ↑ Negligible n.c. n.c. n.c. n.c. Negligible n.c. n.c.
18–21 DAF Vernalization ↑ ↑ ↑ ↓ ↓ ↓ ↑ ↑ ↓ ↓ ↓ ↓ ↑ ↑ ↑ ↓ ↓ ↑ ↓ ↓
21–22 DAF Germination ↑ ↑ ↑ ↓ ↓ ↑ ↑ ↑ n.c. ↑ n.c. ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓
cell metabolism for germination (Araujo et al., 2011). The declining levels of almost all identified amino acids (Fig. 4a) during early imbibition-lag phase suggest that they were exploited for energy generation (Hildebrandt et al., 2015). Alanine, glycine, serine, threonine and aspartate (Fig. 4a) for instance may be converted into pyruvate and metabolized in TCA cycle to yield some energy or consumed for nitrogen metabolism (Hildebrandt et al., 2015), and utilized for seedling growth as seen in recalcitrant seed of Ocotea catharinensis (Dias et al., 2009).
seed (Alhadi et al., 2012), glutamate level decrease during germination and is thought to have novel signalling function during amino acid interconversion. Both C. acuminata and P. granatum prefer arginine conversion and this may suggest the same for mangosteen, although further investigation to target and identify this amino acid in mangosteen seed is needed in future study. Amino acid metabolism is imperative in germinating seeds as they, in coherence with stored carbohydrates and sugars are necessary to drive
Fig. 5. Simplified depiction of metabolic pathways for mangosteen seed germination. Accumulation of metabolites is denoted by heatmap with scale of between −2 (green) to 2 (red) that indicates low and high-level buildup respectively. Consecutive arrows indicate multiple reactions. Dashed arrow indicates alternative pathway (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 232
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Acknowledgements
Additionally, amino acids might have some importance during seedling establishment. The general decrease of amino acids was inferred to support seedling growth in recalcitrant Cedrela fissilis (Aragão et al., 2015) after germination while developing to be an autotroph (Bove et al., 2002). In mangosteen, amino acids most probably assist in the expansion of procambium ring towards radicle and plumule protrusion (D3–D9, Fig. 1 and Table 1). Comparatively, Arabidopsis seed showed the opposite trend for most amino acids (increasing during germination sensu stricto) (Fait et al., 2006). This allude that recalcitrant seed such as mangosteen may utilize amino acids for growth and development differently during germination, in contrast to the orthodox seeds. Even so, it should be noted that the increase in some amino acid levels might be due to the need for alternative energy metabolism (Rosental et al., 2014). In short, the recalcitrant mangosteen seed might employ a unique mechanism for energy supply and amino acid metabolism during seed germination compared to orthodox Arabidopsis seed.
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Fait, A., Angelovici, R., Less, H., Ohad, I., Urbanczyk-Wochniak, E., Fernie, A.R., Galili, G., 2006. Arabidopsis seed development and germination is associated with temporally distinct metabolic switches. Plant Physiol. 142, 839–854. Fernie, A.R., Carrari, F., Sweetlove, L.J., 2004. Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport. Curr. Opin. Plant Biol. 7, 254–261. Frick, K.M., Kamphuis, L.G., Siddique, K.H.M., Singh, K.B., Foley, R.C., 2017. Quinolizidine alkaloid biosynthesis in Lupins and prospects for grain quality improvement. Front. Plant Sci. 8, 87. Fuller, M.P., Hamza, J.H., Rihan, H.Z., Al-Issawi, M., 2012. Germination of primed seed under NaCl stress in wheat. ISRN Bot. 2012, 1–5. Galili, G., 2011. The aspartate-family pathway of plants: linking production of essential amino acids with energy and stress regulation. Plant Signal. Behav. 6, 192–195. Galili, G., Avin-Wittenberg, T., Angelovici, R., Fernie, A.R., 2014. The role of photosynthesis and amino acid metabolism in the energy status during seed development. Front. Plant Sci. 5 (447), 1–6. Gardner, D.R., Pfister, J.A., 2009. HPLC-MS analysis of toxic norditerpenoid alkaloids: refinement of toxicity assessment of low larkspurs (Delphinium spp.). Phytochem. Anal. 20, 104–113. Glauser, G., Veyrat, N., Rochat, B., Wolfender, J.L., Turlings, T.C., 2013. Ultra-high
4.2. Secondary metabolites in seed defense during germination Germination period may pose seeds vulnerable to stresses such as pathogen infection and herbivory. Hence, secondary metabolites may be utilized for seed defense (Fuller et al., 2012; Li et al., 2013; Ramegowda and Senthil-Kumar, 2015). The antibacterial and antimicrobial properties of alkaloids and flavonoids are vital for seed to fend off against herbivores and pathogen-borne diseases (Wink, 1988; Shirley, 1998; Mierziak et al., 2014). In this study, the increase of both alkaloids (anopterine and DMMBP, Fig. 3a) and flavonoid (kuwanon Z, Fig. 3c) were observed particularly during radicle protrusion (D3) and onwards, suggesting their importance during mangosteen seed germination. Furthermore, the transient increase of alpinine, an alkaloid throughout germination stages (Fig. 3a) might also support seedlings defense against herbivory by insects and animals as the alkaloid can be irritant to them as seen in Delphinium (Gardner and Pfister, 2009; Cook et al., 2011) and Lupinus (Frick et al., 2017) plants. During early seed germination, free radicals can be produced as the by-products of metabolism and respiration, and the levels may elevate in response to stress (Fernie et al., 2004; van Dongen et al., 2011). As free radicals can damage cells and impede germination if their levels are high, compounds with antioxidant activity such as xanthones can be elevated to neutralize and limit their deleterious effect. In this study, a xanthone called garcimangosone D was found highly increased in the germinating mangosteen seed particularly during D1–D5 (Fig. 3e). Previous reports have also shown its presence in mangosteen fruit hull or pericarp (Huang et al., 2001; Yoshimura et al., 2015) and may confer protection against free radicals. Furthermore, garcimangosone D may be part of a synthesis pathway that involved 2,4,6-triHB as a precursor for synthesizing other xanthones such as mangostin and mangiferin (Schmidt and Beerhues, 1997; El-Awaad et al., 2016). The increase in both garcimangosone D and 2,4,6-triHB as well as other secondary metabolites during mangosteen seed germination may imply a seed defense strategy against metabolic stress. Nonetheless, further analysis that specifically targets xanthones and other putatively identified metabolites are needed to clarify their functions in mangosteen seed germination.
5. Conclusion This study has elucidated the metabolic profiles of mangosteen seed germination. Primary metabolites such as sugars and amino acids were mostly reduced during the initial phase of mangosteen seed germination, suggesting high carbon utilization to drive the process. Subsequently, secondary metabolites such as alkaloids and xanthones generally increased throughout the later phases of seed germination, highlighting the importance of seed defense during this period.
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