Comprehensive Multiphase (CMP) NMR Monitoring of the Structural

Jul 20, 2017 - ... were monitored in situ over 4 days. A wide range of metabolites were identified, and after 96 h of germination, the number of metab...
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Comprehensive Multiphase (CMP) NMR Monitoring of the Structural Changes and Molecular Flux Within a Growing Seed Blythe E. Fortier-McGill,† Rudraksha Dutta Majumdar,†,¶ Leayen Lam,†,‡ Ronald Soong,†,‡ Yalda Liaghati-Mobarhan,†,‡ Andre Sutrisno,†,‡ Ries de Visser,§ Myrna J. Simpson,†,‡ Heather L. Wheeler,#,⊥ Malcolm Campbell,#,⊥,| Antonie Gorissen,§ and André J. Simpson*,†,‡ †

Department of Physical and Environment Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, Ontario Canada, M1C 1A4 ‡ Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario Canada, M5S 3H6 § IsoLife BV, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands # Department of Biological Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, Ontario Canada, M1C 1A4 ⊥ Department of Cell Systems and Biology, University of Toronto, 33 Willcocks Street, Toronto, Ontario Canada, M5S 3B2 | Molecular and Cell Biology, Summerlee Science Complex, University of Guelph, Guelph, Ontario Canada, N1G 2W1 S Supporting Information *

ABSTRACT: A relatively recent technique termed comprehensive multiphase (CMP) NMR spectroscopy was used to investigate the growth and associated metabolomic changes of 13C-labeled wheat seeds and germinated seedlings. CMP-NMR enables the study of all phases in intact samples (i.e., liquid, gel-like, semisolid, and solid), by combining all required electronics into a single NMR probe, and can be used for investigating biological processes such as seed germination. All components, from the most liquid-like (i.e., dissolved metabolites) to the most rigid or solid-like (seed coat) were monitored in situ over 4 days. A wide range of metabolites were identified, and after 96 h of germination, the number of metabolites in the mobile phase more than doubled in comparison to 0 h (dry seed). This work represents the first application of CMP-NMR to follow biological processes in plants. KEYWORDS: wheat, Triticum aestivum, comprehensive multiphase, CMP, NMR, HSQC, 13C NMR, germination, seed, early seedling growth, metabolites

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INTRODUCTION Nuclear magnetic resonance (NMR) spectroscopy is a powerful molecular-level technique that can probe complex chemical structures and interactions in plants. For example, NMR T2 relaxation measurements have been used to characterize the changes of water status and/or content/uptake in germinating wheat,1 soybeans,2 rice,3 tomato,4 and lupine seeds.5 NMR has also been used to monitor variations in water uptake and lipid consumption in sesame seeds6 and to monitor mobilization of oil reserves following seed germination and during early seedling growth in intact seeds of soybean.7 However, all of these studies have been restricted to specific phases (i.e., liquid, gel-like, or solid components) within the seeds. Comprehensive multiphase (CMP) NMR probes were introduced in 2012 and combine all the electronics and hardware of solution-state NMR (spectrometer lock, susceptibility matched components for sharpest line shape), gel-state NMR (magic angle spinning and magic angle pulsed field gradients) and solid-state NMR (high power circuitry) into a single NMR probe.8 When the technology is combined with spectral editing approaches,8 all components (liquids, gels, and solids) can be fully differentiated in situ. Furthermore, as the full range of solid-state, gel-state and liquid-state NMR experiments are conveniently available within the same probe, all NMR studies including molecular interactions (dynamics, bond © XXXX American Chemical Society

distances, conformation) and kinetic transport (diffusion, phase change) can potentially be performed. However, these measurements are nontrivial and are still subject to the inherent limitations of NMR, mainly its sensitivity, spectral overcrowding, and cost. To date, CMP-NMR has been applied to study basic structure in seeds9 and between different genotypes of Arabidopsis.10 However, studies of a biological process have not been performed. CMP-NMR is conveniently suited for determining both the chemical entities themselves and how they undergo change over time, in all the phases. For example, the conversion of a soluble precursor into a solid biopolymer, swelling/drying, or the transport and conversion of nutrients across phases. Such processes are critical to understanding plant growth in general. This work focuses on the germination and early growth of 13C-labeled wheat seeds (Triticum aestivum) as a demonstration of the potential of CMP-NMR for studying plant processes. The changes, measured at 24 h time increments from a seed to a seedling of isotopically 13C-labeled growing seeds, presented here offer an avenue to establish Received: Revised: Accepted: Published: A

May 24, 2017 July 19, 2017 July 20, 2017 July 20, 2017 DOI: 10.1021/acs.jafc.7b02421 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Decoupling was used in both one-dimensional (1D) and twodimensional (2D) experiments to remove 1H−13C coupling from the labeled samples. The GARP decoupling approach was used for the 1H observed liquid/gel-state experiments, while low-power WALTZ16 decoupling was used for 13C observed experiments in the same phase. The solid-state cross-polarization magic angle spinning (CP-MAS) experiments used high-power SPINAL64 decoupling. This is not an in vivo NMR study but rather using multiphase NMR to follow holistically the various phases and chemical components during development. If the goal was to follow in vivo growth this should be possible at lower speeds, given that in vivo NMR of an aquatic organism has been recently performed for up to 12 h at 2.5 kHz and 5 °C.13 1D NMR Spectroscopy. 1 H spectra were acquired using presaturation for water suppression and the 90° excitation pulse was calibrated for each sample. A standard inversion recovery approach was used for measuring T1 time for each sample, and the recycle delay was set to 5 times the measured T1 value. Spectral width was 20 ppm, with 8192 time-domain points, and 512 scans were recorded. Spectra were processed by applying an exponential multiplication factor of 0.3 Hz to the FIDs, followed by Fourier transformation. All 13C spectra (except for CP) were acquired using a spectral width (SW) of 400 ppm, 16384 time-domain points (NP) (∼164 ms acquisition time) (which provided sufficient digital resolution DR = 2*SW/NP), 2048−4096 scans (as required) and inverse gated 1H decoupling. A standard inversion recovery approach was used for measuring T1 time for each sample; a 5 s recycle delay was employed, which was ∼5 times the measured T1 value. Since the samples were 13 C-labeled, the T1 relaxation times were significantly shorter than what is generally expected from natural isotopic abundance samples, owing to enhanced 13C−13C coupling. Processing was done using an exponential function corresponding to a line broadening of 5 Hz for 13 C inverse gated spectra and 25 Hz for diffusion edited spectra. The same parameters were used for CP-MAS experiments except that it used a spectral width of 300 ppm, 1024 time-domain points, a contact time of 1 ms, and a line broadening corresponding to 25 Hz. Spectral Editing and Scaling. 1H diffusion-based editing was performed with a bipolar pulse pair longitudinal encode-decode (BPPLED) sequence. Scans were collected using encoding/decoding gradients of 1.8 ms at ∼50 gauss/cm and a diffusion time of 180 ms. CPMG (Carr−Purcell−Meiboom−Gill) filtering was achieved using a total delay of 120 ms, with the exception of T2-filtered CP-MAS which employed 2 echoes separated by 7.5 μs prior to cross-polarization. Inverse diffusion editing (IDE), recovery of relaxation losses arising from diffusion editing (RADE) and inverse T2-filtered 13C CP/MAS was done by appropriate spectral subtraction as previously described.8 2D NMR Spectroscopy. 1H−13C heteronuclear single-quantum coherence (HSQC) correlation experiments were collected in phase sensitive mode using Echo/Antiecho encoding and gradients for coherence selection. A total of 256 scans were collected for each of the 256 increments in the F1 dimension. F2 was processed using an exponential function corresponding to line broadening of 15 Hz and F1 using sine-squared functions with a π/2 phase shift and a zero filling factor of 2. 2D correlation spectroscopy (COSY) spectra were acquired on select samples to confirm HSQC NMR assignments of metabolites using the Bioreference databases version 2.0.0 to 2.0.3 and AMIX (Analysis of MIXtures software package, version 3.9.3, Bruker BioSpin, Rheinstetten, Germany). The COSY NMR experiments were done in nonphase-sensitive mode, using gradients for coherence selection. In total, 128 scans and 2048 data points were collected for each of the 196 increments in the F1. Both dimensions were processed using an unshifted sine-squared function and zero filling factor of 2. A magnitude mode was used for projection. Compound Identification and Quantitation. Pattern matching of both 1D and 2D experiments were performed using AMIX against the Bioreference Compound Database using a procedure previously developed for complex mixtures.14 Compounds with a greater than 80% confidence match from automated searches were further selected for detailed manual inspection. Only assignments that exhibited an R2

complementary unaltered metabolite-profiling for the examination of the roles of various biological pathways.

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MATERIALS AND METHODS

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C-Labeled Wheat Seeds. The uniformly 13C-labeled (>97% total carbon content) wheat (Triticum aestivum) seeds were provided by IsoLife (Wageningen, The Netherlands). Using specially designed, airtight high-irradiance growth chambers, with the closed atmosphere containing 97 atom % 13CO2 from pressurized cylinders (Isotec Inc., Miamisburg, OH) for growth. Plants were grown from the seedling stage until full maturity in this atmosphere, which then produced 13Clabeled seeds. Pollination of the wheat was ensured by having efficient internal wind speed. Mineral nutrients were made available as Hoagland-type solutions with micronutrients and iron.11,12 The climate conditions were as follows: irradiance (PPFD) 600 μmol/ m2/s (HPI) during a 16-h day, day/night temperature 24/16 °C, relative humidity 75/85%. Germination. Six 13C-labeled wheat seeds were selected for germination, the mean seed dry weight was 0.0283 g, and variability in seed dry weight was ≤5.75%. The seeds were sandwiched between two sheets of filter paper moist with distilled water in a Petri dish. There was 100% germination of the seeds which was performed in the dark. Sample Preparation. NMR samples were prepared every 24 h for 96 h, which was as long as the seedlings would fit into the 4 mm diameter zirconia rotor (rotor length ∼17 mm). At 0 h, the dry seed was used, and 1 new seedling was used at each time point. For the 5 time points, 0, 24, 48, 72, and 96 h, the specimen measured 0.5, 0.6, 1.1, 3.0, and 3.3 cm, respectively. These measurements were extracted from photographs of the specimens aligned with a 4.8 cm ruler with 0.1 cm increments (Figure 1). The seedlings were measured from the

Figure 1. (A) Intact 13C-labeled wheat seed and seeds germinated for (B) 24 h, (C) 48 h, (D) 72 h, and (E) 96 h. tip of the shoot to the end of the longest root. Montmorillonite clay powder from Source Clays Repository (Chantilly, VA) was prepared by mass in a 1:5 mixture with D2O, this was used to protect the seed/ seedling and ensure it remained intact while spinning. The 4-mm zirconium rotor was filled with the montmorillonite/D2O mixture, and the whole and intact seed/seedling was placed directly into the rotor. The rotor was sealed using a Kel-F top insert, a Kel-F sealing screw, and Kel-F cap. Kel-F is the typical fluorinated (to avoid 1H background signal) thermoplastic used for these MAS accessories. NMR Spectroscopy. All NMR spectra were acquired using an Bruker Avance III 500 MHz 1H spectrometer, fitted with a prototype CMP-MAS 4 -mm 1H−13C−19F−2H probe (Bruker Biospin, Billerica, MA) with an actively shielded z-gradient, at a spinning speed of 6666 Hz. A spinning speed of 6666 Hz results in a rotor period of 150 μs allowing the rotor synchronization of pulses and delays to be easily performed. Spinning faster than 7 kHz is not recommended as commercial seals often leak at higher speeds. All experiments were performed at room temperature and locked on D2O. This lock was maintained for all experiments including solid-state experiments. B

DOI: 10.1021/acs.jafc.7b02421 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry correlation >0.99 between the observed and database shifts were retained as assignments. Where possible, correlations were also confirmed with COSY. While absolute quantitation is theoretically possible from 1D NMR for individual species considerable spectral overlap made it extremely difficult to perform in the present study. Instead relative quantitation of the metabolites and general classes were also performed to indicate how components in the seed change over time. Using the multiintegrate tool in AMIX 1H NMR spectra were normalized over 0−9 ppm to account for seed/seedling differences. The 1H carbohydrate region was defined as 3.10−5.57 ppm and aliphatic lipids defined as 0.57−2.88 ppm. 13C spectra were normalized over the 0−200 ppm region to account for seed/seedling differences. Herein, when changes in triacylglycerides (TAG) and carbohydrates are discussed, they are relative and have been integrated based on the most characteristic and resolved regions. The region most resolved and characteristic of TAG is the double bonds defined as 125−137 ppm. For carbohydrates, the sum of the anomeric carbons were used and defined as 93−103 ppm. The percentage changes were calculated as

%change =

(x − 0 h) × 100% 0h

Figure 2. Carbon spectra obtained for a single 13C-labeled wheat seed at 0 h (dry) and a seedling at 96 h (after imbibition). (A,B) 13C 1D profile. (C,D) Components with unrestricted diffusion (IDE). 1: carbohydrate region, 2: aliphatic amino acids, 3: double bond of TAG, anomeric carbons: AC1, AC2, and AC3.

where x is the normalized integration at a given time point and 0 h is the normalized integration at time 0 h. Individual compounds could not be resolved because the anomeric carbons of several saccharides overlap. However, it was possible to define ranges resulting for a combination of saccharides. To reduce redundancy, the following naming system is used to identify the sugars with anomeric signals overlapping in each spectroscopic range: AC1 for melibiose and Draffinose anomeric carbon signals defined as 101−103 ppm; AC2 for D-glucose, melibiose, and D-xylose anomeric carbons defined as 97−99 ppm; and AC3 for D-glucose, melibiose, D-raffinose, sucrose, and Dxylose anomeric carbons defined as 94−96 ppm. While HSQC shows much higher spectral dispersion and thus reduced spectral overlap when compared to 1D NMR, it cannot be used to derive absolute concentration as 1JCH, which is the basis for coherence transfer in the experiment, varies between structural units. However, if HSQC correlation plots are all acquired under identical conditions, as is the case here, the approach permits relative change across a range of spectra to be monitored. Integration of HSQC was performed using the multi-integrate tool in AMIX after normalization to the total area.

h, respectively. The goal of inverse gated decoupling is to provide representative signal areas in the 13C{1H} spectra by preventing nuclear Overhauser enhancement from 1H. The experiments were deliberately performed using low-power decoupling. Therefore, protons in the solid phase with line width greater than the decoupling field will not be effectively decoupled. Thus, the signals of the spectra in Figure 2 are from the plant components in most phases but signals of the solid phase are suppressed. Upon first inspection, the spectra at 0 and 96 h, respectively (Figure 2A,B) show two noticeable differences. The first is that the sample at 0 h, the dry seed, has some sharp signals that are superimposed on a quite pronounced broader spectral profile. This broad profile is characteristic of gels and semisolids, as well as dynamic amorphous solids, as they all exhibit minimal dynamics compared to truly dissolved metabolites or those in a dynamic gel-like environment, which give rise to sharper signals. For the spectra of the seedling 96 h after first contact of the seed with water, it is clear that it had swollen considerably and the broad profile is less pronounced. The second difference is that there are many more sharp signals in the 96 h spectrum than in the 0 h spectrum, especially in the carbohydrate region and amino acid side chain/aliphatic region. Spectral editing can further emphasize components from specific phases in the seed structure. Inverse Diffusion Editing (IDE) is a difference approach that selects only the components that exhibit unrestricted diffusion (i.e., truly dissolved species).8 Here, unrestricted diffusion is defined as molecules that move physical position (>1 μm) within the sample over the diffusion time of 180 ms, which will include molecules that are truly dissolved. Figure 2C emphasizes the most dynamic components prior to germination in the original seed, whereas Figure 2D shows the mobile components after 4 days of germination which are dominated by mobile carbohydrates. Relative quantitation that permits intercomparison between samples is possible for resolved signals in the 13C{1H} data. This is simply expressed as an approximate relative percentage increase or decrease of a specific signal region at a given time

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RESULTS AND DISCUSSION CMP-NMR spectroscopy was performed every 24 h to study the transformation of an intact 13C-labeled wheat (Triticum aestivum) seed to seedling (Figure 1). The growth of monocotyledonous plants is complemented by physical changes to the seed involving extension of the embryo and the development of cotyledon, radicle, hypocotyls, and shoot. At 24 h (Figure 1B), the radicle can be seen protruding through the bottom of the seed. The length of the wheat embryo/ seedling at 0 h was 0.5 cm and increased during the germination process to 3.3 cm after 96 h, measured from the tip of the shoot to the end of the longest root (Figure 1). For easy visualization, all spectra are presented such that the largest peak in each spectrum is the same size. This was found to be the most useful and logical approach to display a series of samples where the dominant components in the spectrum change with time. For 1D NMR, absolute quantitation was possible for a handful of well-resolved peaks. From 2D NMR, it is not possible to generate absolute concentrations, and only relative percentage changes can be reported. 1D 13C NMR: Unrestricted and Restricted Diffusion Components. Components with Unrestricted Diffusion (Dissolved Components). Figure 2A,B show the 13C{1H} spectra with inverse gated 1H decoupling at 0 h compared to 96 C

DOI: 10.1021/acs.jafc.7b02421 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry relative to the observed signal at 0 h (dry seed) and is most meaningful for minimally overlapping relatively intense signals. The IDE spectrum shows an increase in intensity in the anomeric carbohydrate region (90−110 ppm) at 96 h (roughly 260%), whereas the major form of plant lipids, TAG, decreased about 66% (Figure 2C,D). This is in agreement with literature that suggests that TAG lipids are being mobilized and consumed prior to successful germination.15,16 The increase in carbohydrates and decrease in lipids is a consistent trend in this study and is in accordance with the wider literature.16−20 The signals resulting from anomeric carbons of the carbohydrates are much more defined after 96 h (Figure 2D). Specifically, the AC1, AC2, and AC3 signals at 96 h compared to the 0 h spectrum increased by 500%, 600%, and 50%, respectively, indicating they are released in the solution phase. Based on 2D 1H−13C spectra, see later in this manuscript, the anomeric carbon resonances AC1 and AC3 both have contributions from D-raffinose. It is commonly documented that the raffinose family oligosaccharides (RFOs), which are linear chains of galactosyl residues attached to the glucose moiety of sucrose via an α-(1 → 6) glycosidic linkages, are significantly reduced within hours of germination.21 The liberated galactose is considered to be a critical component of the saccharide signaling pathway in seeds in addition to providing energy and the building blocks for structural components.22 Typically RFOs can make up as much as 16% dry mass of the seed.21 Based on the literature, it would be expected that the total amount of D-raffinose would decrease in the seedling compared to the dry seed. However, the anomeric carbon AC3 is also consistent with sucrose, D-glucose, and Dxylose, besides D-raffinose, while the AC2 signal, is dominated by the contributions from D-glucose and D-xylose, a precursor to hemicelluloses.23 Therefore, the 600% increase in the intensity of the AC2 signal is primarily associated with increase in D-glucose and D-xylose. Since the AC3 signal only increased by 50% it can be inferred that there was a reduction in Draffinose (and/or sucrose) along with the increase of either Dxylose and/or D-glucose, as expected. The signal-to-noise ratios increase between 0 and 96 h is indicative of more metabolites entering the soluble phase. At 0 h the dry seed will not have much contribution to the soluble phase, but as it imbibes/swells and as the seedling grows, it will have a higher contribution to the soluble phase than before. In addition, at 96 h, the roots and shoot, which have a higher water content compared to the dry seed (0 h), will provide additional signals to the freely diffusing species (IDE spectrum) along with newly synthesized metabolites. For completion, Figure 3 (and additional discussion in the supporting information) shows the 1H NMR spectra of the same samples at the different time points (0−96 h). These spectra represent all components present with the exception of true solids. As such, changes in these spectra provide a more comprehensive overview of the changes in the whole seed/ seedling, whereas spectral editing provides targeted information on the individual subphases within the organisms. Besides the obvious increase in carbohydrates, clear increase in aromatic amino acids such as tryptophan, tyrosine, phenylalanine, phenylethylamine, histidine, and the glycoside arbutin, were observed. Components with Restricted Diffusion. Diffusion editing (DE) emphasizes the NMR signals from molecules that have restricted/slow diffusion. Components with restricted diffusion is defined here as compounds that remain in the same

Figure 3. 1H spectra of the 13C-labeled wheat seed/seedling at time (A) 0 h, (B) 24 h, (C) 48 h, (D) 72 h, and (E) 96 h. (1) Aromatic, (2) tryptophan, (3,4) phenylalanine and phenylethylamine; (5) histidine, phenylalanine, phenylethylamine, and tryptophan; (6) tryptophan and tyrosine; (7) arbutin, tyramine, and tyrosine; (8) alkene; 9) anomeric; 10) sucrose and D-raffinose; (11) D-glucose, melibiose, and D-xylose; (12) melibiose and D-raffinose; (13) D-glucose, melibiose, and Dxylose; (14) overlapping carbohydrate; (15) aliphatic. Where more than one metabolite is listed, it means they are overlapping. Residual water remaining after water suppression occurs as distortions from ∼4.5−5.5 ppm.

place throughout the diffusion delay (i.e., move less than ∼1 μm in 180 ms) and whose signals are refocused at the end of the diffusion period. This includes structural components such as cell walls, smaller molecules bound to surfaces, and very large macromolecules that hardly exhibit any translational diffusion. The dominant signals showing restricted diffusion are consistent with lipids, specifically those of TAG (Figure 4). The signals consistent with a generic form of TAG are confirmed by 2D spectroscopy. The generic form of TAG consists of long chained molecules varying in acyl chain-lengths and functionality.24,25 TAG are the primary fuel source for the transformation of seed to seedling by its breakdown, to serve as both an energy and a carbon source.26,27 At 96 h (Figure 4B), the TAG signal decreased by about 34% (Figure 4A) and the anomeric carbohydrates increased by about 390% (Figure 4B). These relative changes are consistent with the trend seen in the IDE spectra earlier. These TAG signals can be easily identified by 1H−13C HSQC. The editing techniques show lipids in many different forms, from liquids all the way to semisolids. 1 H -13C HSQC: Components with Unrestricted and Restricted Diffusion. Since 1D NMR spectra have a considerable number of overlapping signals in complex natural samples such as seeds, it becomes necessary to acquire 2D 1H −13C HSQC (heteronuclear single quantum coherence) experiments as well, to help provide a more detailed assignment. HSQC provide connectivity information between directly bonded 1H and 13C nuclei as well as additional spectral D

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oleaginous seeds mobilize their reserves of TAG during germination.30 Although the major nutrient reserve is starch, the TAGs are a major nutrient reserve in some tissues and can act as both a substrate (source of carbon) and energy source during germination before nutrients are mobilized from the endosperm.30 A whole wheat grain contains 2−4 wt % lipid about half of which is TAG.30 For an African grass the free fatty acid formed 0.1% of the total seed lipid and increased to no more than 0.5% during germination while the TAG content decreased from 68% to 31% of the total lipid content after germination in the dark.28 Phosphatides are another contributor to the lipid content.28 All lipids produce equivalent CH2 and CH3 signals, contributing to spectral overlap. In addition, this 2D HSQC experiment detects the total mobile TAG content and thus excludes any semisolid and solid-phase TAGs, which are detected by the 1D 13C RADE and CP-MAS experiments. The reported concentration of TAG, in the African grass seeds and seedlings germinated in the dark for 3 days, is 58.8 mg/g and 16.7 mg/g, respectively,28 which is a 72% decrease. Our measured 58% change of mobile TAG content of the wheat seed is lower than the percentage change of the total TAG content of the African grass seeds. It is likely that there are TAGs in the gel and/or solid phase that are not detected by this experiment, which would be expected more so for the dry seed compared to the seedling imbibed for 96 h. Percentage change measurements of TAG signals in the germinated wheat seed in this study are relatively consistent with but slightly lower than the reported change of TAG content in the African grass seeds,.28 Carbohydrates. It was observed that a positive percentage change of D-glucose while fructose and sucrose displayed a negative percentage change. This is in contrast to the reported concentrations of these sugars within germinating Arabidopsis seeds31 which show no clear trend. Sugars are metabolized for growth and used as a source material for building cell walls.32 Generally, the TAG lipids are considered to be consumed for energy via the Krebs cycle, but endospermic TAG lipids can be consumed via glucogenesis, to fuel skotomorphogenesis, a process where seedlings grown in the dark develop long hypocotyls.33 The seeds in this study were never exposed to light, in contrast to the reported germinating Arabidopsis seeds, which were exposed to light in the last 24 h of the 96 h imbibition period. The consumption of TAG via glucogenesis to fuel skotomorphogenesis could explain our observed increase of the D-glucose signal in contrast to the reported germinating Arabidopsis seeds,31 which show no clear trend. Shikimate-Derived Aromatic Amino Acids. Like other shikimate-derived aromatic amino acids, phenylalanine shows a positive percentage change (Figure 6). This is consistent with the changes observed in germinating Arabidopsis seeds.31 Aromatic amino acids are involved in organogenesis34 and are known to form structural components like lignin. In addition, aromatic amino acids are the base of energy compounds, including ubiquinone and those that are catabolised by the Krebs cycle.35 Glutamine and Asparagine. Typically, germinating seeds accumulate relatively large amounts of either glutamine or asparagine (nitrogen-containing compounds), while only present in relatively low quantities in the dry seed; specifically, the wheat seed has been observed to be of the glutamineproducing group.30 The results here are consistent with these observations given that over 100% positive increase of the glutamine signals was measured, while there was no observed

Figure 4. Diffusion edited (DE) carbon spectra of components with restricted diffusion for a single 13C-labeled wheat seed at (A) 0 h and (B) 96 h. 1: double bond of TAG, AC: anomeric carbons.

dispersion due to the added dimension. HSQC is not an editing experiment per se, however it does contain relatively long delays during which signals from solids, and likely most semisolids, will relax. As such, HSQC will emphasize the mobile components (dissolved, and dynamic solids such as gels and swollen materials). Identification of Metabolites in HSQC. The additional dispersion reduces overlap considerably which permits the use of NMR databases to aid with assignments. At 0 h (Figure 5A,C) 20 metabolites were identified but at 96 h (Figure 5B and D) over 40 metabolites were identified, some of which were further confirmed through 1 H− 1 H COSY NMR correlations. Where possible, relative quantitation of the wellresolved metabolites was performed and are summarized in Figure 6. TAG. Along with small soluble metabolites, the plant lipids known as TAGs are prominently seen in the HSQC of the 13Clabeled seedling at 96 h. These signals were assigned to a TAG consisting of 1.5 linoleic acids (18:2), 1 oleic acid (18:1), and 0.5 (16:0) palmitic acid based on experimental and theoretical percent mole fraction measurements of these principle fatty acid moieties within the triglycerides of germinating Andropogon gayanus seeds,28 and the spectral simulation and comparison of a sum of two TAG structures, giving these fatty acid moiety ratios, and are consistent with literature findings.29 All the resonances consistent with a TAG structure show a greater than 50% measured decrease between 0 and 96 h (Figure 6). This decrease is expected since wheat grains like all E

DOI: 10.1021/acs.jafc.7b02421 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 5. 1H−13C HSQC spectral regions of (A,C) a single dry 13C labeled wheat seed (0 h), (B,D) an early growth (96 h) seedling. All the identified resonances are marked with a square color coded to its corresponding metabolite. The resonance contours consistent with a generic TAG structure have been thickened.

delays. A RADE spectrum emphasizes semisolid components, specifically signals with fast relaxation and broad lines. Lipids, dominated by TAG again, were consumed over time, while the carbohydrate signals show an increase with time (Figure 7). Specifically, there is a 37% decrease of the TAG signal in the spectrum at 96 h relative to that at 0 h (Figure 7), whereas the carbohydrates signals increase by 380%. This is consistent with the trend seen in IDE and DE, demonstrating that it is not only the metabolites that are changing but also semisolid components of the seed/seedling. There is an increase of the carbohydrate anomeric carbons for all phases at 48 h, whereas the decrease of the TAG signal occurs in the semisolid phase at 48 h (Figure 8). In seeds, TAGs are stored into subcellular droplets called storage lipid bodies surrounded by a monolayer of phospholipids. It has been proposed that in mature seeds, once oleosin (a protein found in storage lipid bodies) interacts and retains the phospholipids and TAGs, the TAG lipids become less mobile.36 This may partially explain why some TAG lipids are present in a semisolid form within the seeds. RADE is an experiment that helps recover signals from semisolid components that could otherwise be missed when using a diffusion-based editing scheme. However, as RADE

change of asparagine signal. The results support the conclusion that glutamine is involved in the complex mechanisms that induces and controls TAG metabolism in the storage tissues of germinating wheat seeds.30 Limitations of HSQC. It is not possible to differentiate whether the metabolite signal was originally undetectable because of its state (solid vs soluble), or if its concentration was below detection the detection limit (≪ 0.1 mmol/L), or because it was originally absent altogether. HSQC NMR detects components with liquid- or gel-like properties but will not detect rigid solids which will relax during the relatively long evolution periods in the 2D experiment. All the metabolites present at 0 h were also present at 96 h. 1D 13C NMR: Semisolid Components. One disadvantage of diffusion editing is the relatively long delays required to encode/decode self-diffusion. During these delays signal components with fast relaxation can be strongly attenuated. Therefore, if IDE and DE are used alone, components with fast relaxation such as semisolids may be underestimated. As introduced by Courtier-Murias et al.8 an experimental approach termed recovery of relaxation losses arising from diffusion editing (RADE), can recover the signal lost during these long F

DOI: 10.1021/acs.jafc.7b02421 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 6. Integral intensities from the HSQC spectra, normalized to the integration of the total spectrum (to compare values of two different spectra), divided by total protons of the assigned functional group (to equate all functional group intensities, ex: methyl (3 protons) is 3/2× more intense than a methylene (2 protons)) and multiplied by a factor to obtain rounded integer values for comparison. The integral intensity is displayed as either the average of all the assigned metabolite’s resonances (Avg) or single resonance of the metabolite. The error bars represent the standard deviation of triplicate integrations or the propagation of these standard deviation errors for the average values. Abbreviations include TAG, triacylglycerides; glc, glucose; fru, fructose; suc, sucrose; glu, glutamic acid; meli, melibiose; Lys, Lysine; Gln, glutamine; Ile, L-isoleucine; Nle, norleucine; raf, raffinose; GABA, γ-aminobutyric acid; PHOS, phosphoethanolamine; Arg, Arginine; Cys, Cystiene; Val, valine; Thr, threonine; Ala, alanine; xyl, D-xylose; Pro, proline; Leu, Leucine; Met, methionine; Gly, glycine; β-Ala, β-Alanine; Tyr, Tyrosine; Phe, Phenylalanine.

Figure 7. 13C spectra of semisolid components (RADE) of labeled wheat seed/seedling at time (A) 0 h, (B) 24 h, (C) 48 h, (D) 72 h, and (E) 96 h. 1: double bond of TAG, AC: anomeric carbons.

Figure 8. Percentage change of both the anomeric carbohydrate signals (93−103 ppm) and the TAG (125−137 ppm) and from the edited spectra: IDE (dissolved small freely diffusing), DE (rigid gels semisolids with restrictive diffusion), RADE (large fast relaxing semisolids), at time 24, 48, 72, and 96 h relative to the dry seed at 0 h. Three integration files were created to obtain mean values with standard deviations, used to derive the error bars.

employs low power decoupling, the signals of true solids are underrepresented. To efficiently detect true solids, crosspolarization magic angle spinning (CP-MAS) is required. The intensity of the CP signals depends on the strength and number of the dipolar interactions between a carbon and its surrounding 1H nuclei, where the strength is attenuated by molecular motion. As a result, the signals of true solids are selected most efficiently. Unrestricted and restricted diffusing components are not detected as they are too dynamic, whereas dynamic solids and semisolids may be selected to some extent depending on the rigidity of the H−C bonds.8 Additional

editing which combines relaxation filters with CP can help differentiate signals from restricted/semisolids and true solids. 1D 13C NMR: Rigid Components. CP-MAS emphasizes the rigid components in the seed/seedling. As signals in the solid-state tend to be broader than their more dynamic G

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consumption of the dynamic solid components. There is a drop in the percentage change of the starch signal at 72 h, which correlates with the initial growth of the shoot (Figure 1). The cellulose signal shows a small decrease with time, which could be indicative of a degradation of cell walls after programmed cell death.40,41 In addition to structural information, this approach of using a variety of CP-MAS NMR experiments provides the physical state of the components within the plant. Specifically, the spectral editing of the solid-state data demonstrates that the dynamic solids and the truly rigid solids are composed mainly of cellulose, hemicellulose and starch, which is the main component in the seed endosperm.42,43 Potential of CMP-NMR as a Tool To Follow Biological Plant Processes. In summary, CMP-NMR was used to elucidate changes in intact samples of 13C labeled wheat seeds and germinated seedlings. It was demonstrated that changes in the major metabolites, such as the aromatic amino acids that are precursors to a large variety of secondary metabolites. However, other relevant metabolites such as hormones are below the limits of detection of this technique, while nucleobases, such as adenine and uridine, are just above the limit. The spectral changes at the 24 h increments consistently show an increase in the carbohydrate signal, accompanied by a decrease in lipids (catabolism of TAG) in all experiments. Relative quantitation based on HSQC revealed that a third of the identified 47 observable mobile and gel phase metabolites decreased during the growth period from 0 to 96h, including TAG, fructose, sucrose, cysteine, and betaine. Another third more than doubled, including, D-glucose, glutamine, and melibiose (Figure 6). Components with restricted diffusion (TAG) recovered by RADE and rigid components (starch dominant) were discussed in terms of TAG interaction with the protein oleosin, which possibly explains its existence in this semisolid/restricted state and starch production and consumption during and after germination. The use of CMP-NMR to study germination and early stage growth for the first time adds an extra dimension to the collaborative effort to understand plant physiology in conjunction with genomics, proteomics, and classic metabolomics.

counterparts in the gel/liquid states, solid-state NMR spectra are often less resolved. However, when combined with other CMP-NMR experiments (e.g., IDE for soluble, DE for components with restricted diffusion), cross-assignment is possible which in turn helps identify components in the solid-state. As changes in the solid-state spectra of the seeds are less prominent with time, only major changes between the 0 and 96 h samples are compared. Even for the same experiment, the 0 h spectrum compared to that at 96 h appears similar; indicating that the solid profile does not change significantly over time (Figure 9A,B). To further investigate changes, a 1H

Figure 9. 13C CP-MAS spectra obtained for a single 13C-labeled wheat seed at 0 h (dry seed) to a seedling at 96 h (after swelling/ germination). (A,B) CP-MAS, (C,D) dynamic solids (T2 filtered), (F) most rigid (rigid solids). As labeled on above, 1: C6 of starch and cellulose, 2: C4 of noncrystalline starch 3: C6 of starch, 4: C6 of cellulose, 5: mostly −CH2− of TAG. Finally, (G) the percentage change, relative to the dry seed (0 h), of the C6 signals from both the starch and cellulose within the dynamic solids (T2 filtered) 13C spectra (C,D) at time 24, 48, 72, and 96 h. An average of three fits with greater than 94% overlap were used to obtain mean values with standard deviations, from which the error bars were derived. Assignment of the starch signals were based on literature.37,38

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ASSOCIATED CONTENT

S Supporting Information *

T2 relaxation filter was applied prior to the CP experiment which emphasizes dynamic solid components (Figure 9C,D). In turn the difference spectrum provides a subspectrum of the rigid-solid components (Figure 9E,F). The noncrystalline C4carbon signal37 in starch (Figure 9D) assigned according to the literature,37,38 is most prominent in the T2-filtered spectra (Figure 9C,D). Likewise, the CH2 peak of aliphatic TAG at ∼33 ppm, are most prominent in the T2-filtered spectra. The same peak, however, does not appear in the rigid solids experiment obtained from spectral editing (Figure 9E,F), suggesting that little, if any, TAG lipids are present in a crystalline solid-state, as expected. The C6 from starch and cellulose appears as one signal in the CP-MAS spectra (Figure 9A,B), and is split in the T2-filtered spectra (Figure 9C,D). This differentiation occurs because starch is less rigid/crystalline than cellulose and is thus emphasized in the mobile-solids spectra (T2 filtered).39 From the spectral deconvolution volumes of these C6 T2-filtered 13C signals, the percentage change for both the starch to cellulose signals were calculated. The flux of the C6 signals from both the starch and cellulose, at 24 h increments suggests a periodic production and

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b02421. 13 C spectra at the different time points, the simulated HSQC of TAG, and a COSYof the seedling at 96 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +1 416-287-7547. Fax: +1 416-287-7279. ORCID

Rudraksha Dutta Majumdar: 0000-0001-9232-7331 Myrna J. Simpson: 0000-0002-8084-411X André J. Simpson: 0000-0002-8247-5450 Present Address ¶

(R.D.M.) Bruker Ltd., 555 Steeles Avenue E, Milton, ON, Canada L9T 1Y6

Notes

The authors declare no competing financial interest. H

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ACKNOWLEDGMENTS We would like to thank the Strategic (STPGP 494273-16) and Discovery Programs (RGPIN-2014-05423), the Canada Foundation for Innovation (CFI), the Ontario Ministry of Research and Innovation (MRI), and the Krembil Foundation for providing funding. A.S. would like to thank the Government of Ontario for an Early Researcher Award. The authors thank E. Koning and R.J.P. Baan (Koppert Cress BV) for kindly providing nonlabeled seeds of Brassica oleracea L. from which the 13C-labeled plants and seeds were produced. The Dutch Innovation Program Food and Nutrition Delta is acknowledged for financial support (Project FND09002).

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ABBREVIATIONS USED BPPLED, Bipolar Pulse Pair Longitudinal Encode-Decode; CMP, Comprehensive Multi-Phase; DE, Diffusion Editing; HPI, High Power Illuminator; IDE, Inverse Diffusion Editing; PPFD, Photosynthetic Photon Flux Density; RADE, Relaxation Losses Arising from Diffusion Editing; RFO, Raffinose Family Oligosaccharides; TAG, Triacylglycerides

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