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Characterization and Germination of 13C Labeled Seedsby Comprehensive Multiphase NMR Spectroscopy
by
Leayen Lam
A thesis submitted in conformity with the requirementsfor the degree of Master of Science
Department of ChemistryUniversity of Toronto
© Copyright by Leayen Lam (2014)
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Characterization and Germination of 13C Labeled Seeds by
Comprehensive Multiphase NMR Spectroscopy
Leayen Lam
Master of Science
Department of ChemistryUniversity of Toronto
2014
Abstract
Seeds are complex entities, within which the intricate processes of germination and
early growth occur. We describe here a novel technique (initially developed by members
of our group in 2012) termed Comprehensive Multiphase (CMP)-NMR spectroscopy,
which is capable of simultaneous solution-, gel-, and solid-state analysis. CMP-NMR
was applied to intact seeds where all components are studied and differentiated in situ.
Characterization, germination and early growth of seeds were studied by variety of 1D
and 2D 1H and 13C CMP-NMR experiments. Various metabolites, lipids, carbohydrate
biopolymers and structural carbohydrates were first identified and further studied in
germination and early growth stages. This research demonstrates the utility of CMP-
NMR as a powerful tool to better understand the composition of seeds and processes
underlying early seed growth.
iii
Acknowledgments
I am most grateful to my supervisor, Prof. Andre Simpson, for giving me this wonderful
opportunity and providing me with assistance and insightfulness throughout my
research experience. "Thanks loads" Andre! To Prof. Myrna Simpson for being my
second reader of this thesis and contributing valuable and thorough comments and
corrections. To Prof. Kagan Kerman for being on my committee and guiding me with
continued optimism. Thank you all.
Many thanks to past and present lab mates in the A. Simpson, M. Simpson, K. Kerman
groups and other lab groups for all the help, advice and friendship. I would also like to
thank the UTSC community; the environmental chemistry faculty and graduate students;
and University of Toronto as a whole because completing my M.Sc. would not have
been possible without all their support.
Lastly, I would like to thank Anthony Veloso a thousand times over for being there when
I doubted myself and encouraging me to carry on.
To all: "Happiness held is the seed; happiness shared is the flower"
- John Harrigan
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Table of Contents
Acknowledgments........................................................................................................... iii
Table of Contents............................................................................................................ iv
List of Tables ................................................................................................................ viii
List of Figures ................................................................................................................. ix
List of Abbreviations........................................................................................................ xi
List of Appendices.......................................................................................................... xii
Preface ......................................................................................................................... xiii
Chapter 1. Introduction.................................................................................................1
1.1 Nuclear Magnetic Resonance (NMR) Spectroscopy .............................................1
1.1.1 Traditional NMR..........................................................................................2
1.1.1.1 Solution-state NMR .......................................................................2
1.1.1.2 Magic Angle Spinning (MAS).........................................................4
1.1.1.2.1 HR-MAS for Gels ................................................................... 5
1.1.1.2.2 CP-MAS for Solids ................................................................. 6
1.1.2 CMP-NMR ..................................................................................................7
1.1.2.1 Spectral Editing .............................................................................7
1.1.2.1.1 Inverse Diffusion Editing (IDE) ............................................... 8
1.1.2.1.2 Diffusion Editing (DE)............................................................. 8
1.1.2.1.3 Recovering relaxation losses Arising from Diffusion Editing(RADE)................. ................................................................................... 8
1.1.2.1.4 T2 filtered CP-MAS................................................................. 9
1.1.2.1.5 Inverse T2 filtered CP-MAS .................................................... 9
1.1.2.2 Potential use of CMP-NMR............................................................9
1.2 Seeds..................................................................................................................10
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1.2.1 Importance of studying seeds...................................................................10
1.2.2 Germination..............................................................................................11
1.3 Previous studies on seeds ..................................................................................12
1.3.1 Sample preparation for liquid chromatography mass spectrometrymass spectrometry (LC-MS/MS) ..............................................................12
1.3.2 Sample preparation for gas chromatography flame ionization detection(GC-FID)...................................................................................................13
1.3.3 Advantages of using CMP-NMR...............................................................14
1.4 Isotopic labeling ..................................................................................................14
1.5 Objectives of this study .......................................................................................15
Chapter 2. Comprehensive Multiphase NMR Spectroscopy of Intact 13C-LabeledSeeds.............. ..........................................................................................................17
2.1 Abstract ...............................................................................................................17
2.2 Introduction .........................................................................................................18
2.3 Materials and methods........................................................................................21
2.3.1 13C Labeling of the seeds .........................................................................21
2.3.2 Sample preparation ..................................................................................22
2.3.3 1D NMR spectroscopy..............................................................................23
2.3.4 Spectral Editing ........................................................................................24
2.3.5 2D NMR Spectroscopy .............................................................................25
2.3.6 NMR Spectroscopy of non-labeled samples.............................................26
2.3.7 Compound Identification...........................................................................26
2.4 Results and discussion .......................................................................................27
2.4.1 Comprehensive Multiphase (CMP)-NMR spectroscopy ...........................27
2.4.2 Detailed Analysis of Wheat Seed .............................................................28
2.4.2.1 Components with unrestricted diffusion (Soluble components) ...28
vi
2.4.2.2 Components with restricted diffusion and semi-solidcomponents via diffusion based editing .......................................31
2.4.2.3 Solid components ........................................................................35
2.4.3 Comparing wheat, broccoli and corn seeds..............................................38
2.4.4 Other considerations ................................................................................41
Chapter 3. Elucidating structural and metabolic change during germination andearly growth of 13C labeled seeds through Comprehensive Multiphase NMRspectroscopy .............................................................................................................45
3.1 Abstract ...............................................................................................................45
3.2 Introduction .........................................................................................................46
3.3 Materials and methods........................................................................................48
3.3.1 13C labeled wheat seeds...........................................................................48
3.3.2 Germination..............................................................................................49
3.3.3 Sample preparation ..................................................................................49
3.3.4 NMR Spectroscopy...................................................................................49
3.3.4.1 1D NMR spectroscopy.................................................................50
3.3.4.2 Spectral editing and scaling.........................................................51
3.3.4.3 2D NMR spectroscopy.................................................................51
3.3.5 Compound Identification and quantification..............................................51
3.4 Results and discussion .......................................................................................53
3.4.1 1D 13C NMR: Components with unrestricted and restricted diffusion .......56
3.4.1.1 Components with unrestricted diffusion .......................................56
3.4.1.2 Components with restricted diffusion...........................................59
3.4.2 2D 1H -13C HSQC: components with unrestricted and restricteddiffusion ....................................................................................................60
3.4.2.1 Components with unrestricted diffusion.......................................60
3.4.2.2 Components with restricted diffusion ...........................................62
vii
3.4.3 1D 13C NMR: Semi-solid components ......................................................64
3.4.4 1D 13C NMR: Rigid components...............................................................66
3.5 Future directions .................................................................................................68
3.5.1 ERETIC II .................................................................................................68
3.5.2 Larger diameter CMP probes ...................................................................68
3.6 Conclusion ..........................................................................................................69
Chapter 4. Conclusions and future directions ............................................................71
4.1 Conclusions.........................................................................................................71
4.2 Future directions .................................................................................................73
4.2.1 Future seed research ...............................................................................74
4.2.2 In vivo studies...........................................................................................74
4.2.3 Larger probes ...........................................................................................75
4.2.4 Cryoprobe technology ..............................................................................75
4.2.5 31P and 15N NMR......................................................................................75
4.2.6 Phytoremediation......................................................................................76
4.2.7 Potential CMP experimental design..........................................................77
References ....................................................................................................................78
Appendices ....................................................................................................................89
viii
List of Tables
Table 2-1 Proton (1H) and carbon (13C) chemical shift assignment of fatty acid and
lipidic components of single 13C labeled (97 atom %) wheat seed................................ 32
ix
List of Figures
Figure 2-1 13C NMR spectra of a single 13C labeled wheat seed. a) 1D carbon profile. 1:
Carbonyls, 2: guanidine group carbon of arginine, 3: aromatic, 4: double bonds, 5:
ethylene, 6: anomeric carbons, 7: overlapping carbohydrate and amino acids, 8:
methanol, 9: amino acids, 10: aliphatic. b) Spectrum showing the components with
unrestricted diffusion (soluble) (by IDE). 11: Small sugars. c) HSQC spectrum showing
color-coded soluble/mobile species as determined by AMIX Bruker Bio-reference
spectra database. See Appendix Figure A 2 for an expansion. .................................... 30
Figure 2-2 13C NMR spectra of a single 13C labeled wheat seed. a) Components with
restricted diffusion (by DE). b) Semi-solids (by RADE). c) HSQC with TAG signals in
black and all others signals grey. d) ACD/Labs HSQC simulation of generic TAG
structure (overlaid). ....................................................................................................... 34
Figure 2-3 13C NMR spectra of a single 13C labeled wheat seed. a) True solids (by CP-
MAS), 1: carbonyls in lignins, hemicelluloses and proteins, 2: double bonds (lignins), 3:
C1 of cellulose and hemicelluloses, 4: C1 of starch (anomeric carbon) 5: C4 of
crystalline cellulose, 6: C4 of amorphous cellulose, hemicelluloses and/or starch, 7: C2,
C3, C5 in celluloses, hemicelluloses and starch and C6 starch branch points, 8: C6 in
celluloses, hemicelluloses and starch, 9: amorphous CH2, 10: aliphatic. b) Spectral
editing to emphasize rigid solids. c) T2 filtered CP-MAS to emphasize dynamic solids d)
DARR to highlight connectivities between carbons for the truly solid components ....... 37
Figure 2-4 13C NMR spectra comparing 13C labelled wheat, broccoli and corn seeds. a-
c) unrestricted diffusion (soluble) components (by IDE). d-f) restricted diffusion (by DE).
g-i) Semi-solids (by RADE). j-l) True solids (by CP-MAS). Figure is labeled as follows:
the -CH3 of TAG is marked with an *, 1: dominated by fructose in corn; 2:
triacylglycerides; 3: carbonyls (result of increased protein content); 4: aromatic (result of
increased protein content); 5: α carbon of amino acids; 6: dominated by aliphatic amino
acids; 7: aliphatic -(CH2)- (dominated by TAG) ............................................................. 40
x
Figure 3-1. a) Intact 13C labeled wheat seed and seeds germinated for b) 24 hrs, c) 48
hrs, d) 72 hrs and e) 96 hrs........................................................................................... 54
Figure 3-2. Comparison of carbon spectra obtained for a single 13C labeled wheat seed
at 0 hour to a seedling at 96 hour. a-b) 13C 1D profile, c-d) Components with
unrestricted diffusion (IDE). As labeled on above, 1: carbohydrate region, 2: aliphatic
amino acids, 3: double bond of TAG, anomeric carbons: AC1, AC2 & AC3. ................ 56
Figure 3-3. Comparison of carbon spectra of components with restricted diffusion (DE)
for a single 13C labeled wheat seed at a) 0 hour and b) 96 hour. As labeled on above, 1:
double bond of TAG, AC: anomeric carbons. ............................................................... 59
Figure 3-4. Comparison of 1H-13C HSQC of a) a single 13C labeled wheat seed at 0
hour to b) a seedling at 96 hour. ................................................................................... 61
Figure 3-5. 1H-13C HSQC at 96 h with the TAG signals highlighted in black. From 1D
editing approaches it can be seen that TAG is most gel-like of all the components
detected by HSQC NMR............................................................................................... 63
Figure 3-6. 13C spectra of semi-solid 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. As labeled on above,
1: double bond of TAG, AC: anomeric carbons. ........................................................... 64
Figure 3-7. Comparison of CP-MAS spectra obtained for a single 13C labeled wheat
seed at 0 hour to a seedling at 96 hour. a-b) CP-MAS, c-d) dynamic solids (T2 filtered),
e-f) most rigid (rigid solids). As labeled on above, 1: C6 of starch & cellulose, 2:non-
crystalline material for C4 of starch 3: C6 of starch, 4: C6 of cellulose, 5: mostly -CH2- of
TAG .............................................................................................................................. 66
xi
List of Abbreviations
AMIX Analysis of MIXtures
ASE Accelerated Solvent Extraction
BPLED Bipolar Pulse pair Longitudinal Encode-Decode
CMP-NMR Comprehensive MultiPhase Nuclear Magnetic Resonance
COSY COrrelation SpectroscopY
CP-MAS Cross Polarization Magic Angle Spinning
CPMG Carr Purcell Meiboom Gill
DARR Dipolar Assisted Rotational Resonance
DE Diffusing Editing
ERETIC Electronic REference To access In vivo Concentrations
FID Free Induction Decay
GC-FID Gas Chromatography Flame Ionization Detector HR-MAS
HR-MAS High Resolution Magic Angle Spinning
HSQC Heteronuclear Single Quantum Coherence
IDE Inverse Diffusion Editing
LC-MS/MS Liquid Chromatography Mass Spectrometry Mass Spectrometry
MAS Magic Angle Spinning
NMR Nuclear Magnetic Resonance
NOESY Nuclear Overhauser Effect SpectroscopY
NS Number of Scans
PURGE Presaturation Utilizing Relaxation Gradients and Echoes
RADE Relaxation recovery Arising from Diffusion Editing
RAMP CP-MAS RAMPed amplitude cross polarization magic angle spinning
RF Radio Frequency
SFE Supercritical Fluid Extraction
TAG TriAcylGlyceride
TOCSY TOtal Correlation SpectroscopY
xii
List of Appendices
Figure A 1 a) Generic triacyclglyceride (TAG) structure with assignments labeled. b) and
c) are example 1H-1H COSY and 1H-1H TOCSY spectra, respectively, both scaled to
show assignments of the dominant TAG structures...................................................... 89
Figure A 2 . Expansion of Figure 2-1c, Heteronuclear single quantum coherence
(HSQC) spectrum showing as assignments of metabolites determined by AMIX Bruker
Bio-reference spectra database. ................................................................................... 90
Figure A 3. CP-MAS of single wheat seed. a) 13C labeled, number of scans (NS) = 2K.
b) Natural abundance, NS=19K .................................................................................... 91
Figure A 4. 1D 13C profile of single wheat seed. a) 13C labeled, NS= 2K. b) Natural
abundance, NS=10K..................................................................................................... 92
Figure A 5. HSQC of single wheat seed. a) 13C labeled, NS=12. b) Natural abundance,
NS=400,........................................................................................................................ 93
Figure A 6. 1H spectra of the 13C labeled wheat seed/seedling using presaturation to
suppress the water signal at time a) 0 h. b) 24 h, c) 48 h, d) 72 h and e) 96 h. Spectra
labeled as follows: 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 D-xylose, 14) overlapping carbohydrate, 15)
aliphatic. Where more than one metabolite is listed means they are overlapping.
Residual water remaining after water suppression occurs as distortions from ~4.5-5.5
ppm and is most prominent in the 24hr and 48hr samples. This likely in part arises from
the water in these samples being broader (i.e. soaking into drier material more and
being inhomogeneous) and thus, more challenging to suppress .................................. 94
xiii
Preface
Two chapters of this thesis are manuscripts that have been either published in a peer-
reviewed journal (Chapter 2) or is being prepared for submission (Chapter 3). Thus, this
thesis may contain unavoidable repetition.
1
Chapter 1. Introduction1
1.1 Nuclear Magnetic Resonance (NMR) Spectroscopy
Nuclear Magnetic Resonance (NMR) spectroscopy is based on the magnetic properties
of atomic nuclei. Specifically, when magnetically active nuclei (e.g. 1H, 13C, 19F) are
placed in a magnetic field most of the magnetic moments align with the field at a lower
energy but some of the magnetic moments align against the field at a higher energy
(Bruice, 2007). Upon irradiation with a radio frequency (RF) pulse that corresponds to
the energy gap between the lower and higher energy levels (ΔE), some of the spin
states in the lower energy are promoted to that of higher energy. Because the RF pulse
covers a range of frequencies, individual nuclei will absorb the ΔE each requires to be
promoted to the higher energy level. When the spin returns to their original energy level,
energy is emitted at the same frequency absorbed and is interpreted by a detector. This
produces a complex signal known as the free induction decay (FID) at a frequency
corresponding to ΔE on a time domain. A mathematical operation called Fourier
transform is applied to the FID to convert the time domain to a frequency domain
(Keeler, 2011). Traditional NMR spectroscopy is limited to single-state sample
characterization, which may require extraction of soluble components for solution-state
NMR or separation and treatment of solid materials for solid-state NMR. Recently in
2012, members of our group developed a new probe and termed it Comprehensive
Multiphase (CMP) NMR which is capable of executing all aspects of solution, gel and
solid NMR experiments on an intact sample so that all organic components in all phases
1 Written by Leayen Lam with critical comments from Myrna J. Simpson and André J. Simpson.
2
can be studied simultaneously (Courtier-Murias et al., 2012). Before discussing CMP in
detail, traditional NMR (solution, High Resolution Magic Angle Spinning (HR-MAS) for
gels and solid-state) needs to be addressed first.
1.1.1 Traditional NMR
1.1.1.1 Solution-state NMRSolution-state NMR was probably was the first type of NMR spectroscopy developed
and is still most common today. Solution-state probes are designed with low-power
electronics, a lock channel and gradients (Simpson et al., 2013). Sample preparation is
often required to extract the soluble components prior to analysis. Usually, solution-
state experiments are performed using a borosilicate sample tube 1.7 or 5 mm in
diameter.
It is inevitable that solvent signals will appear in the acquired spectra since protons are
common in many solvents, but these signals are reduced with the use of deuterated
solvents. Deuterium is an isotope of 1H, also known as heavy hydrogen, 2H or D, which
is not magnetically active and thus, does not appear in 1H NMR spectra. Deuterated
solvents (e.g. deuterated water, D2O) also allow the spectrometer to lock onto the
deuterium signal to adjust for the natural drift of the magnetic field (Richards et al.,
2010). The electronic surroundings around nuclei are generally anisotropic (meaning
the electrons are not evenly distributed around the nucleus) but varies on different sides
of the nucleus with reference to the magnetic field. Consequently, the chemical shift is
also anisotropic and changes as the orientation of the molecule with respect to the
magnetic field changes. In solution-state NMR however, the anisotropy of the chemical
shift is averaged out by fast molecular tumbling and thus, only a single isotropic
3
chemical shift value is observed (Harris et al., 2012). Since solution-state NMR gives
high resolution spectra due to the averaging of anisotropic interactions, significant
structural information can be obtained. Even information about interactions within
complex samples, such as environmental samples like dissolved organic matter can be
determined (Simpson et al., 2011)
A wide range of NMR experiments can be obtained through solution-state NMR,
including a variety of one-dimensional (1D) and two-dimensional (2D) experiments. The
main 1D experiments are 1H and 13C NMR which each give an overview of the proton
(for 1H NMR) or carbon (for 13C NMR) distribution within a sample. 1H detection is most
widespread because of its high natural abundance in organic structures and high
sensitivity. When water (H2O) is used as a solvent, water suppression techniques are
needed to suppress the water signal which could distort and mask the signals of
interest. The simplest method is called presaturation which is a two pulse experiment.
With presaturation, a relatively long (seconds), low power RF pulse whose excitation
profile is very narrow (due to its long length) selectively saturates the water frequency.
Then, a non-selective 45-90° RF pulse with a wide excitation profile (because of its
short length) is used to excite the remaining resonances of interest (McKay, 2009).
Pulsed field gradients are used for an experiment called Diffusion Editing (DE) 1H NMR
which selects for large molecules with restricted diffusion. Gradients can also be utilized
in many methods of water suppression one example being Presaturation Utilizing
Relaxation Gradients and Echoes (PURGE) This technique uses z pulsed field
gradients. (Simpson et al., 2005).
4
2D experiments provide correlation information about the bonds in the sample which
assist in determining the structure. 1H-1H Correlation Spectroscopy (COSY) gives
connectivity information between protons on adjacent carbons. 1H-1H Total Correlation
Spectroscopy (TOCSY) not only provides connectivity information on directly adjacent
carbons but also between long range protons. Both COSY and TOCSY measure
couplings through bonds. Nuclear Overhauser Effect Spectroscopy (NOESY) on the
other hand, measures coupling through space. 1H-13C Heteronuclear Single Quantum
Coherence (HSQC) provides connectivity information identifying which 1H is directly
connected to which 13C resonance. In addition the carbon dimension provides additional
spectral dispersion that is critical to reduce spectral overlap in complex natural samples.
This 2D spectrum is extremely useful, for example, in determining the metabolites
present by pattern matching since the HSQC spectra of different metabolites should be
fairly unique (Simpson et al., 2011).
1.1.1.2 Magic Angle Spinning (MAS)
Static gel/solid-state NMR spectroscopy has very broad line width due to anisotropic
interactions in media with little or no mobility. MAS is a technique where the sample is
spun at an angle of about 54.7° with respect to the magnetic field. In doing so, three
major interactions that cause the lines to be wide are reduced, those are: dipolar,
chemical shift anisotropy and quadrupolar interactions (Duer, 2004). This means that
the three interactions are averaged such that the peak broadness is reduced and line
shape is greatly improved. Spinning a gel/solid sample at the magic angle mimics the
narrow lines of a solution-state NMR spectrum (Stejskal et al., 1977). The disadvantage
of spinning the sample is the appearance of spinning sidebands on either side of the
5
signal that is continuously X ppm away from each other (the ppm separation of the
sidebands is proportional to the spinning speed (in Hz) ) which could complicate the
spectra if a true signal is hidden by an overlapping spinning sideband (Herzfeld et al.,
1980). Samples for probes with MAS are placed in a zirconium rotor, no longer than an
inch and can vary in diameter, most commonly it is 4 or 7 mm.
There are two main types of probes that use MAS, HR-MAS NMR which looks at more
gel-like components and cross polarization magic angle spinning (CP-MAS) NMR which
looks at the rigid components.
1.1.1.2.1 HR-MAS for Gels
HR-MAS NMR probes are usually susceptibility matched and fitted with a lock and
gradient. HR-MAS NMR is usually applied to study gel-like samples or samples that can
be swollen by a solvent. This solvent is added to make contact with the components in
the sample, thus allowing them to be detected (Courtier-Murias et al., 2012). These
samples often have some internal molecular motion, but the dipole-dipole interactions
are inadequately averaged by internal motion alone. HR-MAS NMR can considerably
average out the dipole-dipole broadening and result in spectra similar to high resolution
solution-state NMR. (Simpson et al., 2011)
HR-MAS NMR is able to identify any signals that are not in a true solid-state. HR-MAS
NMR has been applied, and not limited to, studying seeds (Seefeldt et al., 2008), plants
(Santos et al., 2012), soils (Spence et al., 2011) and biological tissues (Beckonert et al.,
2010). For dry samples, like soil, a solvent can be added before it is transferred to the
rotor. Samples which naturally have solvent, for example plant tissue, do not
necessarily require additional solvent to be added. Since the HR-MAS NMR probe is
6
built with a lock, using a deuterated solvent, like in solution-state NMR, would prevent
magnetic field drift.
The experiments and water suppression conducted with HR-MAS NMR are the same as
those explained in Section 1.1.1.1 Solution-state NMR. Unfortunately, since the HR-
MAS probes have low power circuitry they are incapable of handling the high-power
required for CP experiments to detect for most rigid bonds.(Simpson et al., 2013)
1.1.1.2.2 CP-MAS for Solids
Solid-state NMR probes have MAS and circuitry that can handle high power.
Conversely, they do not have a lock built in since the superconducting magnet is stable
enough to handle solid-state NMR experiments (Braun et al., 1998).
With proton, the homonuclear dipolar coupling is very large, and so detecting for proton
is not common in solid-state NMR due to the broadening caused by dipolar coupling.
13C (being less abundant) on the other hand, does not have a problem with
homonuclear dipolar coupling since the nuclei are more distant from each other. Hence,
13C is most commonly used for detection (Simpson et al., 2011).
Most typical solid-state experiment is called CP-MAS which involves the transference of
polarization from an abundant and high sensitivity nuclei (e.g. 1H) to the less abundant
nuclei (e.g. 13C), this enhances the C signal by up to a factor of 4. CP-MAS involving 1H-
13C, 1H-15N and 1H-31P is applied heavily in soil science since its good at studying
relative changes in carbon distribution across a series of samples (Simpson et al.,
2011).
7
Samples for solid-state NMR are often dried and ground so that they can be more
uniformly packed throughout the rotor. This enables the rotor to spin at faster speeds
and moving the spinning sidebands farther away from the signal region
1.1.2 CMP-NMR
Samples can be complex heterogeneous mixtures composed of liquid, gel, semi-solid
and solid components. Conventional solution and solid-state NMR spectroscopy is
limited to single-phase sample characterization, which requires extraction of soluble
components for solution-state NMR or separation and treatment of solids materials for
solid-state NMR spectroscopy, as already described. This sample phase separation
changes the natural chemical and physical interactions that affect relevant properties
such as analyte kinetics across phase boundaries. In 2012, members in our group were
the first to develop novel technology termed CMP-NMR spectroscopy which allows for
simultaneous analysis of all phases in its natural state (Courtier-Murias et al., 2012)
Like solid-state NMR, the sample is prepared in a rotor but can be placed into the rotor
as is without the need for any sample preparation. The multiphase probe has high
power electronics to handle solid-state experiments; is susceptibility matched and spins
at the magic angle to improve line shape. It also has lock channel and pulse field
gradients for use in diffusion editing; obtaining 2D NMR and water suppression which
are generally features only found on solution-state probes.
1.1.2.1 Spectral Editing
The CMP probe is capable of a wide range of solution, gel (HR-MAS) and solid
experiments. Spectra editing capabilities of the CMP probe are described thoroughly by
8
Courtier et al. (2012). Briefly, starting from the most liquid-like through to the most solid-
like, these experiments can be described as follows.
1.1.2.1.1 Inverse Diffusion Editing (IDE)
IDE is a difference based approach that selects molecules that have unrestricted
tumbling, these can be soluble/truly dissolved molecules and liquids. In this thesis these
components will be referred to as "components with unrestricted diffusion".
1.1.2.1.2 Diffusion Editing (DE)
DE selects molecules with restricted diffusion, and will include swollen biopolymers,
mobile gels and smaller molecules that are trapped or sorbed. In this thesis these
components will be referred to as “components with restricted diffusion”. Understand
that there is not a clear cut diffusivity limit that separates all dissolved molecules from all
those with restricted diffusion. Instead, the experiments should be considered as a
continuum with the “fast diffusing molecules contained in IDE” and generally “the
restricted molecules” being in the diffusion editing. The strength of the diffusion editing
has been developed on standard samples to give the best distinction between truly
dissolved molecules from entrapped molecules and gels (Courtier-Murias et al., 2012).
1.1.2.1.3 Recovering relaxation losses Arising from Diffusion Editing(RADE)
RADE is an experiment that compensates for signals that otherwise may be lost through
relaxation during diffusion editing. RADE selects semi-solid components that may
include rigid gels, and possibly some very dynamic solids. In this thesis these
components will be referred to as “semi-solids”.
9
1.1.2.1.4 T2 filtered CP-MAS
This experiment selects the more mobile “true-solids” this may include very rigid gels
and solids that exhibit some dynamics. In this
thesis these components will be referred to as “dynamic solids”. It is important to note
that while no components are missed, some components may be observed twice by 1H
RADE and 13C T2 CP-MAS (Courtier-Murias et al., 2012).
1.1.2.1.5 Inverse T2 filtered CP-MAS
This experiment is a difference approach that selects just the truly rigid solids that show
little to no dynamics. Throughout this thesis, these components will be referred to as
“rigid solids”.
1.1.2.2 Potential use of CMP-NMR
CMP-NMR has great potential in all fields of research since it is capable of studying all
bonds in all phases in intact samples. Heterogeneous samples such as plants,
sediments, soils and biological tissue can finally be analyzed as a whole and not have
to be examined in parts (solution, gels, solids). Separating the phases perturb the native
interactions between phases of the sample; an element that is important to
understanding the sample as a whole. CMP-NMR would also make in vivo studies
possible, monitoring real-time changes of, for example, germination of a seed. This
thesis details the first research into examining seeds and elucidating changes during
early growth using CMP-NMR.
10
1.2 Seeds
Simply, a seed is a self-contained vessel that encompasses the embryonic plant
surrounded by food reserves which are all protected by a seed coat. The first step of
seed development is the reproductive cycle, which is initiated with pollination and
fertilization. Seed formation is the product of post-fertilization and the last step in the
reproductive process of the parent seed-bearing plants (Chaudhury et al., 2001; Weber
et al., 2005). The purposes of seeds include plant dispersal, propagation, and most
importantly, seeds possess a high desiccation tolerance to preserve parent genetic
materials until the seed reaches favorable environment to germinate and grow.
1.2.1 Importance of studying seeds
In addition to being the reproductive system of seed-bearing plants, seeds are essential
because of their many different uses in today’s society. They can be consumed as is;
sown to be grown into fruits and vegetables; processed into edible products like flour
and oil or non-edible products such as biofuels. There are several types of seeds with
the three main categories being cereal grains (e.g. corn, rice, wheat), nuts (e.g.
almonds, cashew, walnut) and legume seeds (e.g. beans, peas and soybean)
(Berdanier et al., 2007). Herein, the focus will be on cereal grains as they are a major
global crop; specifically wheat because the size of the wheat seed matches the NMR
rotor size permitting a single seed to be analyzed. This opens the door for future studies
to look at germination and early growth in a single seed using CMP-NMR
Approximately 12,000 years ago, the agricultural revolution marked a wide scale
departure of many cultures from a lifestyle of hunting and gathering towards crop
cultivation. Technological developments such as irrigation, food storage techniques and
11
milling enabled certain raw seed material, such as cereal grains, to be processed into
flour and starch allowing for the expansion of seed-based foods to a larger component
of the human diet (Bocquet-Appel, 2011)
Cereal grains alone constitute approximately 87% of all cultivated seeds and are staple
foods in many continents (Kawakatsu et al., 2010). Canada is the fifth largest producer
of agricultural goods, with exports valued at $35.5 billion in 2010 indicating a substantial
global dependency on Canadian agricultural resources (Canada, 2012). Factors such
as the increasing global population, climate change, the limited availability of arable
land and fresh water increase the demand for agricultural goods.
1.2.2 Germination
Germination is a critical process central to agricultural productivity and plant growth.
Seed growth is initiated by germination, a process characterized by water uptake
(imbibition) into a dry seed and is successfully completed following the emergence of an
embryonic axis (or radicle) that extends through external seed layer (seed coat)
resulting in a seedling (Bewley, 1997). Imbibition denotes a significant alteration to seed
storage metabolism that favours growth, in which storage reserves are processed for
biosynthesis and energy. Failed germination generally occurs due to an absent or
dysfunctional embryo. Alternatively, a given seed could undergo all cellular and
metabolic events occur correctly, however, unsuccessful penetration of the seed coat by
the radicle results in a non-viable or damaged seed state (Nonogaki et al., 2010). The
germination rate is one of the fundamental factors that dictate the annual production of
crops and therefore a comprehensive understanding of its underlying processes is
paramount.
12
1.3 Previous studies on seeds
Various aspects of seeds have been studied in the past by different methods and
instruments. Examples of interests in seed analysis are (1) lipids/oil content in
pomegranate (Parashar et al., 2010), rapeseed (Hu et al., 2013) and sunflower seeds
(Troncoso-Ponce et al., 2010). (2) Protein content in rice (Ohdaira et al., 2010), pea
(Burstin et al., 2007) and bean seeds (Subagio, 2006). (3) Carbohydrate content in
wheat (Barron et al., 2007), lentil (Tahir et al., 2011) and lupine seeds (Gdala, 1996).
The following sections 1.3.1-1.3.3 will describe briefly the sample preparation for the
two common instruments (gas chromatography (GC) and liquid chromatography (LC))
for seed analysis and how CMP-NMR can be advantageous and provide highly
complementary information.
1.3.1 Sample preparation for liquid chromatography mass spectrometrymass spectrometry (LC-MS/MS)
Phytoestrogens are a group of secondary plant metabolites that are referred to as
“dietary estrogen” for humans because of their structural resemblance to estradiol, a
form of estrogen; they have the capability to cause antiestrogenic and/or estrogenic
changes. Two types of phytoestrogens are isoflavones and lignans (Usui, 2006;
Winuthayanon et al., 2009). Kuhnle and co-workers (2008) described a study where
isoflavones and lignan content was quantified in seeds (in addition to coffee, tea,
alcoholic beverages and oils) such as Brazil nuts, pistachios, pumpkin and sunflower
seeds amongst many more. The experimental procedure described seeds that were
frozen then freeze dried. 100 mg of freeze dried sample was extracted 3 times with 2
mL of 10% methanol in sodium acetate and deconjugated with a hydrolysis reagent
consisting of purified H. pomatia juice (β-glucuronidase), cellulase, and β-glucosidase.
13
Deconjugated samples were then extracted using Strata C-18E SPE cartridges, dried,
redissolved in 40% aqueous methanol and quantified using LC-MS/MS and 13C3 labeled
standards (Kuhnle et al., 2008). Though this study was able to quantify the
phytoestrogen content, the laborious sample preparation may have greatly perturbed
the analytes of interest and it’s possible that the structural features of the
phytoestrogens may have become altered from its natural state in the seeds.
1.3.2 Sample preparation for gas chromatography flame ionizationdetection (GC-FID)
Stevenson and co-workers (2007) studied oil content and fatty acid composition of
pumpkin seeds since they are not a popular source for vegetable oil but hold great
potential for industrial applications and in enhancing the well-being of the population
since it can be the source of many nutritional benefits. In these experiments, whole
pumpkin seeds were ground and oil was extracted using both supercritical fluid
extraction (SFE) and accelerated solvent extraction (ASE). Any substance at a pressure
and temperature above its critical point, where the liquid and gas phases cannot be
differentiated, is called a supercritical fluid (Sharif et al., 2014). SFE is the technique of
separating the matrix from the desired component utilizing supercritical fluids as the
extraction solvent. ASE is similar to SFE but uses common solvents with increased
temperatures and pressures. Once the oil was extracted the fatty acid content could be
characterized using gas chromatogram fitted with a flame ionization detector (FID)
(Stevenson et al., 2007).
Using this technique, they were able to quantify and determine the exact fatty acid
composition (e.g. lauric acid, myristic acid, etc.) of the seed sample. However, like the
14
previous sample preparation requirements described in section 1.3.1 using LC-MS/MS,
this study also required relatively extensive sample preparation to extract the oil content
which disturbs the natural state and probably alters the results.
1.3.3 Advantages of using CMP-NMR
CMP-NMR is advantageous since it can observe molecular interactions of a sample in
its native and intact state. All the phases are kept as is and not extracted so kinetics
across phase boundaries and interaction of phases can be studied in the intact
unaltered sample. It can be complimentary to LC-MS/MS, which was used in the study
of phytoestrogens by Kuhnle and co-workers (2008) or to GC-FID which was used in
the study of oil content by Stevenson and co-workers (2007). Using CMP-NMR gives
the complete story of the sample, which is necessary to fully understand the seed
composition and the process of germination.
1.4 Isotopic labeling
Proton (1H) is naturally abundant at 99.9% while the major isotope of carbon (12C) is not
NMR active because the spin quantum number is zero. NMR active carbon-13 (13C) is
only present at 1.07% so it is much less sensitive (Böhlke et al., 2005); Carbon-13 is
about 4 orders of magnitude lower than 1H when it comes to receptivity (Webster,
2006). In the case of complex samples, like seeds and seedlings, the spectra can be
overwhelmingly complicated if only 1H was studied. Using 13C not only simplifies the
spectra by spreading it over a wider frequency range, but the likelihood of coupling
between carbons causing splitting (which can complicate spectra) can be ignored due to
the low natural abundance of 13C. In the research described in this thesis 13C labeled
samples were used since this was the first-ever application of CMP-NMR to
15
seeds/seedlings and the focus of the thesis was on the application of the technique and
not having to encounter difficulties obtaining data. Labeled samples also allowed for
uncommon2D experiments (e.g. dipolar assisted rotational resonance (DARR)) to be
performed that would otherwise be nearly impossible to acquire data in a reasonable
amount of time with natural abundance samples
1.5 Objectives of this study
Seeds are important globally as they provide a source of life in many aspects. Seeds
can flourish into plants which are at the bottom of the food chain and support many
animals above. Seeds themselves can be eaten raw or cooked providing many
nutritional benefits. For these reasons, seeds, the process of germination and early
seedling growth should be studied to further understand their structure. Research
questions associated with this study are:
a. Can CMP-NMR be used to comprehensively study seeds in their native state?
b. What information can be gained from applying CMP-NMR to seeds?
c. Can CMP-NMR be used to study seeds during germination?
d. What information can be gained from applying CMP-NMR to seed germination?
These research questions a-d are discussed and addressed by the objectives of this
thesis, listed below:
1. Demonstrate the utility of CMP-NMR probe. This study also serves to validate the
utility of CMP-NMR in the application of seeds and agriculture since this is the
first time use in its field.
16
2. Characterize the components of the broccoli, corn and wheat seed components
using Comprehensive Multiphase (CMP) NMR which can study all bonds in all
phases (components with unrestricted and restricted diffusion, semi-solid and
solid components).
3. To elucidate changes in the wheat seed during germination and early seedling
growth using CMP-NMR.
Objectives 2 and 3 will correspond to chapters 2 and 3, respectively, and objective 1
will be discussed throughout both chapters.
17
Chapter 2. Comprehensive Multiphase NMRSpectroscopy of Intact 13C-Labeled Seeds1,2,3
2.1 Abstract
Seeds are complex entities comprised of liquids, gels and solids. NMR spectroscopy is
a powerful tool for studying molecular structure, but has evolved into two fields, solution-
and solid-state. Comprehensive Multi-phase (CMP)-NMR spectroscopy is capable of
liquid, gel, and solid-state experiments for studying intact samples where all organic
components are studied and differentiated in-situ. Herein, intact 13C labeled seeds were
studied by a variety of 1D/2D 1H/13C experiments. In the mobile phase, an assortment of
metabolites in a single 13C labeled wheat seed were identified; the gel phase was
dominated by triacylglycerides; the semi-solid phase was composed largely of
carbohydrate biopolymers and the solid phase was greatly influenced by starchy
endosperm signals. Subsequently, the seeds were compared and relative similarities
and differences between seed types discussed. This study represents the first
application of CMP-NMR to food chemistry and demonstrates its general utility and
feasibility for studying intact heterogeneous samples.
1 The samples were provided by IsoLife (Wageningen, The Netherlands). The experimental design wascreated by Leayen Lam and André J. Simpson. The lab experiments were conducted by Leayen Lam withguidance from André J. Simpson. Data interpretation was performed by Leayen Lam with guidance fromAndré J. Simpson. The manuscript was written by Leayen Lam with critical comments from André J.Simpson, Heather L. Wheeler, Malcolm Campbell, Ries de Visser and Myrna J. Simpson.2 Published as: Lam, L., R. Soong, A. Sutrisno, R. De Visser, M. J. Simpson, H. Wheeler, M. Campbell,W. E. Maas, M. Fey, A. Gorissen, H. Hutchins, B. Andrew, J. O. Struppe, S. Krishnamurthy, R. Kumar, M.Monette, H. Stronks, A. Hume and A. J. Simpson (2013). "Comprehensive Multiphase NMR Spectroscopyof Intact 13C Labeled Seeds." Journal of Agricultural and Food Chemistry [Just Accepted], DOI:10.1021/jf4045638, Published online: Dec 19, 20133 Reprinted with permission from Journal of Agricultural and Food Chemistry, 2013, DOI:10.1021/jf4045638. Copyright 2013 American Chemical Society.
18
2.2 Introduction
Seeds are integral to world nutrition as they not only serve as a direct source of food
rich with essential vitamins, fibre, sterols and antioxidants but carry the potential to be
cultivated into fruit- and vegetable-bearing plants (Byers et al., 2002; Yang et al., 2009;
Elleuch et al., 2011; Poutanen, 2012). Plant sterols have been shown to reduce low-
density lipoprotein cholesterol absorption, whereby the structurally analogous sterols
compete with cholesterol absorption sites in the intestine (Ostlund, 2007). Similarly, high
antioxidant intake has demonstrated protective effects against chronic diseases such as
cancer, cardiovascular disease, osteoporosis and diabetes by mitigating the damaging
effects related to oxidative stress (Byers et al., 2002; Yang et al., 2009; Poutanen,
2012). β-carotene, a precursor to vitamin A, and other antioxidants such as vitamin C
are not produced natively by the human body and must be obtained from extrinsic
dietary sources, for which fruits and vegetables are naturally abundant (Montel-Hagen
et al., 2008; Lobo et al., 2012).
Historically, NMR spectroscopy has evolved into two separate fields, namely solution-
state and solid-state NMR. Seed components can be extracted for solution-state NMR;
but this process is destructive and selective toward only a subset of chemicals present.
Solid-state NMR can be used to study intact seeds but in the case of soluble/gel
components, the lack of pulse field gradients and a spectrometer lock restrict the type of
experiments and information that can be extracted directly. Recently, NMR
spectroscopy has been applied in seed analysis for the study of oil and protein
composition, but often requires extensive and time-consuming sample preparation (Jiao
et al., 2012; Kouame et al., 2012). One study used wheat seeds that were initially milled
19
into a flour, dissolved in a buffer solution, centrifuged and supernatant collected prior to
NMR analysis (Lamanna et al., 2011). Such methods of sample preparation can be
detrimental as they potentially perturb the structure and native chemical and physical
interactions that influence analyte kinetics across phase boundaries, which are
important for analysis.
The first 1H measurements of intact seeds were likely performed in 1963, in which the
oil content was determined for a variety of seeds (Conway et al., 1963). This was
followed by the first high resolution 13C measurements conducted in 1974, whereby the
oil composition was measured for a single soybean (Schaefer et al., 1974). Previous
analysis of intact seeds has been performed by High Resolution Magic Angle Spinning
(HR-MAS) of canola seeds to determine seed oil composition (Hutton et al., 1999);
Cross Polarization (CP)-MAS and HR-MAS to characterize Arabidopsis, pea and lettuce
seeds (Bardet et al., 2001); metabolite profiling to assess conifer seed quality (Terskikh
et al., 2005) as well as measuring moisture content of garden cress seeds (Rachocki et
al., 2012). In all cases, although intact seeds were used, only select phases (liquid, gel
or solid) were studied in a given experiment.
Traditional solution-state NMR probes use low power electronics, a lock channel, pulsed
field gradients, and provides excellent line-shape but only for dissolved samples. HR-
MAS probes were introduced in 1996 and employ magic angle spinning, a magic angle
gradient and susceptibility matched stators (Maas et al., 1996). HR-MAS probes permit
the study of swellable and liquid components. However, HR-MAS probes are designed
using low power circuitry; as such, they cannot generate the RF field required for high
power decoupling or cross-polarization, elements essential to the majority of solid-state
20
NMR experiments. Solid-state probes on the other hand are designed to generate high
RF fields, but as solid-state NMR spectroscopy has been predominantly reserved for
the study of true solids, they lack a lock and gradients which are required for the
efficient study of liquid and gel components. Comprehensive Multiphase NMR (CMP)-
NMR, introduced in 2012, incorporates all of the aforementioned aspects, including
magic angle spinning, a magic angle gradient, a lock, full susceptibility matching, and
solid-state circuitry to permit high power handling. Therefore, it is built to study unaltered
samples where all organic components can be observed and differentiated in-situ,
resulting in a universal approach (Courtier-Murias et al., 2012).
The use of separate probes to achieve the same goal is only an option for the most
simple, structural studies where the sample does not change. Even in this case, it
important to stress that in large part, due to independent development of liquids and
solid state NMR, very few labs in the world would have separate liquid, HR-MAS and
solids probes, and even if they did, scheduling all to be available at the same time
would be extremely challenging. More importantly, any study involving kinetics transfer
between phases (e.g. growth, contaminant sequestration), or changes of one phase into
another (for example soil swelling/drying, feeding phenylalanine to follow lignin
formation) will require a CMP probe as such studies are impossible to perform using
separate probes, this is discussed in more detail later in this study.
CMP-NMR has been, thus far, used to determine the fate and binding of contaminants
in soil (Courtier-Murias et al., 2012; Longstaffe et al., 2012). Here, we introduce CMP-
NMR to applications in food and agriculture. This study focuses on structural information
that can be obtained from 1H and 13C CMP-NMR characterization of intact broccoli, corn
21
and wheat seeds. Wheat and corn were selected as they represent major global crops,
while broccoli seeds were included as an example of a legume. The different seed types
will be compared to each other for relative differences and similarities as well as further
considerations of CMP probes (including quantification; need for labelling and future
potential) will be discussed towards the end of this study.
Seeds are used here an example which serves to demonstrate the general applicability
of CMP-NMR for the analysis of all organic components in all phases in whole,
unaltered samples. CMP-NMR is likely to find widespread application in the agricultural
and food sciences due to versatility and ability to provide unsurpassed molecular detail
on intact samples. CMP-NMR has potential to understand intact structure, processes
that are involved in phase changes (drying, swelling), and molecular interactions (for
example, between an herbicide and plant tissue) and thus, has considerable potential
for the analysis of food, soil, sediments, plants and seeds.
2.3 Materials and methods
2.3.1 13C Labeling of the seeds
Uniformly 13C-enriched seeds of broccoli, corn and wheat (Brassica oleracea
var. botrytis 'Broccoli’, Zea mays, Triticum aestivum respectively) were produced in
specially designed, air-tight, high-irradiance growth chambers (Gorissen et al., 2011)
(IsoLife, Wageningen, The Netherlands). Plants were grown from 13C-labeled seeds in a
closed atmosphere containing 97 atom % 13CO2 (from pressurised cylinders; Isotec,
Inc., Miamisburg, OH) from the seedling stage until full maturity. Internal wind speed
ensured efficient pollination of the corn and wheat. Pollination of broccoli flowers was
ensured by combining compatible parent plants and introducing bluebottle flies into the
22
chambers. Mineral nutrients were supplied as Hoagland-type solutions with
micronutrients and iron (Smakman et al., 1982; De Visser et al., 1997). Climate
conditions were: irradiance (PPFD) 600 mol m–2 s–1 (HPI) during a 16 h day, day/night
temperature 24/16 °C, relative humidity 75/85%.
2.3.2 Sample preparation
Uniformly 13C labelled (>97% total carbon content) and non-labelled (<1.2% total carbon
content) seeds of broccoli, corn and wheat (Brassica oleracea var. botrytis 'Broccoli’,
Zea mays, Triticum aestivum respectively) were used. The whole and intact seeds were
placed directly into a 4 mm zirconium rotor and filled with D2O (Andover,
Massachusetts) as a lock solvent. The seeds, which contained 6.7 and 6.6, 6.8 and 7.2,
8.6 and 7.5% water for labelled and non-labelled wheat, labelled and non-labelled corn,
labelled and non-labelled broccoli respectively, were not swollen beforehand. Since the
goal of the study was to study seed structure and not the process of germination, 99.8%
D2O was deliberately used since seeds are found to either not germinate or germinate
but do not continue to grow afterwards when imbibed with D2O (Siegel et al., 1964;
Blake et al., 1967). No significant differences were noted over each 24 hr acquisition
period; all spectra datasets were consistent with each other. In addition, during longer
experiments CP-MAS, direct carbon and 2D NMR the spectra (collected over many
hours) will represent the average state of the seeds during that time period and hence
give a representative overview of the overall structural state. Seeds remained intact and
were not damaged during the spinning process. In the case of corn, which was cut in
half to fit into the rotor, the D2O solvent may have been more likely to leach components
from the seed into the aqueous solvent. The goal of this study was only to look at
23
overall structural state; future studies that employ H2O as the solvent (with an external
D2O lock) and kinetic experiments with high temporal resolution could be used to
understand the molecular processes behind germination.
The rotor was sealed using a top insert made from Kel-F, a Kel-F sealing screw and
Kel-F cap. For wheat, only one seed was used. For broccoli, two seeds were used for
proton studies and ten seeds for anything that required carbon to increase sensitivity.
Lastly for corn, the seed had to be cut into half due to its size before being placed in the
rotor. The non-labeled equivalents were prepared identically.
2.3.3 1D NMR spectroscopy
All NMR measurements were performed on a 500 MHz Bruker Avance III Spectrometer
at a spinning speed of 6666 Hz using a prototype CMP MAS 4 mm 1H–13C–19F–2H
probe fitted with an actively shielded Z gradient (Bruker BioSpin) at 298K. All
experiments were locked on D2O and the lock was maintained for all experiments
including the solid-state experiments. Decoupling was used in all 1D and 2D
experiments to remove 1H-13C coupling from the labeled sample. For low power
experiments garp was used for proton observe and waltz16 for carbon observe. For
high power decoupling spinal-64 was used for carbon observe.
All 1H NMR spectra were recorded using presaturation for water suppression except for
when presaturation utilizing relaxation gradients and echoes (PURGE) was employed
(Simpson et al., 2005). The 90° pulse was calibrated for each sample in the study. A
spectral width of 20 ppm was used, 2 K scans were acquired and 8 K time domain
points. T1 times were measured using the standard inversion recovery approach and
the recycle delay set at 5 times the measured T1 value. Spectra were processed using a
24
zero filling factor of 2 and an exponential function corresponding to a line broadening of
2 Hz.
One dimensional 13C NMR spectra were recorded with a spectral width of 400 ppm
using inverse gate decoupling. Scans ranged from 2 K- 12 K and 16 K time domain
points. 13C RAMPed-amplitude cross polarization magic angle spinning (13C RAMP CP-
MAS) (Metz et al., 1994) was acquired with 2K scans, a 1 ms contact time and a 2 s
carbon recycle delay. T1 times were measured using the standard inversion recovery
approach and the recycle delay set at 5 times the measured T1 value. Spectra were
processed using a zero filling factor of 2 and an exponential function corresponding to a
line broadening of 5 Hz, (conventional carbon) and 40 Hz (CP-MAS).
2.3.4 Spectral Editing
Diffusion edited proton and carbon spectra were produced using a bipolar pulse pair
longitudinal encode-decode (BPLED) sequence (Wu et al., 1995) with inverse gated
decoupling. Scans were collected using encoding/decoding gradients of 1.8 ms at 50
gauss/cm and a diffusion time of 180 ms. Inverse Diffusion Edited (IDE) and Recovering
relaxation losses Arising from Diffusion Editing (RADE) were created via difference from
the appropriate controls as previously described (Courtier-Murias et al., 2012). Using
CPMG (T2 filtered) experiments to obtain the liquid phase components the T2 delay was
rotor synchronized and set at 600 µs and the pulse train was repeated 100 times,
yielding a total pulse train length of 120 ms (Courtier-Murias et al., 2012). To remove
solid components, 2 CPMG echoes of 7.5 µs were inserted prior to cross polarization.
During this short period, signals from extremely broad 1H signals (i.e. true crystalline
solids) relax (or are not efficiently refocused) while signals from more mobile solids
25
remain along the XY plane for subsequent cross polarization. The approach has been
discussed in detail by Courtier-Murias et al. (2012). For spectral editing, the spectra
were scaled until the dominant component being subtracted was nulled leaving a
difference spectrum containing positive peaks (Courtier-Murias et al., 2012).
2.3.5 2D NMR Spectroscopy1H Total COrrelation SpectroscopY (TOCSY) spectra were acquired in the phase
sensitive mode, using a mixing sequence with rotor synchronized constant adiabatic
WURST–2 pulses within an X_M16 mixing scheme (Peti et al., 2000). Scans (128) were
collected for each of the 196 increments in the F1 dimension. Data points (2048) were
collected in F2 at a mixing time of 120 ms. Both dimensions were processed using sine-
squared functions with a π/2 phase shift and a zero filling factor of 2. Nuclear
Overhauser Effect SpectroscopY (NOESY) spectra were acquired in phase-sensitive
mode, using time proportional phase incrementation. Scans (128) and data points
(2048) were collected for each of the 196 increments in the F1 dimension at a mixing
time of 300 ms. Both dimensions were processed using sine-squared functions with a
π/2 phase shift and a zero filling factor of 2. The COrrelation SpectroscopY (COSY)
spectra were acquired in non-phase-sensitive mode, using gradients for selection.
Scans (128) and data points (2048) were collected for each of the 196 increments in the
F1. Both dimensions were processed using unshifted sine-squared function, a zero
filling factor of 2, and a magnitude mode for projection.
1H–13C Heteronuclear Single-Quantum Coherence (HSQC) spectra were collected in
phase sensitive mode using Echo/Antiecho-TPPI gradient selection. Scans (128) were
collected for each of the 196 increments in the F1 dimension. Data points (2048) were
26
collected in F2 and a 1J 1H–13C of 145 Hz. Both dimensions were processed using sine-
squared functions with a π/2 phase shift and a zero filling factor of 2. Dipolar Assisted
Rotational Resonance (DARR) spectroscopy was acquired using 256 scans for each of
96 increments in F1 and a mixing time of 200 ms. F2 was processed using an
exponential function corresponding to 35 Hz line broadening and F1 using a sine-
squared function with a π/2 phase shift. Both dimensions were zero filled by a factor of
2.
2.3.6 NMR Spectroscopy of non-labeled samples
For non-labeled samples, only basic 1D 13C spectra and CP-MAS were acquired.
Experiments such as diffusion edited carbon and DARR are very insensitive and time
prohibitive on a non-labeled sample.
2.3.7 Compound Identification
Spectra were calibrated against a range of known compounds in the Bruker Biofluid
Reference Compound Database (v 2.0.3). Pattern matching of both 1D and 2D spectra
was performed using Analysis of MIXtures (AMIX, version 3.9.3, Bruker BioSpin)
against the Bruker Biofluid Reference Compound Database (v 2-0-0 to v 2-0-3) using a
procedure previously developed for complex mixtures (Woods et al., 2011). Compounds
with a greater than 80 % confidence match (from automated searches) were further
selected for detailed manual inspection. Only compounds that showed near perfect
matches in all spectral regions were retained as assignments. The chemical shifts of the
identified compounds were compared with database values (r2 = 0.99, σ = 0.01). For
quantification, spectra were also normalized to total intensity over the 200-0 ppm region
in the carbon spectra. The multi-integration tool in AMIX (version 3.9.3, Bruker BioSpin)
27
was used to compare the relative quantity of carbonyl protein (defined as 190-170 ppm),
carbohydrates (defined as 110-50 ppm), lipids/fatty acids (defined as 183-164, 135-121
and 44-3 ppm) and aliphatic proteins (defined as 60-40 ppm)
2.4 Results and discussion
2.4.1 Comprehensive Multiphase (CMP)-NMR spectroscopy
In this study a number of spectral editing approaches have been used. These have
been discussed in detail by Courtier-Murias et al (2012). Briefly, starting from the most
liquid-like through to the most solid-like, these experiments can be described as follows.
1) Inverse Diffusion Editing (IDE) is a difference based approach that selects molecules
that have unrestricted tumbling (i.e. truly dissolved molecules). Here, these components
will be referred to as “components with unrestricted diffusion”. 2) Diffusion Editing (DE)
selects molecules with restricted diffusion, and will include swollen biopolymers, mobile
gels and smaller molecules that are trapped or sorbed. In this study these components
will be referred to as “components with restricted diffusion”. There is not clear cut
diffusivity that separates all dissolved molecules from all those with restricted diffusion.
Instead, the experiments should be considered as a continuum with the “fast diffusing
molecules contained in IDE” and generally “the restricted molecules” being in DE. The
strength of the diffusion editing has been developed on standard samples to give the
best distinction between truly dissolved molecules from entrapped molecules and gels
(Courtier-Murias et al., 2012). 3) Recovering relaxation losses Arising from Diffusion
Editing (RADE) is an experiment that compensates for signals that otherwise may be
lost through relaxation during diffusion editing. RADE selects semi-solid components
that may include gels and possibly some very dynamic solids. In this study these
28
components will be referred to as “semi-solids”. 4) T2 filtered CP-MAS selects the more
mobile “true-solids” this may include very rigid gels and solids that exhibit some
dynamics. In this study these components will be referred to as “dynamic solids”. It is
important to note that while no components are missed, some components may be
observed twice by 1H RADE and 13C T2 CP-MAS (Courtier-Murias et al., 2012). 5)
Finally, inverse T2 filtered CP-MAS is a difference approach that selects just the truly
rigid solids that show little to no dynamics. In this study these components will be
referred to as “rigid solids”.
2.4.2 Detailed Analysis of Wheat Seed
2.4.2.1 Components with unrestricted diffusion (Soluble components)
A conventional 1D carbon profile of the single 13C labeled (97 atom %) wheat seed was
acquired with low power waltz-16 decoupling (Figure 2-1a). In this experiment carbons
from true solid components are strongly attenuated as the lower power decoupling is
insufficient to decouple broad proton resonances that are characteristic in solids. As
such, the spectrum contained signals from components with and without restricted
diffusion and semi-solids but signals from true solids are largely suppressed (Figure
2-1a). Due to the complex nature of the seeds, the conventional 13C spectrum showed
considerable overlap which was reduced by spectral editing to obtain the inverse
diffusion editing (IDE) spectrum (Figure 2-1b). IDE only shows molecules that
demonstrate unrestricted diffusion in solution. It requires the subtraction of the diffusion
edited spectrum (contains molecules with restricted diffusion) from a reference
spectrum (without diffusion weighting, i.e. defocusing/refocusing gradients set to zero
power) but otherwise acquired under identical conditions. The IDE spectrum (Figure
29
2-1b) emphasized a range of small molecules but identification from the IDE spectrum
alone is challenging. Two-dimensional HSQC correlations provided additional spectral
dispersion as well as one bond 1H-13C connectivity information. Database matching
against the AMIX Bruker Bio-reference spectral database of the HSQC data along with
1H-1H COSY, 1H-1H TOCSY and 1H-1H NOESY (see Appendix Figure A 1 for example
COSY and TOCSY data) confirmed a wide range of metabolites present, Figure 2-1c
(see Appendix Figure A 2 for an expansion of Figure 2-1c).
30
Figure 2-1 13C NMR spectra of a single 13C labeled wheat seed. a) 1D carbon profile. 1: Carbonyls, 2: guanidine groupcarbon of arginine, 3: aromatic, 4: double bonds, 5: ethylene, 6: anomeric carbons, 7: overlapping carbohydrate andamino acids, 8: methanol, 9: amino acids, 10: aliphatic. b) Spectrum showing the components with unrestricteddiffusion (soluble) (by IDE). 11: Small sugars. c) HSQC spectrum showing color-coded soluble/mobile species asdetermined by AMIX Bruker Bio-reference spectra database. See Appendix Figure A 2 for an expansion.
31
After identification from the 2D spectrum, metabolite assignments were transferred to
the 1D carbon spectrum (Figure 2-1a). Major regions can be summarized as: carbonyls
from proteins and lipids (approximately 190-170 ppm); guanidine group of arginine (159
ppm); conjugated double bonds (135-130 ppm) from triacylglycerides (TAG, see next
section); ethylene (sharp signal at 128.1 ppm); anomeric carbons of carbohydrates (90-
110ppm); various overlapping carbohydrate and amino acid signals (90-50ppm);
methanol (56.40 ppm) and amino acids and various metabolites (42-46 ppm including 4-
aminobutyric acid, arginine, cadaverine, lysine, ornithine, putrescine) and aliphatics (50-
5 ppm, mainly TAG, see next section).
As discussed above, the IDE spectrum (Figure 2-1b) highlighted the molecules that are
most liquid-like or dynamic within the seed. To our knowledge, this is the first report of
ethylene, a gas within the seed (Matilla et al., 2008), detected by NMR spectroscopy.
Strong signals from methanol and a range of small sugars dominated, indicating that
these are present in a dissolved or dynamic state. Conversely, the aliphatic signals
hardly contribute at all to the IDE spectrum and suggest many of these are in a more
restricted environment, this will be discussed in the next section.
2.4.2.2 Components with restricted diffusion and semi-solid componentsvia diffusion based editing
Diffusion editing encodes the spatial position of a signal at the start of the experiment
and decodes it at the end. If the molecule physically changes position during the
experiment it is not refocused and is attenuated. The result is a spectrum that contains
signals from molecules that show very slow/no diffusion, such as macromolecules,
32
swollen polymers and small molecules trapped in an environment that prevents free
diffusion.
Figure 2-2a shows the 13C diffusion edited (DE) spectrum of the wheat seed. Small
signals from carbohydrate were present indicating that some carbohydrates were
present in a restricted diffusion like state. These could be swollen carbohydrate
polymers (for example, starch) or smaller entities sorbed to other larger components.
The dominant signals in the DE spectrum arose from triacylglycerides (TAG). The
HSQC of the single 13C labeled wheat seed (Figure 2-2c) matches extremely well with
the ACD/Labs HSQC simulation of the generic TAG structure overlaid (Figure 2-2d).
While generic TAG structures clearly dominate the lipid profile individual TAG molecules
could not be distinguished. Detailed assignments are present in Table 2-1 which are
consistent with COSY, TOCSY, NOESY and with the previous work by Sacco and co-
workers (1998) .
Table 2-1 Proton (1H) and carbon (13C) chemical shift assignment of fatty acid andlipidic components of single 13C labeled (97 atom %) wheat seedComponent 1H (ppm) 13C (ppm)CH3-(CH2)n- 0.90 16.60CH3-(CH2)n- 1.31 25.30-(CH2)n-CH2-CH2 1.31 32.20-(CH2)n-CH2-CH2 1.31 34.20-(CH2)-CH2-CO-O- 1.59 27.60-CH2-CH=CH- 2.05 29.80-CH2-CH2-CO-O- 2.25 36.40-CH=CH-CH2-CH=CH- 2.76 28.20-CH2O- 4.08-4.28 64.40-CHO- 5.21 71.50-CH2-CH=CH- 5.32 130.90-132.20
33
Complete TAG oxidation yields twice the amount of energy that could be generated
from protein or carbohydrates (Theodoulou et al., 2012). Thus, TAGs are considered
highly compact energy reserves and are therefore an efficient way to maximize energy
storage within the confines of a seed (Theodoulou et al., 2012). Most triglycerides exist
as a “solid fat” somewhat analogous to butter, as such it is logical that TAG within the
seeds display restricted diffusion like properties rather than a true liquid or solid.
34
Figure 2-2 13C NMR spectra of a single 13C labeled wheat seed. a) Components with restricted diffusion (by DE). b)Semi-solids (by RADE). c) HSQC with TAG signals in black and all others signals grey. d) ACD/Labs HSQCsimulation of generic TAG structure (overlaid).
35
Diffusion editing is a useful experiment to emphasize components with restricted
movement. However, the experiment uses a relatively long diffusion delay which could
potentially lead to the loss of signal from very large or semi-solid components that
exhibit fast relaxation. This rapidly relaxing signal can be fully recovered using an
experiment termed Recovering relaxation losses Arising from Diffusion Editing (RADE)
the technicalities of which are discussed by Courtier-Murias and co-workers (2012). In
simple terms, RADE accounts for signals from semi-solids which otherwise could be
missed by diffusion based spectral editing alone. Figure 2-2b shows the RADE
spectrum for the 13C labeled wheat seed. The spectrum contains signals from the
components that relax extremely fast and have a semi-solid character. As with the
spectrum of components with restricted diffusion, there is a strong signature from TAG
suggesting that some of the TAG has a semi-solid like character as expected. Other
lipid signals may arise from membrane structures within the seed. In addition to the
aliphatic species, the RADE spectrum also contains significant carbohydrate signal,
indicating strong contribution from carbohydrate polymers. These could arise from the
starchy endosperm of wheat seeds (Calucci et al., 2004) as well as cell walls and
possibly more flexible components of the seed coat itself. These carbohydrate signals
are strongly emphasized by CP-MAS (Figure 2-3a) which emphasizes the true solid in
the seed coat (Oliveira et al., 2001).
2.4.2.3 Solid components
The above experiments focused on the unrestricted diffusion (soluble), restricted
diffusion and semi-solid components of 13C labeled seeds. As CMP-NMR probes can
handle the high power RF requirements required for modern solid-state experiments,
36
the solid components of the seeds can also be studied. Cross polarization magic angle
spinning (CP-MAS) is an excellent filter for the 13C detection of true solid components
(Courtier-Murias et al., 2012). During CP, magnetization is passed from proton on
carbon via a strong dipole network. As such, CP is not efficient for dynamic systems (for
example solutions, mobile gels). However, in the true solids static H-C dipoles provide a
useful framework for CP and the process is highly efficient. The result is that CP
provides a strong bias toward the components in the sample with the most solid-like
character. The solid state NMR spectral profile is shown in Figure 2-3a and is
dominated by starch, cellulose and hemicellulose signals. Assignments for all peaks
have previously been reported (Bardet et al., 2001; Jiang et al., 2012) and are labeled in
the Figure 2-3a. Absolute quantification is challenging because of overlap of signals and
additional in-depth research is required to understand how the editing procedure affects
quantification. It is unclear how the different editing steps influence absolute
quantification, however, it is possible to do relative quantification between the species
(see next section) to estimate crude changes or differences.. T2 relaxation editing can
be used to further edit the solid components with dynamics from the true solid domains.
The proton T2 filter is utilized prior to CP so that protons in the most solid environment
with short T2 will preferentially relax. This leaves only the resonances from the
unrestricted diffusion or semi-mobile materials (Figure 2-3c). The most rigid signals with
short T2 can be recovered with spectral subtraction of the T2-filtered CP-MAS spectrum
(dynamic solids) from the regular CP-MAS spectrum (everything) leaving a spectrum
containing the most rigid resonances (Figure 2-3b). For example, the peak for
amorphous CH2 does appear in T2 filtered CP-MAS (labelled "9" in Figure 2-3c)
demonstrating these chains exhibit motion and likely exist as restricted diffusion/semi-
37
solid state. Conversely, the signal labelled “5” (Figure 2-3b) arose from crystalline
cellulose. Specifically, this signal represents the C4 position in crystalline cellulose and
is the only signal that can be clearly resolved due to overlap. Crystalline cellulose
behaves like a true solid as such it is strongly emphasized along with the most rigid
components (Figure 2-3b)
Figure 2-3 13C NMR spectra of a single 13C labeled wheat seed. a) True solids (byCP-MAS), 1: carbonyls in lignins, hemicelluloses and proteins, 2: double bonds(lignins), 3: C1 of cellulose and hemicelluloses, 4: C1 of starch (anomeric carbon)5: C4 of crystalline cellulose, 6: C4 of amorphous cellulose, hemicellulosesand/or starch, 7: C2, C3, C5 in celluloses, hemicelluloses and starch and C6starch branch points, 8: C6 in celluloses, hemicelluloses and starch, 9:amorphous CH2, 10: aliphatic. b) Spectral editing to emphasize rigid solids. c) T2filtered CP-MAS to emphasize dynamic solids d) DARR to highlight connectivitiesbetween carbons for the truly solid components
38
Other carbohydrate signals, mainly starch, are prominent in all spectra and come from
the starchy endosperm of wheat seeds (Calucci et al., 2004) and from the seed coat
(Oliveira et al., 2001). Next to TAGs, starch and storage protein are the main storage
reserves of cereal seeds. Higher plants produce starches which are a critical nutritional
source for humans (Tetlow, 2011). Dipolar Assisted Rotational Resonance (DARR) is
able to identify 13C-13C correlations through space (Figure 2-3d) and is a very useful
experiment to confirm and help assign structure. DARR confirms that, the signals at 62-
73 ppm, 62-100 ppm and 73-100 ppm are correlated in the same structure consistent
with that of carbohydrates in general. DARR also confirms that the TAG at 30 ppm and
carbonyl at 130 ppm (not shown) each are not correlated to carbohydrate signals.
2.4.3 Comparing wheat, broccoli and corn seeds
For unrestricted diffusion (soluble) components (IDE), corn has the most signals in the
free carbohydrate area (about 10% more than wheat and 55% more than broccoli based
on integration); with the majority of signals arising from glucose and fructose which is
consistent with literature (Figure 2-4c) (Whistler et al., 1957; García-Pérez et al., 2013).
It is important to note that in the case of corn, the seed was cut in half and it is possible
that some of this additional signal intensity from soluble molecules could arise from D2O
leaching components from the exposed interior of the seed. The IDE (unrestricted
diffusion (soluble) components, Figure 2-4b) and DE (restricted diffusion components,
Figure 2-4e) for broccoli looked quite similar and both were dominated by lipids in large
part because of the lower carbohydrate levels compared to the other two species.
Broccoli had the highest lipid content (about 10% more than wheat and corn based on
integration) and these findings are consistent with trends found in literature (U.S.; Gu et
39
al., 2011). The actual location of these small molecules within the seeds is unclear,
previous MRI based studies have indicated that water has some mobility within the
endosperm (Callaghan et al., 1979; Ishida et al., 1995) and it is possible that some of
these relatively free molecules are associated with this water. The RADE spectrum
(semi-solids) of corn and wheat (Figure 2-4i) emphasized a considerable contribution of
carbohydrates (U.S.). Conversely, the broccoli shows very few carbohydrates in the
semi-solid phase. Note that in wheat, broccoli and corn the CH3 signal of lipids (marked
with an * in Figure 2-4, a-f) appeared in the IDE and DE but disappeared in the RADE
spectrum. This likely resulted from the local motion of the CH3 terminal groups that
leads to longer relaxation times compared with other parts of the lipid/TAG molecules.
40
Figure 2-4 13C NMR spectra comparing 13C labelled wheat, broccoli and cornseeds. a-c) unrestricted diffusion (soluble) components (by IDE). d-f) restricteddiffusion (by DE). g-i) Semi-solids (by RADE). j-l) True solids (by CP-MAS). Figureis labeled as follows: the -CH3 of TAG is marked with an *, 1: dominated byfructose in corn; 2: triacylglycerides; 3: carbonyls (result of increased proteincontent); 4: aromatic (result of increased protein content); 5: α carbon of aminoacids; 6: dominated by aliphatic amino acids; 7: aliphatic -(CH2)- (dominated byTAG)
The CP-MAS of broccoli (Figure 2-4k) was very different than that of wheat (Figure 2-4j)
mainly because it had stronger signals from proteins (about 2.5 times more intense
based on integration) and from long chain fatty acyl groups than the wheat spectrum.
41
This is consistent with literature that lists protein in broccoli seeds to be ~21 g/100 g (Gu
et al., 2011) and wheat to be ~10 g/100 g (U.S.). The 60-0 ppm region mainly arises
from aliphatic amino acids with the strong signals from long chain aliphatics (CH2)n
superimposed at ~30ppm. α-carbons from protein resonate in a band from ~60-40 ppm
centered at ~50-55 ppm and aromatic amino acids add to the aromatic region and are
most prominent from 110-120 ppm. The carbonyl signals at 190-160 ppm arise from
proteins, lignins, lipids and hemicelluloses. The carbonyls are likely much more intense
in the broccoli compared to the wheat (roughly 5.5 times more based on integration) in
large part due to the additional protein and oil content (U.S.; Gu et al., 2011). The
presence of a relatively strong (CH2)n superimposed at ~30ppm in the broccoli is
interesting. This suggests that in the broccoli, at least some of the aliphatic components
are more solid-like than in the other seeds. As the characteristic double bonds from
TAG are also present (~125ppm) it is most likely that truly solid aliphatic component is a
portion of TAG stored in the more solid form when compared to the other seeds.
However, it is also possible that some of this additional CH2 intensity also arises from
lipo-protein, free fatty acids or lipids other than TAG which are known to be present in
seeds (Terskikh et al., 2005; Austin et al., 2006; Bréhélin et al., 2007).
2.4.4 Other considerations
CMP-NMR provides a unique insight into both chemical and physical attributes of
molecular structure inside unaltered natural samples. Seeds here were used to
exemplify the approach to natural samples in general. Isotopic labelling is beneficial but
not essential for CMP-NMR. Generally, labelling is necessary when less sensitive multi-
dimensional spectra (for example the DARR in this study) are being obtained in order to
42
abbreviate spectrometer time with enhanced signal to noise. Natural abundance of 13C
can be used to acquire NMR spectra but will increase sampling time considerably. The
13C labelled CP of wheat was performed over a period of 1 hour 43 minutes for 2K
scans (signal to noise: 690), whereas the unlabelled equivalent containing 13C at the
natural abundance took 16 hours and 23 minutes for 20K scans (signal to noise: 69).
Comparison of the 13C labelled spectra to natural abundance spectra are provided in the
appendix (see Figure A 3-A5). In the future, the use of 7mm rotor diameters will allow
~4-5 times the sample volume to be introduced, this should substantially increase signal
from unlabelled samples and should make similar studies on unlabelled material more
feasible. The main drawback of CMP-NMR probes in comparison to a dedicated HR-
MAS or solids probe is loss of sensitivity. As discussed by Courtier Murias et al. (2012)
the 4 channel prototype probe used here suffers from a loss of ~40% when compared to
a two channel solids probe. This loss mainly arises due to single circuit being
quadrupley tuned in the 4 channel design. If identical probes could be compared (note,
no 2 channel CMP-NMR probes have been built to date), it is predicted that the loss in
sensitivity would be ~10-15% (mainly associated with the addition of the gradient coil).
Due to the sensitivity losses CMP-NMR probes should be restricted to studies of native
sample where the study of the components in their native phase is critical. In such
cases the potential for CMP-NMR to provide otherwise inaccessible molecular-level
information in-situ is considerable. In studies following developmental changes it may
be important to study the development of the solid, gel, and liquid components over
time. Here it may be important to interleave solids, gel and liquid NMR experiments and
measure, not just the phases independently, but kinetic transfer between the phases.
Such studies would be impossible using two or three separate NMR probes as it takes
43
several hours to change and properly calibrate an NMR probe. Similarly, studies that
follow the conversion of a molecule from the liquid, to gel, to solid phases (for example
feeding phenylalanine to follow lignin growth, or the binding and sequestration of a
contaminant) also require the kinetic transfer between phases to be monitored and may
find CMP-NMR probes useful in the future. These examples, indicate a future potential
of CMP-NMR in seed/plant/food research. Finally, as the CMP-NMR probes combine all
aspects of solution, HR-MAS and solids, they provide the potential to develop novel
NMR experiments (for example solids using gradients) to better select and study
structure and interaction in-situ.
Finally, it is important to note that quantification in complex systems such as seeds are
complicated by spectral overlap. Spectral editing can extract molecules in the liquid, gel,
semi-solid or solid state and theoretically if the carbons were detected in exactly the
same manner it would be possible to quantify the distribution between the different
phases. However, in this study, this has been complicated by the fact CP-MAS was
used for solid components, which enhances certain signals more than others making
this comparison inaccurate. Commonly, for quantification in solids, direct polarization
magic angle spinning (DP-MAS) (Keeler et al., 2003) would be used. With regard to
CMP-NMR, the cross polarization (CP) element was required to select the rigid bonds
(i.e. spectral editing to select the solid components) and so further complicating the
issue. It may possible to quantify in the future but this would require advanced
techniques such as spin counting (Smernik et al., 2000).
While the focus of this study has been to demonstrate the general applicability of CMP-
NMR to the chemical structures of seeds, non-labelled broccoli seeds were collected
44
after spinning to test if they would germinate. They successfully germinated after being
transferred to a Petri-dish with a filter paper moist with water. This indicates that the
seeds were still alive during spinning and the potential for monitoring them while
germinating the seed exists. CMP-NMR holds potential to provide an unprecedented
window into the germination process itself. This avenue opens potential for research in
fields such as physiology (germination, vernalization processes), agriculture (seed
viability) and food safety (pathogen test, purity). Specifically, applications such as
selecting viable seeds (current methods are mutagenic, destructive or time consuming)
or for seed selection breeding programs which all require detailed yet non-destructive
molecular analysis (Terry et al., 2003).
45
Chapter 3. Elucidating structural and metabolic changeduring germination and early growth of 13C labeledseeds through Comprehensive Multiphase NMR
spectroscopy1
3.1 Abstract
Nuclear Magnetic Resonance (NMR) spectroscopy has been used before to monitor
germination and early growth but has historically developed as two separate fields,
solid-state and liquid-state NMR. In this study, a novel technique, introduced in 2012
termed Comprehensive Multiphase (CMP) NMR spectroscopy, was used. CMP-NMR is
capable of studying all phases: liquid, gel-like, semi-solid and solid, in intact samples by
combining all required electronics into a single NMR probe and is ideal for investigating
biological processes such as seed germination. In this study 13C labeled wheat seeds
were used to demonstrate the applicability of CMP-NMR for studying germination and
early growth. All components, from the most liquid like (i.e. metabolites) to the most rigid
(seed coat) are monitored in-situ. After 96 h, the number of metabolites in the mobile
phase more than doubled in comparison to 0 h. Relative quantification revealed the
carbohydrates increased >265% in each of the mobile, gel and semi-solid phases.
Lipids (dominated by triacylglycerides) decreased 63, 41 and 36% in the mobile, gel and
semi-solid phases, respectively, which is in agreement with literature as the growing
seedling metabolizes the lipids for energy. The true solid component is dominated by
1 The samples were provided by IsoLife (Wageningen, The Netherlands). The experimental design wascreated by Leayen Lam and André J. Simpson. The lab experiments were conducted by Leayen Lam withguidance from André J. Simpson. Data interpretation was performed by Leayen Lam with guidance fromAndré J. Simpson. The manuscript was written by Leayen Lam with critical comments from AndréJ.Simpson and Myrna J. Simpson
46
structural carbohydrates. This article demonstrates the applicability of CMP-NMR to
understand early seed growth; CMP-NMR in general represents a powerful tool with
wide spread applicability to unravel complex biological processes in general.
3.2 Introduction
The typical propagation and dispersal method of angiosperms (flowering plants) is
through the use of seeds. Seeds are complex reproductive organs that upon maturation
from the parent plant can endure extreme desiccation to tolerate unfavorable
environmental conditions for extended amounts of time prior to germination (Angelovici
et al., 2010; Graeber et al., 2012). The typical structure of an angiosperm seed consists
of an embryo, endosperm and a seed coat. The embryo is comprised of the cotyledon
(embryonic leaves), radicle (embryonic root) and hypocotyl (connects the cotyledon to
the radicle) (Finch-Savage et al., 2006; Linkies et al., 2010). The starchy endosperm
provides the embryo with sustenance during germination and early growth. Until the
seedling can start photosynthesizing the embryo relies on the endosperm for food
(Calucci et al., 2004; Shewry et al., 2012). The seed coat protects it from physical
damage and from desiccation (Patrick et al., 2010).
Germination is an intricate procedure involving complex processes including structural
distribution of various hormones, action of the hormones within the seed, structural and
metabolic change, gene expression and response to environmental cues (Allen et al.,
2010). It commences with imbibition which is when the dry, quiescent seed takes up
water compelled by low matrix potential (Garnczarska et al., 2007). Once swollen with
water, the seed increases metabolic activity and hydrolysis of triacylglycerol (TAG),
protein and starch reserves to feed the embryo. When the radicle protrudes out through
47
the seed coat, germination is said to be complete (De-La-Cruz Chacón et al., 2013) and
thus, early seedling growth begins.
Nuclear magnetic resonance (NMR) spectroscopy is a powerful molecular-level
technique that allows examination individual nuclei and bonding in a sample to probe
complex chemical structures and interactions. It has been used for various aspects of
germination and early seedling growth to characterize the changes in germinating wheat
(Krishnan et al., 2004a), soybeans (Krishnan et al., 2004b), rice (Ishibashi et al., 2005),
tomato (Nagarajan et al., 2005) and lupine seeds (Garnczarska et al., 2007) by
hydration using T2 relaxation times. NMR has also been used to monitor variations in
water uptake and lipid consumption in sesame seeds (Sarkar et al., 2009) and to
monitor mobilization of oil reserves following seed germination and during early seedling
growth in intact seeds of soybean (Terskikh et al., 2011). However, all of these NMR
studies have been restricted to the study of specific phases (i.e. liquid, gel-like or solid
components) within the seeds.
Comprehensive Multiphase (CMP) NMR was introduced in 2012 and combines all the
electronics 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 (Courtier-Murias et al., 2012). The resulting technology permits
uncompromised NMR studies of components with and without unrestricted diffusion
(liquid and gel, respectively) and solid components in intact and unaltered samples. The
approach provides in-depth information on structure and interaction within- and between
phases in situ (Courtier-Murias et al., 2012).
48
As such this approach is ideally suited, not just for determining the chemical entities
themselves, but how processes such as swelling, drying, etc., affect the molecular
structure. Such processes are critical to understanding seed germination and early
growth, which is initiated by the uptake of water. Elucidating events following initial
water uptake, should provide an unprecedented window into the molecular events
behind seedling growth and the overall process of germination
This study represents the first application of CMP-NMR to study seed germination and
early growth. In this study, 13C labeled wheat seeds (Triticum aestivum) were
germinated and changes over time elucidated using CMP-NMR. As CMP-NMR provides
an unprecedented window into molecular phenomena occurring within intact biological
samples, it is likely that CMP-NMR will become an indispensable tool for understanding
natural processes.
3.3 Materials and methods
3.3.1 13C labeled wheat seeds
Specially designed, air-tight high-irradiance growth chambers were used to produce
uniformly 13C labelled (>97% total carbon content) wheat (Triticum aestivum) seeds
provided (IsoLife, Wageningen, The Netherlands). In a closed atmosphere containing
97 atom% 13CO2 (from pressurised cylinders; Isotec, Inc., Miamisburg, OH, USA),
plants were grown from (13C-labeled) seed from the seedling stage until full maturity.
Pollination of the wheat was certified by having efficient internal wind speed. Mineral
nutrients were made available as Hoagland-type solutions with micronutrients and iron
(Smakman et al., 1982; De Visser et al., 1997). The climate conditions were as follows:
49
irradiance (PPFD) 600 mol m–2 s–1 (HPI) during a 16-h day, D/N temperature 24/16°C,
relative humidity 75/85%.
3.3.2 Germination
Six 13C labeled wheat seeds were selected for germination, average seed dry wt was
0.0283 g and variability in seed dry wt 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 to prevent
photosynthesis from beginning.
3.3.3 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 zirconium rotor (length of rotor is ~1.7 cm) . At 0 h the dry seed
was used and 1 new seedling was used at each time point. Montmorillionite clay powder
from Source Clays Repository (Indiana, USA) was prepared by mass in a 1:5 mixture
with D2O (Andover, Massachusetts), 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 then the whole and intact seed/seedling was placed
directly into the rotor. The rotor was sealed using a top insert made from Kel-F, a Kel-F
sealing screw and Kel-F cap
3.3.4 NMR Spectroscopy
All NMR spectra were acquired using a Bruker BioSpin Avance 500 MHz 500 MHz
Bruker Avance III Spectrometer at a spinning speed of 6666 Hz fitted with a prototype
CMP MAS 4 mm 1H–13C–19F–2H probe that has an actively shielded Z gradient (Bruker
50
BioSpin). All experiments were performed at room temperature and locked on D2O. This
lock was maintained for all experiments including solid-state experiments
Decoupling was used in both one-dimensional (1D) and two-dimensional 2D
experiments to removed 1H-13C coupling from the labeled sample. Garp decoupling was
used for proton observe, low power waltz16 decoupling for carbon observe and spinal64
for high power decoupling (Cross Polarization Magic Angle Spinning (CP-MAS) NMR
experiments).
3.3.4.1 1D NMR spectroscopy
Proton spectra were obtained using presaturation for water suppression and 90° pulse
calibrated for each sample. Standard inversion approach was used for measuring T1
time for each sample; the recycle delay was set to 5 times the measured T1 value.
Spectral width was 20 ppm, 8192 time domain points, 512 number of scans. Processing
was done using an exponential function corresponding to a line broadening of 0.3 Hz.
All carbon spectra (with the exception of CP) were obtained using spectral width of 400
ppm, 16384 time domain points, 2048-4096 number of scans and inverse gated 1H
decoupling. A Standard inversion recovery approach was used for measuring T1 time
for each sample; the recycle delay was set to 5 times the measured T1 value.
Processing was done using an exponential function corresponding to a line broadening
of 5 Hz for 13C 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 and a contact time of 1ms and a
line broadening corresponding to 25Hz.
51
3.3.4.2 Spectral editing and scaling
Inverse diffusion editing (IDE), relaxation Recover Arising from Diffusion Editing (RADE)
and inverse T2 filtered 13C CP-MAS was done by appropriate spectral subtraction as
previously described (Courtier-Murias et al., 2012).
3.3.4.3 2D NMR spectroscopy1H–13C Heteronuclear Single-Quantum Coherence (HSQC) spectra were collected in
phase sensitive mode using Echo/Antiecho-TPPI gradient selection. Number of scans
was 256 and was collected for each of the 256 increments in the F1 dimension. 1024
time domain points were collected in F2 and a 1J 1H–13C of 145 Hz. 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 was acquired on select samples to
confirm HSQC NMR assignments of metabolites using Bruker’s Bioreference databases
version 2.0.0 to 2.0.3 and AMIX (Analysis of MIXtures software package, version 3.9.3,
Bruker BioSpin). The COSY NMR experiments were done in non-phase-sensitive mode,
using gradients for coherence selection and low power 13C garp decoupling throughout.
128 scans and 2048 data points were collected for each of the 196 increments in the
F1. Both dimensions were processed using unshifted sine-squared function, zero filling
factor of 2 and a magnitude mode was used for projection. COSY spectra not presented
in this manuscript.
3.3.5 Compound Identification and quantification
Spectra were calibrated against a range of known compounds in the Bruker Biofluid
Reference Compound Database (v 2.0.3). Pattern matching of both 1D and 2D spectra
52
was performed using AMIX (version 3.9.3, Bruker BioSpin) against the Bruker
Bioreference Compound Database (v 2-0-0 to v 2-0-3) using a procedure previously
developed for complex mixtures (Woods et al., 2011). Compounds with a greater than
80% confidence match from automated searches were further selected for detailed
manual inspection. Only assignments that exhibited a greater than an R2 correlation
>0.99 between the observed and database shifts were retained as assignments. Where
possible, correlations were also confirmed with COSY.
Relative quantification of the metabolites and general classes were also performed with
the multi-integrate tool in AMIX to give the reader an idea of how components in the
seed change over time. Proton NMR spectra were normalized over 9-0 ppm to account
for seed/seedling differences. The proton carbohydrate region was defined as 5.57-3.10
ppm and aliphatic lipids defined as 2.88-0.57 ppm. Carbon spectra were normalized
over the 200-0 ppm region to account for seed/seedling differences. Herein, when
changes in 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 were the double bonds defined as 136 – 128 ppm.
For carbohydrates the sum of the anomeric carbons were used and defined as 103-94
ppm. Individual sugars could not be resolved because the anomeric carbons of several
sugars overlap however, it was possible to define ranges resulting for a combination of
sugars To reduce redundancy in the article, the following naming system will be used to
identify the sugars who anomeric signals overlap in each spectral range: AC1 for
melibiose and D-raffinose anomeric carbon signals defined as 103 -101 ppm, AC2 for
D-glucose, melibiose and D-xylose anomeric carbons defined as 99-97 ppm and AC3
53
for D-glucose, melibiose, D-raffinose, sucrose and D-xylose anomeric carbons defined
as 96-94 ppm.
3.4 Results and discussion
CMP-NMR spectroscopy was performed to study the transformation of intact seed to
seedling of 13C labeled wheat every 24 hours until the seedling was too tall to fit into the
NMR rotor . Photographs of the wheat seeds at different developmental stages are
shown in Figure 3-1. The growth of monocotyledonous plants are 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 3-1b) the radicle can be seen
protruding through the bottom of the seed. The length of the wheat seed at 0 h was ~0.5
cm (Figure 3-1a) and increased during the course of the germination process to ~3 cm
after 96 h (Figure 3-1e), measured from the tip of the shoot to the end of the longest
root.
54
Figure 3-1. a) Intact 13C labeled wheat seed and seeds germinated for b) 24 hrs, c)48 hrs, d) 72 hrs and e) 96 hrs.
For easy visualization all spectra are presented in this chapter such that the largest
peak in each spectrum is the same size. We found this most useful way and logical way
to display a series of sample where the dominant spectral components change with
time. Even though the selected seeds were within 5.75% dry wt of each other, scaling to
noise was not an option because the signal-to-noise depends on the proximity and
orientation of the sample with respect to the detection coil. Note the detection coil is
slightly shorter than the rotor and the highest sensitivity and homogeneity is obtained at
the center of the coil. As the sample changed size and shape during the study it was
impossible to position the seed in exactly the same place. For example, for the later
time points the seeds had to be positioned slightly lower to allow room for the shoots.
Furthermore, it was also impossible to guarantee that the specimens, while surrounded
55
with montmorillonite clay, did not move slightly on spinning. Given all these challenges,
quantification was done based on normalization to the total spectral area. Such
normalization is common in metabolomics studies and permits comparison of extracts of
organisms with differing weights (McKelvie et al., 2009). Absolute quantification was not
possible, due to spectral overlap, sample positioning, variations in water content, and
complications arising from the use of spectral editing approaches to separate the
various components with and without restricted diffusion (liquid and gel, respectively)
and semi-solid and solid components. For example, Courtier-Murias and co-workers
(2012) reported that while CMP-NMR can detect all components, some gel components
tended to contribute to both the gel-like spectrum and the semi-solid like spectrum and
some components may be detected twice. As such, any attempts to provide absolute
quantification at this early stage of CMP-NMR development was not justifiable.
Overtime and with in-depth characterization of the experiments along with a detailed
understanding of structural overlap in the CMP-NMR of plants and seeds, absolute
quantification may become feasible. This is discussed in more detail in section 3.5for
future directions.
Here, relative quantification that permits inter-comparison between samples is
presented. This is simply expressed as an approximate relative percentage (%)
increase or decrease of a specific signal between samples and can only be performed
for resolved signals. This is included such that readers can gauge the comparative
changes during growth and should be considered an estimate rather than exact values..
The NMR data will be introduced such that the components with unrestricted diffusion
56
are considered first followed by components with restricted diffusion, semi-solid and
finally solid components in the seeds/seedlings.
3.4.1 1D 13C NMR: Components with unrestricted and restricteddiffusion
3.4.1.1 Components with unrestricted diffusion
Figure 3-2. Comparison of carbon spectra obtained for a single 13C labeled wheatseed at 0 hour to a seedling at 96 hour. a-b) 13C 1D profile, c-d) Components withunrestricted diffusion (IDE). As labeled on above, 1: carbohydrate region, 2:aliphatic amino acids, 3: double bond of TAG, anomeric carbons: AC1, AC2 &AC3.
Figure 3-2a and b show the 13C carbon inverse gated spectrum at 0 h compared to 96 h
respectively. In this case the 13C spectra have been acquired using inverse-gated
57
decoupling and the recycle has been set at 5 x T1 value to provide a quantitative
overview. However, the experiment has been deliberately collected using low power
decoupling. As such carbons that are in a true solid domains (i.e. true glassy solid) will
be attached to protons with extremely wide spectral profile (caused by 1H-1H dipolar
coupling in a true solid-state) and will not be efficiently decoupled using lower power
coupling. The result is that carbons in the solid phase will be strongly attenuated in the
spectrum. As such the spectrum represents a roughly quantitative overview of the
components with unrestricted diffusion, restricted diffusion, semi-solid in the sample
with the true solids strongly attenuated.
Upon first observation of the 13C carbon inverse gated spectrum at 0 h compared to 96
h (Figure 3-2a and b respectively) two noticeable differences dominate. The first is that
at 0 h (dry seed) has some sharp peaks but these are superimposed upon a quite
pronounced broader spectral profile. This broad profile is characteristic of signals that
exhibit little dynamics and could arise from gels, semi-solids and maybe even some of
the more dynamic amorphous solids. The sharper signals in contrast arise from mobile
carbon such as those truly dissolved or in a dynamic gel-like environment. By 96 h it is
clear that the seed has swollen considerably and producing range of sharper signals
dominate with a less pronounced broad profile.
The second difference is that there are many more sharp signals in the 96 h then the 0
h spectrum, especially in the carbohydrate region (labeled "1" in Figure 3-2b) and amino
acid side chain/ aliphatic region (labeled "2" in Figure 3-2b). Detailed assignments of
these amino acids, carbohydrates and other metabolites will be discussed in further
detail in section 3.4.2 which introduces 2D NMR.
58
Spectral editing that can further emphasize components from specific phases in the
seed structure is possible. IDE is a difference approach that selects only the
components that exhibit unrestricted diffusion (i.e. truly dissolved species; (Courtier-
Murias et al., 2012). The IDE experiment shows an increased in intensity in the
carbohydrate region at 96 hours (roughly 265%) whereas the major plant lipid, TAG,
have decreased (about 63%; Figure 3-2c and d).
This is in agreement with literature that concludes that TAG is mobilized and consumed
prior to successful germination (Bewley, 1997; Allen et al., 2010). TAG will be further
discussed in section 3.4.1.2 and 3.4.2.2 describing restricted diffusion. This increase in
carbohydrates and decrease in lipids is a consistent trend that will be observed through
all spectra in this chapter and is consistent with the wider literature (Bewley, 1997;
Vensel et al., 2005; Finch-Savage et al., 2006; Nonogaki et al., 2010; Nelson et al.,
2013). The anomeric carbons of the carbohydrates are much more defined after 96hrs (
labels AC1, AC2, AC3 in Figure 3-2d). Specifically, the AC1, AC2 and AC3 signals at 96
h have increased by 1860, 1.29 x 104 and 74% compared to the 0 h spectrum.
The signal-to-noise ratios have also increased between 0 and 96 h; this 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 is imbibed, takes up water and as the seedling
grows, it will have a higher contribution to the soluble phase than it did before. In
addition, at 96hrs, contributions from the roots and shoot which have a higher water
content will contribute additional signals to freely diffusing species (IDE spectrum) along
with newly synthesized metabolites.
59
3.4.1.2 Components with restricted diffusion
DE emphasizes molecules that have restricted/slow diffusion; this could include
dissolved macromolecules, gel-like species or sorbed molecules. Signals with restricted
diffusion are dominated by what appears to be, lipid signals, specifically TAG. The TAG
signals are confirmed by 2D spectroscopy in section 3.4.2.2. Briefly, TAGs are long
chained molecules varying in acyl chain-lengths and functionality (Nascimento et al.,
2007; Purkrtova et al., 2008). They are the primary fuel source for the transformation of
seed to seedling by the breakdown of TAG to serve as both an energy source and a
carbon skeleton (van der Schoot et al., 2011; Theodoulou et al., 2012).
Figure 3-3. Comparison of carbon spectra of components with restricted diffusion(DE) for a single 13C labeled wheat seed at a) 0 hour and b) 96 hour. As labeled onabove, 1: double bond of TAG, AC: anomeric carbons.
At 96 h (Figure 3-3b) the TAG has decreased by about 41% (double bond of TAG
labeled as "1" in Figure 3-3a) and the carbohydrates have increased by about 486%
(labeled "AC" in Figure 3-3b). These relative changes are consistent with the trend seen
in the IDE spectra in section 3.4.1.1. These TAG signals can be easily identified by
60
reference to the 1H-13C HSQC NMR spectrum (Figure 3-5) in section Error! Reference
source not found. for 2D analysis of components with restricted diffusion. The editing
shows lipids in many different forms, from liquid in section 3.4.1.2 all the way to semi-
solid which is discussed in section 3.4.3.
3.4.2 2D 1H -13C HSQC: components with unrestricted and restricteddiffusion
1D NMR spectra have considerable amount of overlap complex natural samples such
as seeds, so it becomes necessary for more detailed spectral assignment to acquire 2D
HSQC spectra. HSQC disperses the chemical shift information into two dimensions as
well as providing additional one bond H-C connectivity information. In simple terms a
“molecular map” that describes the H-C bonds in the sample results. A correlation
appears for each H-C bond that in the horizontal plane represents the 1H chemical shift
and in the vertical plane represents the 13C chemical shift.
3.4.2.1 Components with unrestricted diffusion
The additional dispersion reduces overlap considerably which permits the use of
accurate pH adjusted databases to make spectral assignments. Here, the Bruker
Bioreference Databases version 2.0.0 to 2.0.3 where used in conjunction with AMIX
(version 3.9.3, Bruker BioSpin) software package in assigning of metabolites.
61
Figure 3-4. Comparison of 1H-13C HSQC of a) a single 13C labeled wheat seed at 0 hour to b) a seedling at 96 hour.
62
At 0 h (Figure 3-4a) 19 metabolites were identified but at 96 h (Figure 3-4b) 41
metabolites were identified using the Bruker Bioreference database, confirmed through
COSY NMR correlations (where possible)using the Bruker Bioreference database .
While it is possible many of the additional metabolites in the solution phase may have
been synthesized in the seedling it is also possible that some were present but in the
drier state that could not be detected by HSQC NMR. 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
are also present at 96 h. Where possible select assignments have been transferred
from the HSQC NMR to the 1H NMR spectra (see Figure A 6 in the appendices). While
the overlap in 1H is much greater than in 13C or HSQC data, example 1H spectra are
provided as studies using 1H detection can be performed without the need to
isotopically label samples.
3.4.2.2 Components with restricted diffusion
Along with small, soluble metabolites, the plant lipid TAG is also prominently seen in the
HSQC of the 13C labeled seedling at 96 h (Figure 3-5).
63
Figure 3-5. 1H-13C HSQC at 96 h with the TAG signals highlighted in black. From1D editing approaches it can be seen that TAG is most gel-like of all thecomponents detected by HSQC NMR.
These signals have been assigned to TAG because it matches very well with ACD/Labs
HSQC simulation of the generic TAG structure (Figure 2-2d) and is consistent with
literature findings (Sacco et al., 1998). Figure 3-5 is the same HSQC in Figure 3-4b but
has been rescaled to show the intense contours which result from TAG. The 0 h HSQC
spectrum is not shown because the TAG does not change significantly over time.
64
3.4.3 1D 13C NMR: Semi-solid components
One disadvantage of diffusion editing is the relatively long delays required to
encode/decode self-diffusion. During these delays signal components with very fast
relaxation can be strongly attenuated and if IDE and DE are used alone it is possible
components with fast relaxation such as semi-solids may be underestimated. Luckily, as
introduced by Courtier-Murias et al. (2012) there is an experimental approach to recover
signal from semi-solids which is termed RADE. For the purpose of this manuscript
readers can think of the RADE spectrum as the emphasis of semi-solids components
within the sample.
Figure 3-6. 13C spectra of semi-solid components (RADE) of labeled wheatseed/seedling at time a) 0 h, b) 24 h, c) 48 h, d) 72 h and e) 96 h. As labeled onabove, 1: double bond of TAG, AC: anomeric carbons.
65
Figure 3-6 shows the RADE spectrum at 24 h increments. Lipids, dominated by TAG
again, are being consumed over time, the relative change between 0 & 96 h is a
decrease of about 36% (labeled "1" in Figure 3-6) whereas carbohydrates are being
synthesized with a relative overall increase of approximately 410%. This is consistent
with the trend seen in IDE and DE. This shows that it’s not just the metabolites that are
changing but also semi-solid components of the seed/seedling. Here we see changes
and differences in semi-solid carbohydrates which are probably from swollen
biopolymers like lignin, cellulose and hemi-cellulose (Barron et al., 2007; Singh et al.,
2011; Brouns et al., 2013). In seeds, TAG is stored into subcellular droplets called
storage lipid bodies surrounded by a monolayer of phospholipids. It has been proposed
that in mature seeds once the oleosin (a proteins found in storage lipid bodies) retains
the phospholipid and TAG, the TAG becomes less mobile (Huang, 1992). This may
partially explain why some TAG is present in a semi-solid form within the seeds
RADE is an excellent experiment that helps recover signals from semi-solid
components that could otherwise could be missed be missed when using a diffusion
based editing scheme. However, RADE still only employs low power decoupling and as
such will strongly discriminate against true solids. To detect true solids in an efficient
manner CP-MAS is required. CP select carbons that exhibit strong H-C dipolar coupling.
The result is that true solids are selected efficiently. Unrestricted and restricted
components are not detected as they are too dynamic, whereas dynamic solids and
semi-solids may be selected to some extent depending on the rigidity of the H-C bonds
(Courtier-Murias et al., 2012). Further additional editing which combined relaxation
66
filters with CP (discussed in further detail in the next section 3.4.4 which addresses rigid
components) can help differentiate signals from restricted/semi-solids and true solids.
3.4.4 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 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 help identity components in the
solid-state. As changes in the solid-state spectra of the seeds are less prominent with
time here we focus on the major changes between the 0 h and 96 h samples only.
Figure 3-7. Comparison of CP-MAS spectra obtained for a single 13C labeledwheat seed at 0 hour to a seedling at 96 hour. a-b) CP-MAS, c-d) dynamic solids(T2 filtered), e-f) most rigid (rigid solids). As labeled on above, 1: C6 of starch &cellulose, 2:non-crystalline material for C4 of starch 3: C6 of starch, 4: C6 ofcellulose, 5: mostly -CH2- of TAG
67
Even comparing 0 to the 96 h spectrum of the same experiment appears pretty similar;
indicating that the solid profile does not change that much over time (Figure 3-7a and
b). To further investigate changes, a T2 filtered was applied to the CP experiment which
emphasizes dynamic solid components (Figure 3-6c and d). In turn the difference
spectrum provides a sub-spectrum of the rigid-solids components (Figure 3-6e and f;
(Courtier-Murias et al., 2012).
The C6 from starch & cellulose appears as one peak in the CP-MAS spectra (Figure
3-6a and b) but becomes a split peak where you can differentiate the starch from the
cellulose signal in the T2 filtered spectra (Figure 3-6c and d). This differentiation occurs
because starch is less rigid/crystalline than cellulose and so appears as separate peaks
in the mobile-solids spectra (Deguchi et al., 2006). At 96 h the splitting of starch and
cellulose signals is more prominent compared to 0 h suggesting that the starch in the
seed has become more swollen with water and thus more mobile. Also, non-crystalline
material for C4 in starch is most prominent in the T2 filtered spectra which is labeled
accordingly (Peng et al., 2011).
Similarly, in the spectrum of the T2 filtered or dynamic solids we see the CH2 peak of
aliphatic TAG at ~33 ppm but the same peak does not appear in the rigid solids
experiment obtained from spectral editing (Figure 3-6e and f). This suggests that little if
any TAG is present in a true crystalline solid-state.
This approach using a variety of CP-MAS NMR experiments doesn’t just give structural
information but also information about the physical state of the components within the
plant. We have learned that there are some dynamic solids and truly rigid, both
68
dominated by cellulose, hemicellulose and starch which is the main component in the
seed endosperm (Calucci et al., 2004; Shewry et al., 2012).
3.5 Future directions
3.5.1 ERETIC II
Accurate absolute quantification of metabolites maybe possible in plants and seed by
combining using Electronic REference To access In vivo Concentrations (ERETIC) and
spin counting approaches. ERETIC provides a reference signal, synthesized by an
electronic device (or software generated) which can be calibrated against absolute
concentrations. This removes the need of adding any internal standard. In addition spin
counting experiments are in-depth quantitation experiments that try to account for the
absolute number of nuclei detected. Using spin counting standards of difference phases
(gel, liquids etc) could help optimize the editing approaches such that certain
components (for example some semi-solids detected in both RADE and CP) in specific
samples leading to more accurate absolute quantitation. Unfortunately, the largest
stumbling block is not the NMR experiments themselves, which at least in theory can be
made fully quantitative, but the complex and heterogeneous character of natural
samples themselves (Akoka et al., 1999).
3.5.2 Larger diameter CMP probes
This study was in large part only possible due to availability of 13C labeled seeds which
increases the 13C signal close to 100 fold. Without labeling much of the carbon detected
experiment would have been impossible or challenging to collect in a reasonable
amount of time. From experience it is possible to just obtain a 13C CP-MAS NMR
spectrum of a fully swollen seedling overnight. In comparison using a standard solid-
69
state approach it is possible to collect a spectrum of dried plant biomass within an hour.
The difference arises not from the probe but the sample itself. In traditional solid-state
NMR the sample is dried and ground and ~100mg packed into a rotor. However, if take
a single seedling it may only weigh 20mg, of which 90% could be water, and of the
remaining biomass only 50% be a solid. This means only 1mg of true solid may be
present. The results of course are not identical the CMP-NMR approach provides
information of the solid-components in their truly natural swollen state whereas drying
and grinding converts naturally and swollen materials into the solid phase for detection.
One solution is to increase the amount of sample and build larger diameter CMP-NMR.
In a 7mm rotor ~4-5 samples can be used. This will not only permit larger specimens to
be studied but also a 5 fold amount of sample would decreases the time required to
collect the data by 25 fold (signal-to noise ratio of an NMR experiment increases with
the square root of number of scans). In turn this should make collecting direct carbon
with 1hr possible opening up the door to advanced growth studies using CMP-NMR on
samples without isotopic labeling.
3.6 Conclusion
CMP-NMR has successfully been used to elucidate changes on intact samples of 13C
labeled wheat seeds and germinated seedlings. In summary, from the 0 h dry seed to
the 96 h seedling, there has been a consistent increase in carbohydrate (sugars: D-
glucose, D-raffinose, D-xylose, melibiose and sucrose) accompanied by a decrease in
lipids (catabolism of TAG) in all experiments. Throughout this chapter, there has been a
shift from small, soluble metabolites to more structural components. At 96 h the number
of metabolites (amino acids among others) detected increased by more than double.
70
Components with restricted diffusion (TAG), semi-solid signals (biopolymers) recovered
by RADE and rigid components (starch dominant) were discussed.
It is interesting to note, that if CP (common in most solid-state NMR studies is used
alone) the mobile components are missed and the gel/semi-solid components
underestimated. Conversely, if solution-state or gel-state (HR-MAS) NMR technology is
used mobile and gel components are detected well, but semi-solids and solids are
missed. As such for analysis of natural samples that contain components distributed
across a continuous array of physical states a CMP-NMR probe is ideal as it provides
information on all components in all different states in addition to key information on the
physical state, and dynamics of these components in response to a process such as
swelling. Furthermore as kinetics and interactions can be measured across and
between interfaces CMP-NMR has great potential for understanding the metabolism of
specific residue (i.e. selective feeding of a 13C labeled precursor) following its
incorporation in biopolymers. Or the behavior and binding of a small molecule such as a
pesticide or the mechanisms behind metal phytoremediation. Such studies have already
proved very powerful for understanding the fate of contaminants in soil (Longstaffe et
al., 2012). As such CMP introduced here for the first time to study germination and early
stage growth has potential for wide spread application across many areas of plant and
biological research.
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Chapter 4. Conclusions and future directions1
4.1 Conclusions
Nuclear magnetic resonance (NMR) spectroscopy is a very powerful analytical tool that
can measure bond characteristics and help determine structural features in a sample
(Bruice, 2007; Keeler, 2011). Traditionally, only solution and solid state NMR
spectroscopy have been common place, which can be inadequate since they are
restricted to the analysis of single phases and most often require sample preparation.
Sample preparation can disrupt the natural bonds in the specimen and interactions
between phases, which can give a false representation of results. Furthermore drying or
extract prevents studying natural samples in their most biologically active state. This
problem can now be circumvent with the advent of Comprehensive Multiphase (CMP)
NMR, which allows for the simultaneous study all bonds in all phases of an intact
sample (Courtier-Murias et al., 2012). CMP-NMR requires is minimal to no sample
preparation and is thus, the perfect tool to study seeds in their entirety. Seeds are an
important source of nourishment and fuel. Cereal grains represent about 87% of all
harvested seeds and are staple foods in many countries (Kawakatsu et al., 2010).
Therefore, seed research should be emphasized and prioritized in order to better
understand the nature of seeds and germination processes, which would allow for
agricultural improvements in the future. In this regard, CMP-NMR would serve has a
highly utile approach.
1 Written by Leayen Lam with critical comments from Myrna J. Simpson and André J. Simpson
72
In Chapter 2, all detectable seed components were characterized by CMP-NMR in a
single 13C labeled wheat seed and results compared to 13C labeled broccoli and corn
seeds. A range of metabolites in the Heteronuclear Single Quantum Coherence (HSQC)
of 13C labeled wheat seed were identified by the Analysis of MIXtures (AMIX) program
using the Bruker Bio-reference database. Ethylene, a gas within the seed, was to our
knowledge detected for the first time by NMR spectroscopy. The diffusion editing (DE)
spectrum emphasized plant lipids, also known as triacylglycerides (TAG). The TAG
structure was confirmed in the HSQC where it matched well with both an ACD/Labs
simulation of an HSQC of a generic TAG structure and previous literature studies
(Sacco et al., 1998). The semi-solid, or Relaxation recovery Arising from Diffusion
Editing (RADE) spectrum, showed some TAG and carbohydrates biopolymers. Lastly, in
the cross polarization magic angle spinning (CP-MAS) spectrum, which emphasized
rigid components of the seed, show strong signals from the starchy endosperm and
seed coat (Oliveira et al., 2001; Calucci et al., 2004), hemicelluloses and cellulose
signals (Bardet et al., 2001; Jiang et al., 2012). Labeled material was not necessary for
this study but allowed for uncommon two dimensional (2D) spectra (e.g. dipolar assisted
rotational resonance (DARR)) to be collected in a reasonable amount of time. Without
the NMR database, identifying metabolites in the HSQC would have been extremely
difficult. However, our database is limited to approximately 650 small molecules with no
biopolymer data. The spectral complexity and spectral editing also made the
quantification of components difficult. For instance in the gel-like and solid spectra,
signals are never missed but it is possible for a signal to be counted twice. However,
alternative techniques exist like direct polarization magic angle spinning (DP-MAS).
However, DP-MAS NMR spectra do not contain a cross polarization filter making it
73
impossible to select just the solid components using this approach (Keeler et al., 2003).
Spin counting (Smernik et al., 2000) or Electronic REference To access In vivo
Concentrations (ERETIC) (Akoka et al., 1999) that would resolve such problems.
NMR spectroscopy has been previously used to monitor germination and early growth
but never in a method where all the phases could be observed in a single sample. In
Chapter 3, the application of CMP-NMR to the germination of 13C labeled wheat seeds
is discussed. All components, from the most liquid like (i.e. metabolites) to the most rigid
(seed coat) are monitored in situ. After 96 h, the number of metabolites in the mobile
phase were more than double in comparison to 0 h. Relative quantification revealed that
the carbohydrates increased >265% in each of the unrestricted diffusion (mobile),
restricted diffusion (gel) and semi-solid phases. Lipids (dominated by TAG) decreased
63, 41 and 36% in the mobile, gel and semi-solid phases, respectively, which is in
agreement with literature as the growing seedling metabolizes the lipids for energy. The
true solid component is dominated by structural carbohydrates. This chapter
demonstrates the applicability of CMP-NMR as an ideal technique for investigating
biological processes such as seed germination and early seed growth. CMP-NMR in
general represents a powerful tool with wide spread applicability to unravel complex
biological processes.
4.2 Future directions
As CMP-NMR was only introduced in 2012 it remains quite new; applications for this
novel technology are just beginning to unfold. There are numerous promising areas in
agricultural, food, biological, environmental etc. research where CMP-NMR can be
applied.
74
4.2.1 Future seed research
There are many practical uses possible for CMP-NMR research in agriculture. CMP-
NMR could be used to study the physiology of seeds and plants. Specifically, in process
like vernalization which is the plants ability to flower. In seed viability, CMP-NMR could
be used to select for viable seeds and identify diseased seeds since current methods
are destructive, mutagenic or time consuming (Terry et al., 2003). Or, CMP-NMR could
be used in food safety; determining purity and presence of pathogens in edibles. CMP-
NMR is especially powerful for looking at interactions within intact samples and has the
potential to study molecular orientation, binding, sequestration diffusion etc.
Applications could include the binding of herbicides to plants or the sequestration and
transport of contaminant in soil. These are just some examples of potential applications
of CMP-NMR in the future. However, CMP-NMR is not limited only to agriculture but is
applicable to a variety of research topics across all fields.
4.2.2 In vivo studies
Several trials were done where broccoli seeds (2-3 seeds) were placed a rotor in
10/90% D2O/H2O mixture and spun in the CMP-NMR probe at 6666 Hz for 2-3 hours.
After spinning, they were transferred out of the rotor to a kim wipe moist with H2O. The
next day, 100% of the broccoli seeds germinated, this means that the seeds remained
alive during spinning. This opens the doors to potential for germination to be monitored
in vivo by CMP-NMR. Even studies involving whole organisms in a rotor, such as
Daphnia magna (a key global species for aquatic toxicity tests) are possible (Simpson et
al., 2013).
75
4.2.3 Larger probes
Larger diameter probes will definitely be a key step to achieving greater
accomplishments with CMP-NMR. With a 7 mm rotor, about 5 times more volume of
sample will be able to fit compared to the 4 mm rotor. This makes using natural
abundance more feasible since the acquisition time will decrease by about 25 fold and
signal to noise increases with the square root of the number of scans. Also, the larger
probe could increase the chances of studying organisms in vivo, as there will be more
space to work with in optimizing the experiment to keep the organism alive during data
acquisition.
4.2.4 Cryoprobe technology
Cryoprobes are cryogenically cooled probes that significantly increase NMR sensitivity
enabling scientists to study sample amounts that were considered too small. Cooling
the probe coil to around 20 K with liquid helium, reduces the level of thermal noise
generated by electronic circuits, thus, increasing sensitivity by about 4 times compared
to a room temperature probe (Simpson et al., 2011). If this cryoprobe technology was to
be combined with CMP-NMR technology, the possibilities would be vast as it could be
applied to studying very small organisms or samples where only a limited amount was
obtained.
4.2.5 31P and 15N NMR
The current CMP-NMR probe only has the four RF channels (1H, 19F, 13C, 2H), if a
separate probe was fitted with phosphorous (31P) and nitrogen (15N) channels it could
have additional application to biological sciences. 31P CMP-NMR could have great
implications in, for example, studying biological membranes, DNA/RNA, energy cycles
76
(ATP, ADP etc) in a natural state. Specifically, in phospholipid bilayers regarding
aspects such as the lipid bilayer packing configuration, contaminant partitioning or the
results of protein binding (Dubinnyi et al., 2006). Likewise, 15N would be very useful in
proteomics and medical research such as DNA replication and protein binding (Liu et
al., 2010).
4.2.6 Phytoremediation
Phytoremediation is the approach of using seeds and plants to alleviate environmental
issues by bypassing the requirement to unearth the contaminant material and discard it
elsewhere. Phytoremediation can be applied to air, soil or water and contaminants can
range from crude oils, metals, and pesticides (Cunningham et al., 1995; Pilon-Smits,
2005). Since we apply CMP-NMR here to characterize the wheat seed, germination and
early growth stages, we can further extend analysis to study wheat seeds in the
presence of contaminants using the data presented in this thesis as a benchmark for
evaluation. Wheat seeds and seedlings have already been used in phytoremediation of
pesticides (Macek et al., 2000; Chaudhry et al., 2002; Harvey et al., 2002) and metals
(Cheng, 2003; Alkorta et al., 2004; Saifullah et al., 2010) but the effects have never
been studied by CMP-NMR. If lead (207Pb), mercury (199Hg) and cadmium (111Cd/113Cd)
channel probes were available, this would provide additional information to how those
specific metal contaminants interact with the seed and plant, this demonstrates the
potential versatility of CMP-NMR. CMP-NMR may help to elucidate how plants interact
with contaminants and has promising potential to optimize seeds and plants for more
effective usage in phytoremediation.
77
4.2.7 Potential CMP experimental design
Consider, for example, growth studies where the goal was to study how sugars are
utilized by a plant. The plant could be supplied 13C labeled sugars as the carbon source
and their utilization and conversion of the labeled sugars inside all phases of the plant
could be monitored This would include all processes from being taken up by the roots,
used in growth and thereafter degraded by the plant. In that example, it is important to
study the development of the solid, gel, and liquid components over time. In doing so it
is important to leave solids, gel and liquid NMR experiments as is and measure, not just
the phases independently, but kinetic transfer between the phases. Such studies would
be impossible using two or three separate NMR probes as it takes many hours to
change and properly calibrate a NMR probe during which time the sample will change.
Another experiment following the conversion of a molecule from the liquid, to gel, to
solid phases would be beneficial. For example, studying the transformation of biomass
into biofuel (Simpson et al., 2013) also requires the kinetic transfer between phases to
be monitored and therefore will require CMP-NMR probes in the future. These
examples, are mentioned simply to demonstrate the considerable future potential of
CMP-NMR in not only seed/plant/food research, but in all fields of science.
78
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Appendices
Figures A1-A5 are Supplementary information for Chapter 2 1, 2
Figure A 1 a) Generic triacyclglyceride (TAG) structure with assignments labeled.b) and c) are example 1H-1H COSY and 1H-1H TOCSY spectra, respectively, bothscaled to show assignments of the dominant TAG structures
1 Published as: Lam, L., R. Soong, A. Sutrisno, R. De Visser, M. J. Simpson, H. Wheeler, M. Campbell,W. E. Maas, M. Fey, A. Gorissen, H. Hutchins, B. Andrew, J. O. Struppe, S. Krishnamurthy, R. Kumar, M.Monette, H. Stronks, A. Hume and A. J. Simpson (2013). "Comprehensive Multiphase NMR Spectroscopyof Intact 13C Labeled Seeds." Journal of Agricultural and Food Chemistry [Just Accepted], DOI:10.1021/jf4045638, Published online: Dec 19, 20132 Reprinted with permission from Journal of Agricultural and Food Chemistry, 2013, DOI:10.1021/jf4045638. Copyright 2013 American Chemical Society
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Figure A 2 . Expansion of Figure 2-1c, Heteronuclear single quantum coherence(HSQC) spectrum showing as assignments of metabolites determined by AMIXBruker Bio-reference spectra database.
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Figure A 3. CP-MAS of single wheat seed. a) 13C labeled, number of scans (NS) =2K. b) Natural abundance, NS=19K
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Figure A 4. 1D 13C profile of single wheat seed. a) 13C labeled, NS= 2K. b) Naturalabundance, NS=10K
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Figure A 5. HSQC of single wheat seed. a) 13C labeled, NS=12. b) Natural abundance, NS=400,
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Figure A 6. 1H spectra of the 13C labeled wheat seed/seedling using presaturationto suppress the water signal at time a) 0 h. b) 24 h, c) 48 h, d) 72 h and e) 96 h.Spectra labeled as follows: 1) aromatic, 2) tryptophan, 3 & 4) phenylalanine andphenylethylamine, 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 D-xylose, 14)overlapping carbohydrate, 15) aliphatic. Where more than one metabolite is listedmeans they are overlapping. Residual water remaining after water suppression
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occurs as distortions from ~4.5-5.5 ppm and is most prominent in the 24hr and48hr samples. This likely in part arises from the water in these samples beingbroader (i.e. soaking into drier material more and being inhomogeneous) andthus, more challenging to suppress
