Final Report
WNMRC
Research Infrastructure Wageningen NMR Centre:
NMR in Agriculture, Food and Biology
Transnational Access
implemented as
Specific Support Action
Contract number: RITA-026164
Project coordinator: Dr. Henk Van As, Wageningen University
Project website: www.wnmrc.nl and www.wnmrc.wur.nl
Project Duration : 42 months from 01/02/2006 to 01/08/2009
Project funded by the European Community
under the “Structuring the European Research Area” specific programme
Research Infrastructures Action
A. ACTIVITY REPORT
Project objectives (www.wnmrc.nl/UK/LSF)
A minimum of 475 measurement days on different NMR and MRI spectrometers available
under this contract will be provided for short research projects and training of Users. These
spectrometers cover a wide and rare range of possibilities for analytical and (bio)physical
research and applications in biology, agriculture, food sciences and technology, environmental
sciences, soils sciences, biotechnology and biophysics.
It is estimated that around 120 users within 32 user groups will benefit under this contract. On
average a user group spends 70 days at the infrastructure and in that time up to a maximum of
20 spectrometer days can be used per project. Training, instructions, data analysis and
processing, simulations, interpretation and reporting (by publications preferably) are used for
the remaining time.
The different NMR, MRI and ESR spectrometers that are available within the contract are:
 0.2 –0.75 T variable field NMR spectrometer (VARNMR)
 0.25-0.75 T NMR imager (LF-MRI)
 3 T, 70 cm- vertical bore MRI system (3T-MRI)
 7 T wide bore supercon NMR spectrometer (7T-WB-NMR)
 9.4 T narrow bore supercon NMR spectrometer (9.4T-LC-NMR)
 11.7 T two- and three-dimensional high-resolution NMR spectrometer (11.7T-NMR)
 14.1 T LC-NMR-MS (metabolic profiler with high sensitivity cryo NMR probe) in
combination with LC-FT-MS
 SuperX high sensitivity ESR
The VARNMR, 3T-MRI as well as the LC-NMR-MS systems are rather unique and cover a
number of new applications. A large number of software packages and programmes is
available for data analysis and simulation. For metabonomics applications specific databases
are available.
This facility is flanked by analytical facilities (for example advanced mass spectrometry and
optical (micro)spectroscopy) (for a description of the advanced optical spectroscopic
techniques available see: http://www.mcswu.nl/ ) and computer facilities.
Contractors involved
Wageningen University, Department of Agrotechnology and Food Sciences, Wageningen
NMR Centre, Wageningen The Netherlands.
Work performed
The work has been performed as described in Annex I of the contract.
End results
Important scientific results of the user projects. The project numbers and descriptions can be
found at http://www.wnmrc.wur.nl/UK/LSF/Accepted+EU+proposals/
Plant Sciences/Environmental Sciences
Despite decades of studies, there are still fundamental unanswered questions related to phloem
flow in natural and semi-natural ecosystems. Phloem flow in plants has hardly been studied
due to lack of proper methods. It is important to answer these questions, however, in order to
improve our knowledge of the carbon cycle, of relevance for eco-physiological plant studies in
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relation to climate and climate change. The unique intact plant 3T-MRI system has been used
for long distance transport studies in intact plants and trees. The effects of root and shoot
anoxia on source and sink of water flow and solute transport in the transport phloem of
Ricinus was studied to answer the question if phloem flux is determined by sink or source
strength. The results give new insights in the regulation of phloem flow (06-002). MRI flow
imaging has been used as a reference for flow measurements by a new non-invasive heat pulse
method. The dynamics in flow conducting area could be studied by MRI and the
consequences for flux quantification by the classical heat pulse method were revealed (06001). Sensors based on heat pulse techniques are in use in eco-physiological investigations.
Therefore this is totally new information of importance for water management and tree
functioning in response to global warming and water management.
The quantitative performance of a non-invasive, laser-based heat pulse technique for
measuring phloem sap flow in broadleaf saplings was compared to MRI flow imaging data.
The comparison with MRI flow imaging allowed for the identification of issues inherent to
the surface technique: overestimation of phloem velocities due to a thermal artefact arising at
relatively high transpiration rates, and uncertainties regarding the directionality of the flow
measured by a surface thermometric approach in the case of a bi-directional flow system (07003).
MRI flow imaging has been used to study the effect of a reduction in the phloem flow
conducting area on the phloem flow velocity using castor bean, pine, and maize as model
systems (08-003). Even under the most rigorous phloem area reductions the flow velocity did
not become higher than 1 mm/s, suggesting that there indeed is a maximum flow velocity that
the phloem can sustain. These findings can hardly been explained by current hypotheses of
phloem transport mechanisms.
The course of early wood (xylem) vessel formation and initiation of water transport in was
studied by MRI in oaks (Quercus robur) in relation to spring phenology (08-015). In stems of
oak during xylem recovery after winter dormancy an active re-hydration of the cambial zone
was observed before bud swelling. Water transport starts more substantially when the first
large early wood vessels are fully lignified during leaf unfolding. Most of water transport
occurs in the most recent annual ring – the one which is formed in the actual season. Phloem
transport could be detected when the leaves were fully developed. Tissue related analyses
supply important qualitative information and a needed foundation for further analyses on oak
trees.
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P NMR saturation transfer techniques have been used to investigate motion and organisation
in thylakoid membranes. This information is used to test the hypothesis that there is a dynamic
equilibrium between the membrane and the sequestered non-lamellar phase, which in turn can
regulate the lipid:protein ratio in the membrane. Maintaining the lipid:protein ratio in a
relatively narrow range probably has great physiological significance in energy-converting
membranes and can ensure high stability of their components and the membrane structure
(required for their optimum operation), and at the same time a structural flexibility can be
ensured (required for fine adjustments and regulatory functions, e.g. in response to
environmental conditions) (06-003).
The generation of pH gradient across the thylakoid membrane upon actinic illumination in
cyanobacterial and algae cells have been studied by 31P NMR (08-010). Suspension of intact,
photosynthetically active Syneochocystis cells showed a Pi peak that in dark shifts to acidic
values and ended up to pH of 6.9. In the light the Pi peak shifts to more alkaline position - pH
7.8. Most probably the acidic pH corresponds to the pH in the lumen, the alkaline one to the
stroma. The coexistence of these two peaks was not clearly detected. Probably upon
illumination the Pi migrated to the stroma (alkaline pH) where it is used up for ATP synthesis.
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This is supported by the fact that the peaks corresponding to ATP are increasing upon
illumination.
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P in vivo NMR has been used to characterize the effects of antifungal toxin phosphite (Phi)
on plant cell metabolism. Cell suspension cultures of tobacco (BY2) cell lines turned out to be
very suitable for this purpose, remaining viable over more than 20 h. It was demonstrated that
phosphite inhibited phosphate uptake in a competitive manner. These studies allowed for the
first time to follow Phi flux processes in an in vivo setting in plants. The external Pi nutrition
status and the metabolic state of the cells had a strong influence on the intracellular
compartmentalization of xenobiotic Phi (06-005).
Metabolomics on (medicinal) plant extracts, food extracts and body fluids by use of LC(SPE)-NMR/MS and LC-MS
A number of publications as a result of former User projects has been published directed
towards the optimization and application of hyphenated LC-NMR-MS techniques for the
analysis of different extracts, e.g. with well know or potential medicinal use or in relation to
plant metabolomics. This method has been used by a number of Users, e.g. for flavonoid
search.
Flavonoids are important bioactive dietary compounds. The chemical nature of the grain
pigments of different types of pigmented rices (differently colored) has been analyzed.
Complete metabolic profiles of extracts from five rice samples, corresponding to different
pigmented types. In combination with genetic profiles of the rices (determined at the CRAGPG, Italy) full characterization of the rices has been obtained. In this way a better
understanding of the regulation of the flavonoid pathway in rice has been achieved (08-002).
Flavonoids induce electrophile-responsive element (EpRE)-mediated expression of enzymes,
including major defense enzymes against electrophilic toxicants and oxidative stress. The prooxidant chemistry of flavonoids (anthocyanidins and catechin metabolites) has been studied
(08-005). These compounds can be converted in electrophilic quinones which can be
damaging to the cell. Glutathione can protect the cell by reacting with these quinones forming
adducts. The two glutathione adducts formed from cyanidin have been identified on the basis
of 1H NMR and LC-MS-MS analysis. Based on 1H NMR identification, two hypothetic
mechanisms for the reaction have been proposed. Moreover, two diglutathionyl adducts of
catechins were identified. Finally, Epigallocatechin Gallaat (EGCG) was incubated with rat
liver microsomes in the presence of UDP-glucuronyl transferases to form the EGCG
glucuronidated metabolites. Individual EGCG-glucuronides have been tentatively identified in
the incubation mixture. Two of these metabolites were separated, purified and tested for their
ability to induce EpRE-mediated gene expression.
The possibilities of HPLC-SPE(separation based on MS)-NMR has been explored to identify
new unknown functional compounds present at low concentrations in different plant extracts.
Several species from the Teucrium genus are reported as neo-clerodane diterpene producers,
being one of the richest sources of these secondary metabolites. This type of compounds is
insect antifeedants, hepatotoxic, cytotoxic, and anti-inflammatory. Four unstudied species
from this genus have been collected from Morocco, and pre-purified in order to determine the
presence of neo-clerodane diterpenes (08-007). Finally six peaks were isolated and identified,
uncluding some totally new compounds.
Three extracts the Drosera intermedia (methanol, water and hexane) were previously tested
against several microbial strains and showed remarkable antimicrobial activities. The hexane
extract showed much greater activity than the methanol and water extracts. However the
methanol extract showed activity against Pseudomonas aureginosa, which was tolerant to the
hexane extract, indicating that different compounds are involved in the antimicrobial activity
of D. intermedia. HPLC-SPE-NMR results indicated that most of the chromatographic peaks
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were successfully separated and trapped, and in some cases 2D experiments were possible
(08-011). The major compounds of the methanol extract of D. intermedia turned out to be
flavonoids, more specifically quercetin derivatives. The information gathered in this project
will be used to compare the production of secondary metabolites in different organs of D.
intermedia in order to determine if the compounds with antimicrobial potential are
preferentially secreted in one determined region.
The identification of minor phenoloic compounds and their glycoside derivatives in
methanolic and methanolic/water plant extracts of O. dictamnus was done (08-014). Sixteen
different fractions could be isolated. The fractions usually contained one or two components
in considerable quantity and smaller amount of other compounds. Fourteen compounds were
identified. From these compounds, only two have previously been reported in the literature to
exist in O. dictamnus extracts. All the other compounds were identified for the first time, three
of which were identified as totally new and are derivatives of rosmarinic acid, a well known
compound for its anti-HIV properties.
Green tea represents a promising source of health-protecting photochemicals. It contains the
unique set of polyphenolic compounds. These compounds can be converted in electrophilic
quinones which can be damaging to the cell. Glutathione can protect the cell by reacting with
these quinones forming adducts. 1H NMR in combination with MS has been used to identify
glutathione adducts resulting from pro-oxidant quinone chemistry. From the NMR data it can
be concluded that GSH is preferentially reacting with the C2’/C6’ carbon centers in all
catechin studied. In the case of anthocyanins the situation is more complicated. Here hemiacetal structures are formed as based on NMR data analysis. Based on the NMR results a
reaction mechanism has been be proposed (07-002).
For he first time disturbed metabolic pathways in dairy cows with abomasal displacement, the
most important disease complex associated with the abomasum in cattle, was studied using a
1H NMR-based metabolomics approach to find biomarkers for abomasal displacement (08013). The most striking metabolites found were keton bodies. There were clear keton bodies
in cows with LDA in which ketosis could not be determined clinically. Metabolites such as
choline, betaine and leucine were decreased in cows with LDA. Cows with RDA in which
there was no clear ketosis had a little bit more lactate and glucose, and less lipoproteins
compared to controls. It was concluded that NMR evaluation on the blood of dairy cows with
abomasal displacement and ketosis will provide important mechanistic insight into cellular
metabolism in steatotic liver disease.
Soil and Environmental Sciences.
As compared to conventional soil water characteristics the NMR relaxometry method has
been reported to have limited precision to characterize the pore size distribution in soil
samples. Now a new approach has been developed to reliably estimate pore size distribution,
mean pore diameter, retention function and corresponding hydraulic properties and to
discriminate between changes of pore sizes and changes of surface properties in the course of
soil organic matter swelling (06-006). This is key information in a wide field of applications.
Root water uptake belongs to the most challenging topics in soil science. Multi-slice-multiecho approach (MSME) was applied to monitor the root water uptake by ricinus and maize
plants in a model soil under progressive water depletion over a period of about two weeks. By
co-registration with the root architecture, which was visualized by a 3D fast spin echo
sequence, it was concluded that the largest water content changes occurred in the
neighborhood of the roots and in the upper layers of the soil (07-001).
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Bio- and environmental technology
Free radical forming reactions are of importance e.g. in water pollution and soil remediation
treatments. Here radical forming mechanism and kinetics of peracetic acid decomposition
over heterogeneous MnO2 catalyst has been studied by means of a unique combined ESR and
NMR spin trapping technique (08-006).
During the project various spin traps were studied for conventional (Fenton) and target
systems. In addition, a reliable method of interpretation of ESR spectra by a combination of
simulation-based fitting and artificial neural network was developed (08-012). In this way, the
types of free radicals detected by ESR and NMR spin trapping allowed to establish the
chemical reaction, occurring in the system. The obtained results allowed to expand previous
study and gave new insight into newly developed system. Along with the study of proposed
free radical system, the main aspects of ESR and NMR spectroscopy along with fluorine
containing spin trap (FDMPO) have been investigated. The study of FDMPO is very
important due to outstanding properties of the spin trap, which can be used for study of
complex mixtures and have potential application in free radical biology and anti-oxidant
capacity of complex mixtures/plant extracts.
The characterization of the fluid dynamics and the substrate transport, in the bulk phase and in
the biomass matrix, is necessary for the elaboration and verification of a mathematical model
of Upflow Anaerobic Substrate Bioreactors (UASB). MRI has been used to study the local
flow velocities and bed expansion in a laboratory scale UASB reactor to validate a
mathematical model, able to predict the reactor performances (07-005 and 08-001).
The relation between the inner structure of the methanogenic granules of UASB reactors and
its effect on metal transport within the granular matrix was studied by other users (08-009).
The inner structure of these granules is considerably complex. The MRI results were
compared with Scanning Electron Microscopy (SEM) images. These indicated that the
diffusion resistant zones may correspond with the regions where death biomass occurs
(disintegrated microbial cells). Transport of non-complexed metals within the granules was
substantially faster than transport of complexed metals. An accumulation of metals and an
existence of a reactive barrier were observed in the granules exposed to the non-complexed
metal solutions.
A combination of analytical techniques for Cultural heritage
Parts of the ancient Greek diet in the ancient Upper Macedonia region, northern Greece, as
found in organic pottery residues (crusts) provided by the local archaeological authorities, was
studied (08-004). A combination of analytical techniques as solid state NMR, proton NMR,
FTIR, LC-MS, GC-MS and HPLC-SPE-NMR/MS was used to characterize three
characteristic residues. Aromatics were persistent in the carbonized (charred) remains, while
non-charred material (cream-coloured) had both aliphatic and aromatic moieties. 1H NMR
indicates the presence of fatty acids. The combination of techniques provided important
information about the origin of the residues.
Structure determination of membrane proteins by using novel probes in site-directed spin
labeling
Membrane proteins play an important role in many biological systems. To understand their
mode of action, it is important to know in detail the structural and dynamic properties of these
proteins. Such large proteins are hard to study by conventional spectroscopic methods. A new
electron paramagnetic resonance spectroscopy (EPR) methodology for the determination of
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intermolecular distances in large molecules has been developed and successfully tested on
model transmembrane segments of membrane proteins (07-007, 08-008).
Engineering
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C and 23Na NMR has been used to study the alkali chemistry and char structure in biomass
chars, to gather information about the changes in chemical composition of biofuels under
thermal treatment in pyrolysis. A number of biomass samples, pre-treated biomass samples as
well as their chars obtained by pyrolysis using different heating rates at the end temperature of
800 ºC were characterized. No evidence was found that the leaching process could affect the
carbon composition of the biomass samples. Only aromatic groups of carbon was left in the
pyrolysis char samples. According to the results the origin of biomass, the leaching procedure
and the different heating rates do not affect the char carbon composition. Therefore, it could
be assumed that the carbon structure does not affect the reactivity of the char. 23Na NMR
spectroscopy proved that leaching affects the fuel properties, i.e. sodium chemistry in a
biomass sample, and therefore it could be assumed that the char reactivity is affected (06007).
Access provided, supported users and user projects
Table summarizing access provided, supported users and user projects
Particip
ant no.
1
Organiation
short name
WU
Short name
of the
Infrastrucu
tre
WNMRC
Installation(s)
Number(s)
Short
name(s)
1
Unit cost,
based on cost
model UF
Unit of
access
€ 1510
One day
For the whole duration of the project
Quantity of
access
provided
715
Number of
users
Number of
user groups
108
29
The quantity of access days provided is clearly more than the minimum of 475 days promised.
The number of user groups and users is slightly lower than estimated: 29 and 108 actual, 32
and 120 estimated, respectively. The definition of a user group is not strict: most applicants
only mentioned the supervisor(s) or direct collaborators, but the effective groups are much
bigger. 17 Users have spent less than 1 month at the infrastructure, 8 between 1 and 2 months
and 3 more than 2 months. On average a User group used 24.6 measurement days.
The User groups came from 14 different EU countries, well spread over Europe:
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Distribution of User groups over the different countries (see map): BG: 1, CH: 2, CS: 2, DE:
6, EL: 2, ES: 1, ET: 1, FI: 2, HU: 1, TR: 2, PL: 2, PT: 2, PL: 2, IT: 3.
Impact on the European scientific community
E.g. new installations, industrial contacts.
A number of (user) meetings have been (co)organised:
- the 3rd European Symposium on NMR Spectroscopy in Soil, Geo and Environmental
Sciences was held August 6-9, 2006, in Freising, Germany with 65 participants, five potential
new users
- the 9th International Conference on Magnetic Resonance Microscopy in combination with
the 7th Colloquium on Mobile Magnetic Resonance will be held September 3-7, 2007, in
Aachen, Germany. Number of participants: around 130.
- a WNMRC User meeting in combination with the start of the spin-off company Biqualys, on
November 29-30, 2007, in Wageningen. Number of participants: around 60.
- a VLAG thematic Meeting on NMR and MRI applications in food systems, on October 1,
2008 in Wageningen. Number of participants: 30.
- a two-days WNMRC User meeting in combination with the spin-off company Biqualys, on
June 18-19, 2009, in Oosterbeek, NL. Number of participants: 45.
Publications as a follow up of (user) meetings co-organized by WNMRC:
- a large number of papers in a issue of European Journal of Soil Science, representing the
proceedings of the 3rd European Symposium on NMR Spectroscopy in Soil, Geo and
Environmental Sciences, held August 6-9, 2006, in Freising, Germany (Volume 59,
Issue 3, June 2008)
- book on NMR Microscopy as a follow up of the 9th International Conference on Magnetic
Resonance Microscopy in combination with the 7th Colloquium on Mobile Magnetic Resonance,
held September 3-7, 2007, in Aachen, Germany (Magnetic Resonance Microscopy / Codd, S.L.,
Seymour, J.D., eds - Weinheim: Wiley-VCH, 2009 (Spatially Resolved NMR Techniques and
Applications) - ISBN 978-3-527-32008-0 - p. 315 - 330).
As is evident from the users background we have served a large amount of users from different
scientific disciplines and countries. Results obtained via this project have been reported in over
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40 publications in diverse peer reviewed scientific journals, serving a very broad audience. In
addition, they have contributed to 5 PhD and 1 Habilitations theses. Results have been
reported in oral and poster presentations in a large number of different scientific
meetings/congresses covering diverse fields of applications, and will be followed by more
full publications.
The Helmholtz Association of German Research Centres has granted an application for a Virtual
Institute for Portable NMR (VIP-NMR; see www.portable-nmr.eu ). WNMRC is one of the
partners because of the worldwide leading position and infrastructure for intact plant research.
This VIP-NMR will expand the possibilities for portable NMR applications for the users of
WNMRC, allowing in situ field measurements.
On the basis of the contacts and results two new EU projects have been defined and submitted:
I3 Nuclear Magnetic Resonance for sustainable bioeconomy (4F NMR), a combination of
collaborative project and coordination and support action for integrating activities (as a follow
up of present RITA contract, see 1.2), and SPROBE-MQ, a small collaborative project in work
program topic Exploring the microstructures in foods (10 partners). Further, two new DFG grant
applications have at least partly been based on the cooperation and results of this project.
By the newly developed HPLC-(SPE)-MS-NMR method new natural compounds with have
been found and the structure elucidated. Some of these are of interest for industry for medical
applications, others as functional food components. A new method for soil characterization
has been developed, which has big potential for practical use.
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