This chapter includes (1) a description of the technical equipment used for the global 13C- and 15N- labelling in detail; (2) a presentation of the cultivation procedure adapted to produce globally labelled wheat from seedling to flowering stage and (3) a sample preparation workflow for the applied untargeted LC-HRMS based metabolomics approach.
Description of the labelling equipment
This section is related to the functional description of the cultivation process to produce globally 13C and 15N labelled plants using the PhytolabelBox equipment (ECH, Halle, Germany). All symbols contained in this section are related to Figure 5.
The plants were cultivated in two specially designed cultivation chambers, so called labelboxes ①. Each of these 214-L Plexiglas labelboxes is equipped with two internal fans as well as a sensor assembly S for temperature, relative humidity and overpressure measurement. Each labelbox also contains 4 openings tightly sealed with gloves (Glove Box Glove Jugitec® H, chlorosulfonated polyethylene, JUNG RUBBERTEC) allowing plant manipulation during cultivation. Control and regulation of cultivation parameters (e.g. CO2 supply, overpressure and atmospheric humidity), which are essential to achieve and maintain adequate conditions for plant growth were implemented by different modules, which are together with the labelbox termed as PhytolabelBox equipment. These modules are navigated by the control unit ② based on the user predefined parameter setpoints via the software on the PC ③. Separate from this, the nutrient supply is manually carried over the irrigation delivery module ⑥.
Gas supply and control module: CO2 is supplied from the gas bottles ④ which are connected via stainless steel and copper lines to the labelbox ①. Since carbon labelling was realised in one of the boxes (13C-labelbox), this is supplied with 13CO2 gas while the 15N-labelbox was operated with native 12CO2. Each gas bottle is equipped with a pressure reducer, which was set to 1 bar. The amount of the introduced CO2 is balanced by dosage loops 2b (ca. 2 ml at 1 bar) located in the control unit ②. The dosage loops 2b are covered with magnet valves 2a,c from both sides (in- and outlet). These valves are opened reciprocally in short time intervals during gas dosing operation. The pressure difference between the pressure reducer at the gas bottle and the interior of the labelbox forces the CO2 into the loop space and further into the labelbox ①. The CO2 [ppmv] measurement was performed by IR absorption using separate cuvettes 2e for 13CO2 and 12CO2. Simultaneously, the O2 [%] is also measured with an electrochemical sensor 2f, which is located in the control unit. Immediately before measurement, the sensor and cuvettes are rinsed with air from the labelbox to achieve a homogeneous gas sample. The labelbox is always operated at slight overpressure (the difference between ambient and inner pressure) of 10 ± 2 mbar to prevent ambient air from entering the closed and controlled atmosphere.
The overpressure levels are constantly recorded by the integrated sensor S in each labelbox ① and the pressure signal status is continuously transferred via the control unit ② to the PC ③. If the overpressure level decreases below the allowed minimum setpoint (slight gas leaks are expected and occur), the dosing of synthetic air (mixture of N2 + O2) is activated. To this end, a magnet valve 2d, which is located in the control unit opens and the synthetic air is dosed into the labelbox ① until the overpressure level reaches the setpoint again. If the pressure in the cultivation box exceeds the maximum allowed overpressure of 12 mbar, a magnet valve ⑧ opens to release excess air. This mechanism is controlled by the software and is additionally used for the air exchange in case CO2 and/or O2 levels exceed the defined maximum values. To prevent damage to the plexiglas labelboxes by excessive overpressure, a power-independent mechanism preventing too high pressure in the closed labelboxes is installed (overpressure limitation unit ⑦). For this, a water-filled bottle connected to the labelbox provides a robust safety tool, as by a simple physical mechanism, overpressure > 15mbar is automatically released.
Humidity control module: To regulate the maximum humidity levels, each cultivation chamber contains an internal relative humidity (polymer-based capacitive) sensor S, an external membrane pump 5a and Peltier element 5b as well as a condensate bottle 5c. A permanent gas circulation (30 L/min) is maintained throughout the whole cultivation period passing the Peltier element 5b by use of a membrane pump 5a. In such a way, not only continuous homogenous air is provided to the plants but also relative humidity levels can be limited. The increase of relative humidity above a specified value caused by plant transpiration activates an increase of Peltier current in the drying module. Peltier current forces one side of the block to cool down, which enables water condensation from the air coming from the labelbox. The condensate is further collected in separate bottles whereas the dried air is guided back to the cultivation chambers.
Irrigation system: The irrigation system ⑥ consisted of two external bottles with nutrient solutions (for the growing period, Table 2) connected over hoses and pipes to the plant pots in the labelboxes. Depending on the labelling regime the two boxes were supplied with different nutrient solutions 6b,c. The labelbox used for global 15N-labelling was supplied with salts enriched with 15N 6c. To prevent contamination of the air in the boxes during watering, the airspace in the nutrient solution bottles was connected with labelboxes to allow pressure compensation with air from inside the boxes.
CO2 scrubber: At the beginning of the cultivation, before placing the seedlings into the labelbox, CO2 was removed from air inside the labelbox. This is achieved by the use of CO2 scrubber which is attached to the gas hose between the membrane pump 5a and labelbox ①. It is filled with CO2 adsorbent (3-4 mm diameter, Soda Lime Carbon Dioxide adsorbent spherical granules, SpherasorbTM, Intersurgical Ltd., Wokingham, UK) which traps CO2 after navigating the dry air from the labelbox through the CO2 scrubber. In the presented setup, switching of the valve to direct the gas flow through the CO2 scrubber has to be done manually.
Chemicals
Methanol (MeOH, LC-MS CHROMASOLV®), acetonitrile (ACN, LC-MS CHROMASOLV®) and formic acid (FA, MS grade, ~ 98% purity) were purchased from Riedel-de Haën, Honeywell (Seelze, Germany). The ultra-pure water was obtained from an ELGA Purelab system Veolia Water (Ultra AN MK2, Vienna, Austria). The salts KOH (≥99.5%), NH4NO3 (≥99%), Na2MoO4*2H2O, KH2PO4 (≥99.8%), KNO3 (65%) were obtained from Merck (Darmstadt, Germany) and MgSO4*7H2O, ZnSO4*7H2O, Ca(NO3)2*4H2O, Ferric sodium - EDTA (C10H12N2NaFeO8), MnCl2*4H2O, ZnSO4*7H2O, CuSO4*5H2O (>98%) from Sigma-Aldrich (Steinheim, Germany). NH4NO3 (15N, 98 atom%), Ca(NO3)2 (15N, 98 atom%), KNO3 (15N, 98 atom%) and 13CO2 (99 % purity) was purchased from Eurisotop (St-Aubin, France) while CO2 and synthetic air were obtained from Messer (Gumpoldskirchen, Austria).
Plant material
Wheat genotypes Karur (T.durum), Remus (T.aestivum) and Apogee (a dwarf cultivar of T. aestivum) were generated and used in native and labelled (13C and 15N) form. The native seeds of Karur and Remus were obtained from - and grown in the glasshouse at - the Institute of Biotechnology in Plant Production (University of Natural Resources and Life Sciences, Vienna, Department of Agrobiotechnology, IFA-Tulln, Austria). The native Apogee seeds were obtained from the Department of Applied Genetics and Cell Biology (University of Natural Resources and Life Sciences, Vienna, University Research Center Tulln, Austria) and grown in the labelbox while supplied with non-labelled substrates. The 13C- and 15N-labelled material of each genotype was produced in the labelbox from native seeds.
Experimental description
The cultivation of T.durum plants lasted 87 days in total and comprised following steps: 1. preparation of the nutrient solution, wheat seedlings and labelboxes; 2. cultivation and 3. harvest
Preparation of the nutrient solution
To generate globally 13C- and 15N-labelled T.durum plants, the seedlings were grown in a perlite substrate using nutrient solutions adapted from Hoagland (1950) (45) and Bugbee, Spanarkel (1994) (46) (Table 2). The salt concentration was adapted to the growing stage of the plants resulting in two nutrient solutions (one for the germination and one for the growing period respectively, Table 2). Both labelling regimes were provided with the same nutrient solutions, with the only difference that the 15N labelling experiment was carried out with highly 15N-enriched salts. From two stock solutions, a total of 4 different nutrient solutions were prepared for two developmental periods, i.e. 2 with labelled 15N (98-99 atom% enriched) and 2 with native 14N salts.
Table 2 Adapted Hoagland solution. *Salts substituted with highly 15N enriched analogues in the 15N-labellling experiment
Salts
|
Hoagland Stock solutions [g/l]
|
Germination period [mg/l]
|
Growing period [mg/l]
|
KNO3*
|
202
|
101
|
404
|
Ca(NO3)2x4H2O*
|
236
|
472
|
472
|
C10H12N2NaFeO8 (Ferric sodium – EDTA)
|
15
|
3.75
|
0.975
|
MgSO4x7H2O
|
493
|
123.3
|
98.6
|
NH4NO3*
|
80
|
8
|
8
|
H3BO3
|
2.86
|
0.143
|
0.072
|
MnCl2x4H2O
|
1.18
|
0.59
|
1.18
|
ZnSO4x7H2O
|
0.22
|
0.88
|
0.33
|
CuSO4
|
0.051
|
0.051
|
0.051
|
Na2MoO4x2H2O
|
0.12
|
0.024
|
0.0072
|
KH2PO4
|
136
|
68
|
68
|
All 4 nutrient solution types were prepared from the Hoagland stock solutions (composition in Table 2) in cool (< 10°C) ultra-pure autoclaved water and stirred for ca. 10 min on a magnet stirrer. Low amounts of 12C as well as 14N were present in the nutrient solutions to provide iron uptake in the form of ethylenediamine tetraacetic acid (EDTA) chelate complex.
Preparation of wheat seedings
Before placement into the labelboxes, wheat seedlings were prepared according to the following three steps: germination, vernalisation and planting.
Germination: Wheat seeds were placed in blocks of rock wool (4x4 cm, Grodan) with the embryo upwards so that each block contained three seeds. The seed-contained blocks were placed in two separate darkly shaded boxes and watered with solutions for the germination period containing either 15N or 14N salts until saturation of the rock wool. Boxes were closed and the seeds germinated at room temperature in the darkness for two days.
Vernalisation: Germinated seedlings were proceeded to vernalisation in the dark for 2 days at 4°C in the cooling room to promote shoot yield and acceleration of the flowering process (47). Under these conditions, previous experiments have shown to result in 4-6 shoots per T.durum plant during cultivation.
Planting of seedlings: Plant pots (~1L) were wrapped in aluminum foil as depicted in Figure 6, to prevent access of light to the medium and thus growth of algae. Pots were filled with perlite and nutrition solutions for the growth period (Table 2). For 15N-labelling nutrient solution containing 15N salts was used. Germinated and vernalised seedlings were transferred from rock wool blocks into corresponding prepared plant pots. After the placement of seedlings, a layer of rock wool was added on top of the pot, to reduce water evaporation from the nutrient solution and prevent access of light during plant cultivation.
Preparation of the PhytolabelBox equipment
Prior to placement of plants into the labelboxes, the technical performance of the equipment was verified.
First, the tightness of labelboxes was tested. The labelboxes are operated with slight overpressure which is regulated at (10 ± 2) mbar above the ambient atmospheric pressure. The ability to keep the overpressure at the setpoint range can be taken as a measure of tightness of the system including the labelboxes. To check this, the pressure cycle consisting of the pressure drop from 10 mbar to 8 mbar and build up back to 10 mbar by dosage of synthetic air was monitored, resulting in pressure cycle periods of 10 min for the 15N- and 20 min for the 13C-labelbox respectively. These values slightly increased with the progress of the cultivation.
Second, the increase of CO2 in the labelbox per dosed loop was determined. This is required for the estimation of the CO2 consumption rate of plants after the cultivation is finished. Tests showed that per dosed loop of CO2, the concentration of CO2 in the labelbox increased by (4.6 ± 0.3) ppm 12CO2 in the 15N-labelbox and (9.7 ± 0.3) ppm in 13C-labelbox when the pressure reducing device on the CO2 bottles was set to 1 bar each.
Cultivation of plants
Start of the cultivation
- The pots with seedlings were placed in the corresponding 15N- and 13C-labelboxes.
- The labelbox cover at the back was tightly sealed with screws. Note: Too tightly sealed screws may damage the Plexiglas housing. To promote tightness a silicone paste was applied on the sealing gums of the labelboxes.
- The atmospheric CO2 in the 13C-labelbox was removed by passing the air through the CO2 Further, 13CO2 gas was manually dosed to reach a level of 400 ppm in the labelbox.
- The setpoint values of CO2, O2, overpressure and humidity were defined in the software (Table 3).
- The cultivation was initiated by starting the measurement in the software. All user predefined setpoints were automatically monitored and regulated by the system during the whole cultivation period (Table 3).
Table 3 Setpoint values for the recorded and regulated atmospheric parameters for both labelboxes.
Regulated parameters
|
Set point values
|
Allowed parameter range
|
Min.
|
Max.
|
CO2 [ppmv]
|
400
|
300
|
1600
|
O2 [%]
|
20
|
-
|
-
|
Overpressure [mbar]
|
10
|
8
|
12
|
Humidity [%]
|
variable
|
-
|
70
|
CO2, O2 and overpressure were maintained at setpoints by the control unit while values below the minimum led to the activation of gas dosing system. The maximum allowed setpoint for relative humidity was defined while levels below depended on the plant biomass and thus was increasing with growth (see Figure 1).
|
Cultivation conditions
The temperature and light-duration setpoints outside the labelbox for the day/night cycle were realised over an external control unit of the climate chamber, in which the labelboxes were located throughout the cultivation period. These setpoint values are shown in Table 4. The light intensity remained constant during the whole cultivation period. The light intensity at simulated day conditions was estimated with the Li-COR sensor (LI-190SA Quantum Sensor, Li-COR, Germany) and was measured on top of the rock wool immediately after placing the pots with seedlings into the labelbox to be 206 µmol/(m2·s) in the 13C-labelbox and 220 µmol/(m2·s) in 15N-labelbox respectively.
Table 4 Light and temperature intervals in the climate chamber during the cultivation process
Cultivation days
|
Light cycle
day [h]/night [h]
|
Temperature
day [°C]/night [°C]
|
Growth stage
(Zadoks scale (48))
|
0 - 28
|
12/12
|
12/10
|
Z1 and Z2
|
28 - 31
|
12/12
|
14/12
|
Z2 and Z3
|
31 - 38
|
14/10
|
16/14
|
38 - 86
|
14/10
|
18/16
|
Z3, Z4 and Z6
|
Zadoks scale: Z1- seedling development, Z2 - tillering, Z3 - stem elongation, Z4 - booting and Z6 - anthesis
|
Irrigation
Irrigation rhythm was adapted to the growth stage of the plants. In the first three weeks of cultivation, the plants were irrigated once a week and from day 28 until harvest twice a week. Irrigation was accomplished manually by an external peristaltic pump (S20, Vederflex® smart, UK) and a flow velocity of 100 ml/min. The volume of nutrient solution applied during watering was recorded. Both labelboxes were watered with the respective nutrient solution by the use of separate hoses.
Harvest
Each ear and the adjacent stems, leaves and roots inside the 13C-labelbox were harvested separately 144 h after the first anthers had appeared on the ear, respectively. The sample material was removed via the gate compartment with an air lock system and immediately shock frozen in liquid nitrogen outside the labelboxes. The time between cutting the samples and freezing was kept as short as possible. The 15N-plants were harvested under similar experimental conditions while the 15N-labelbox was open, as the ambient atmosphere does not disrupt the 15N-labelling process.
Freeze-drying and sample storage
Sample material was freeze-dried (FreeZone 6Plus, Labconco, Kansas City, MO, USA) to <5% rest moisture for long term storage at -80°C. To this end, wheat ears were dried for 6 days, stems for 1 day and leaves for 2 days at -80°C and ~0.4 mbar. Rest moisture was estimated with the infrared moisture analyzer (LC 4800P-OOV1, Sartorius, Göttingen, Germany).
Sample preparation
Two set of samples were prepared for the measurement. One is used for the determination of the degree of enrichment and the other for the annotation of the global C and N metabolome. The procedure for milling and extraction as well as the solvent composition of samples at the time point of measurement was the same in both cases. The only difference is in the composition of the sample extracts. For annotation of the global metabolome, wheat ear extracts of freshly sampled native wheat from glasshouse were mixed with either freeze-dried 12C15N or freeze-dried 13C14N ears from the labelbox. For the determination of the degree of enrichment, extracts of either native, 12C15N- or 13C14N- wheat ears were used.
The wheat ears were milled to a fine powder with a ball mill (MM400, Retsch, Haan, Germany) while being kept in frozen condition. The extraction was performed similarly as reported in (8). In short, 100 mg of fresh native wheat powder and 30 mg of dried wheat powder was extracted separately with 1 ml MeOH/ACN/H2O (1.5/1.5/1 v/v/v) + 0.1 % formic acid (FA). 70µl of H2O was added to the extraction solvent of dried wheat in order to compensate for the loss of water during the drying process. For the detection of the global C and N metabolomes, native extracts were mixed 1:1 (v/v) with 13C14N or 12C15N extracts and diluted with H2O + 0.1% FA in order to obtain 1:1 organic: water ratio prior to LC-HRMS (8). For determination of the degree of enrichment, the labelled and native extracts were diluted with H2O + 0.1% FA individually.
LC-HRMS measurement
The LC-HRMS measurement was performed in positive ionisation mode on an Orbitrap mass spectrometer (QExactiveHF, Thermo Fisher Scientific, Bremen, Germany) coupled to a Vanquish uHPLC (Thermo Fisher Scientific, Bremen, Germany) with the method described in (49).
Data processing
Data processing was performed with the AllExtract module in MetExtract II software (14) LC-HRMS spectra of the sample set for global C and N metabolome annotation were considered. riefly summarised, the MetExtract II software was set up to search for pairs of chromatographic peaks, which originate from co-eluting native and (13C- or 15N-) labelled metabolite ion forms. Native and labelled metabolite forms must show their distinctive and mirror-symmetric isotopologue patterns. Moreover, their chromatographic peak shapes must be highly similar and show perfect coelution. Such detected metabolite ions were then aligned across all samples of the either 13C- or 15N-labelling experiment.
The measured LC-HRMS raw files were converted into mzXML format using the MSConvertGUI (version 3.0.19166-cc86d1f56) from Proteowizard (50), and further loaded in the MetExtract II. Data processing parameters of MetExtract II were: Intensity threshold for M and M’: 10000 counts; Chromatography start and end time: 3-36 minutes; Chromatographic peak scales: 7-21; Maximum allowed deviation for signal pairs: ±3 ppm; Maximum isotopologue abundance error: ± 15%; Minimum peak correlation: 0.85 (Pearson correlation); MZ-delta between native and labelled metabolite ion form: 1.00335484 for the 13C-labelling experiment and 0.99703000 for the 15N-labelling experiment; Isotopologue purity: 0.9893 for the native metabolite form, 0.9850 for the 13C-labelled metabolite ion form, 0.9951 for the 15N-labelled metabolite ion form; Maximum MZ deviation of consecutive signal pairs: ±8 ppm; Number of isotopologues checked: 2; Number of carbon atoms searched for in the 13C-labelling experiment: 3-60; Number of nitrogen atoms searched for in the 15N-labelling experiment: 1-12. Parameters for the combination of the 13C- and the 15N-labelling experiment: Maximum allowed MZ deviation: 5 ppm; Maximum allowed retention time shift: ± 0.15 minutes.