Bachelor Thesis Scheikunde
Isolation of solanoeclepin A from endogenous potato root
exudate and subsequent phenotypical analysis on
Arabidopsis thaliana
door
Yannick Ricky van Dijk
23 september 2019
Studentnummer:
Verantwoordelijk docent:
11342749
Harro Bouwmeester
Onderzoeksinstituut:
Begeleiders:
Universiteit van Amsterdam
Aleksandra Chojnacka,
Onderzoeksgroep:
Kristyna Flokova, Lieke
Abstract
Solanoeclepin A is an important factor for cyst nematode hatching and damages crops of potato and tomato plants causing reduction in the yield of food production. Solanoeclepin A research on plants and cyst nematodes requires a high purity for determination of the influence on these organisms. In this research we developed a method for solanoeclepin A isolation from endogenous potato root exudate. Multiple SPE methods were examined to determine the best candidate for extraction and purification. Fractionation of extracted root exudates at different stages of purity influenced the solanoeclepin A detection signal. In this manner, four isolation routes were established with different amounts of solanoeclepin A in each route. These isolates of different concentrations solanoeclepin A, a positive control and a negative control were applied to Arabidopsis thaliana. This resulted in an effect on the root development which is not linearly with the amount of applied solanoeclepin A.
1. Introduction
Plants communicate chemically with underground living organisms using primary and secondary metabolites. Arbuscular mycorrhizal (AM) fungi is an example of such an organism and communicates with more than 80% of land plants, in which this communication is essential for completion of the fungi lifecycle.(1,2) The
rhizosphere of plants is typically a small region of soil around the roots that are influenced by secretion of metabolites by the plant and associated soil microorganisms known as the root microbiome. These regions contain a high variety of nematodes, bacteria, fungi, and arthropod herbivores.(3) Metabolomic analysis
on root exudate provides information about the compounds that are secreted by the plant and corresponding organisms in the microbiome.(4)
Even exudates of a simple model plant such as
Arabidopsis thaliana can already contain over
100 different metabolites and more are being
discovered.(5) Arabidopsis is considered the organism of choice in a wide range of plant studies due to
the convenience for performing plant biology experiments and for addressing fundamental questions of biological structure and function.(6) Some organisms, such as Rhizobia, live by an endosymbiosis
relationship with plants to codependently fixate nitrogen from the air to sustain themselves. Conversely, organisms that are dependent on a plant host in which the plant is detrimentally affected are parasites. Cyst nematodes are examples of these types of parasites and often result in destructive damage to the host plants, causing for harvest los in over 50 countries already. (7) Potato cyst nematode
(PCN) egg hatching depend on the presence of hatching factor (HF) solanoeclepin A (figure 1), which is exuded near the root tips of the plant.(8) The hatching process initiates after first stage juveniles hatch
from their eggs upon the presence of solanoeclepin A. Subsequently the juveniles penetrate the roots and mate followed by encapsulation of the fertilized female which forms the new cyst. These flask-shaped cysts act as a protective covering for the juveniles resisting dryness and frost and are able to survive in the soil for over 20 years in absence of solanoeclepin A. In contrast, hatched juveniles die within several weeks in absence of the host plant.(9)
Structure-activity relationships (SAR) of solanoeclepin A-hatching Structure-activity have not been reported although model compounds, which show substantial similarities, have indicated that the carboxylic acid group present at solanoeclepin A has a high probability to be essential for the hatching factor function for potato cyst nematodes.(10) Furthermore, SAR studies for a similar
compound, glycinoeclepin A (figure 2), concluded that a carboxylic acid group is also essential for the hatching activity of soybean cyst nematodes.(11) Next to the structural similarities of
glycinoeclepin A compared to solanoeclepin A, it functions as hatch factor for soybean cyst nematodes in a similar way.(12)
Brassinolide (figure 3), a plant growing hormone, also shows
Figure 1. Solanoeclepin A
Figure 2. Glycinoeclepin A
similar characteristics in structure and the biosynthetic pathway of the molecule can possibly be closely related to the biosynthetic pathway of solanoeclepin A. Solanoeclepin A was first identified in 1999 by Schenk et al. with empirical formula C27H30O9 and the total synthesis of the molecule was first reported
in 2011 using 52 synthetic steps starting from 3-methylcyclohexenone with a yield of 0.18% .(9,13) It is
unclear why plants secrete solanoeclepin A in the rhizospheres and more research is needed to determine this behaviour. In addition, no reports have yet shown the effect solanoeclepin A exerts on the shoot and root system of model plants. Changes in the behaviour of plants after applying solanoeclepin A can be determined only by using a pure compound. Since root exudate contains a very complex mixture of different compounds, an important part of this research will be dedicated to isolation of solanoeclepin A. Isolation off glycinoeclepin A was performed using successive fractionation of active fractions on multiple columns.(12) It was concluded before that Desirée potato
secretes the highest amount of solanoeclepin A in the soil by evaluation of different genotypes of potato plants. Considering that a reasonable amount of solanoeclepin A was needed for the project, the root exudate of this species was used specifically in our experiments. The molecular structure of solanoeclepin A contains several functional groups such as hydroxy-, carbonyl- and methoxy substituents. In this work we use the presence of a carboxylic acid group as the most important feature for development of a selective separation technique. The carboxylic acid group is a convenient group for separation due to the stable anion that forms upon deprotonation of the group which can be used favourably in ion-exchange based purification methods. For isolation of solanoeclepin A, multiple SPE methods were used for preliminary concentration and purification of Desirée potato root exudate. Fractionation at multiple stages of purity was used as next step to isolate solanoeclepin A. Phenotypical analysis after applying solanoeclepin A on Arabidopsis seedlings identified the effect of the compound on a simple model plant by just looking at the positive and negative control.
2. Materials and Method
3.1
Chemicals
Quantification of solanoeclepin A was performed using multiple ion monitoring mode (MRM) analysis using a standard solutions solanoeclepin A provided by prof. Keiji Tanino (Hokkaido University, Japan)(9). It was used for calibration of the MRM detection apparatus and injected as standard for
calculation of the concentration solanoeclepin A in the purified samples. Furthermore, the standard solution was used to determine the elution time of solanoeclepin A for both a short and long gradient. LC-MS grade water, acetonitrile, methanol formic acid and ammonia solution were supplied by LiChropur®. LC-MS grade ethanol and hydrochloric acid were supplied by Emsure®.
3.2
UHPLC-MS/MS analysis
The targeted analysis of solanoeclepin A was performed using Acquity® UPLC System coupled to triple quadrupole Xevo® TQ XSTM mass spectrometer with electrospray interface (Waters®). The
separation was achieved on Acquity BEH C18 column (2.1x100 mm, 1.7 μm, Waters) at flow rate 0.3 ml/min. The column was thermostated at 40˚C. The binary gradient elution over 10 minutes was set as follows: 0-1 min (95% A), 1-6 min (50% A), 6-8 min (20% A), 8-10 min (5% A) and final column equilibration for 3 min for initial conditions (95% A), where A = 15 mM formic acid/ water and B = 15 mM formic acid/ acetonitrile. The eluate was introduced in the electrospray ion source of mass spectrometer operating at following conditions: ion source/desolvation temperature (150/600˚C), desolvation/cone gas (nitrogen) flow (1000/150 L/hr), collision gas (argon) flow 0.15 ml/min. The compound was analysed in multiple ion monitoring mode (MRM) using optimized MS conditions. The MassLynx software version 4.2 (Waters) was used to operate instrument, acquire and process data.
3.3
Semi-preparative LC-MS
Fullscan analysis and fractionation of solanoeclepin A was performed on Agilent® 1100 series coupled to micrO-TOF Bruker® mass spectrometer. The separation was achieved on Kinetex® C18 column (2.1x100 mm, 2.6 μm) at flow rate 0.3 ml/min and an injection volume of 10 µL. The column was thermostated at 40˚C using a Chrompack® column thermostat. The binary long gradient elution over 40 minutes was set as follows: 0-2 min (95% A), 2-20 min (5% A), 20-30 min (5% A), 30-31 min (95% A), 31-40 min (95% A) and final column equilibration for 30 min for initial conditions (95% A), where A = MilliQ + formic acid (0.1%) and B = Acetonitrile + formic acid (0.1). Fractionation was performed using Waters® fraction collector 2 under constant pressure (200-205 bar). Fractionation initiated at 8.00 min after injection of the sample with a total fractionation time of 10 minutes yielding 20 fractions (30 seconds for each fraction). Agilent® OpenLAB CDS was used to run the HPLC equipment.
3.4 Solid-phase extraction
Low capacity cartridges Discovery® C18 (500 mg, 3 mL, Merck), Oasis® HLB (150 mg, 3 mL, Waters) and Oasis® MAX (60 mg, 3 mL, Waters) were used for optimization. High capacity Discovery® C18 (6 g, 20 mL, Merck), Oasis® HLB (5 g, 20 mL, Waters), Oasis® MAX (500 mg, 10 mL, Waters) and Strata® X-AW (1g, 12 mL, Phenomenex) were used for higher amounts of root exudate and involved additional fractionation steps for isolation of solanoeclepin A. Equilibration steps described below were always performed with twice the amount of volume compared to the other steps.
Low capacity C18 and HLB cartridges were conditioned using methanol (3 mL) followed by equilibration with water (6 mL). Subsequently, the cartridges were loaded with root exudate (10 mL) followed by washing with water (3 mL) and elution using aqueous methanol (3 mL). For the high capacity C18 and HLB protocol, we used 1 L root exudate and steps were performed with 50 mL.
Root exudate acidification preconditioning (BL) was performed using 0.2 M HCl to pH 3.30. MAX (BL samples) were conditioned, equilibrated and loaded similarly compared to C18 and HLB. Washing involved two steps and was performed with 5% aqueous NH4OH (3 mL) followed by methanol (3 mL).
Lastly, elution with 2% formic acid in methanol (3 mL) was used for elution
MAX (AL sample) cartridges were conditioned with methanol followed by activation using 5% aqueous NH4OH (3 mL) and equilibration with water (6 mL). Root exudate was loaded (10 mL) followed by
washing with water (3 mL) and methanol (3 mL). Finally, elution with 0.75% formic acid in methanol was used for elution. High capacity MAX was performed according to the AL method with loading of 16 mL and 4 mL for concentrated and concentrated/fractionated root exudate respectively. High capacity steps involved 10 mL instead of 3 mL.
High capacity Strata X-AW was conditioned with methanol (20 mL) followed by equilibration with water (40 mL). Loading was performed with 4 mL concentrated and fractionated root exudate. Washing was performed with 25 mM ammonia (20 mL) and methanol (20 mL) followed by elution with 5% formic acid in methanol (20 mL). Strata X-AW SPE with concentrated root exudate (16 mL) was performed with 3 mL for the steps which was insufficient to detect solanoeclepin A in MRM analysis. All low capacity SPE procedures were performed dropwise using Visiprep (Supelco). High capacity SPE was performed under vacuum. Replicates of Discovery® C18, Oasis® HLB and Oasis® MAX were dried by evaporating methanol in SpeedVac (Labogene Scan speed 40) and sublimating water with FreezeDrying (Heto powerdry LL1500 freeze dryer). Vials were covered with miracloth (EMD Milipore) for prevention of material loss. The solid material was redissolved in 25% aqueous acetonitrile 100 µL (for low capacity) or 500 µL (for high capacity) and vortexed (IKA® MS2 minishaker) (10 sec.). Finally the samples were filtered using nylon micro spin filter (pore size 0.2μm, Thermo) tubes and centrifuged for 3 minutes at 8000 rcf (Eppendorf centrifuge 5424). Filtered samples were transferred to the glass insert of LC-MS vial (300μl, BGB®) and analysed. HLB and C18 concentrated samples were redissolved in 25% aqueous acetonitrile 500 µL. As solvent was lost in the filtration process, only 400 µL was used for fractionation and further MAX/X-AW purification. The unfractionated 400 µL was redissolved in water (16 mL) and pH was adjusted with 1% aqueous NH4OH to 6.51 for HLB and 6.29 for C18.
Fractionated HLB and C18 root exudate was dried and redissolved in 25% aqueous acetonitrile 100 µL with addition of water (4 mL). pH was again adjusted with 1% aqueous NH4OH to 6.64 for HLB fraction
5, 6.55 for HLB fraction 6 and 6.53 for C18 fraction 6.
3.5 Plant treatment
Dried and purified root exudate sample was redissolved in 5% ethanol in water (2 mL). Plants were treated using a spray bottle which gave 155 µL per spray. Each spray was aimed on the middle of the developed leaves that were present after 11 days. A solution of 5% ethanol in water was used for the negative control and a dissolved amount of solanoeclepin A standard in 5% ethanol in water as positive control. The amount of solanoeclepin A that was sprayed on the plants was calculated from the MRM results (this is not the real amount as ion suppression was still present for the purified root exudate) (figure 16). Highest amount of solanoeclepin A of each purification route was selected for plant treatment. Figure 14 displays solanoeclepin A in multiple fractions and is not directly related to the amount sprayed on the plants.
3.6 Growing conditions
Desirèe potato plants were grown on soil 3 and root exudate was collected weekly by pouring demi-water through the soil and filtered (WhatmanTM Glass microfiber filter) on a Buchner funnel
under vacuum. Plants were watered every 2 days and kept at 19-21 ⁰C with 15hrs light – 9hrs dark. Sterilisation of the Arabidopsis seeds was performed with bleach+tween20 under intermittent shaking (10 min). Seeds were accumulated by spinning (1000 rpm) and bleach was removed. Seeds were rinsed with water six times according to the same procedure. Agar solution (approx. 7 plates) contained demi-water (250 mL), Murashige & Skoog medium (basal salt mixture) (0.54 g), sucrose crystallized (2.5 g) and Daishin agar (2 g) (all solid materials were supplied by Duchefa Biochemiebv). The agar solution was sterilized in an autoclave (30 min. at 15 bar) and poured on sterile square plates with a length of 11.7 cm x 11.7 cm. Arabidopsis seeds were planted on agar plates (10 on each plate) in a sterile environment (Scanlaf Mars safety class 2) and grown in a climate room at 22 ⁰C with 16hrs light – 8hrs dark.
3.7 Root surface determination using ImageJ
Root surface area (2D) was quantified using imageJ version 1.52a. Arabidopsis leaves were erased from the picture followed by applying the make binary (8-bit) function under the tab process (figure 4). Subsequently, black (pixel value 255) pixels were counted using the histogram function under the tab analyse. The area of the plate was calculated in pixels (7743191) and cm2 (136.89) which resulted in the ratio:1 cm2 = 56565 pixels. Number of black pixels were
divided by 56565 to find the total surface area in each
picture (figure 16) Figure 4. ImageJ example of binary (8-bit) with
erased leaves of plant treatment with C18->MAX->Fractionation (→)
3. Results and discussion
2.1 Optimization of extraction protocol
The main aim for extraction of root exudates is to reduce the sample complexity by retaining compounds on a cartridge with subsequent washing and elution (SPE). Target molecule analysis and profiling may be hindered due to suppression which is caused by strong matrix effects. Root exudate contains large quantities of different molecules such as salts, proteins, carbohydrates, pigments and lipids.4 To extract and concentrate Desirée potato root exudate, we optimized the elution solvent used
to extract a-polar molecules from the sample while maintaining a minimal loss of the target molecule: solanoeclepin A. First, different aqueous solvents of methanol were used to determine the elution strength necessary to release solanoeclepin A, pre-concentrated on the reverse-phase cartridge. Desirée potato root exudate was loaded on C18 Discovery® cartridges under low pressure and salts together with non-retained polar compounds were washed out using water. Elution was performed sequentially using methanol and its aqueous solutions of different percentages (40%, 60%, 80%, 100%) with commercially recommended volume. The endogenous solanoeclepin A was detected by MRM in the fraction of 40% and 60% methanol (figure 5 a, b, c) with no distinct difference of background signal in the area of interest (5.9 min), obtained by fullscan measurements using the HPLC-qTOF-MS (figure 5 d, e).
(a)
(b)
(c)
(d)
(e)
Figure 5. Short gradient HPLC-qTOF-MS chromatogram of solanoeclepin A standard with elution time 5.9 min
(d) and total ion current of endogenous solanoeclepin A: 40% fraction (green); 60 % fraction (purple) (e). UHPLC-MRM chromatogram of endogenous solanoeclepin A: 80% (c); 60% (d); 40% (e). Concentration level of eluted endogenous solanoeclepin A in different fractions (f). Quoted values are means ± SD (n = 3).
Concluded from these results, at least a 60% aqueous methanol solution was needed to elute compound from C18 cartridges (figure 6). However, time consuming evaporation of large volume of water-containing elution solvent is not convenient, especially for the reason of possible compound degradation. Therefore, we were looking for balance between yield, acceptable sample purity and duration of isolation procedure.
Recent developments in solid-phase extraction presented new polymer-based sorbents with higher capacity that can combine several separation mechanisms.14 The
commercially available HLB Oasis® (Waters) with both hydrophilic and lipophilic retention character was tested for solanoeclepin A extraction as an alternative to
silica-based C18 Discovery® cartridges. Loaded samples were eluted separately using aqueous methanol (60%, 70%, 80%, 90%, 100%). Strong matrix effects may hinder the detection of solanoeclepin A which was observed for 90% and 100% methanol elution (figure 7).
In addition, the standard deviation of the solanoeclepin A detection was higher for elution with 100% methanol compared to usage of 90% aqueous methanol. To minimize the loss of solanoeclepin A and to gain the maximum effect with this extraction procedure, 90% aqueous methanol with an average recovery of 0.097 pmol for C18 and HLB was determined to be the ideal elution solvent for extracting root exudate. The Q-TOF-MS chromatogram of the C18 and HLB extracted samples showed no significant difference in the area of interest (10.9 min) (figure 8).Consequently, all subsequent experiments which involved a-polar based SPE extraction were performed using 90% aqueous methanol (figure 9 a).
0 0,01 0,02 0,03 0,04 0,05 40 60 So la n o ie cl ep in A (p m o l)
Elution strength (% MeOH) Figure 6. Concentration of eluted endogenous
solanoeclepin A in different fractions (f). Quoted values are means ± SD (n = 3).
Figure 7. Elution of endogenous solanoeclepin A from silicabased C18 sorbent and polymer based HLB sorbent
by increasing elution strength (% MeOH). Concentration level of eluted endogenous solanoeclepin A of different elution strengths is shown. Quoted values are means ± SD (n = 2).
0 0,02 0,04 0,06 0,08 0,1 0,12 60 70 80 90 100 So la n o e cl ep in A (p mo l)
Elution strength (% MeOH)
C18 HLB
Figure 8. The HPLC-qTOF-MS chromatogram of solanoeclepin A standard with longer elution time 10.9 min (a).
Comparison of background, pre-concentrated by silica-based C18 (green) and polymer based HLB (red).
0 5 10 15 20 25 30 35 Time [min] 0.0 0.5 1.0 1.5 2.0 6 x10 Intens.
20190528_eclep000002.d: TIC +All MS 20190528_eclep000003.d: TIC +All MS
(a)
Pour demi-water over potato plant soil
Collect root exudate
Dry extracted samples and redissolve in 25% ACN in
H2O
Filter collected root exudate with vacuum
filtration
Filter sample using filter tubes
HPLC MS/MS
Figure 9. Optimal extraction method for a-polar SPE (C18 and HLB) using 90% aqueous methanol as elution solvent
(a). Purification method with another ion exchange SPE methodusing 5% formic acid in methanol as elution solvent (X-AW) (b). Optimal purification method for ion exchange SPE (MAX) using 0.75% formic acid in methanol as elution solvent (c). HLB->Fractionation->X-AW (→); HLB->X-AW-> Fractionation (→); C18->Fractionation->MAX (→); C18->MAX->Fractionation (→). Washing steps always involved 2x the volume of the other steps.
H
LB
H
LB
C
1
8
C
1
8
FractionationX
-AW
X
-AW
M
AX
M
AX
Fractionation Fractionation Fractionation Fractionation Fractionation Fractionation Fractionation a b c2.2 Optimization purification protocol
Purification with SPE was performed based on the favourable stable carboxylic acid group in its protonated and deprotonated form. Sample purification was performed with ion-exchange based extraction using Oasis® MAX cartridges, combining both reverse-phase and anion-exchange retention mechanisms.
Two different loading conditions were compared using a different starting pH of the crude root exudate. The first method involved conditioning by sample acidification before loading (pH = 3.30, BL). The second method involved a similar retention mechanism and starts with root exudate without pH adjustments (pH = 6.49, AL). Anion carrying compounds, a-polar groups and groups for hydrogen bonding retain well on the MAX sorbent. The aim of MAX purification was to utilize the (cationic) pockets located in the MAX sorbent to isolate an anionic charged compound, solanoeclepin A. The HPLC-qTOF-MS total ion current chromatogram (figure 10 b) shows the comparison of sample background when using different loading conditions. BL results in a more complex background compared to AL. Possibly, the neutrally charged analyte is retained together with all molecules that contain an a-polar moiety or via hydrogen bridge, so ion-exchange potential of the column is not used and capacity is decreased. Washing steps were not collected and therefore not analysed. The full scan measurement was sensitive to detect pure standard of solanoeclepin A (figure 10 a).
The endogenous compound was determined and quantified for both loading conditions using targeted UPLC-MS/MS (figure 11). MAX cartridges were conditioned using methanol followed by activation with 5% aqueous NH4OH. In this way we
made sure that the cationic pocket in the cartridge was attainable for anion containing molecules. As the root exudate of the BL method was acidified to a pH of 3.30, we could not conclude that all the solanoeclepin A was retained on the first purification step. To gain best retention the pH of root exudate should be at least 2 units below the pKa of the carboxylic acid group (4.7). Therefore, a similar experiment was performed using root exudate acidified to pH 2.00, 2.50 and 3.00. This
(a)
(b)
Figure 10. Elution of endogenous solanoeclepin A from ion-exchange mixed mode (MAX) sorbent by elution with
formic acid in MeOH. Short gradient HPLC-qTOF-MS chromatogram of solanoeclepin A standard with elution time 5.9 min (a) and total ion current of endogenous solanoeclepin A: Before loading (orange); After loading (blue) (b).
Figure 11. Concentration of eluted endogenous
solanoeclepin A before loading (BL) and after loading (AL)). Quoted values are means ± SD (n = 4).
0 0,05 0,1 0,15 0,2 So la n o ec le p in A ( p m o l) BL AL
did not yield in higher amounts of isolated solanoeclepin A (results not shown). The AL method selectively purifies carboxylic acid directly as it is loaded in its ionized form. In this way, capacity is not exceeded which presumably results in higher amounts of solanoeclepin A in MRM analysis. Another possible explanation for this difference could be that the BL method MRM analysis was hindered by signal suppression as more compounds were detected with Q-TOF-MS.
Washing with 5% aqueous NH4OH raises the pH of the solvent in the cartridges and consequently
changes the retention mechanisms for compounds with groups that have a moderately low pKa (3-5) in the BL method. The AL method already has solanoeclepin A in the deprotonated form before loading and washing with 5% aqueous NH4OH does not change the mechanism purification. Additional
methanolic wash flushes out the a-polar uncharged compounds while solanoeclepin A, as an anion, should stay retained on to the sorbent.
Elution using 0.75% formic acid in methanol causes protonation and desertion of solanoeclepin A from the cartridge. Considering that the AL method provides a higher purity (for early eluting compounds) and more presence of solanoeclepin A compared to BL, this method was determined to be most favourable for purification (figure 10, 11). Therefore, all subsequent experiments which involved ion exchange MAX SPE extraction were performed using the AL method (figure 9 b). Comparison of the Q-TOF-MS chromatogram of nonselective preconcentration and selective purification indicates a less polluted background for the selective purification method (figure 12). For further methods of isolation of solanoeclepin A, nonselective preconcentration was used in succession with selective purification to assure that the capacity of MAX columns is not exceeded.
Phenomenex® Strata-X-AW was also used as an alternative for MAX in further purification procedures to determine if this increases the solanoeclepin A isolation. As Strata-X-AW has a similar mechanism for retention as MAX but it is a cheaper alternative, we used it in the purification procedure described in: 2.3 fractionation.
(a)
(b)
Figure 12. The HPLC-qTOF-MS chromatogram of solanoeclepin A standard with shorter elution time 5.9 min (a)
2.3 Fractionation
Extraction was repeated using high capacity C18 and HLB cartridges. HLB and C18 extracted samples were compared by their 20x diluted solutions and analysed by MRM to detect ion suppression (figure 13).
It is obvious that sample dilution helped to decrease the background noise, therefore the concentration level of detected compound was ~7 times higher than theoretical expectations. The C18 and HLB pre-concentrated samples were further fractionated. Due to fractionation the ion suppression effect was reduced causing a 1.97-fold higher MRM signal compared to unfractionated samples. Simultaneously C18 and HLB concentrated samples were also purified by an additional second step using MAX and Strata-X-AW cartridges respectively. These two step SPE purified samples were analysed or additionally fractionated (figure 9). To ensure that there is no difference in the order of purification or fractionation, another set of C18 and HLB extracted samples were fractionated before purification (figure 9). In this way, four routes for isolation of solanoeclepin A were established in which the main difference between each route is the order of the purification method. MRM analysis indicated no significant difference in the different routes in which we used MAX as purification step (figure 14). 0 0,02 0,04 0,06 0,08 0,1 0,12 C18 C18; 20x diluted HLB HLB; 20x diluted C18 and fractionation HLB and fractionation So la n o ec le p in A (p m o l)
Figure 13. The amount of solanoeclepin A in root exudates concentrated with: high capacity C18 (a); high capacity
C18 – sample 20x diluted(b), high capacity HLB (c); high capacity HLB – sample 20x diluted (d); high capacity C18 and fractionation (e); high capacity HLB and fractionation (f).
(a)
(b)
(c)
(d)
(e)
(f)
0 0,050,1 0,150,2 0,250,3 0,350,4 0,450,5 So la n o ec lep in A (p mo l)Figure 14. The amount of solanoeclepin A in pre-concentrated root exudates and purified with: MAX and
fractionation(→) (a); MAX (b); MAX – sample 20x diluted (c); fractionation followed by high capacity MAX (→)
HLB->X-AW->Fractionation was performed with insufficient elution volume for the ion exchange mode (5% formic acid in methanol) which resulted in no detection of solanoeclepin A. Solanoeclepin A was detected in the methanol wash of HLB->Fractionation->X-AW. MAX as ion-exchange purification in the second step resulted in the highest amount of solanoeclepin A and was considered the best method for purification. Fractionation of samples additionally purified with MAX was considered to be most applicable as this method injects a sample of better purity onto the semipreparative column and improves detection of solanoeclepin A 4.6 times compared to the fraction of only pre-concentrated sample.
2.4 Plant treatment
In order to examine the biological effect of solanoeclepin A on the shoot and root system of a model plant, Arabidopsis seeds were sterilised and planted on agar plates under climate room conditions. Arabidopsis seedlings were treated with samples processed by four established purification routes, a negative control and a positive control after growing for 11 days. Pictures captured the influence of the treatments after an extra 11 days (figure 15).
Results show no direct correlation between the amount of solanoeclepin A applied and the root/shoot development 11 days after applying each route of purification to Arabidopsis plants (figure 16). It seems from the negative and positive control that the amount of solanoeclepin A results in a higher production of root. However, only one plate for each treatment was used for determining differences in Arabidopsis development. Therefore, the bias and the influence of genetic variation is unknown.15
In addition, the absolute concentration (due to ion suppression) and purity of solanoeclepin A in each purification method is unknown. Therefore, it is not certain that solanoeclepin A is singularly involved in the effect on root development. Root area development of HLB->X-AW-> Fractionation (→) was not analysed due to pollution of fungi on the agar plate (figure 15 e).
(a)
(b)
(c)
(d)
(e)
(f)
Figure 15. Plant treatment with C18->Fractionation->MAX (→) (a); HLB->X-AW->Fractionation (→) (b); positive control (c); C18->MAX->Fractionation (→) (d); HLB->Fractionation->X-AW (→) (e); negative control (f).
4. Conclusion
The method for solanoeclepin A isolation from potato root exudates was successfully developed. We have compared four routes of isolation. Elution solvents for the isolation methods involved 90% aqueous methanol for reversed phase SPE, 0.75% formic acid in methanol for MAX and 5% formic acid in methanol for Strata-X-AW. C18 extraction followed by MAX purification and C18 fractionation was determined to be the most effective route for collection of the highest amount of solanoeclepin A. No direct correlation of solanoeclepin A with respect to root development of Arabidopsis could be determined after application of the isolates. Solanoeclepin A is responsible for an increase in root development when comparing the positive and negative control, but this cannot be directly concluded as the bias and the influence of genetic variation was not tested.
5. Future prospects
Solanoeclepin A was isolated with unknown purity and we are positive that it can be improved by addition of another basis of separation. In this research, only one method of fractionation was performed using C18 sorbent. Offline 2D fractionation using a sorbent which separates on different chemical moieties should lead to a higher purity of solanoeclepin A. Sielc® Primesep SB is a good candidate as this sorbent is reverse-phase with strong embedded basic ion-pairing groups that are most probably reachable for the carboxylic acid group of solanoeclepin A. This column is also compatible for both acidic and alkaline conditions which are required for the deprotonation of the carboxylic acid group.
Furthermore, an improvement of the Arabidopsis treatment should lead to a better understanding of the influence of solanoeclepin A on the shoot and root system. In our experiments, only one plate was used for each treatment with only one timepoint of application on the plants. Solanoeclepin A could have a different effect on the plant if it is applied on different stages of growth. Therefore, an experiment allowing more variables should exclude what effect those variables have on variations in plant development. 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0 5 10 15 20 25 30
pos_control C18_frac6_MAX C18_MAX_frac5 HLB_X-AW_frac6 neg_control
So la n o ec le p in A (p m o l) Su rf ac e area (c m ^2 )
surface area roots amount sprayed on leaves
Figure 16. Root surface area with corresponding solanoeclepin A concentration applied fortreatments positive control (a); C18->Fractionation->MAX (→) (b); C18->MAX->Fractionation (→) (c);HLB->X-AW-> Fractionation (→)
(d); negative control (e).
(a)
(b)
(c)
(d)
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