Jasmonic Acid Effect on the Fatty Acid and Terpenoid Indole Alkaloid Accumulation in Cell Suspension Cultures of Catharanthus roseus

The stress response after jasmonic acid (JA) treatment was studied in cell suspension cultures of Catharanthus roseus. The effect of JA on the primary and secondary metabolism was based on changes in profiles of fatty acids (FA) and terpenoid indole alkaloids (TIA). According to multivariate data analyses (MVDA), three major time events were observed and characterized according to the variations of specific FA and TIA: after 0–30 min of induction FA such as C18:1, C20:0, C22:0 and C24:0 were highly induced by JA; 90–360 min after treatment was characterized by variations of C14:0 and C15:0; and 1440 min after induction JA had the largest effect on both group of metabolites were C18:1, C18:2, C18:3, C16:0, C20:0, C22:0, C24:0, catharanthine, tabersonine-like 1, serpentine, tabersonine and ajmalicine-like had the most significant variations. These results unambiguously demonstrate the profound effect of JA particularly on the accumulation of its own precursor, C18:3 and the accumulation of TIA, which can be considered as late stress response events to JA since they occurred only after 1440 min. These observations show that the early events in the JA response do not involve the de novo biosynthesis of neither its own precursor nor TIA, but is due to an already present biochemical system.


Introduction
Fatty acids (FAs) are the most abundant form of reduced carbon chains available from nature and plant FAs represent a large reservoir of diversity with at least 200 different types that occur mainly in plants. In eukaryotic organisms like plants, they consist almost exclusively of 16 and 18-carbon FAs, being palmitic acid (C16:0) the major saturated FA followed by the unsaturated linolenic (C18:3), linoleic (C18:2) and oleic (C18:1) and these are often referred to as the common FAs [1] (Table 1). Fatty acids are essential molecules present in all living organisms. They serve as major source of reserve energy by being part of complex lipids and are essential components of cellular membranes [2]. Moreover, they are also key molecules that participate in various biological processes [3]. In plants, composition and turnover of intracellular lipids and FAs are frequently altered during development and are among the first targets of environmental cues [4]. For example, the polyunsaturation of FAs has proven to be correlated to adaptation when plants are challenged with high or low temperatures [5,6] or high salinity [7] and consequently they modulate a variety of responses to biotic and abiotic stresses.
One of the best-characterized FA-derived signal molecules is jasmonic acid (JA) along with its derivatives, collectively known as oxylipins or jasmonates (JAs). They play a role of master switch in many plant responses to biotic and abiotic factors such as wound response after herbivore attack, ultraviolet light or ozone, drought, but they also modulate flower, seed and fruit development, seed germination, pollen viability, anthocyanin accumulation, fruit ripening [8], tuberization in Solanum, tendril coiling in Bryonia and promotion of leaf senescence [9][10][11]. Jasmonic acid and its methyl ester (MeJA) have also a positive effect on the signal transduction chain leading to the accumulation of highly valued terpenoid indole alkaloids (TIA) in C. roseus. Both JAs induce most of the known TIA pathway genes [12], which results in higher levels of TIA in cell suspensions of C. roseus [13].
The coordinated expression of biosynthetic genes is mediated through the octadecanoid-responsive Catharanthus AP2-domain proteins ORCA2 and ORCA3, which are both induced by JA [14,15].
The accumulation of TIA can be considered as a late response in the jasmonate-mediated stress response in C. roseus where the expression of biosynthetic genes such as geraniol 10-hydroxylase (G10H), tryptophan decarboxylase (TDC), strictosidine synthase (STR) and strictosidine β-D-glucosidase (SGD) (Scheme 1) takes place after 2 h of induction [12,16] and the accumulation of TIA are detected in significant amounts only after 4-24 h of induction [17,18]. Therefore, since the rapid burst of jasmonates after wounding and feeding with JA takes place within 30 s [19], it is considered as an early response that precedes any transcriptional activity. Further events that involve the de novo biosynthesis of precursors, the de novo production of enzymes or secondary metabolites is considered as a late event in the JA-mediated stress response. As a part of an integrative study on the fast jasmonate response, we attempted to contribute to the knowledge on FA and TIA accumulation after treating cell suspensions of C. roseus with JA to establish a time frame where all these responses occur both in primary and secondary metabolism. Scheme 1. Biosynthetic pathway of TIA in C. roseus (modified from [20]). Broken arrows indicate multiple enzymatic reactions. Enzymes with their corresponding cloned gene are indicated. Genes previously reported to be regulated by ORCA3 are indicated in gray boxes.
According to the principal component analysis (PCA) scores plot of PC1 vs. PC2 ( Figure 1A), a clear separation was achieved by PCA score plot where two main groups were observed: 0-5-30 min and 90-360-1440 min after elicitation. Examination of PCA loading plot of JA-treated cells showed that C16:0, C20:0, C18:3, C18:2, C22:0 and C18:1 and C24:0 ( Figure 1B) were the most influential FA for the separation with the highest variation in the model.
These observations suggest that JA is able to induce the biosynthesis of its own precursor, that is C18:3, but only at later time points i.e., 1440 min after induction which also means that changes in the FA composition of the membrane are not involved in the fast response after induction with JA.
A variation in FA profile after JA-elicitation suggests a remodeling in the lipid architecture of the membrane where the cell has to cope first with non-physiological amounts of JA with an efficient and fast mechanism of signal attenuation mainly accomplished by hydroxylation [34], epimerization or methyl esterification [35]. In wound-induced tissues, further responses involve the release of precursors from the membrane mediated by lipases to generate the substrates for JA synthesis as occurred for wound-induced leaves of tomato, where 15-fold increase of levels of C18:3 peaked 1 h after wounding, which correlated with an increase in JA [36,37]. These results differ from ours, where levels of C18:3 had significant variations after 1440 min of treatment suggesting that at least in C. roseus, the JA-mediated response does not involve the relocation of precursors like C18:3 but the induction of the stress response pathway as it has been demonstrated in previous experiments where the exogenous application of C18:2 and C18:3 as elicitors, leads to the enhanced accumulation of secondary metabolites in root cultures of Panax ginseng [38,39], cell suspension cultures of Lycopersicon esculentum, Tinospora cordifolia, Erythrina cristagalli and Eschscholtzia californica [40] and LOX activity in N. tabacum cell cultures after 1-2 h of induction [41] suggesting the de novo biosynthesis of precursors.  Jasmonic acid was applied to cell suspension cultures of C. roseus on the 4th day after subculture. Seven peaks were noticed and distinguished only by their different retention times (α-TIA-1-7). According to their absorbance under ultraviolet (UV) light, three maxima were observed at 225, 300, 325 nm, which is characteristic of alkaloids possessing an α-methylene indoline chromophore, constituted by a double bond conjugated with a carbonyl group in the α position of the indoline nitrogen atom, like present in tabersonine. However, the aim of this work was not to identify each trace alkaloid present in our cell system but to get a more general overview of the main TIA accumulated after JA treatment.

C D
The most significant effects were observed on the accumulation of α-TIA-1 and tabersonine after 360 min of induction whereas catharanthine and serpentine were induced after 1440 min of elicitation ( Figure 3). Levels of catharanthine, α-TIA-1 and tabersonine transiently increased after 5 and 360 min of elicitation, respectively, reaching a maximum after 1440 min where their accumulation was clearly induced by JA. Previous investigations on cell suspensions of C. roseus treated with 100 µM of MeJA, have shown a 300% increase in alkaloid accumulation like ajmalicine and serpentine [14] and strictosidine [42]. This is in contrast with our observations and previous reports on the same cell line [43] where only serpentine but not strictosidine was induced by JA.  [44]. This inability is mainly due to the lack of expression of D4H and the transcriptional inactivation of DAT [45,46]. This can be explained with the tight regulation in the biosynthesis of vindoline where the enzymes are located in different cellular compartments. The first step in the conversion from tabersonine to vindoline is its hydroxylation [47]. Regardless the fine-tuned regulation in vindoline biosynthesis, some transcripts specific to its biosynthesis have been detected in hairy roots and cell suspension cultures of C. roseus [48]. However, exposure of cell cultures of C. roseus to light increased the expression of T16H within 22-28 h of treatment [49] and was also found along with 16OMT to be expressed in cell suspensions cultures in 20% lower levels than in young leaves of C. roseus [50]. In addition, activities of T16H and O-methyltransferase but not that of NMT, D4H and DAT are present in cell suspensions of C. roseus indicating a degree of separation in the regulation of the first two and last four steps in the conversion of tabersonine to vindoline [50].

Jasmonic acid exerts well-documented differential responses both in cell systems and in plants in the stress response. Wound-induced experiments in
Arabidopsis have shown that the accumulation of JA starts within 120 s after wounding and spreading the signal to long distance unwounded leaves at an average velocity of 3.4-4.5 cm min −1 [19]. Induction of JA accumulation in cell suspension cultures of Rauvolfia canescens takes place within 20-30 min after exposure to a biotic elicitor, reaching a maximum concentration of the free acid after 90 to 240 min [37,51], which is in complete agreement with our results. These results suggest that de novo biosynthesis of JA starting from the release of C18:3 from thylakoids is very unlikely since lipoxygenase 2 (LOX2) is known to contribute to almost 75% of JA measured at 24 h after wounding leaves [52]. Mutants in the LOX pathway such as lox2-1 and lox6A, were still able to accumulate low levels of JA and JA-Ile in the first 5 min upon wounding but caused > 99% reduction in the concentrations of 12-oxo-phytodienoic acid (OPDA) and dinor-12oxo-phytodienoic acid (dnOPDA)-containing arabidopsides in wounded leaves [19] and reduction of JAZ10 expression [53] only indicating the redundancy of this family of enzymes but confirming that the fast response is not mediated by them.
Interestingly, when the ability of the precursor of JA i.e., C18:3 to induce TIA accumulation in hairy roots of C. roseus was tested and compared to that of JA, it was found that the octadecanoid pathway is not responsible of the production of TIA [54]. Thus, the effects of feeding JA on TIA production to any system are related to pathway induction and not to the relocation of precursors. In contrast, ajmalicine production is highly inducible by MeJA in cell suspension cultures of C. roseus where the highest yields (4.75 mg/L) was observed when cells were treated with 3 mM of CaCl 2 and 100 µM of MeJA suggesting that cells were dependent on a low intracellular Ca 2+ concentration [55], which is in contrast with the previous knowledge that Ca 2+ and MeJA induce the production of the transcriptional factors CrBPF-1 (Catharanthus roseus Box P Binding Factor-like protein 1) and ORCAs that regulate the expression of STR [56].
The very fast burst of JA accumulation excludes any transcriptional activation assuming that the endogenous JA is formed from a readily and abundantly precursor and released through a constitutively enzymatic process. A likely scenario includes the liberation and conversion of OPDA and/or dnOPDA both esterified to a monogalactosyldiacylglyceride (MGDG) and/or digalactosyldiacylgliceride (DGDG) into JA, a system so far only found in Arabidopsis [57,58].
The time line of events including primary and secondary metabolites e.g. fatty acids and alkaloids after JA-induction, places their accumulation into a late response since significant increases in C18:3 along with tabersonine, catharanthine, serpentine and α-TIA-1 were observed only after 360 min of JA induction with peaking levels after 1440 min. Based on these observations and previous knowledge where C18:3 does not induce neither JA nor TIA accumulation in hairy roots of C. roseus, we support the idea that two independent time-events occur after JA induction where in the early response i.e., after 5-30 min of induction, responses include a rapid propagation and intensification of the signal as an "alert-state" to accumulate enough endogenous amounts of JA to enter into a late response i.e. after 6-24 h, where the de novo biosynthesis of precursors like C18:3 and the accumulation of TIA are the result of gene expression mediated through the JA-mediated signaling pathway.

Cell Cultures and Elicitation with Jasmonic Acid
Cell suspension cultures of C. roseus cell line CRPP were grown in 250 mL Erlenmeyer flasks containing 50 mL of Gamborg B5 [59] medium supplemented with 30 g/L sucrose and 1.86 mg/L 1-naphthaleneacetic acid (NAA) and adjusted to pH 5.8 with 0.1 N KOH [43]. Cell cultures were propagated on a rotary shaker (110 rpm) at 25 °C under continuous light (500-1500 lx) and were subcultured every three weeks. Four-day-old cell suspension cultures were treated with 101.9 µM/70 mL of culture medium of JA (Sigma-Aldrich, St. Louis, MO, USA) or 150 µL of 40% v/v EtOH (mock) or nothing (untreated) and were harvested in quadruplicates at 0, 5, 30, 90, 360 and 1440 min after elicitation. Cells were filtered on Whatman filter paper under partial vacuum and biomass and media samples were immediately frozen in liquid nitrogen and kept at −80 °C until further analysis.

Chemicals Used for Fatty Acid Determination and Alkaloid Standards
The mixture of 37 FA methyl esters (37 Component FAME Mix) was obtained from Supelco (Sigma-Aldrich; Bellefonte, PA, USA). All other chemicals and solvents were of analytical grade and purchased from common sources. Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, Brea, CA, USA). Strictosidine and secologanin were provided by Phytoconsult (Leiden, The Netherlands); loganic acid, tabersonine and vindoline were purchased from PhytoLab (Vestenbergsgreuth, Germany); tryptamine was purchased from Sigma-Aldrich Chemical (Milwaukee, WI, USA) tryptophan and ajmalicine were purchased from Sigma-Aldrich; serpentine was purchased from Roth (Karlsruhe, Germany) and catharanthine were kindly gifted by Pierre Fabre (Gaillac, France).

Fatty Acid Extraction
Samples of 10 mg of freeze-dried cells were spiked with 50 µg of C17:0 as internal standard and then subjected to hydrolysis with 1 mL of 1M of KOH in 95% EtOH. The suspension was ultrasonicated for 10 min followed by heating the closed tube at 80 °C for 30 min. After cooling at room temperature, 1 mL of Milli Q water was added followed by two times extraction with 1 mL of n-hexane containing 0.01% butylated hydroxytoluene (BHT; Sigma-Aldrich), after vigorously vortexing, samples were centrifuged for 10 min at 3,500 rpm and the upper hexane layer was removed and discarded. The aqueous layer was acidified with 6 M HCl and extracted twice with 1 mL n-hexane (0.01% BHT) and after centrifugation for 10 min at 3,500 rpm upper layers were pooled and completely dried under a gentle flow of N 2 gas. To the residues 1 mL of boron trifluoride (BF 3 ; Sigma-Aldrich) in MeOH was added and the closed tubes were heated for 15 min at 80 °C and then cooled down at room temperature and 1 mL of n-hexane (0.01% BHT) was added and after centrifugation, the upper layer (300 µL) was used in GC-MS analysis.  Table 1. The 37 Component FAME Mix was used as a control for possible retention time shifts and mass spectra ion identification.

Alkaloid and Precursor Extraction for HPLC
A modified extraction protocol was followed after [60]. Briefly, freezed-dried samples of 50 mg were extracted twice with 5 mL of MeOH, vortexed for 1 min, sonicated for 20 min and then centrifuged for 30 min at 3,500 rpm. Pooled samples were reduced to dryness under reduced pressure. To the dried extract 500 µL of 1 M H 3 PO 4 was added and the suspension was thoroughly homogenized and then transferred to an Eppendorf tube for centrifugation for 10 min at 13,000 rpm. Extracts were filtered with a 0.2 µm PTFE membrane and then 50 µL were analyzed by HPLC.

HPLC Analysis
Chromatographic separations were carried out on a 250 mm × 4.60 mm, 5 µm Gemini-NX C18 (Phenomenex Inc., Torrance, CA, USA) column and a guard column filled with RSil C 18 HL (Zeochem AG, Uetikon, Switzerland) at room temperature. Elutions were performed at a flow rate of 1.5 mL/min. Solvents used for TIA were 80% 5mM Na 2 HPO 4 pH 7 adjusted with H 3 PO 4 ; (Solvent A) and 20% ACN (Solvent B) and for precursors 88% 0.01M H 3 PO 4 (Solvent A) and 12% ACN (Solvent B). Gradient analyses modified from [61] were as follows: 80:20 (A:B) to 20:80 in 31 min for TIA and 88:12 (A:B) to 30:70 and then to 88:12 in 25 min for precursors, injection volume was 50 µL. The HPLC system was equipped with an Agilent 1200 Series coupled to an Agilent G1315D photodiode-array detector. It consisted on of a G1310A binary pump, a G1329A autosampler, a G1322A degasser and a G1315D photo-diode array detector controlled by ChemStation software (Agilent v. 03.02; all from Agilent Technologies Inc.). Spectroscopic data from all peaks were accumulated in the range of 220-320 nm and chromatograms were recorded at 220, 254, 280, 306 and 320 nm. Detection and quantification was achieved by comparison of their absorbance with that of reference standards.

Data Handling
Differences between treatments, among time points and groups of TIA, regarding contents of each individual TIA in the positive controls were assessed using the Mann-Whitney non-parametric test using R-Project software (v 3.0.0). A p-value less than 0.05 was considered statistically significant.
Fatty acids were identified as FAME with the help of the National Institute of Standards and Technology (NIST) library version 2.0f (Agilent Technologies Inc.). Quantification was done by normalizing the peak area of each FA species with that of the internal standard (C17:0). Differences between days and between TIC and SIM, as well as between groups of FA, regarding contents of each individual FA in the positive controls were assessed using the Mann-Whitney non-parametric test using R-Project software (v 3.0.0). A p-value less than 0.05 was considered statistically significant. Principal component analysis (PCA) was performed with the SIMCA-P software (v 13.0, Umetrics, Umeå, Sweden). Normalized peak area data was scaled using unit variance (UV). The output from the PCA consisted of a scores plot, which gave an indication of the discrimination of the profile of 13 different FA and a loadings plot, which allowed the identification of those FA with significant influence on the separation of the clustering data.

Conclusions
Using an MS-based targeted profile, we demonstrate a clear effect of jasmonic acid over time on the FA and TIA accumulation in cell suspensions of C. roseus where compounds such as C18:3, C18:2, tabersonine, catharanthine and serpentine had the highest variations. It is very interesting to note that JA applied as an exogenous elicitor induced the accumulation of its own precursor C18:3 but only after 90 min of treatment which cannot explain the fast response seen in the accumulation of JAs. This can only be explained by a larger pool of an intermediate or precursor of JA that is released and immediately activated by a constitutive enzyme system into JA.