{"paper_id":"4aed8a5c-bfb3-48f2-a3e0-0583ed3fca0e","body_text":"Modulating stilbenes in peanut cells: A novel approach with metabolic modulators | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Modulating stilbenes in peanut cells: A novel approach with metabolic modulators Hajer Ben Ghozlen, Sven Mangelinkcx, Stefaan P.O. Werbrouck This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6375460/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Stilbenes, including resveratrol, piceatannol and piceid, are often limited in terms of bioproduction yield. This study represents the first attempt to modulate stilbene production pathways in peanut ( Arachis hypogaea ) cells. We investigated the potential of L-phenylalanine, sodium malonate dibasic, and cerulenin as metabolic modulators to promote stilbene biosynthesis. These modulators were tested at different concentrations and time points in both peanut callus cultures and cell suspension cultures. The effects of these modulators on cell growth and stilbene production were assessed. The results revealed that metabolic modulators significantly influence the production patterns of resveratrol, piceid, and piceatannol in peanut cells. Interestingly, both static and suspension cultures displayed distinct responses, with the specific metabolite produced and its level depending on the growth phase, modulator concentration, and incubation time. Our findings showed that 0.2 µM cerulenin was the most effective modulator, resulting in more than tenfold increase in resveratrol production in callus cultures. In cell suspension cultures, 0.5 mM sodium malonate dibasic also enhanced the production of resveratrol during the lag phase whereas piceatannol and piceid were more prominently produced during the stationary phase. This effect was more significant than that observed with phenylalanine and cerulenin. This research provided valuable insights into the modulation of metabolic pathways within this novel host system and established peanut cells as a viable platform for future stilbene production. Stilbene peanut cells metabolic modulators bioproduction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Key Message Peanut cell cultures are a sustainable platform for the production of resveratrol, piceatannol and piceid, enhanced by metabolic modulation, offering a promising method for wider availability of these valuable compounds. Introduction Biotechnological production of specialized metabolites using in vitro plant cultures, including culturing of plant tissues, organs, or cells, has the potential to overcome the limitations and overexploitation associated with traditional plant-based methods (Rattan et al. 2022 ). Advances in plant cell culture methods, particularly callus and suspension cultures, present promising avenues for the large-scale production of stilbenes (Jeandet et al. 2021 ). It is important to note that while recent studies have shown successful production of resveratrol and various derivatives in a few plants like Vitis vinifera and Morus alba , such production has been rare in peanuts. By supplementing specific precursors and manipulating metabolic pathways, these strategies may offer promising solutions to bioproduction bottlenecks. Given the key role of phenylalanine as a direct precursor in the stilbene biosynthesis pathway, supplementing cultures with phenylalanine will lead to maximize the utilization of intracellular phenylalanine pools and potentially enhance stilbene production. Therefore, increasing the resveratrol content in peanut by adding phenylalanine could have important effects, as shown in sprouts (Yu et al. 2016 ) and hairy roots culture (Tothong et al. 2023 ). Metabolic pathways modulation within cells can be strategically enhanced by using compounds like malonate and cerulenin. These known modulators of plant metabolism offer valuable tools for researchers aiming to redirect cellular pathways towards the stilbene production (Shrestha et al. 2019 ). Malonyl-CoA is a metabolic node at the intersection of both the flavonoid and the stilbenoid pathways as well as of the fatty acid biosynthesis. Studies have shown that increasing the intracellular pool of malonyl-CoA is a key strategy to overproduce stilbenes in engineered microorganisms by redirecting the carbon flux into this compound (Jeandet et al. 2018 ). Two main approaches, the regulation of the upstream malonyl-CoA biosynthetic pathway and the control of the downstream malonyl-CoA utilization pathway, are often used to achieve this redirection. Malonyl-CoA is produced mainly from acetyl-CoA by the irreversible action of the acetyl-CoA carboxylase (ACC) complex. Thus, overexpression of the malonate assimilation pathway (malonyl-CoA synthetase [MatB] and malonate carrier protein [MatC]) or inhibition of fatty acid synthesis increases malonyl-CoA concentration (Hu et al. 2024 ). Researchers have looked at supplementing the culture medium directly with malonate (malonyl-CoA's precursor). However, this feeding approach often requires genetic modification of the microorganisms for efficient uptake. Supplementation of the culture medium with malonate, a compound chemically known as disodium malonate, at varying ratios has been shown to successfully increase resveratrol and piceatannol content (Wu et al. 2013 ; Wu et al. 2017 ; Shrestha et al. 2018 ). Shrestha et al. ( 2018 ) confirmed that the addition of disodium malonate was assimilated readily in different strains, leading to an increase in resveratrol and piceatannol yield. Cerulenin, on the other hand, is a natural product derived from fungi. It functions as a potent inhibitor of fatty acid synthesis by repressing FabB and FabF genes, thereby blocking the elongation of fatty acid chains (Kallscheuer et al. 2016 ; Hu et al. 2024 ). Modulation of malonyl-CoA synthesis by cerulenin addition has emerged as a potential means of influencing stilbene production exclusively in microorganisms. Building on previous experience in studying the effects of metabolic modulators on microorganisms, we present a novel approach to modulate peanut ( Arachis hypogaea ) metabolism to significantly increase the production of important stilbenes, including resveratrol, piceatannol and piceid. This is the first attempt to manipulate these pathways into peanut cells. The study will evaluate the effects of three candidate pathway modulators: L-phenylalanine, sodium malonate dibasic and cerulenin. These modulators will be tested at different concentrations and time points in both peanut callus cultures and cell suspension cultures. Materials and methods Callus culture of peanut Callus tissue culture of A. hypogaea was maintained in solid Murashige and Skoog (MS) medium supplemented with 2.5 g L − 1 gelrite, 30 g L − 1 sucrose, 5.0 mg picloram, 0.1 g L − 1 myo-inositol and 1.0 g L − 1 casein hydrolysate, with a pH level of 5.8 (adjusted with KOH, prior to autoclaving at 121°C for 20 min) (Ghozlen et al. 2023 ). The cultures were incubated at 26°C in dark. The stock callus cultures were maintained under the same physical conditions as described above. Callus tissue pieces were subcultured at the 30-day intervals in 100 × 20 mm diameter Petri dishes containing 25 mL of solid medium. Fresh mass, morphology and growth index were recorded every 4 weeks for 4 cycles. Growth index (GI) = ((Final weight of callus - Initial weight of callus)/ Initial weight of callus) *100 Establishment of peanut cell suspension Cell suspensions were initiated by placing 3 g of friable calli in 250 ml flasks containing 40 ml of liquid MS medium supplemented with 5 mg/L picloram and 20 g/L sucrose, without gelling agent. A cell batch cultures for pathway regulation was carried out in the dark at 25°C with orbital shaking at 115 rpm. Metabolic modulators preparation L-phenylalanine (≥ 98%; Sigma-Aldrich), cerulenin (≥ 98.0%; Sigma-Aldrich) and sodium malonate dibasic (≥ 97.0% ; Sigma-Aldrich) were used for the stilbenes induction in callus cultures and cell suspensions. A stock solution was prepared for each modulator. Cerulenin was dissolved in DMSO at a concentration of 0.1mg/ml, while phenylalanine and sodium malonate dibasic were dissolved in ultrapure water only at a concentration of 10mg/ml. All the solutions were filter sterilized through a membrane filter of 0.22 µm pore size and incorporated into the MS medium at the desired concentrations. All modulators were added individually to the culture medium at the following final concentrations: Phenylalanine (Phe): 0.2 mM (P1), 1mM (P2) and 5mM (P3); Cerulenin (Cr): 0.2 µM (Cr1), 1 µM (Cr2) and 5 µM (Cr3); sodium malonate dibasic (M): 0.5 mM (M1), 2.5 mM (M2) and 12.5 mM (M3). Treatment of callus tissue In order to optimize stilbene production, two-week-old A. hypogaea callus cultures (10 g) were co-cultured with previously prepared stock solutions of modulators and maintained under the aforementioned culture conditions for varying durations: 2 h, 8 h, 16 h, 32 h, and 144 h. To remove excess moisture following exposure to metabolic modulators, callus from all treatments was transferred to filter paper and dried for 1 hour. Gentle pressing facilitated this process and the fresh weight (FW) of each callus sample was measured. The samples were lyophilized to remove all moisture and the dry weight (DW) was recorded. These freeze-dried callus samples were then used for subsequent stilbene quantification analysis. All experiments were performed in triplicate. Treatment of cell culture Three sets of batch cultures were initiated simultaneously and grown over a period of 28 days. Substrate addition occured at specific intervals based on the growth pattern of the cell suspension: on Day 0 to initiate the lag phase, on Day 10 during the early exponential phase, and on Day 20 at the stationary phase. Three replicates for each treatment were performed and the sampling was conducted at various intervals after modulator addition (2, 4, 8, 16, 32, 72, 96, and 144 h), with 3 mL taken from each flask. The collected suspensions were used to determine fresh cell mass, dry cell mass, and stilbene content. Staining cell viability The following protocol was used for assessment of cell viability using fluorescein diacetate (FDA). First, a 1:10 dilution of a 1 mg/mL FDA stock solution was made in phosphate-buffered saline (PBS) to give a 100 µg/mL FDA working solution (WS). The culture medium was then discarded from the cell cultures and the cell pellets were resuspended in 100 µL of the prepared WS sample. The cells were incubated with FDA for 15 minutes in the dark at room temperature. The supernatant was then carefully removed by centrifugation at 11,000 rpm for 3 minutes. The cells were then washed twice with PBS to remove any excess FDA. Finally, the cells were resuspended in a fresh aliquot of PBS for visualization by ultraviolet light with an inverted microscope Nikon ECLIPSE TE2000-S. Stilbene quantification by LC-MS analysis Standard solutions A fresh stock solution of 1 mg/mL resveratrol in methanol was prepared and linearity was demonstrated with the internal standard, resveratrol-(4-hydroxyphenyl- 13 C 6 ), used during the validation process. The calibration curve was assessed over a wide concentration range from 50 to 5000 ng/mL. The stock solution of the internal standard was prepared in methanol at a concentration of 1 mg/mL and stored at -20°C. A working standard solution (WS1) was prepared by dilution with methanol to a concentration of 13.3 ng/mL. Calli samples were spiked with 30 µL of WS1. Stock solutions of piceatannol and piceid were prepared in methanol at a concentration of 0.1 mg/mL. Sample preparation and extraction The lyophilized calli samples were ground to powder and stored at 4°C for further use. 0.5 g dry weight of the material from each treatment was extracted into 5 mL of absolute methanol. The solutions were gently stirred and heated in a water bath at 51°C for 57 min. Then 12 mL of methanol was added followed by ultrasonic extraction for 5 min (Xu et al. 2020 ). The crude extract was then centrifuged (Sigma 3-18ks) at 4000 rpm for 10 min at room temperature. 8 mL of the supernatant was collected and dried under vacuum at 55°C (BUCHI Rotavapor R-124). The dried samples were redissolved in 1 mL of methanol and vigorously vortexed. To analyse the extracellular production, 0.2 mL of culture medium was extracted with 0.8 mL of methanol spiked with 20 µl of WS1 (Komaikul et al. 2019 ). Following vigorous vortexing, samples were filtered through a 0.22 µm filter and analysed by LC-MS. Liquid chromatography-mass spectrometry The methanol extract was filtered through a 0.22 µm filter prior to analysis by liquid chromatography-mass spectrometry (LC-MS) (1260 Infinity II LC). A Kinetex C18 column 2.6µm 100 Å (150 mm x 4.60 mm; Phenomenex USA) was used at 35°C. The mobile phase consisted of (A) ultrapure water containing 0.05% acetic acid and (B) acetonitrile. The injection volume for all samples was 20 µl and the flow rate was 0.5 ml/min. The solvent gradient was as follows 0–2 min 95% solvent A- 5% solvent B; 2–14 min 100% solvent B; 14–16 min 95% solvent A- 5% solvent B. The MS operated in negative ion mode and was equipped with an electrospray ionization source. The setup parameters were as follows MS heater, 100°C for the single quadrupole analyzer; voltage, + 4000V, -3500V; curtain gas: hot nitrogen gas 350°C at 13L min − 1 at a flow rate of 1 ml min − 1 . The chromatogram was recorded at 306 nm. Resveratrol content was determined by comparing the retention time and m/z value with those of the standard under similar conditions and then quantified using a standard curve. All samples were run in triplicate. Statistical analysis Statistical analysis of the data obtained was performed using MINITAB statistical program version 18 (Minitab Inc.). The identification of differences between groups was performed using one-way ANOVA and Tukey's post hoc test for multiple comparisons with statistical significance at the 95% confidence level (p < 0.05). Results Analytical method performance assessment The LC-MS analysis successfully separated the main compounds of interest. The analysis identified resveratrol at a retention time of 10.27 minutes, piceatannol at 9.7 minutes, piceid at 9.2 minutes and the internal standard at 10.27 minutes. Proliferation of Arachis hypogaea calli All leaf explants had expanded and developed callus on their surface after 8 days of culture in the dark. All calli obtained were transferred to the same medium every 4 weeks and the callus growth index peaked during the second subculture, reaching an increase of 152% (Table 1 ). However, the growth index decreased in subsequent subcultures, resulting in friable, vigorously growing and homogeneous tissues (Fig. 1 ). Table 1 Effect of picloram on A. hypogaea callus fresh weight, growth index (GI) and morphology in proliferation medium for 3 subculture cycles Subculture cycle PGR Second Third Fourth Mass (g) GI (%) Texture Mass (g) GI (%) Texture Mass (g) GI (%) Texture 5mg Pic 8.91 ± 1.3 156.2 Friable, off-white 14.24 ± 2.4 66.3 Friable, off-white 24.33 ± 2.6 64.1 Friable, off-white Effect of metabolic modulators on the cell biomass in callus culture To assess the effects of the modulators, calli per treatment were transferred to culture medium containing different concentrations. Biomass accumulation as fresh and dry weight was monitored at multiple time points (2, 8, 16, 32 and 144 hours) as shown in Fig. 2 . Fresh weight (FW) patterns in treated calli were generally similar. M1 treatment yielded the highest FW, while untreated calli showed minimal changes with a slight decrease at 32 hours. Interestingly, unlike the other treatments, M1 and high Phe concentrations maintained their FW at 144 hours. Conversely, dry weight was significantly lower in all Cr-treated calli compared to the untreated control. Similar to FW, M1 treatment resulted in the most pronounced increase in dry weight biomass. Effect of metabolic modulators on the production of resveratrol, piceid and piceatannol in callus culture Our experiments across all treatment groups revealed significant variations in the concentration of the targeted stilbenes (resveratrol, piceatannol, and piceid). These variations depended on both the treatment concentration applied and the incubation time, as shown in Tables 2 – 4 . In the untreated control group, callus tissue maintained a constant level of resveratrol and piceatannol production until the 32 h mark, at which point a statistically significant increase was observed. However, piceid showed a distinct pattern. Its content peaked after a short 2 h incubation period, followed by a subsequent decline. These results highlight the influence of treatments on stilbene production dynamics in peanut callus. Effect of precursor feeding with L-phenylalanine Resveratrol content peaked after 32 h when callus cultures were treated with the lowest Phe concentration. This level was nearly three times higher compared to the control group. Interestingly, the highest Phe concentration induced an almost six-fold increase in resveratrol content compared to the control, but this peak occurred at an earlier time point 16 h (Table 2 ). This trend of early peak with higher Phe concentration was also observed for piceatannol content. Piceatannol levels peaked after 16 h when exposed to Phe3, exhibiting a six-fold increase compared to the control. In contrast to resveratrol and piceatannol, piceid showed a different pattern. Piceid levels consistently peaked after 8 h for all Phe concentrations tested. Effect of sodium malonate dibasic The addition of malonate at a low concentration resulted in higher resveratrol levels compared to the control after 16 h. A higher concentration of malonate led to an even earlier peak in resveratrol accumulation, after 8 h. The best concentration of malonate to increase resveratrol production was M3, which resulted in an accumulation of up to 19.71 µg/g DW. This represents an almost tenfold increase compared to the control group. Piceid production followed a similar trend, with an almost threefold increase compared to the control. Piceatannol, however, showed a different response. Its content peaked after 32 h, reaching a level of 7.44 µg/g DW. Effect of cerulenin As illustrated in Table 2 – 4 , results revealed distinct patterns of stilbene accumulation in response to cerulenin treatment. Resveratrol content peaked at 8 h after treatment with Cr1, reaching a level of 23.42 µg/g DW, followed by a decline. Similar trends were observed for its derivatives, piceatannol and piceid. Both exhibited increases after 8 h when treated with the same cerulenin concentration. Interestingly, piceid content consistently peaked at 8 h regardless of the cerulenin concentration used. In contrast, resveratrol continued to increase progressively until 16 h when exposed to Cr3. Piceatannol, however, differed the most, with the highest accumulation observed at just 2 h after treatment with Cr2 up to almost five times greater than the control. Table 2 Effect of metabolic modulators treatment, sodium malonate dibasic (M1: 0.5mM; M2: 2.5 mM; M3: 12.5 mM) phenylalanine (P1: 0.2 mM; P2: 1mM; P3: 5mM) and cerulenin (Cr1: 0.2 µM ; Cr2: 1µM; Cr3: 5µM) on resveratrol production in peanut callus culture (µg/g) at different incubation times (T1: 2h; T2: 8h; T3: 16h; T4: 32 h; T5: 144 h) Treatment T1 T2 T3 T4 T5 C 2.15 ± 0.22 a−d 2.20 ± 0.10 f 2.40 ± 0.21 d 6.62 ± 0.28 c 1.39 ± 0.12 a M1 1.98 ± 0.66 b−e 3.99 ± 0.91 ef 5.07 ± 0.82 cd 1.85 ± 0.47 d 1.18 ± 0.12 a M2 0.89 ± 0.11 e 12.93 ± 1.90 bc 11.68 ± 1.29 b 12.41 ± 1.24 b 0.93 ± 0.16 a M3 1.76 ± 0.08 cde 19.71 ± 1.97 a 5.61 ± 1.25 cd 13.07 ± 2.44 b 1.07 ± 0.20 a P1 1.23 ±0.15 de 14.44 ± 2.30 b 7.08 ± 0.91 c 17.16 ± 1.56 a 1.37 ± 0.30 a P2 1.46 ±0.22 de 3.54 ± 0.15 ef 4.33 ± 0.41 cd 3.73 ± 0.26 cd 1.34 ± 0.11 a P3 2.28 ± 0.22 a−d 10.49 ± 0.35 bcd 13.60 ± 1.26 b 2.68 ± 0.68 d 1.30 ± 0.21 a Cr1 2.97 ± 0.07 ab 23.42 ± 3.41 a 3.90 ± 0.78 cd 3.26 ± 0.47 d 1.28 ± 0.29 a Cr2 3.12 ± 0.76 a 7.61 ± 0.45 de 3.24 ± 0.24 cd 2.52 ± 0.25 d 0.89 ± 0.13 a Cr3 2.72 ±0.53 abc 9.02 ± 0.73 cd 21.72 ± 3.86 a 1.74 ± 0.44 d 1.44 ± 0.33 a Different letters in the same column indicate significantly different values per p < 0.05 between different treatments for the same incubation time. Table 3 Effect of metabolic modulators treatment (Trt), sodium malonate dibasic (M1: 0.5mM; M2: 2.5 mM; M3: 12.5 mM) phenylalanine (P1: 0.2 mM; P2: 1mM; P3: 5mM) and cerulenin (Cr1: 0.2 µM ; Cr2: 1µM; Cr3: 5µM) on piceid production in peanut callus culture (µg/g) at different incubation times (T1: 2h; T2: 8h; T3: 16h; T4: 32 h; T5: 144 h) Treatment T1 T2 T3 T4 T5 C 5.69 ± 0.61 abc 3.07 ± 0.13 d 2.83 ± 0.77 bc 2.58 ± 0.50 d 1.52 ± 0.16 cd M1 5.98 ± 0.66 abc 5.69 ± 0.28 c 2.16 ± 0.67 c 1.50 ± 0.37 e 1.06 ± 0.06 d M2 5.34 ± 0.74 abc 7.32 ± 0.23 abc 2.99 ± 0.77 bc 3.18 ± 0.21 cd 2.23 ± 0.16 a M3 5.88 ± 0.44 abc 8.58 ± 1.25 ab 2.87 ± 0.27 bc 2.93 ± 0.12 cd 2.10 ± 0.17 ab P1 4.47 ± 1.78 bc 6.26 ± 1.96 bc 3.17 ± 0.43 bc 0.76 ± 0.15 e 1.52 ± 0.06 cd P2 5.38 ± 0.19 abc 8.09 ± 1.07 abc 3.16 ± 0.08 bc 3.06 ± 0.15 cd 2.27 ± 0.16 a P3 3.97 ± 0.44 c 7.19 ± 0.46 abc 4.84 ± 0.42 a 3.72 ± 0.61 bc 1.67 ± 0.14 bc Cr1 5.87 ± 0.18 abc 6.85 ± 0.68 abc 4.37 ± 0.99 ab 4.99 ± 0.28 a 1.84 ± 0.13 abc Cr2 6.64 ± 1.05 ab 8.85 ± 0.25 a 3.34 ± 0.26 abc 3.45 ± 0.22 bcd 1.48 ± 0.35 cd Cr3 6.81 ± 0.57 a 7.10 ± 0.70 abc 3.07 ± 0.14 bc 4.30 ± 0.54 ab 1.50 ± 0.23 cd Different letters in the same column indicate significantly different values per p < 0.05 between different treatments for the same incubation time. Table 4 Effect of metabolic modulators treatment (Trt), sodium malonate dibasic (M1: 0.5mM; M2: 2.5 mM; M3: 12.5 mM) phenylalanine (P1: 0.2 mM; P2: 1mM; P3: 5mM) and cerulenin (Cr1: 0.2 µM ; Cr2: 1µM; Cr3: 5µM) on piceatannol production in peanut callus culture (µg/g) at different incubation times (T1: 2h; T2: 8h; T3: 16h; T4: 32 h; T5: 144 h) Treatment T1 T2 T3 T4 T5 C 0.97 ± 0.013 ef 0.96 ± 0.03 e 0.96 ± 0.07 g 3.10 ± 0.11 b 0.26 ± 0.05 c M1 1.29 ±0.24 def 1.80 ± 0.09 d 1.99 ± 0.04 cde 1.19 ± 0.19 de 0.28 ± 0.02 c M2 0.77 ±0.05 f 2.66 ± 0.16 bc 4.15 ± 0.05 b 1.80 ± 0.26 cd 0.47 ± 0.03 c M3 1.23 ±0.25 def 4.27 ± 0.19 a 1.26 ± 0.25 fg 7.45 ± 0.43 a 1.65 ± 0.29 a P1 1.39 ±0.25 de 2.92 ± 0.32 b 1.25 ± 0.11 fg 1.21 ± 0.23 de 0.28 ± 0.01 c P2 1.52 ±0.13 d 1.66 ± 0.29 de 2.35 ± 0.29 cd 1.34 ± 0.14 de 0.39 ± 0.03 c P3 3.21 ±0.16 b 2.64 ± 0.27 bc 6.66 ± 0.57 a 1.48 ± 0.26 cde 0.39 ± 0.03 c Cr1 3.16 ±0.31 b 4.29 ± 0.46 a 1.81 ± 0.12 def 2.02 ± 0.02 c 0.41 ± 0.01 c Cr2 4.76 ±0.01 a 1.95 ± 0.09 cd 1.62 ± 0.05 efg 1.31 ± 0.12 de 0.46 ± 0.02 c Cr3 2.22 ±0.12 c 3.05 ± 0.29 b 2.64 ± 0.13 c 1.11 ± 0.10 e 0.77 ± 0.02 b Different letters in the same column indicate significantly different values per p < 0.05 between different treatments for the same incubation time. Establishment of peanut cell suspension The peanut cell suspension cultures were established using calli derived from leaves. Viable and actively growing peanut cell suspension cultures were successfully obtained and cell viability was evaluated using the FDA staining method. The intensity of the fluorescence emitted by the stained cells reflects the proportion of living cells in the sample. Higher fluorescence intensity observed at day 20 (Fig. 3 ) indicates a greater number of viable cells. This information allows us to identify the optimal cell growth stage for further investigation into its correlation with stilbene content. This provides the basis for subsequent experiments aimed at optimizing stilbene production. Effect of metabolic modulators on the cell biomass in cell suspension The effect of the metabolic modulators on cell growth was assessed by measuring both cell fresh weight and dry weight. Figures 4 , 5 and 6 show that between day 0 and 6, covering the lag growth phase, all cell cultures exhibited similar growth patterns. Both untreated and treated cells maintained a constant weight until day 5, when the exponential growth phase began. During this subsequent phase, all groups continued to show exponential growth. Exceptionally, cells treated with different concentrations of phenylalanine showed a significant weight jump after T7 of addition (Fig. 5 ). Other treatments resulted in a less pronounced increase, but still significantly higher than the control group (Fig. S1 &S2). Transitioning into the stationary phase, untreated cells showed a steady increase in weight, reaching a plateau after T5 of sampling. Similar observations were made for cells treated with cerulenin and the phenylalanine group (except for Phe1). In these groups, biomass started to decline after 72 hours, indicating of cell death, with a more severe effect observed in the Cr3 group (Fig. 6). Conversely, cells treated with malonate and Phe1 showed a renewed increase in weight after reaching a plateau at 144 hours. Effect of metabolic modulators on the extracellular production of stilbenes in peanut cell suspension Effect on resveratrol production During the lag phase, resveratrol was the only stilbene detected in the culture medium. In untreated control cultures, extracellular resveratrol production remained stable until T6. At this time, a significant increase was observed. After the addition of metabolic modulators, resveratrol was found in the medium after only 2 h (T1). However, in some treatments the level decreased before increasing again. In all treatments, resveratrol levels peaked at 32 h. Among the modulators tested, M1 was the most effective and significant in inducing resveratrol production (Fig. S3), leading to a maximum concentration of 554.13 µg/L, which is five times higher compared to the control (Fig. 7 ). Similarly, P1 and Cr3 also induced a two-fold increase in resveratrol content, followed by a decrease. Interestingly, untreated peanut cell suspensions continued to produce resveratrol even after entering the exponential growth phase, following a different pattern as observed during the lag phase. The highest amount was detected after 4 h up to 118.34 µg/L. However, the addition of metabolic modulators on day 10 triggered a distinct response in extracellular resveratrol production. In most treatments, resveratrol showed a progressive rise in the culture medium starting from 2 hours. Notably, different concentrations of the three modulators tested resulted in varying optimal incubation times for resveratrol accumulation. Treatment with M3 yielded the highest resveratrol content at 32 h (T5), reaching 126.28 µg/L, similar to P1 and Cr3, which also peaked at 32 h. The pattern of resveratrol production shifted during the pre-stationary phase. Resveratrol became detectable only after 8 hours of sampling in both treated and untreated cell suspensions. Notably, all groups showed a similar trend, with resveratrol levels increasing until 32 hours, when they peaked before declining. As observed in previous phases, M3 and P1 remained the most effective modulators for enhancing resveratrol content to 206.69 µg/L and 203.59 µg/L, respectively. However, Cr2 was the most effective concentration within the cerulenin treatments. Effect on piceid production Piceid, unlike resveratrol, was detectable throughout the exponential and stationary phases (Fig. 8 ). Untreated cells consistently produced piceid during the exponential phase at all incubation times tested. Piceid content in untreated cultures showed a significant increase compared to treated suspensions at both 2 h and 18 h (Fig. S4). In contrast to resveratrol, piceid peaked earlier, reaching its highest level after only 2 h in most treatment groups. However, M3 and Cr2 treatments showed a delayed peak for piceid content, reaching their highest levels at 32 h. Interestingly, the addition of P3 significantly increased piceid content, reaching a maximum of 75.69 µg/L, almost three times higher than the control. In contrast to resveratrol, piceid production was more prominent during the stationary phase. Similar to the exponential phase, piceid accumulation in the medium started as early as 2 h of incubation. Remarkably, the graphs revealed an inverse relationship between resveratrol and piceid levels for some treatments. Cells treated with the most effective modulators of resveratrol production (M1, M3, and P1) showed a decrease in resveratrol but a subsequent increase in piceid content later at 72h. This trend was also observed with M2 and Cr3. Piceid in these groups peaked at 8 h (T3) and then decreased, coinciding with the peak of resveratrol. Treatments that induced the highest piceid levels at the same incubation time as resveratrol after 32 h showed a more sustained level of piceid compared to the quantified resveratrol levels. Effect on piceatannol production Piceatannol was only detected in the culture medium during the pre-stationary phase, appearing after 18 h of incubation as shown in Fig. 9 . Untreated control cells initiated piceatannol production after 18 h, with statistically significant levels compared to treated cells, except for M2 and M3 (Fig. S5). This production continued to increase until 72 h, reaching a significant peak of 88.92 µg/L. Similar to resveratrol, M3 treatment demonstrated the most pronounced effect on piceatannol content, reaching a maximum after 32 h. As shown in the figure, both untreated and M3-treated cells produced piceatannol after 96 h of incubation. For Phe treatments, piceatannol content peaked after 32 h but decreased subsequently by 72 h. Interestingly, P2 and P3 treatments resulted in undetectable levels of piceatannol beyond 72 h. Finally, piceatannol was only observed in Cr-treated cultures after 32 h. Discussion Effect of phenylalanine feeding on resveratrol, piceatannol and piceid contents in peanut cells Currently, a promising approach to resveratrol production involves biotechnological techniques that use building blocks to enhance its natural biosynthesis in peanuts. The biosynthesis of stilbenes starts with two aromatic amino acids, phenylalanine and tyrosine, which are mainly obtained via the shikimate pathway, although the malonate pathway may play an indirect role by supplying precursor molecules. Molecular engineering of the resveratrol pathway was reported in plants in the 1990s for disease resistance and functional foods (Jeandet et al. 2018 ). Precursor feeding is a powerful technique to stimulate plant secondary metabolite production. Supplying phenylalanine is likely to increase the metabolic flux towards the desired target, leading to enhanced resveratrol production in peanut sprouts or hairy roots (Yu et al. 2016 ; Tothong et al. 2023 ). Phe may act as an early metabolic regulator in plant cells. Phe supplementation could influence the biosynthesis of enzymes involved in the pathway leading to stilbenes and possibly regulate signalling pathways associated with plant defence response (Tothong et al. 2023 ). Researchers further investigated the effects of supplementing phenylalanine or tyrosine in Morus alba root culture and Vitis vinifera cell suspension to increase resveratrol and piceid production (Andi et al. 2019 ; Inyai et al. 2020). Kumari et al. ( 2023 ) reported that varying Phe concentrations can boost phenylpropanoid production in sterile cell cultures. Grapes cells can efficiently take up and metabolize large amounts of externally added Phe, converting it into stilbenes, both within the cell and excreted into the medium, enabling high overall production. These findings support our results showing that phenylalanine supplementation successfully increased the production of resveratrol, piceid and piceatannol in both callus and cell suspension cultures. Phenylalanine ammonia lyase (PAL) could be a major regulatory enzyme in response to supplemental Phe in the stilbene biosynthesis pathway. In peanut hairy root cultures, the identified PAL gene family (including PAL, PAL2, MYB36, ERF3, PR2, and STH2) showed significantly higher expression levels at 24 hours after elicitation with 5 mM Phe compared to cultures without Phe supplementation. Tothong et al. ( 2023 ) suggested that a combined approach of Phe feeding and elicitor treatment might accelerate the plant's defense response by rapidly inducing early response genes, potentially leading to a faster stilbene biosynthesis response. Effect of sodium malonate dibasic on resveratrol, piceatannol and piceid contents in peanut cells Phenylalanine feeding remains a popular strategy to boost resveratrol production through the well-defined phenylpropanoid pathway in plants. However, exploring alternative or complementary pathways, similar to successful approaches in microorganisms, holds promise for further optimizing resveratrol production. An exceptional case of malonate's ability to enhance resveratrol production comes from Langcake et al. (1977), who reported the only documented instance of its successful use in grapevine berries and leaves. Tyrosine incorporation was minimal in all experiments. Conversely, phenylalanine and acetate acted as effective precursors, while malonate incorporation showed exceptionally high levels. Our observations agree with previous findings where malonate feeding during the lag phase of peanut cell suspension cultures resulted in a 2-fold increase in resveratrol production compared to cells fed with phenylalanine. Interestingly, malonate also enhanced the production of piceatannol and piceid during the stationary phase, with a more pronounced effect than phenylalanine. The observed stimulation of stilbene production by malonate addition suggests the cultures' ability to effectively take up malonate and convert it into its active form, malonyl-CoA. The increased resveratrol observed in peanut cells might be due to the combined effect of overexpressing matB and matC genes along with malonate supplementation as suggested by Thapa et al. ( 2019 ). This combined approach likely leads to higher intracellular levels of malonyl-CoA, the essential substrate used by STS enzymes for stilbene biosynthesis. Similarly, in Rhamnus purshiana cell cultures, 12.5 mM malonate significantly increased anthra-derivative production by a factor of 2.7, whereas the same concentration of acetate had no effect (Van den berg et al. 1988 ). Effect of cerulenin on resveratrol, piceatannol and piceid contents in peanut cells Alternatively, other strategies involve the use of the antibiotic cerulenin, which binds to the β-ketoacyl carrier protein (ACP) synthase of the fatty acid synthesis machinery, thereby inhibiting its enzymatic activity. For instance, Liu et al. (2022) reported this approach by adding cerulenin to boost intracellular levels of malonyl-CoA, with in vivo evolution of Pinus strobus stilbene synthase for increased pinosylvin biosynthetic activity in engineered E. coli . Interestingly, application of cerulenin at 200 µM increased the intracellular malonyl-CoA pool by a factor of 50. Similar results were observed using a genetically engineered E. coli strain, resulting in a significant increase in resveratrol production (from 1.3 g/L to 2.3 g/L). However, it's important to note that this success is not universal (Jeantdet et al. 2021). Low concentrations of cerulenin (up to 5 µM) effectively increased resveratrol and piceid content in peanut cells suspension. In contrast to our findings, Kallscheuer et al. ( 2016 ) found that higher cerulenin concentrations (5, 10, and 25 mM) significantly increased resveratrol production. However, further escalation (50 to 150 mM) did not lead to comparable gains in resveratrol levels, suggesting a possible threshold effect. Our results on the successful increase of resveratrol derivatives like piceid and piceatannol in the static peanut cell culture align well with reports of similar effects in Corynebacterium glutamicum cultures, where cerulenin addition also promoted piceatannol formation (Thapa et al. 2019 ). This achievement may be linked to a creation of a roadblock in fatty acid synthesis by adding cerulenin. By binding to the active site of β-Ketoacyl-acyl carrier protein synthase (KAS), a key enzyme, cerulenin inhibits its function and effectively stalls the entire pathway at that step. This disruption triggers feedback mechanisms within the cell, which might not sense a need for further fatty acids production. Signaling cascades can be initiated, ultimately leading to a decrease in the activity and changes in the overall expression levels of some fatty acid synthesis genes, fab operon (fabD, fabB, fab F, fabH, fabI). With fatty acid synthesis partially blocked, cells can potentially redirect resources towards other pathways where a crucial precursor molecule, malonyl-CoA, might be accumulated. Effect of the cell density on resveratrol, piceatannol and piceid contents While limited research has documented resveratrol production in peanut cell suspensions, this study successfully demonstrates its induction alongside piceatannol and piceid. Notably, the production levels and their proportions differed significantly compared to callus static cultures, and also varied across different growth stages within the cell suspension. Eliciting grapevine cell cultures with specific compounds like methyl jasmonate (MeJA) and cyclodextrins can advantageously trigger the production and secretion of trans -resveratrol and piceid extracellularly (Lambert et al. 2019 ; Jeong et al. 2020 ; Vera-Urbina et al. 2023 ). This approach makes grapevine cells promising factories for resveratrol production, as these compounds accumulate in the culture medium, facilitating their extraction. Lambert et al. ( 2019 ) found a positive correlation between resveratrol production and biomass concentration. Their study suggests that the exponential growth phase might be the optimal time for elicitation, as cultures with the highest biomass yielded the highest resveratrol content. A critical, yet often overlooked factor in optimizing stilbene production through cell cultures is the timing of elicitor application. The physiological state of the cells, influenced by their growth phase (lag, exponential, and stationary), is worthy of consideration. Studies generally suggest that adding elicitors during the mid-to-late exponential growth phase yields the best response in terms of secondary metabolite production, compared to earlier or later stages (Vera-Urbina et al. 2023 ). Interestingly, our results in peanut cell cultures differ from some previous observations. While our study showed higher resveratrol production at lower cell densities, Belchi-Navarro et al. (2012) observed similar trends in Monastrell grapevine cell cultures treated with elicitors. In their study, extracellular resveratrol production also peaked at lower initial cell densities. The variations observed in resveratrol production depending on the cell density in peanut cell cultures might be due to how cell density influences the response to the added compounds. In cultures with lower cell density, each cell could be more receptive to the effects of phenylalanine, cerulenin or malonate added. A proportionally larger share of the metabolic machinery might be available for stilbene biosynthesis compared to cultures with higher cell densities, leading to a more efficient utilization of the added compounds by individual cells. This explanation is consistent with our findings of higher resveratrol levels at lower cell densities. However, the response may be different for other stilbenes. The production of piceid and piceatannol, which we observed to be higher in later growth phases, may not be as directly influenced by the initial availability of added modulators. Other factors within the specific pathways for piceid and piceatannol may play a more important role in regulating their production at different growth stages, as their specific biosynthetic steps may be less dependent on the initial boost in the phenylalanine or malonyl-Coa pool. Piceid and piceatannol production differed from that of resveratrol. Their levels peaked when modulators were added in the late exponential phase, consistent with observations by Vera-Urbina et al. ( 2023 ). This suggests that the regulation of piceid and piceatannol biosynthesis may be mediated by higher cell density. Higher cell density might play a role in regulating piceid and piceatannol production. Increased biomass could potentially lead to a shift in cellular metabolism towards the production of a wider range of stilbenes, not just resveratrol. Conclusion In conclusion, the aim of this study was to increase knowledge of the bioproduction of resveratrol and its derivatives in peanut cells through biotechnological approaches. We have successfully established a method for inducing the production of resveratrol, piceatannol, and piceid in peanut cell suspension cultures. The levels and proportions of these stilbenes differed significantly from those observed in peanut callus cultures and varied across the cell suspension growth stages. Our findings highlight the potential of feeding and metabolic modulation on producing these valuable stilbenes in peanut cell cultures. By providing the basis for a sustainable and efficient method, this research sets the stage for wider availability of these valuable compounds. Moreover, the scale-up of culture volume without loss in growth open exciting avenues for further research. Declarations Author contribution statement Hajer Ben Ghozlen conceived, performed the experiments and drafted the manuscript. Sven Mangelinckx participated in data analysis. Stefaan Werbrouck and Sven Mangelinckx revised and supervised the manuscript. All authors contributed to the article and approved the version submitted. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Declaration of Competing Interest The authors have no relevant financial or non-financial interests to disclose. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-6375460\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":441523652,\"identity\":\"a7e9f173-5eb2-42b9-9d79-940debafaa01\",\"order_by\":0,\"name\":\"Hajer Ben Ghozlen\",\"email\":\"data:image/png;base64,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\",\"orcid\":\"https://orcid.org/0000-0001-7037-2684\",\"institution\":\"Ghent University: Universiteit Gent\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Hajer\",\"middleName\":\"Ben\",\"lastName\":\"Ghozlen\",\"suffix\":\"\"},{\"id\":441523653,\"identity\":\"8390589f-7234-4958-a236-3e06aa6887c6\",\"order_by\":1,\"name\":\"Sven Mangelinkcx\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ghent University: Universiteit Gent\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Sven\",\"middleName\":\"\",\"lastName\":\"Mangelinkcx\",\"suffix\":\"\"},{\"id\":441523654,\"identity\":\"ba490035-61d0-4827-bc59-cdcd45e439be\",\"order_by\":2,\"name\":\"Stefaan P.O. Werbrouck\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Ghent University: Universiteit Gent\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Stefaan\",\"middleName\":\"P.O.\",\"lastName\":\"Werbrouck\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-04-04 10:48:15\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6375460/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6375460/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":80763396,\"identity\":\"14f7d18f-ce5a-4a5e-a913-366d56fcdd81\",\"added_by\":\"auto\",\"created_at\":\"2025-04-16 20:27:36\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":29587,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eProliferated peanut calli on MS medium with 5 mg/L picloram. Scale bar in = 1cm\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6375460/v1/d4091b9ef31c4202debcbd91.jpg\"},{\"id\":80763900,\"identity\":\"797b5b22-721d-4fd9-95ae-cbd9cea641f1\",\"added_by\":\"auto\",\"created_at\":\"2025-04-16 20:43:36\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":57883,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTreatment\\u003cem\\u003e \\u003c/em\\u003eeffect on peanut callus biomass\\u003cem\\u003e (f\\u003c/em\\u003eresh weight\\u003cem\\u003e and\\u003c/em\\u003e \\u003cem\\u003ed\\u003c/em\\u003ery weight\\u003cem\\u003e)\\u003c/em\\u003eat different concentrations\\u003cem\\u003e: \\u003c/em\\u003econtrol (C\\u003cem\\u003e), \\u003c/em\\u003eSodium malonate dibasic \\u003cem\\u003e(\\u003c/em\\u003eM1: 0.5mM; M2: 2.5 mM; M3: 12.5 mM)\\u003cem\\u003e, \\u003c/em\\u003ePhenylalanine feeding\\u003cem\\u003e (\\u003c/em\\u003eP1: 0.2mM; P2: 1 mM; P3: 5 mM)\\u003cem\\u003e,\\u003c/em\\u003e Cerulenin\\u003cem\\u003e (\\u003c/em\\u003eCr1: 0.2 µM; Cr: 1 µM; Cr3: 5 µM)\\u003cem\\u003e, \\u003c/em\\u003efor multiple incubation times (T1: 2 h; T2: 8 h; T3: 16 h; T4: 32h; T5: 144 h). Values within a histogram baseline followed by the same letter do not differ significantly for all treatment at the same time (p \\u0026lt; 0.05), while values within a row above the line followed by the same letter do not differ significantly for all treatment at the same time (p \\u0026lt; 0.05)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6375460/v1/676eb38ec2f072fd3b192210.jpg\"},{\"id\":80763589,\"identity\":\"73aebcfa-272b-49ea-b12b-7431b623df62\",\"added_by\":\"auto\",\"created_at\":\"2025-04-16 20:35:36\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":44332,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePeanut cell suspension growth stages at A-B) 10 days and C-D) 20 days, with corresponding FDA vital staining to assess cell viability\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6375460/v1/82cb231c16ebb50d610b3166.jpg\"},{\"id\":80763398,\"identity\":\"bdb0dd3f-a1a7-4bb7-b637-8a55440543c1\",\"added_by\":\"auto\",\"created_at\":\"2025-04-16 20:27:36\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":82255,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSodium malonate dibasic effect on peanut suspension growth curve during 3 different phases separately \\u003cstrong\\u003eA.\\u003c/strong\\u003e Fresh weight \\u003cstrong\\u003eB.\\u003c/strong\\u003eDry weight at different concentrations (C: 0; M1: 0.5 mM; M2: 2.5 mM; M3: 12.5 mM) for different incubation times (T1: 2 h; T2: 4 h; T3: 8 h; T4: 18h; T5: 32h; T6: 72h; T7: 96h; T8: 144 h).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6375460/v1/0f835e98a9f7a7ef2ba4277e.jpg\"},{\"id\":80763591,\"identity\":\"1eb960c9-1228-4f8c-ad08-a9feaec8b96a\",\"added_by\":\"auto\",\"created_at\":\"2025-04-16 20:35:36\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":84347,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePhenylalanine feeding effect on peanut suspension growth curve during 3 different phases separately\\u003cstrong\\u003e A.\\u003c/strong\\u003e Fresh weight \\u003cstrong\\u003eB.\\u003c/strong\\u003eDry weight at different concentrations (C: 0; P1: 0.2 mM; M2: 1 mM; M3: 5 mM) for multiple incubation times (T1: 2 h; T2: 4 h; T3: 8 h; T4: 18h; T5: 32h; T6: 72h; T7: 96h; T8: 144 h).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6375460/v1/8792e02dcf13033cf4e944b8.jpg\"},{\"id\":80763400,\"identity\":\"a84f3fb4-2e83-4c15-bdee-1caf712bc40c\",\"added_by\":\"auto\",\"created_at\":\"2025-04-16 20:27:36\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":81508,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCerulenin effect on peanut suspension growth curve during 3 different phases separately\\u003cstrong\\u003e A.\\u003c/strong\\u003e Fresh weight \\u003cstrong\\u003eB.\\u003c/strong\\u003e Dry weight at different concentrations (C: 0; Cr1: 0.2 µM; Cr2: 1 µM; Cr3: 5 µM) for multiple incubation times (T1: 2 h; T2: 4 h; T3: 8 h; T4: 18h; T5: 32h; T6: 72h; T7: 96h; T8: 144 h).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6375460/v1/ef336016fb1a21cd6709e224.jpg\"},{\"id\":80763593,\"identity\":\"0928777f-af6f-4f05-a8f7-d95d3fe5413e\",\"added_by\":\"auto\",\"created_at\":\"2025-04-16 20:35:36\",\"extension\":\"jpg\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":255974,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eTrans\\u003c/em\\u003e-resveratrol content in response to modulators treatments in peanut cell suspension during different growth phases (Phase 1 (Lag phase); Phase 2 (Exponential phase); Phase 3 (Stationary phase)) for various incubation times (T1: 2 h; T2: 4 h; T3: 8 h; T4: 18 h; T5: 32 h; T6: 72 h; T7: 96 h; T8: 144 h). No addition: control;\\u003cstrong\\u003e \\u003c/strong\\u003eSodium malonate dibasic treatment (M1: 0.5mM; M2: 2.5 mM ; M3: 12.5 mM);\\u003cstrong\\u003e \\u003c/strong\\u003eL-phenylalanine treatment\\u003cstrong\\u003e \\u003c/strong\\u003e(P1: 0.2 mM ; P2: 1mM; P3: 5mM ); Cerulenin treatment (Cr1: 0.2 µM ; Cr2: 1µM ; Cr3: 5µM)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6375460/v1/861829de632db879bc799f0c.jpg\"},{\"id\":80763409,\"identity\":\"653471c2-8b57-4689-ad55-2d493bc2812f\",\"added_by\":\"auto\",\"created_at\":\"2025-04-16 20:27:36\",\"extension\":\"jpg\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":234881,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePiceid content in response to modulators treatments in peanut cell suspension during different growth phases (Phase 1 (Lag phase); Phase 2 (Exponential phase); Phase 3 (Stationary phase)) for various incubation times (T1: 2 h; T2: 4 h; T3: 8 h; T4: 18 h; T5: 32 h; T6: 72 h; T7: 96 h; T8: 144 h). No addition: control;\\u003cstrong\\u003e \\u003c/strong\\u003eSodium malonate dibasic treatment (M1: 0.5mM; M2: 2.5 mM ; M3: 12.5 mM);\\u003cstrong\\u003e \\u003c/strong\\u003eL-phenylalanine treatment\\u003cstrong\\u003e \\u003c/strong\\u003e(P1: 0.2 mM ; P2: 1mM; P3: 5mM ); Cerulenin treatment (Cr1: 0.2 µM ; Cr2: 1µM ; Cr3: 5µM)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"8.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6375460/v1/758fbb5e49c30bf4c9b73f35.jpg\"},{\"id\":80763904,\"identity\":\"58e52f5f-4772-41d0-89ad-5a3fdad102fe\",\"added_by\":\"auto\",\"created_at\":\"2025-04-16 20:43:36\",\"extension\":\"jpg\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":240665,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePiceatannol content in response to modulators treatments in peanut cell suspension during different growth phases (Phase 1 (Lag phase); Phase 2 (Exponential phase); Phase 3 (Stationary phase)) for various incubation times (T1: 2 h ; T2: 4 h ; T3: 8 h; T4: 18 h; T5: 32 h; T6: 72 h; T7: 96 h; T8: 144 h). No addition: control;\\u003cstrong\\u003e \\u003c/strong\\u003eSodium malonate dibasic treatment (M1: 0.5mM; M2: 2.5 mM ; M3: 12.5 mM);\\u003cstrong\\u003e \\u003c/strong\\u003eL-phenylalanine treatment\\u003cstrong\\u003e \\u003c/strong\\u003e(P1: 0.2 mM ; P2: 1mM; P3: 5mM ); Cerulenin treatment (Cr1: 0.2 µM ; Cr2: 1µM ; Cr3: 5µM)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"9.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6375460/v1/90c0ddb688d4e36c8281aa64.jpg\"},{\"id\":83948083,\"identity\":\"6916fcc0-6a63-40a2-aab0-3bc0f85d0cd8\",\"added_by\":\"auto\",\"created_at\":\"2025-06-04 22:37:37\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2474128,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6375460/v1/98a199b4-f7d6-4b08-bf52-e77602e3afe9.pdf\"},{\"id\":80763597,\"identity\":\"4bd884b8-d9dc-4a6d-bd40-e954d716baa2\",\"added_by\":\"auto\",\"created_at\":\"2025-04-16 20:35:36\",\"extension\":\"docx\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1237290,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplemantaryfile.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6375460/v1/e4fda7c1783baf15501d2424.docx\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Modulating stilbenes in peanut cells: A novel approach with metabolic modulators\",\"fulltext\":[{\"header\":\"Key Message\",\"content\":\"\\u003cp\\u003ePeanut cell cultures are a sustainable platform for the production of resveratrol, piceatannol and piceid, enhanced by metabolic modulation, offering a promising method for wider availability of these valuable compounds.\\u003c/p\\u003e\"},{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eBiotechnological production of specialized metabolites using \\u003cem\\u003ein vitro\\u003c/em\\u003e plant cultures, including culturing of plant tissues, organs, or cells, has the potential to overcome the limitations and overexploitation associated with traditional plant-based methods (Rattan et al. \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Advances in plant cell culture methods, particularly callus and suspension cultures, present promising avenues for the large-scale production of stilbenes (Jeandet et al. \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). It is important to note that while recent studies have shown successful production of resveratrol and various derivatives in a few plants like \\u003cem\\u003eVitis vinifera\\u003c/em\\u003e and \\u003cem\\u003eMorus alba\\u003c/em\\u003e, such production has been rare in peanuts. By supplementing specific precursors and manipulating metabolic pathways, these strategies may offer promising solutions to bioproduction bottlenecks. Given the key role of phenylalanine as a direct precursor in the stilbene biosynthesis pathway, supplementing cultures with phenylalanine will lead to maximize the utilization of intracellular phenylalanine pools and potentially enhance stilbene production. Therefore, increasing the resveratrol content in peanut by adding phenylalanine could have important effects, as shown in sprouts (Yu et al. \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e) and hairy roots culture (Tothong et al. \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eMetabolic pathways modulation within cells can be strategically enhanced by using compounds like malonate and cerulenin. These known modulators of plant metabolism offer valuable tools for researchers aiming to redirect cellular pathways towards the stilbene production (Shrestha et al. \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Malonyl-CoA is a metabolic node at the intersection of both the flavonoid and the stilbenoid pathways as well as of the fatty acid biosynthesis. Studies have shown that increasing the intracellular pool of malonyl-CoA is a key strategy to overproduce stilbenes in engineered microorganisms by redirecting the carbon flux into this compound (Jeandet et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eTwo main approaches, the regulation of the upstream malonyl-CoA biosynthetic pathway and the control of the downstream malonyl-CoA utilization pathway, are often used to achieve this redirection. Malonyl-CoA is produced mainly from acetyl-CoA by the irreversible action of the acetyl-CoA carboxylase (ACC) complex. Thus, overexpression of the malonate assimilation pathway (malonyl-CoA synthetase [MatB] and malonate carrier protein [MatC]) or inhibition of fatty acid synthesis increases malonyl-CoA concentration (Hu et al. \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Researchers have looked at supplementing the culture medium directly with malonate (malonyl-CoA's precursor). However, this feeding approach often requires genetic modification of the microorganisms for efficient uptake. Supplementation of the culture medium with malonate, a compound chemically known as disodium malonate, at varying ratios has been shown to successfully increase resveratrol and piceatannol content (Wu et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e; Wu et al. \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Shrestha et al. \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Shrestha et al. (\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e) confirmed that the addition of disodium malonate was assimilated readily in different strains, leading to an increase in resveratrol and piceatannol yield. Cerulenin, on the other hand, is a natural product derived from fungi. It functions as a potent inhibitor of fatty acid synthesis by repressing FabB and FabF genes, thereby blocking the elongation of fatty acid chains (Kallscheuer et al. \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Hu et al. \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Modulation of malonyl-CoA synthesis by cerulenin addition has emerged as a potential means of influencing stilbene production exclusively in microorganisms.\\u003c/p\\u003e \\u003cp\\u003eBuilding on previous experience in studying the effects of metabolic modulators on microorganisms, we present a novel approach to modulate peanut (\\u003cem\\u003eArachis hypogaea\\u003c/em\\u003e) metabolism to significantly increase the production of important stilbenes, including resveratrol, piceatannol and piceid. This is the first attempt to manipulate these pathways into peanut cells. The study will evaluate the effects of three candidate pathway modulators: L-phenylalanine, sodium malonate dibasic and cerulenin. These modulators will be tested at different concentrations and time points in both peanut callus cultures and cell suspension cultures.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCallus culture of peanut\\u003c/h2\\u003e \\u003cp\\u003eCallus tissue culture of \\u003cem\\u003eA. hypogaea\\u003c/em\\u003e was maintained in solid Murashige and Skoog (MS) medium supplemented with 2.5 g L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e gelrite, 30 g L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e sucrose, 5.0 mg picloram, 0.1 g L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e myo-inositol and 1.0 g L\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e casein hydrolysate, with a pH level of 5.8 (adjusted with KOH, prior to autoclaving at 121\\u0026deg;C for 20 min) (Ghozlen et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). The cultures were incubated at 26\\u0026deg;C in dark. The stock callus cultures were maintained under the same physical conditions as described above. Callus tissue pieces were subcultured at the 30-day intervals in 100 \\u0026times; 20 mm diameter Petri dishes containing 25 mL of solid medium. Fresh mass, morphology and growth index were recorded every 4 weeks for 4 cycles.\\u003c/p\\u003e \\u003cp\\u003eGrowth index (GI) = ((Final weight of callus - Initial weight of callus)/ Initial weight of callus) *100\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eEstablishment of peanut cell suspension\\u003c/h3\\u003e\\n\\u003cp\\u003eCell suspensions were initiated by placing 3 g of friable calli in 250 ml flasks containing 40 ml of liquid MS medium supplemented with 5 mg/L picloram and 20 g/L sucrose, without gelling agent. A cell batch cultures for pathway regulation was carried out in the dark at 25\\u0026deg;C with orbital shaking at 115 rpm.\\u003c/p\\u003e\\n\\u003ch3\\u003eMetabolic modulators preparation\\u003c/h3\\u003e\\n\\u003cp\\u003eL-phenylalanine (\\u0026ge;\\u0026thinsp;98%; Sigma-Aldrich), cerulenin (\\u0026ge;\\u0026thinsp;98.0%; Sigma-Aldrich) and sodium malonate dibasic (\\u0026ge;\\u0026thinsp;97.0% ; Sigma-Aldrich) were used for the stilbenes induction in callus cultures and cell suspensions. A stock solution was prepared for each modulator. Cerulenin was dissolved in DMSO at a concentration of 0.1mg/ml, while phenylalanine and sodium malonate dibasic were dissolved in ultrapure water only at a concentration of 10mg/ml. All the solutions were filter sterilized through a membrane filter of 0.22 \\u0026micro;m pore size and incorporated into the MS medium at the desired concentrations.\\u003c/p\\u003e \\u003cp\\u003eAll modulators were added individually to the culture medium at the following final concentrations: Phenylalanine (Phe): 0.2 mM (P1), 1mM (P2) and 5mM (P3); Cerulenin (Cr): 0.2 \\u0026micro;M (Cr1), 1 \\u0026micro;M (Cr2) and 5 \\u0026micro;M (Cr3); sodium malonate dibasic (M): 0.5 mM (M1), 2.5 mM (M2) and 12.5 mM (M3).\\u003c/p\\u003e\\n\\u003ch3\\u003eTreatment of callus tissue\\u003c/h3\\u003e\\n\\u003cp\\u003eIn order to optimize stilbene production, two-week-old \\u003cem\\u003eA. hypogaea\\u003c/em\\u003e callus cultures (10 g) were co-cultured with previously prepared stock solutions of modulators and maintained under the aforementioned culture conditions for varying durations: 2 h, 8 h, 16 h, 32 h, and 144 h. To remove excess moisture following exposure to metabolic modulators, callus from all treatments was transferred to filter paper and dried for 1 hour. Gentle pressing facilitated this process and the fresh weight (FW) of each callus sample was measured. The samples were lyophilized to remove all moisture and the dry weight (DW) was recorded. These freeze-dried callus samples were then used for subsequent stilbene quantification analysis. All experiments were performed in triplicate.\\u003c/p\\u003e\\n\\u003ch3\\u003eTreatment of cell culture\\u003c/h3\\u003e\\n\\u003cp\\u003eThree sets of batch cultures were initiated simultaneously and grown over a period of 28 days. Substrate addition occured at specific intervals based on the growth pattern of the cell suspension: on Day 0 to initiate the lag phase, on Day 10 during the early exponential phase, and on Day 20 at the stationary phase. Three replicates for each treatment were performed and the sampling was conducted at various intervals after modulator addition (2, 4, 8, 16, 32, 72, 96, and 144 h), with 3 mL taken from each flask. The collected suspensions were used to determine fresh cell mass, dry cell mass, and stilbene content.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStaining cell viability\\u003c/h2\\u003e \\u003cp\\u003eThe following protocol was used for assessment of cell viability using fluorescein diacetate (FDA). First, a 1:10 dilution of a 1 mg/mL FDA stock solution was made in phosphate-buffered saline (PBS) to give a 100 \\u0026micro;g/mL FDA working solution (WS). The culture medium was then discarded from the cell cultures and the cell pellets were resuspended in 100 \\u0026micro;L of the prepared WS sample. The cells were incubated with FDA for 15 minutes in the dark at room temperature. The supernatant was then carefully removed by centrifugation at 11,000 rpm for 3 minutes. The cells were then washed twice with PBS to remove any excess FDA. Finally, the cells were resuspended in a fresh aliquot of PBS for visualization by ultraviolet light with an inverted microscope Nikon ECLIPSE TE2000-S.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eStilbene quantification by LC-MS analysis\\u003c/h3\\u003e\\n\\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStandard solutions\\u003c/h2\\u003e \\u003cp\\u003eA fresh stock solution of 1 mg/mL resveratrol in methanol was prepared and linearity was demonstrated with the internal standard, resveratrol-(4-hydroxyphenyl-\\u003csup\\u003e13\\u003c/sup\\u003eC\\u003csub\\u003e6\\u003c/sub\\u003e), used during the validation process. The calibration curve was assessed over a wide concentration range from 50 to 5000 ng/mL.\\u003c/p\\u003e \\u003cp\\u003eThe stock solution of the internal standard was prepared in methanol at a concentration of 1 mg/mL and stored at -20\\u0026deg;C. A working standard solution (WS1) was prepared by dilution with methanol to a concentration of 13.3 ng/mL. Calli samples were spiked with 30 \\u0026micro;L of WS1. Stock solutions of piceatannol and piceid were prepared in methanol at a concentration of 0.1 mg/mL.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSample preparation and extraction\\u003c/h2\\u003e \\u003cp\\u003eThe lyophilized calli samples were ground to powder and stored at 4\\u0026deg;C for further use. 0.5 g dry weight of the material from each treatment was extracted into 5 mL of absolute methanol. The solutions were gently stirred and heated in a water bath at 51\\u0026deg;C for 57 min. Then 12 mL of methanol was added followed by ultrasonic extraction for 5 min (Xu et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). The crude extract was then centrifuged (Sigma 3-18ks) at 4000 rpm for 10 min at room temperature. 8 mL of the supernatant was collected and dried under vacuum at 55\\u0026deg;C (BUCHI Rotavapor R-124). The dried samples were redissolved in 1 mL of methanol and vigorously vortexed.\\u003c/p\\u003e \\u003cp\\u003eTo analyse the extracellular production, 0.2 mL of culture medium was extracted with 0.8 mL of methanol spiked with 20 \\u0026micro;l of WS1 (Komaikul et al. \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Following vigorous vortexing, samples were filtered through a 0.22 \\u0026micro;m filter and analysed by LC-MS.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eLiquid chromatography-mass spectrometry\\u003c/h2\\u003e \\u003cp\\u003eThe methanol extract was filtered through a 0.22 \\u0026micro;m filter prior to analysis by liquid chromatography-mass spectrometry (LC-MS) (1260 Infinity II LC). A Kinetex C18 column 2.6\\u0026micro;m 100 \\u0026Aring; (150 mm x 4.60 mm; Phenomenex USA) was used at 35\\u0026deg;C. The mobile phase consisted of (A) ultrapure water containing 0.05% acetic acid and (B) acetonitrile. The injection volume for all samples was 20 \\u0026micro;l and the flow rate was 0.5 ml/min. The solvent gradient was as follows 0\\u0026ndash;2 min 95% solvent A- 5% solvent B; 2\\u0026ndash;14 min 100% solvent B; 14\\u0026ndash;16 min 95% solvent A- 5% solvent B. The MS operated in negative ion mode and was equipped with an electrospray ionization source. The setup parameters were as follows MS heater, 100\\u0026deg;C for the single quadrupole analyzer; voltage, +\\u0026thinsp;4000V, -3500V; curtain gas: hot nitrogen gas 350\\u0026deg;C at 13L min\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e at a flow rate of 1 ml min\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. The chromatogram was recorded at 306 nm. Resveratrol content was determined by comparing the retention time and m/z value with those of the standard under similar conditions and then quantified using a standard curve. All samples were run in triplicate.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eStatistical analysis of the data obtained was performed using MINITAB statistical program version 18 (Minitab Inc.). The identification of differences between groups was performed using one-way ANOVA and Tukey's post hoc test for multiple comparisons with statistical significance at the 95% confidence level (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAnalytical method performance assessment\\u003c/h2\\u003e \\u003cp\\u003eThe LC-MS analysis successfully separated the main compounds of interest. The analysis identified resveratrol at a retention time of 10.27 minutes, piceatannol at 9.7 minutes, piceid at 9.2 minutes and the internal standard at 10.27 minutes.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eProliferation of\\u003c/b\\u003e \\u003cb\\u003eArachis hypogaea\\u003c/b\\u003e \\u003cb\\u003ecalli\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eAll leaf explants had expanded and developed callus on their surface after 8 days of culture in the dark. All calli obtained were transferred to the same medium every 4 weeks and the callus growth index peaked during the second subculture, reaching an increase of 152% (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). However, the growth index decreased in subsequent subcultures, resulting in friable, vigorously growing and homogeneous tissues (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eEffect of picloram on \\u003cem\\u003eA. hypogaea\\u003c/em\\u003e callus fresh weight, growth index (GI) and morphology in proliferation medium for 3 subculture cycles\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"10\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c9\\\" colnum=\\\"9\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c10\\\" colnum=\\\"10\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eSubculture cycle\\u003c/em\\u003e\\u003c/p\\u003e \\u003cp\\u003e\\u003cem\\u003ePGR\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c4\\\" namest=\\\"c2\\\"\\u003e \\u003cp\\u003eSecond\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c7\\\" namest=\\\"c5\\\"\\u003e \\u003cp\\u003eThird\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003eFourth\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c10\\\" namest=\\\"c9\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eMass (g)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eGI (%)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eTexture\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eMass (g)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eGI (%)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eTexture\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eMass (g)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eGI (%)\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c10\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eTexture\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003e5mg Pic\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e8.91\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e156.2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eFriable,\\u003c/p\\u003e \\u003cp\\u003eoff-white\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e14.24\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.4\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e66.3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eFriable,\\u003c/p\\u003e \\u003cp\\u003eoff-white\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e24.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.6\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e64.1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c10\\\"\\u003e \\u003cp\\u003eFriable,\\u003c/p\\u003e \\u003cp\\u003eoff-white\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEffect of metabolic modulators on the cell biomass in callus culture\\u003c/h2\\u003e \\u003cp\\u003eTo assess the effects of the modulators, calli per treatment were transferred to culture medium containing different concentrations. Biomass accumulation as fresh and dry weight was monitored at multiple time points (2, 8, 16, 32 and 144 hours) as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e. Fresh weight (FW) patterns in treated calli were generally similar. M1 treatment yielded the highest FW, while untreated calli showed minimal changes with a slight decrease at 32 hours. Interestingly, unlike the other treatments, M1 and high Phe concentrations maintained their FW at 144 hours. Conversely, dry weight was significantly lower in all Cr-treated calli compared to the untreated control. Similar to FW, M1 treatment resulted in the most pronounced increase in dry weight biomass.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEffect of metabolic modulators on the production of resveratrol, piceid and piceatannol in callus culture\\u003c/h2\\u003e \\u003cp\\u003eOur experiments across all treatment groups revealed significant variations in the concentration of the targeted stilbenes (resveratrol, piceatannol, and piceid). These variations depended on both the treatment concentration applied and the incubation time, as shown in Tables\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e\\u0026ndash;\\u003cspan refid=\\\"Tab4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e. In the untreated control group, callus tissue maintained a constant level of resveratrol and piceatannol production until the 32 h mark, at which point a statistically significant increase was observed. However, piceid showed a distinct pattern. Its content peaked after a short 2 h incubation period, followed by a subsequent decline. These results highlight the influence of treatments on stilbene production dynamics in peanut callus.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEffect of precursor feeding with L-phenylalanine\\u003c/h2\\u003e \\u003cp\\u003eResveratrol content peaked after 32 h when callus cultures were treated with the lowest Phe concentration. This level was nearly three times higher compared to the control group. Interestingly, the highest Phe concentration induced an almost six-fold increase in resveratrol content compared to the control, but this peak occurred at an earlier time point 16 h (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). This trend of early peak with higher Phe concentration was also observed for piceatannol content. Piceatannol levels peaked after 16 h when exposed to Phe3, exhibiting a six-fold increase compared to the control. In contrast to resveratrol and piceatannol, piceid showed a different pattern. Piceid levels consistently peaked after 8 h for all Phe concentrations tested.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEffect of sodium malonate dibasic\\u003c/h2\\u003e \\u003cp\\u003eThe addition of malonate at a low concentration resulted in higher resveratrol levels compared to the control after 16 h. A higher concentration of malonate led to an even earlier peak in resveratrol accumulation, after 8 h. The best concentration of malonate to increase resveratrol production was M3, which resulted in an accumulation of up to 19.71 \\u0026micro;g/g DW. This represents an almost tenfold increase compared to the control group. Piceid production followed a similar trend, with an almost threefold increase compared to the control. Piceatannol, however, showed a different response. Its content peaked after 32 h, reaching a level of 7.44 \\u0026micro;g/g DW.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEffect of cerulenin\\u003c/h2\\u003e \\u003cp\\u003eAs illustrated in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e\\u0026ndash;\\u003cspan refid=\\\"Tab4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, results revealed distinct patterns of stilbene accumulation in response to cerulenin treatment. Resveratrol content peaked at 8 h after treatment with Cr1, reaching a level of 23.42 \\u0026micro;g/g DW, followed by a decline. Similar trends were observed for its derivatives, piceatannol and piceid. Both exhibited increases after 8 h when treated with the same cerulenin concentration. Interestingly, piceid content consistently peaked at 8 h regardless of the cerulenin concentration used. In contrast, resveratrol continued to increase progressively until 16 h when exposed to Cr3. Piceatannol, however, differed the most, with the highest accumulation observed at just 2 h after treatment with Cr2 up to almost five times greater than the control.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eEffect of metabolic modulators treatment, sodium malonate dibasic (M1: 0.5mM; M2: 2.5 mM; M3: 12.5 mM) phenylalanine (P1: 0.2 mM; P2: 1mM; P3: 5mM) and cerulenin (Cr1: 0.2 \\u0026micro;M ; Cr2: 1\\u0026micro;M; Cr3: 5\\u0026micro;M) on resveratrol production in peanut callus culture (\\u0026micro;g/g) at different incubation times (T1: 2h; T2: 8h; T3: 16h; T4: 32 h; T5: 144 h)\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"6\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTreatment\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eT1\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eT2\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eT3\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eT4\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eT5\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e2.15\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.22 \\u003csup\\u003ea\\u0026minus;d\\u003c/sup\\u003e \\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e2.20\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.10\\u003csup\\u003ef\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e2.40\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.21\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e6.62\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.28\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.39\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.12\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eM1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.98\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.66 \\u003csup\\u003eb\\u0026minus;e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e3.99\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.91\\u003csup\\u003eef\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e5.07\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.82 \\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1.85\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.47\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.18\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.12\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eM2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.89\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.11\\u003csup\\u003ee\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e12.93\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.90\\u003csup\\u003ebc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e11.68\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.29 \\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e12.41\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.24\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.93\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.16\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eM3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.76\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.08\\u003csup\\u003ecde\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e19.71\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.97 \\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e5.61\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.25 \\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e13.07\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.44\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.07\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.20\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eP1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.23 \\u0026plusmn;0.15 \\u003csup\\u003ede\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e14.44\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.30 \\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e7.08\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.91 \\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e17.16\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.56\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.37\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.30\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eP2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.46 \\u0026plusmn;0.22 \\u003csup\\u003ede\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e3.54\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.15\\u003csup\\u003eef\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e4.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.41 \\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3.73\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.26\\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.34\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.11\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eP3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e2.28 \\u0026plusmn; 0.22 \\u003csup\\u003ea\\u0026minus;d\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e10.49\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.35\\u003csup\\u003ebcd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e13.60\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.26 \\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2.68\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.68\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.30\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.21 \\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCr1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e2.97\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.07 \\u003csup\\u003eab\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e23.42\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.41\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.90\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.78 \\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3.26\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.47\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.28\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.29 \\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCr2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.12\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.76 \\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e7.61\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.45 \\u003csup\\u003ede\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.24\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.24 \\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2.52\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.25\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.89\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.13 \\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCr3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e2.72 \\u0026plusmn;0.53\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e9.02\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.73\\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e21.72\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.86\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1.74\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.44\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.44\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.33\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eDifferent letters in the same column indicate significantly different values per p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 between different treatments for the same incubation time.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eEffect of metabolic modulators treatment (Trt), sodium malonate dibasic (M1: 0.5mM; M2: 2.5 mM; M3: 12.5 mM) phenylalanine (P1: 0.2 mM; P2: 1mM; P3: 5mM) and cerulenin (Cr1: 0.2 \\u0026micro;M ; Cr2: 1\\u0026micro;M; Cr3: 5\\u0026micro;M) on piceid production in peanut callus culture (\\u0026micro;g/g) at different incubation times (T1: 2h; T2: 8h; T3: 16h; T4: 32 h; T5: 144 h)\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"6\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTreatment\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eT1\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eT2\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eT3\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eT4\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eT5\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5.69\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.61\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e3.07\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.13\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e2.83\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.77\\u003csup\\u003ebc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2.58\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.50\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.52\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.16\\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eM1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5.98\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.66\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5.69\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.28\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e2.16\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.67\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1.50\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.37\\u003csup\\u003ee\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.06\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.06\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eM2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5.34\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.74\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e7.32\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.23\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e2.99\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.77\\u003csup\\u003ebc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3.18\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.21\\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e2.23\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.16\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eM3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5.88\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.44\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e8.58\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.25\\u003csup\\u003eab\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e2.87\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.27\\u003csup\\u003ebc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2.93\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.12\\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e2.10\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.17\\u003csup\\u003eab\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eP1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e4.47\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.78\\u003csup\\u003ebc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e6.26\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.96\\u003csup\\u003ebc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.17\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.43\\u003csup\\u003ebc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.76\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.15\\u003csup\\u003ee\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.52\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.06\\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eP2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5.38\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.19\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e8.09\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.07\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.16\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.08\\u003csup\\u003ebc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3.06\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.15\\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e2.27\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.16\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eP3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.97\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.44\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e7.19\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.46\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e4.84\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.42\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3.72\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.61\\u003csup\\u003ebc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.67\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.14\\u003csup\\u003ebc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCr1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5.87\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.18\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e6.85\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.68\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e4.37\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.99\\u003csup\\u003eab\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e4.99\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.28\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.84\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.13\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCr2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e6.64\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.05\\u003csup\\u003eab\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e8.85\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.25\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.34\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.26\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3.45\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.22\\u003csup\\u003ebcd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.48\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.35\\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCr3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e6.81\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e7.10\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.70\\u003csup\\u003eabc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.07\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.14\\u003csup\\u003ebc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e4.30\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.54\\u003csup\\u003eab\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.50\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.23\\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eDifferent letters in the same column indicate significantly different values per p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 between different treatments for the same incubation time.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab4\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 4\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eEffect of metabolic modulators treatment (Trt), sodium malonate dibasic (M1: 0.5mM; M2: 2.5 mM; M3: 12.5 mM) phenylalanine (P1: 0.2 mM; P2: 1mM; P3: 5mM) and cerulenin (Cr1: 0.2 \\u0026micro;M ; Cr2: 1\\u0026micro;M; Cr3: 5\\u0026micro;M) on piceatannol production in peanut callus culture (\\u0026micro;g/g) at different incubation times (T1: 2h; T2: 8h; T3: 16h; T4: 32 h; T5: 144 h)\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"6\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTreatment\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eT1\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eT2\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eT3\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eT4\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eT5\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eC\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.97\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.013\\u003csup\\u003eef\\u003c/sup\\u003e \\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.96\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.03\\u003csup\\u003ee\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.96\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.07\\u003csup\\u003eg\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3.10\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.11\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.26\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.05\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eM1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.29 \\u0026plusmn;0.24\\u003csup\\u003edef\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.80\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.09\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.99\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.04\\u003csup\\u003ecde\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1.19\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.19\\u003csup\\u003ede\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.28\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.02\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eM2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.77 \\u0026plusmn;0.05\\u003csup\\u003ef\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e2.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.16\\u003csup\\u003ebc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e4.15\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.05\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1.80\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.26\\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.47\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.03\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eM3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.23 \\u0026plusmn;0.25\\u003csup\\u003edef\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e4.27\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.19\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.26\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.25\\u003csup\\u003efg\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e7.45\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.43\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.65\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.29\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eP1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.39 \\u0026plusmn;0.25\\u003csup\\u003ede\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e2.92\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.32\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.25\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.11\\u003csup\\u003efg\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1.21\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.23\\u003csup\\u003ede\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.28\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.01\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eP2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.52 \\u0026plusmn;0.13\\u003csup\\u003ed\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.29\\u003csup\\u003ede\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e2.35\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.29\\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1.34\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.14\\u003csup\\u003ede\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.39\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.03\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eP3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.21 \\u0026plusmn;0.16\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e2.64\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.27\\u003csup\\u003ebc\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e6.66\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1.48\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.26\\u003csup\\u003ecde\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.39\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.03\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCr1\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.16 \\u0026plusmn;0.31\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e4.29\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.46\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.81\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.12\\u003csup\\u003edef\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e2.02\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.02\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.41\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.01\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCr2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e4.76 \\u0026plusmn;0.01\\u003csup\\u003ea\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.95\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.09\\u003csup\\u003ecd\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.62\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.05\\u003csup\\u003eefg\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1.31\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.12\\u003csup\\u003ede\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.46\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.02\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCr3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e2.22 \\u0026plusmn;0.12\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e3.05\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.29\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e2.64\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.13\\u003csup\\u003ec\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1.11\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.10\\u003csup\\u003ee\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.77\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.02\\u003csup\\u003eb\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eDifferent letters in the same column indicate significantly different values per p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 between different treatments for the same incubation time.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEstablishment of peanut cell suspension\\u003c/h2\\u003e \\u003cp\\u003eThe peanut cell suspension cultures were established using calli derived from leaves. Viable and actively growing peanut cell suspension cultures were successfully obtained and cell viability was evaluated using the FDA staining method. The intensity of the fluorescence emitted by the stained cells reflects the proportion of living cells in the sample. Higher fluorescence intensity observed at day 20 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e) indicates a greater number of viable cells. This information allows us to identify the optimal cell growth stage for further investigation into its correlation with stilbene content. This provides the basis for subsequent experiments aimed at optimizing stilbene production.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEffect of metabolic modulators on the cell biomass in cell suspension\\u003c/h2\\u003e \\u003cp\\u003eThe effect of the metabolic modulators on cell growth was assessed by measuring both cell fresh weight and dry weight. Figures\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e and 6 show that between day 0 and 6, covering the lag growth phase, all cell cultures exhibited similar growth patterns. Both untreated and treated cells maintained a constant weight until day 5, when the exponential growth phase began. During this subsequent phase, all groups continued to show exponential growth. Exceptionally, cells treated with different concentrations of phenylalanine showed a significant weight jump after T7 of addition (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). Other treatments resulted in a less pronounced increase, but still significantly higher than the control group (Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e\\u0026amp;S2). Transitioning into the stationary phase, untreated cells showed a steady increase in weight, reaching a plateau after T5 of sampling. Similar observations were made for cells treated with cerulenin and the phenylalanine group (except for Phe1). In these groups, biomass started to decline after 72 hours, indicating of cell death, with a more severe effect observed in the Cr3 group (Fig.\\u0026nbsp;6). Conversely, cells treated with malonate and Phe1 showed a renewed increase in weight after reaching a plateau at 144 hours.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec23\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eEffect of metabolic modulators on the extracellular production of stilbenes in peanut cell suspension\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec24\\\" class=\\\"Section4\\\"\\u003e \\u003ch2\\u003eEffect on resveratrol production\\u003c/h2\\u003e \\u003cp\\u003eDuring the lag phase, resveratrol was the only stilbene detected in the culture medium. In untreated control cultures, extracellular resveratrol production remained stable until T6. At this time, a significant increase was observed. After the addition of metabolic modulators, resveratrol was found in the medium after only 2 h (T1). However, in some treatments the level decreased before increasing again. In all treatments, resveratrol levels peaked at 32 h. Among the modulators tested, M1 was the most effective and significant in inducing resveratrol production (Fig. S3), leading to a maximum concentration of 554.13 \\u0026micro;g/L, which is five times higher compared to the control (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e). Similarly, P1 and Cr3 also induced a two-fold increase in resveratrol content, followed by a decrease. Interestingly, untreated peanut cell suspensions continued to produce resveratrol even after entering the exponential growth phase, following a different pattern as observed during the lag phase. The highest amount was detected after 4 h up to 118.34 \\u0026micro;g/L. However, the addition of metabolic modulators on day 10 triggered a distinct response in extracellular resveratrol production. In most treatments, resveratrol showed a progressive rise in the culture medium starting from 2 hours. Notably, different concentrations of the three modulators tested resulted in varying optimal incubation times for resveratrol accumulation. Treatment with M3 yielded the highest resveratrol content at 32 h (T5), reaching 126.28 \\u0026micro;g/L, similar to P1 and Cr3, which also peaked at 32 h. The pattern of resveratrol production shifted during the pre-stationary phase. Resveratrol became detectable only after 8 hours of sampling in both treated and untreated cell suspensions. Notably, all groups showed a similar trend, with resveratrol levels increasing until 32 hours, when they peaked before declining. As observed in previous phases, M3 and P1 remained the most effective modulators for enhancing resveratrol content to 206.69 \\u0026micro;g/L and 203.59 \\u0026micro;g/L, respectively. However, Cr2 was the most effective concentration within the cerulenin treatments.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec25\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eEffect on piceid production\\u003c/h2\\u003e \\u003cp\\u003ePiceid, unlike resveratrol, was detectable throughout the exponential and stationary phases (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e). Untreated cells consistently produced piceid during the exponential phase at all incubation times tested. Piceid content in untreated cultures showed a significant increase compared to treated suspensions at both 2 h and 18 h (Fig. S4). In contrast to resveratrol, piceid peaked earlier, reaching its highest level after only 2 h in most treatment groups. However, M3 and Cr2 treatments showed a delayed peak for piceid content, reaching their highest levels at 32 h. Interestingly, the addition of P3 significantly increased piceid content, reaching a maximum of 75.69 \\u0026micro;g/L, almost three times higher than the control. In contrast to resveratrol, piceid production was more prominent during the stationary phase. Similar to the exponential phase, piceid accumulation in the medium started as early as 2 h of incubation. Remarkably, the graphs revealed an inverse relationship between resveratrol and piceid levels for some treatments. Cells treated with the most effective modulators of resveratrol production (M1, M3, and P1) showed a decrease in resveratrol but a subsequent increase in piceid content later at 72h. This trend was also observed with M2 and Cr3. Piceid in these groups peaked at 8 h (T3) and then decreased, coinciding with the peak of resveratrol. Treatments that induced the highest piceid levels at the same incubation time as resveratrol after 32 h showed a more sustained level of piceid compared to the quantified resveratrol levels.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec26\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003eEffect on piceatannol production\\u003c/h2\\u003e \\u003cp\\u003ePiceatannol was only detected in the culture medium during the pre-stationary phase, appearing after 18 h of incubation as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003e. Untreated control cells initiated piceatannol production after 18 h, with statistically significant levels compared to treated cells, except for M2 and M3 (Fig. S5). This production continued to increase until 72 h, reaching a significant peak of 88.92 \\u0026micro;g/L. Similar to resveratrol, M3 treatment demonstrated the most pronounced effect on piceatannol content, reaching a maximum after 32 h. As shown in the figure, both untreated and M3-treated cells produced piceatannol after 96 h of incubation. For Phe treatments, piceatannol content peaked after 32 h but decreased subsequently by 72 h. Interestingly, P2 and P3 treatments resulted in undetectable levels of piceatannol beyond 72 h. Finally, piceatannol was only observed in Cr-treated cultures after 32 h.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cdiv id=\\\"Sec28\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEffect of phenylalanine feeding on resveratrol, piceatannol and piceid contents in peanut cells\\u003c/h2\\u003e \\u003cp\\u003eCurrently, a promising approach to resveratrol production involves biotechnological techniques that use building blocks to enhance its natural biosynthesis in peanuts. The biosynthesis of stilbenes starts with two aromatic amino acids, phenylalanine and tyrosine, which are mainly obtained via the shikimate pathway, although the malonate pathway may play an indirect role by supplying precursor molecules. Molecular engineering of the resveratrol pathway was reported in plants in the 1990s for disease resistance and functional foods (Jeandet et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Precursor feeding is a powerful technique to stimulate plant secondary metabolite production. Supplying phenylalanine is likely to increase the metabolic flux towards the desired target, leading to enhanced resveratrol production in peanut sprouts or hairy roots (Yu et al. \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Tothong et al. \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Phe may act as an early metabolic regulator in plant cells. Phe supplementation could influence the biosynthesis of enzymes involved in the pathway leading to stilbenes and possibly regulate signalling pathways associated with plant defence response (Tothong et al. \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Researchers further investigated the effects of supplementing phenylalanine or tyrosine in \\u003cem\\u003eMorus alba\\u003c/em\\u003e root culture and \\u003cem\\u003eVitis vinifera\\u003c/em\\u003e cell suspension to increase resveratrol and piceid production (Andi et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Inyai et al. 2020). Kumari et al. (\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e) reported that varying Phe concentrations can boost phenylpropanoid production in sterile cell cultures. Grapes cells can efficiently take up and metabolize large amounts of externally added Phe, converting it into stilbenes, both within the cell and excreted into the medium, enabling high overall production. These findings support our results showing that phenylalanine supplementation successfully increased the production of resveratrol, piceid and piceatannol in both callus and cell suspension cultures. Phenylalanine ammonia lyase (PAL) could be a major regulatory enzyme in response to supplemental Phe in the stilbene biosynthesis pathway. In peanut hairy root cultures, the identified PAL gene family (including PAL, PAL2, MYB36, ERF3, PR2, and STH2) showed significantly higher expression levels at 24 hours after elicitation with 5 mM Phe compared to cultures without Phe supplementation. Tothong et al. (\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e) suggested that a combined approach of Phe feeding and elicitor treatment might accelerate the plant's defense response by rapidly inducing early response genes, potentially leading to a faster stilbene biosynthesis response.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec29\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEffect of sodium malonate dibasic on resveratrol, piceatannol and piceid contents in peanut cells\\u003c/h2\\u003e \\u003cp\\u003ePhenylalanine feeding remains a popular strategy to boost resveratrol production through the well-defined phenylpropanoid pathway in plants. However, exploring alternative or complementary pathways, similar to successful approaches in microorganisms, holds promise for further optimizing resveratrol production. An exceptional case of malonate's ability to enhance resveratrol production comes from Langcake et al. (1977), who reported the only documented instance of its successful use in grapevine berries and leaves. Tyrosine incorporation was minimal in all experiments. Conversely, phenylalanine and acetate acted as effective precursors, while malonate incorporation showed exceptionally high levels. Our observations agree with previous findings where malonate feeding during the lag phase of peanut cell suspension cultures resulted in a 2-fold increase in resveratrol production compared to cells fed with phenylalanine. Interestingly, malonate also enhanced the production of piceatannol and piceid during the stationary phase, with a more pronounced effect than phenylalanine. The observed stimulation of stilbene production by malonate addition suggests the cultures' ability to effectively take up malonate and convert it into its active form, malonyl-CoA. The increased resveratrol observed in peanut cells might be due to the combined effect of overexpressing matB and matC genes along with malonate supplementation as suggested by Thapa et al. (\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). This combined approach likely leads to higher intracellular levels of malonyl-CoA, the essential substrate used by STS enzymes for stilbene biosynthesis. Similarly, in \\u003cem\\u003eRhamnus purshiana\\u003c/em\\u003e cell cultures, 12.5 mM malonate significantly increased anthra-derivative production by a factor of 2.7, whereas the same concentration of acetate had no effect (Van den berg et al. \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e1988\\u003c/span\\u003e).\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eEffect of cerulenin on resveratrol, piceatannol and piceid contents in peanut cells\\u003c/h3\\u003e\\n\\u003cp\\u003eAlternatively, other strategies involve the use of the antibiotic cerulenin, which binds to the β-ketoacyl carrier protein (ACP) synthase of the fatty acid synthesis machinery, thereby inhibiting its enzymatic activity. For instance, Liu et al. (2022) reported this approach by adding cerulenin to boost intracellular levels of malonyl-CoA, with \\u003cem\\u003ein vivo\\u003c/em\\u003e evolution of \\u003cem\\u003ePinus strobus\\u003c/em\\u003e stilbene synthase for increased pinosylvin biosynthetic activity in engineered \\u003cem\\u003eE. coli\\u003c/em\\u003e. Interestingly, application of cerulenin at 200 \\u0026micro;M increased the intracellular malonyl-CoA pool by a factor of 50. Similar results were observed using a genetically engineered \\u003cem\\u003eE. coli\\u003c/em\\u003e strain, resulting in a significant increase in resveratrol production (from 1.3 g/L to 2.3 g/L). However, it's important to note that this success is not universal (Jeantdet et al. 2021). Low concentrations of cerulenin (up to 5 \\u0026micro;M) effectively increased resveratrol and piceid content in peanut cells suspension. In contrast to our findings, Kallscheuer et al. (\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e) found that higher cerulenin concentrations (5, 10, and 25 mM) significantly increased resveratrol production. However, further escalation (50 to 150 mM) did not lead to comparable gains in resveratrol levels, suggesting a possible threshold effect. Our results on the successful increase of resveratrol derivatives like piceid and piceatannol in the static peanut cell culture align well with reports of similar effects in \\u003cem\\u003eCorynebacterium glutamicum\\u003c/em\\u003e cultures, where cerulenin addition also promoted piceatannol formation (Thapa et al. \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). This achievement may be linked to a creation of a roadblock in fatty acid synthesis by adding cerulenin. By binding to the active site of β-Ketoacyl-acyl carrier protein synthase (KAS), a key enzyme, cerulenin inhibits its function and effectively stalls the entire pathway at that step. This disruption triggers feedback mechanisms within the cell, which might not sense a need for further fatty acids production. Signaling cascades can be initiated, ultimately leading to a decrease in the activity and changes in the overall expression levels of some fatty acid synthesis genes, fab operon (fabD, fabB, fab F, fabH, fabI). With fatty acid synthesis partially blocked, cells can potentially redirect resources towards other pathways where a crucial precursor molecule, malonyl-CoA, might be accumulated.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec31\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEffect of the cell density on resveratrol, piceatannol and piceid contents\\u003c/h2\\u003e \\u003cp\\u003eWhile limited research has documented resveratrol production in peanut cell suspensions, this study successfully demonstrates its induction alongside piceatannol and piceid. Notably, the production levels and their proportions differed significantly compared to callus static cultures, and also varied across different growth stages within the cell suspension. Eliciting grapevine cell cultures with specific compounds like methyl jasmonate (MeJA) and cyclodextrins can advantageously trigger the production and secretion of \\u003cem\\u003etrans\\u003c/em\\u003e-resveratrol and piceid extracellularly (Lambert et al. \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Jeong et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Vera-Urbina et al. \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). This approach makes grapevine cells promising factories for resveratrol production, as these compounds accumulate in the culture medium, facilitating their extraction. Lambert et al. (\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e) found a positive correlation between resveratrol production and biomass concentration. Their study suggests that the exponential growth phase might be the optimal time for elicitation, as cultures with the highest biomass yielded the highest resveratrol content. A critical, yet often overlooked factor in optimizing stilbene production through cell cultures is the timing of elicitor application. The physiological state of the cells, influenced by their growth phase (lag, exponential, and stationary), is worthy of consideration. Studies generally suggest that adding elicitors during the mid-to-late exponential growth phase yields the best response in terms of secondary metabolite production, compared to earlier or later stages (Vera-Urbina et al. \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Interestingly, our results in peanut cell cultures differ from some previous observations. While our study showed higher resveratrol production at lower cell densities, Belchi-Navarro et al. (2012) observed similar trends in Monastrell grapevine cell cultures treated with elicitors. In their study, extracellular resveratrol production also peaked at lower initial cell densities.\\u003c/p\\u003e \\u003cp\\u003eThe variations observed in resveratrol production depending on the cell density in peanut cell cultures might be due to how cell density influences the response to the added compounds. In cultures with lower cell density, each cell could be more receptive to the effects of phenylalanine, cerulenin or malonate added. A proportionally larger share of the metabolic machinery might be available for stilbene biosynthesis compared to cultures with higher cell densities, leading to a more efficient utilization of the added compounds by individual cells. This explanation is consistent with our findings of higher resveratrol levels at lower cell densities. However, the response may be different for other stilbenes. The production of piceid and piceatannol, which we observed to be higher in later growth phases, may not be as directly influenced by the initial availability of added modulators. Other factors within the specific pathways for piceid and piceatannol may play a more important role in regulating their production at different growth stages, as their specific biosynthetic steps may be less dependent on the initial boost in the phenylalanine or malonyl-Coa pool. Piceid and piceatannol production differed from that of resveratrol. Their levels peaked when modulators were added in the late exponential phase, consistent with observations by Vera-Urbina et al. (\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). This suggests that the regulation of piceid and piceatannol biosynthesis may be mediated by higher cell density. Higher cell density might play a role in regulating piceid and piceatannol production. Increased biomass could potentially lead to a shift in cellular metabolism towards the production of a wider range of stilbenes, not just resveratrol.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eIn conclusion, the aim of this study was to increase knowledge of the bioproduction of resveratrol and its derivatives in peanut cells through biotechnological approaches. We have successfully established a method for inducing the production of resveratrol, piceatannol, and piceid in peanut cell suspension cultures. The levels and proportions of these stilbenes differed significantly from those observed in peanut callus cultures and varied across the cell suspension growth stages. Our findings highlight the potential of feeding and metabolic modulation on producing these valuable stilbenes in peanut cell cultures. By providing the basis for a sustainable and efficient method, this research sets the stage for wider availability of these valuable compounds. Moreover, the scale-up of culture volume without loss in growth open exciting avenues for further research.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAuthor contribution statement\\u0026nbsp;\\u003c/strong\\u003eHajer Ben Ghozlen conceived, performed the experiments and drafted the manuscript. Sven Mangelinckx participated in data analysis. Stefaan Werbrouck and Sven Mangelinckx revised and supervised the manuscript. All authors contributed to the article and approved the version submitted.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u0026nbsp;\\u003c/strong\\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDeclaration of Competing Interest\\u0026nbsp;\\u003c/strong\\u003eThe authors have no relevant financial or non-financial interests to disclose.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u0026nbsp;\\u003c/strong\\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical Approval\\u0026nbsp;\\u003c/strong\\u003eThis study does not contain any experiments involving humans or animals.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAndi SA, Gholamib M, Fordc M, Christopher MF (2019) The effect of light, phenylalanine and methyl jasmonate, alone or in combination, on growth and secondary metabolism in cell suspension cultures of \\u003cem\\u003eVitis vinifera\\u003c/em\\u003e. 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J Sep Sci 43(6):1024\\u0026ndash;1031. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1002/jssc.201900915\\u003c/span\\u003e\\u003cspan address=\\\"10.1002/jssc.201900915\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYu M, Liu H, Yang Y, Shi A, Liu L, Hui H, Wang Q (2016) Optimisation for resveratrol accumulation during peanut germination with phenylalanine feeding \\u0026amp; ultrasound-treatment using response surface methodology. J Food Sci Technol 51(4):938\\u0026ndash;945. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1111/ijfs.13036\\u003c/span\\u003e\\u003cspan address=\\\"10.1111/ijfs.13036\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Stilbene, peanut cells, metabolic modulators, bioproduction\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6375460/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6375460/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eStilbenes, including resveratrol, piceatannol and piceid, are often limited in terms of bioproduction yield. This study represents the first attempt to modulate stilbene production pathways in peanut (\\u003cem\\u003eArachis hypogaea\\u003c/em\\u003e) cells. We investigated the potential of L-phenylalanine, sodium malonate dibasic, and cerulenin as metabolic modulators to promote stilbene biosynthesis. These modulators were tested at different concentrations and time points in both peanut callus cultures and cell suspension cultures. The effects of these modulators on cell growth and stilbene production were assessed. The results revealed that metabolic modulators significantly influence the production patterns of resveratrol, piceid, and piceatannol in peanut cells. Interestingly, both static and suspension cultures displayed distinct responses, with the specific metabolite produced and its level depending on the growth phase, modulator concentration, and incubation time. Our findings showed that 0.2 \\u0026micro;M cerulenin was the most effective modulator, resulting in more than tenfold increase in resveratrol production in callus cultures. In cell suspension cultures, 0.5 mM sodium malonate dibasic also enhanced the production of resveratrol during the lag phase whereas piceatannol and piceid were more prominently produced during the stationary phase. This effect was more significant than that observed with phenylalanine and cerulenin. This research provided valuable insights into the modulation of metabolic pathways within this novel host system and established peanut cells as a viable platform for future stilbene production.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Modulating stilbenes in peanut cells: A novel approach with metabolic modulators\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-04-16 20:27:31\",\"doi\":\"10.21203/rs.3.rs-6375460/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"511273e2-66f3-4748-8c68-b1e4c1b4978b\",\"owner\":[],\"postedDate\":\"April 16th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-06-04T22:29:29+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-04-16 20:27:31\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6375460\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6375460\",\"identity\":\"rs-6375460\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}