Pyruvate and α-ketoglutarate precursor feeding combined with methyl jasmonate elicitation increases anthraquinone accumulation in hairy root cultures of Rubia tinctorum

Pyruvate and α-ketoglutarate precursor feeding combined with methyl jasmonate elicitation increases anthraquinone accumulation in hairy root cultures of Rubia tinctorum | 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 Pyruvate and α-ketoglutarate precursor feeding combined with methyl jasmonate elicitation increases anthraquinone accumulation in hairy root cultures of Rubia tinctorum Yazmin Kalapuj, María Perassolo, Julián Marcelo Rodrïguez, Alejandra Beatriz Cardillo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8650732/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Anthraquinones (AQs) are specialized metabolites produced by Rubia tinctorum that have traditionally been used as dyes, but also have important biological activities. The present research studied the effects of the addition of pyruvate (Pyr) and α-ketoglutarate (KG) precursors in combination with methyl jasmonate (MeJa) elicitation, via a full factorial design, on hairy root cultures of R. tinctorum . MeJa was the main factor that triggered AQ accumulation, and the addition of Pyr and KG also significantly enhanced AQ production. Feeding of each precursor resulted in increased intracellular and total AQ production, which revealed that Pyr and KG are limiting precursors of AQ biosynthesis in hairy root cultures of R. tinctorum . The three variables showed a significant interaction effect, resulting in total AQ production of ~ 2022 µM. Moreover, the addition of Pyr and KG increased AQ release into the culture medium. KG was the main factor responsible for the secretion of AQs; combination with MeJa elicitation resulted in 56 µM of AQs in the culture medium. The phenolic and flavonoid contents were also evaluated, and the results showed that MeJa elicitation triggered only flavonoid production. At the same time, precursor addition had no significant effect on phenolic or flavonoid accumulation. Anthraquinones Rubia tinctorum Hairy roots Elicitation Methyl jasmonate Precursor feeding Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key message Pyruvate, α-ketoglutarate and elicitation increase anthraquinone productivity in hairy root cultures of . Pyruvate and α-ketoglutarate addition stimulate anthraquinone release to the culture medium. 1. Introduction Anthraquinones (AQs) are specialized metabolites produced by plants of the Rubiaceae family that have been used as dyes since antiquity. In traditional medicine, crude extracts of R. tinctorum have been used to treat and remove calcium oxalate and phosphate kidney stones. Additionally, interesting biological activities have been attributed to AQs, including antibacterial and antiviral properties, and they have also shown efficacy in photodynamic therapy against cancer cells (Duval et al. 2016 ; Tian 2020; Campora et al. 2021 ). The three-ring base structure can be substituted in different ways, leading to a wide variety of compounds. As shown in Fig. 1 , rings A and B of AQs in Rubiaceae are derived from the condensation of α-ketoglutarate (KG) and isochorismate (Murthy et al. 2023 ). The latter is formed by the action of isochorismate synthase (ICS) on chorismate, the final product of the shikimate pathway. The C ring is formed by incorporating a DMAPP unit that comes from the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The first step in this route is the conversion of glyceraldehyde 3-phosphate (GDP) and pyruvate (Pyr) into deoxyxylulose phosphate (DOXP), catalyzed by the deoxyxylulose phos phate synthase (DXS)(Eichinger et al. 1999 ; Han et al. 2001 ; Murthy et al. 2023 ). Plant cell culture technology has emerged as an alternative platform for the production of specialized metabolites under controlled conditions (complying with GMP standards) and in shorter times. In addition, the cultivation of plant cells in bioreactors avoids unsustainable extractivism, reduces the use of land, herbicides, and insecticides, and decreases the production of gases responsible for the greenhouse effect (Gubser et al. 2021 ; Hasnain et al. 2022 ). Moreover, these bioprocesses can be optimized through different strategies, including culture media design, elicitation, release and in situ product removal (ISPR), precursor feeding, and metabolic engineering (Krasteva et al. 2021 ). Elicitation with methyl jasmonate (MeJa) is one of the most effective strategies for increasing the production of specialized metabolites (Ho et al. 2020 ; Nallakaruppan and Thiagarajan 2024 ). This endogenous elicitor has been successfully applied to increase gingenosides, taxanes, podophyllotoxin, tropane alkaloids, rosmarinic acid, valerenic acid, and artemisinin, among others (Morey and Peebles 2022 ; Wu et al. 2021 ). MeJa also triggers AQ production in suspension and hairy root cultures of R. tinctorum , an effect that is mediated by H 2 O 2 signaling (Perassolo et al. 2011 ; Perassolo et al. 2017 ). Precursor feeding is another strategy to enhance the accumulation of specialized metabolites in plant cell cultures. The exogenous supply of pathway intermediates can also help to elucidate potential carbon flux limitations or regulatory bottlenecks within the metabolic pathway under study ; (Gerszberg and Wiktorek-Smagur, 2022; Rifqi et al. 2024 )). Pyr and KG are key intermediates in the energy metabolism of plant cells and, apart from AQ biosynthesis, are also involved in the synthesis of many amino acids. KG has also been described as a signal molecule and has antioxidant activity. In addition, it participates in many enzymatic reactions mediated by 2-oxoglutarate-dependent dioxygenase (2-OD) in several plant metabolic pathways (Lei and Huang 2022 ; Soubeyrand et al. 2018 ). In the present study, we evaluated the effects of the addition of precursors (Pyr and KG) and MeJa elicitation on the AQ production in hairy root cultures of R. tinctorum . Effects on the phenylpropanoid and flavonoid pathways were also analyzed. 2. Materials and methods 2.1. Plant cell culture Hairy roots were obtained as described by Perassolo et al. ( 2017 ). The cultures were maintained by subculturing the roots every 30 days in Lloyd and McCown’s Woody Plant Medium (WPM) contained in 100 mL Erlenmeyer flasks on a gyratory shaker at 100 rpm. The temperature was set at 24 ± 2°C with a 16-h photoperiod using cool white LED lamps at an intensity of 1.8 W/m 2 . 2.2. MeJa elicitation and precursor addition Elicitation and precursor feeding analysis followed two separate experiments with a full factorial design. In both cases, the experiments were initiated by inoculating approximately 0.25 g (fresh weight, FW) of R. tinctorum hairy roots into 100 mL Erlenmeyer flasks that contained 25 mL of fresh medium, and cultures were harvested at 7 days post elicitation (dpe). The pH was kept constant by the addition of 50 mM MES buffer, pH 5.7, and all the treatments were performed in triplicate. The first experiment tested 2 variables, MeJa and Pyr, at two levels (2 2 ): no addition of MeJa or Pyr was coded as 0, whereas MeJa (100 µM) and Pyr (20 mM) addition were coded as 1 (Table 1 ). After 14 days of culture, MeJa or its diluent (ethanol) was added to the Erlenmeyer flasks and hairy roots were cultured for 2 more days. At that moment Pyr or its diluent (water) was added, and the roots were cultured until harvest time (7dpe). Table 1 Full factorial design (2 2 ) for the experiment combining Pyr and MeJa Experimental Unit Level Codification Concentration Treatment Codification MeJa Pyr MeJa (µM) Pyr (mM) 1 0 0 0 0 M0P0 2 0 1 0 20 M0P1 3 1 0 100 0 M1P0 4 1 1 100 20 M1P1 The second experiment evaluated 3 variables, MeJa, Pyr and KG, at two levels (2 3 ): no addition of MeJa, Pyr or KG was coded as 0, whereas MeJa (100 µM), Pyr (20 mM) and KG (20 mM) addition were coded as 1 (Table 2 ). After 21 days of culture, MeJa or its diluent (ethanol) was added and hairy roots were cultured for 2 days. At that moment, Pyr, KG and/or their diluent (water) were added to the Erlenmeyer flasks and roots were cultured until harvest time (7dpe). Table 2 Full factorial design (2 3 ) for the experiment combining Pyr, KG and MeJa Experimental Unit Level Codification Concentration Treatment Codification MeJa Pyr KG MeJa (µM) Pyr (mM) KG (mM) 1 0 0 0 0 0 0 M0P0K0 2 0 1 0 0 20 0 M0P1K0 3 0 0 1 0 0 20 M0P0K1 4 0 1 1 0 20 20 M0P1K1 5 1 0 0 100 0 0 M1P0K0 6 1 1 0 100 20 0 M1P1K0 7 1 0 1 100 0 20 M1P0K1 8 1 1 1 100 20 20 M1P1K1 2.3. Biomass determination For fresh weight (FW) determination, hairy root cultures were harvested, washed twice with distilled water and vacuum-filtered for 2 minutes. Finally, the root biomass was placed on filter paper for 5 minutes to eliminate the excess of water and then weighed on an analytical balance. The final biomass was expressed as g FW/L, considering the FW determination and the volume of medium in each Erlenmeyer flask (25 mL). Root tissues were subsequently ground to a fine powder with liquid nitrogen and stored at -80°C for further analysis. 2.4. Anthraquinone determination The AQ content was determined according to Perassolo et al. ( 2017 ). For intracellular (IC) AQs, approximately 100 mg of frozen biomass (FW) was extracted several times with 1.5 mL of 80% ethanol for 30 minutes at 80°C. The extracted fractions were collected together and the absorbance was measured spectrophotometrically at 434 nm. The extracellular (EC) AQ content was determined in sample medium, previously centrifuged at 13,000 rpm for 5 minutes. In both cases, the AQ content was estimated using the molar extinction coefficient of alizarin (5.5 mM − 1 cm − 1 ), the most abundant AQ in R. tinctorum , according to Schulte et al. (Schulte et al. 1984 ). IC AQ content was expressed as µmol/g FW, whereas EC AQ content was expressed as µmol/L. Total AQs (µmol/L) were calculated as the addition of EC AQs + IC AQs, considering the final biomass achieved (g FW/L). 2.5. Total phenolic determination The total phenolic (TP) content was determined by the Folin‒Ciocalteu method, according to Shetty et al. ( 2003 ). Briefly, 100 mg of frozen roots were extracted with 100% methanol, and the samples were stored at -20°C for 48–72 hours. Fifty µl of the extracts were mixed with 375 µl of a 0.5 N Folin-Ciocalteu solution and incubated for 5 minutes, and then 375 µl of a 1 M Na 2 CO 3 solution were added. The samples were incubated in the dark for 1 hour, and the absorbance was measured spectrophotometrically at 725 nm (Shetty et al. 2003 ). A standard curve prepared with different concentrations of hydroquinone in 95% ethanol, and the absorbance values were converted to mg TP (hydroquinone equivalents) per gram of biomass (FW). 2.6. Total flavonoid quantification Total flavonoid (TF) determination was performed according to Dewanto et al. (Dewanto et al. 2002 ). A total of 125 µl of the methanolic extracts (obtained as stated in subsection 2.5) were mixed with 630 µl of distilled water and 38 µl of a 5% NaNO 2 solution. The samples were incubated for 6 minutes, and 75 µl of a 10% AlCl 3 .6H 2 O solution was added. After 6 min of incubation, 75 µl of a 1 M NaOH solution was added, the final volume was adjusted to 1.25 mL with distilled water, and the absorbance was recorded at 510 nm. A calibration curve was generated with rutin as a standard, and the absorbance values were converted to mg of flavonoids (as rutin equivalents) per gram of biomass (FW). 2.7. Statistical analysis For both factorial experiments, the significance of the treatment effects was evaluated using two-way ANOVA followed by post hoc analysis using the Duncan´s test. The software used for these analyses was InfoStat 2018 Version (Di Rienzo et al. 2018 ). All experimental data were expressed as mean ± SD of three independent replications. 3. Results 3.1. Pyruvate addition and MeJa elicitation In the first set of experiments (see Table 1 ), hairy root cultures of R. tinctorum were elicited with MeJa on day 14. After two days of elicitation, Pyr was added, and the cultures were collected on day 7 post elicitation. In all the treatments, the pH levels at the end of the experiment were between 5.0 and 5.7. The effects of these treatments on IC AQs (µmol/g FW) are shown in Fig. 2 . IC AQ content when only Pyr was added (M0P1) reached 10.62 µmol/g FW, whereas it was 8.65 µmol/g FW in M0P0 cultures. On the other hand, MeJa-treated cultures resulted in AQ contents of 16.35 µmol/g FW (M1P0) and 17.36 µmol/g FW (M1P1). As two-way ANOVA revealed, the interaction between factors was not significant, but both Pyr and MeJa had positive and significant effects on IC AQ accumulation (Table S1 of Supplementary Information). While Pyr addition accounted only for 3.8% of the total variance, MeJa accounted for almost 91% of the total variance. Similar results were observed for total AQs (Fig. 3 ), which increased up to 73% and 83% in M1P0 and M1P1 treatments, respectively, when compared to M0P0 treatment. However, no significant differences were found between M0P1 and M0P0 treatments, probably due to a slight decrease in hairy root growth after Pyr addition. The analysis of EC AQs (Fig. 3 ) showed that both variables positively affected the release of these specialized metabolites into the culture medium. Two-way ANOVA (Table S2) revealed a significant positive interaction between Pyr and MeJa, so the effects of one variable at each level of the other were analyzed. When MeJa was 0, Pyr addition had a positive effect on EC AQs, increasing from ~ 9.2 to ~ 20.5 µmol/L, but in the presence of MeJa (level 1) EC AQs increased from ~ 22.5 to ~ 46 µmol/L (Fig. 3 ). Similar effects were observed for MeJa at each level of Pyr. These results were also plotted in an interaction graph, showing nonparallel lines (see Figure S1), which indicates that an interaction between the two factors occurred. 3.2. Effects of MeJa elicitation and Pyr and KG addition 3.2.1. Effects on AQ production To gain insight into the effects of feeding precursors on AQ accumulation, the addition of both Pyr and KG after elicitation with MeJa was assayed with a full factorial design (2 3 , see Table 2 ). Hairy root cultures were elicited on day 21, Pyr and KG were added two days after MeJa elicitation, and cultures were harvested at 7 days post-elicitation. For all the treatments, the pH values at the end of the experiment were between 5.0-5.7. The effects of this experiment on biomass production and IC AQs are plotted in Fig. 4 . While MeJa was the only variable that had a significant negative effect on biomass production, the three variables affected AQ production, although double and triple interactions were not significant. MeJa represents 89% of the total variance in the model, whereas KG and Pyr account for 3.6% and 1.9%, respectively. In accordance with this, the increase in IC AQs attributed to Pyr and KG addition was 6.02 and 8.44%, respectively, whereas that of MeJa elicitation was 50.35%. ANOVA results are included as Supplementary Information (Table S3). A multiple comparison study revealed that M1P1K1 treatment resulted in the highest IC AQ production (18.5 µmol/g FW), followed by that of M1P0K1 treatment (17.3 µmol/g FW). Compared with M0P0K0, M1P0K0 and M1P1K0 treatments also resulted in higher IC AQ content (15.9 and 16.5 µmol/gFW, respectively, compared to 10.4 µmol/g FW of M0P0K0). Total and EC AQs are depicted in Fig. 5 . The analysis of total AQs revealed that MeJa elicitation had the highest effect on total AQ accumulation (~ 2020 µmol/L), when Pyr and KG were at the highest level (M1P1K1). KG and Pyr added individually also showed a positive effect on AQ production in the presence of MeJa, with AQ concentrations of ~ 1860 (M1P0K1) and ~ 1820 µmol/L (M1P1K0). When KG and Pyr were at level 0, the addition of MeJa resulted in an AQ content of ~ 1790 µmol/L. At level 0 of MeJa, KG and Pyr alone or combined resulted in increases in total AQ production (~ 1370, ~ 1400, and ~ 1410 µmol/L in M0P0K1, M0P1K0 and M0P1K1, respectively), which were significantly higher than that of M0P0K0 treatment (~ 1240 µM). Triple interaction was significant, which means that the effect observed for any double interaction changes across the levels of the third variable. MeJa explained 87% of the variance of the model, whereas KG, Pyr, and the triple interaction represented 4.6%, 2.3%, and 1.4%, respectively (Table S4). The analysis of double interactions and simple effects revealed that KG-Pyr interaction was not significant at any level of MeJa, but KG showed significant differences at both levels of MeJa, while Pyr effect was significant only at MeJa = 1. Regarding the interactions between MeJa and each precursor, they were significant only at level 1 of the other precursor. In other words, MeJa-KG interaction had a significant effect only at Pyr = 1, whereas MeJa-Pyr interaction had a significant effect at KG = 1. On the other hand, MeJa alone had a significant effect at any combination of KG and Pyr, whereas Pyr had a significant effect in the presence of MeJa, or both MeJa and KG. Regarding KG, it showed significant differences at almost any combination of MeJa and Pyr, except for the case where Pyr = 1 and MeJa was 0. The effects of each factor and the interaction can be more easily perceived in Figure S2. A slightly different response was observed for the accumulation of EC AQs (Fig. 5 ). As shown in Table S5, the ANOVA reveals that although triple interaction was not significant, all the three double interactions (Pyr-KG, Pyr-MeJa and KG-MeJa) were significant. KG addition was the principal factor involved in the release of AQs into culture medium (58% of the total variance). On the other hand, MeJa accounted for 14% of the total variance, whereas Pyr accounted for only 4%. Further analysis of the simple effects revealed that the addition of KG provoked a significant increase in EC AQs at Pyr = 0, and this effect was more pronounced at Pyr = 1. The same behaviour was observed when analyzing simple effects of KG at each level of MeJa, although in this case the synergism resulted in a higher release of AQs. The analysis of Pyr interactions revealed that Pyr addition significantly increased AQ release when KG was also added (for Pyr-KG interaction), but it only increased AQ release when MeJa = 0 (for Pyr-MeJa interaction). Similar results were observed for MeJa: it had a positive impact on AQ release only at KG = 1 (for MeJa-KG interaction), and only when Pyr = 0 (for Pyr-MeJa interaction). M1P0K1 treatment produced the highest EC AQs contents (56 µM), which represented a 6.1-fold increase compared with that in M0P0K0 treatment (9.2 µmol/L, see Fig. 5 ). 3.2.2. Effects on phenylpropanoid and flavonoid pathways Effects of Pyr and KG addition together with MeJa elicitation on total phenolics (TP) and total flavonoids (TF) were also analyzed. The results of specific and volumetric TP production are shown in Fig. 6 . The ANOVA analysis revealed no significant differences between the main variables analyzed and their interactions, for both specific and volumetric TP content (Tables S6 and S7). The study of specific and volumetric TF production revealed that only MeJa had a significant effect on TF content (Fig. 7 and Tables S8 and S9). The treatments where MeJa = 1 showed the highest levels of TF specific production, ranging from 6.9 to 7.1 mg/g FW, while those where MeJa = 0 showed TF contents varying between 3.4 to 3.7 mg/g FW. The volumetric production showed a similar behavior (Fig. 7 ). Effectively, volumetric TF in cultures where MeJa was added (MeJa = 1) ranged from ~ 720 to ~ 780 mg/L, whereas their production was between ~ 390 and 440 mg/L in cultures where MeJa = 0. 4. Discussion The main focus of this research was to study the relationship between precursor addition and MeJa elicitation in hairy root cultures of R. tinctorum . In the first set of experiments, we evaluated the combination of Pyr and MeJa following a 2 2 full factorial design. As mentioned in the introduction, Pyr is involved in the first step of the MEP pathway, and this route generates the DMAPP unit that gives rise to the C-ring of AQs. Our results showed that Pyr alone had a significant but little effect on IC AQ accumulation, whereas MeJa provoked a marked increase in both IC and total AQs. The most interesting finding was the synergism between Pyr and MeJa on EC AQ accumulation. Each factor alone increased AQ release, but the combination of both had a deep impact on EC AQ accumulation (greater than the sum of both effects). These promising results led us to incorporate another AQ precursor to our research. KG was chosen since its condensation with isochorismate is the first step in the biosynthesis of rings A and B of AQs. We tested Pyr and KG addition combined with MeJa by performing a 2 3 full factorial experiment. Our results showed that Pyr and KG in combination with MeJa increased IC and total AQ production. Although MeJa elicitation had the greatest impact on AQ accumulation in R. tinctorum hairy roots, Pyr and KG also triggered AQ production. Effectively, MeJa elicitation and precursor feeding had a significant interaction effect, resulting in a total AQ production of ~ 2020 µM. These effects revealed that Pyr and KG could be limiting substrates in the biosynthetic pathways of AQs in hairy root cultures of R. tinctorum . These results are similar to those obtained with other AQ-producing plant cell cultures. Zenk et al. (Zenk et al. 1975 ) reported that the addition of the precursor ortho-succinylbenzoic acid (OSB) resulted in a 2-fold increase in the AQ content in M. citrifolia cell cultures compared with that in untreated cell cultures. Stalman et al. (Stalman et al. 2003 ) reported that the addition of 20 mM Pyr resulted in a 4-fold increase in AQ accumulation in Morinda citrifolia cell cultures. In related research, the addition of KG produced a 4.8-fold increase in AQ accumulation in M. citrifolia cell suspensions compared with that in control cultures (Sreeranjini and Siril 2019 ). KG addition (30 mg/L) also resulted in a significant increase in alizarin, purpurin and total AQ content in adventitious root cultures of R. cordifolia (Gnanaraj et al. 2025 ). Glutamate, an amino acid that can be converted into KG and proline in plants, was added to cell suspensions of R. tinctorum (5 mM), and resulted in higher AQ content (Perassolo et al. 2011 ). On the other hand, cell suspensions of Cinchona ´Robusta´, a Rubiaceae species, were fed with glyceraldehyde, tectoquinone, ferulic acid, and caffeic acid. The addition of glyceraldehyde, an earlier precursor of the MEP pathway, did not result in any increase in AQ accumulation. However, AQ levels were increased by 48% compared with those in control cultures when caffeic acid was fed to cell cultures, probably due to an indirect effect on the AQ biosynthetic pathway (Han et al. 2002 ). The feeding of Pyr and KG to callus cultures of Cassia angustifolia , a non-Rubiaceae species, resulted in an approximately 30% increase in the Sennoside content (Chetri et al. 2016 ). Another interesting aspect we want to evaluate was the release of AQs to the culture medium. This is a highly relevant topic of research, since metabolite release improves product recovery and opens the opportunity to design strategies such as in situ product removal. We found differences in the effects of Pyr on AQ release between our experiments: it had a synergic effect with MeJa in the first experiment but no synergism between them was detected in the second experiment. One possible explanation is the age of cultures: elicitation and precursor addition occurred between 14–21 days of culture in the first experiment, whereas in the second experiment, they occurred between 21–28 days of culture. Nevertheless, the incorporation of KG in the second experiment led to an interesting outcome: the increased AQ release produced by the addition of both precursors, Pyr and KG. Our research showed that KG resulted in the main factor responsible for the release of AQs into the culture medium. Its combination with MeJa elicitation resulted in the highest EC AQ content (56 µM). The presence of high ketoacid concentrations, such as Pyr, induced an increase in the oxygen consumption rate in pea roots, resulting in hypoxia in the plant roots, even in well-aerated nutrient solutions. Hypoxia has been described as an abiotic stress that could trigger specialized metabolite production in plants and might be another factor that explains the increase in AQ accumulation and release to the culture medium due to Pyr and KG addition (Zabalza et al. 2009 ). Since phenolic and flavonoid biosynthetic pathways compete for carbon precursors with the AQ pathways, we also evaluated the effect of elicitation and precursor addition on the production of these metabolites. In the present study, only MeJa elicitation enhanced total flavonoid accumulation (between 1.6- to 1.8-fold increase). These results are similar to those found in M. citrifolia adventitious root cultures. Effectively, chitosan elicitation resulted in a 2-fold increase on AQ, total phenolic and flavonoid accumulation, compared to control cultures (Baque et al. 2012; Baque et al. 2012b ). MeJa also triggered AQs and phenolics production in adventitious roots of R. tinctorm (Biçer et al. 2017 ). A metabolomic study in MeJa-elicited suspension cultures of Damnacanthus major also showed higher accumulation of the AQ rubiadin and the flavonoid kaempferol, than those of unelicited cultures (Hyeon et al 2024 ). Furthermore, adventitious roots of M. coreia elicited with chitosan showed higher AQ and total phenolic production than non-elicited cultures (Kannan et al. 2020 ). On the contrary, external addition of KG and Pyr did not produce a positive effect on flavonoid accumulation in R. tinctorum hairy roots. In Vitis vinifera cell suspensions, nitrogen limitation raised the intracellular levels of KG, resulting in higher flavonoid and anthocyanin accumulation. KG could be the metabolic signal responsible for this response, since it participates as a reducing agent in many enzymatic steps of the flavonoid pathway (Soubeyrand et al. 2018 ). In summary, the present study reveals that both precursors of the AQ biosynthetic route, Pyr and KG, could be limiting the carbon flux to this specialized metabolic pathway, and this effect is more pronounced after MeJa elicitation. MeJa also stimulated the flavonoid pathway, whereas Pyr and KG did not affect flavonoid production. This paves the way for the development of other biochemical and metabolic engineering strategies to increase the carbon flux to AQ biosynthesis by raising Pyr and KG cellular levels and/or inhibiting the flavonoid pathway. Abbreviations ANOVA Analysis of variance AQs anthraquinones KG α-ketoglutarate DPE days post elicitation DW Dry weight FW Fresh weight MeJa methyl jasmonate Pyr pyruvate TF total flavonoids TP total phenolics WPM Lloyd and McCown’s Woody Plant Medium Declarations Fundings: This work was supported by Agencia Nacional de Promoción Científica y Tecnológica (PICT 2020-02033), Universidad de Buenos Aires (UBACyT 2023- 217BA) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP 2651). MP, ABC, VDB and JRT are researchers from CONICET. YRK is a fellow from Agencia Nacional de Promoción Científica y Tecnológica, and JMR is a fellow from Universidad de Buenos Aires. 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Front Plant Sci 13:1–21. https://doi.org/10.3389/fpls.2022.1009395 Ho T, Murthy HN, Park S (2020) Methyl Jasmonate Induced Oxidative Stress and Accumulation of Secondary Metabolites in Plant Cell and Organ Cultures. Int J Mol Sci. 2020;21(3):716. 10.3390/ijms21030716 . PMID: 31979071; PMCID: PMC7037436 Hyeon H, Jang EB, Kim SC, Yoon S-A, Go B, Lee J-D, Hyun, HB HY-M (2024) Metabolomics Reveals Rubiadin Accumulation and the Effects of. Plants 13:167. https://doi.org/https://doi.org/10.3390/plants13020167 Kannan N, Manokari M, Shekhawat MS (2020) Enhanced production of anthraquinones and phenolic compounds using chitosan from the adventitious roots of Morinda coreia Buck. and Ham. Ind Crops Prod 148:112321. https://doi.org/10.1016/j.indcrop.2020.112321 Krasteva G, Georgiev V, Pavlov A (2021) Recent applications of plant cell culture technology in cosmetics and foods. Eng Life Sci 21:68–76. https://doi.org/10.1002/elsc.202000078 Lei S, Huang B (2022) Metabolic regulation of α-Ketoglutarate associated with heat tolerance in perennial ryegrass. Plant Physiol Biochem 190:164–173. https://doi.org/10.1016/j.plaphy.2022.09.005 Morey KJ, Peebles CAM (2022) Hairy roots: An untapped potential for production of plant products. Front Plant Sci 13:937095. 10.3389/fpls.2022.937095 Murthy HN, Joseph KS, Paek KY, Park SY (2023) Anthraquinone Production from Cell and Organ Cultures of Rubia Species: An Overview. Metabolites 13. https://doi.org/10.3390/metabo13010039 Nallakaruppan N, Thiagarajan K (2024) In vitro elicitation of anthraquinones—a review. Plant Cell, Tissue Organ Cult 156:70. https://doi.org/10.1007/s11240-024-02691-y Perassolo M, Cardillo AB, Mugas ML et al (2017) Enhancement of anthraquinone production and release by combination of culture medium selection and methyl jasmonate elicitation in hairy root cultures of Rubia tinctorum . Ind Crops Prod 105:124–132. https://doi.org/10.1016/j.indcrop.2017.05.010 Perassolo M, Quevedo CV, Giulietti AM, Talou JR (2011) Stimulation of the proline cycle and anthraquinone accumulation in Rubia tinctorum cell suspension cultures in the presence of glutamate and two proline analogs. Plant Cell Tissue Organ Cult 106:153–159. https://doi.org/10.1007/s11240-010-9903-5 Rifqi M, Ramadani N, Jadid N (2024) Review article A comprehensive review of in vitro precursor feeding strategies for the overproduction of high-value plant secondary metabolites. Arab J Chem 17:106018. https://doi.org/10.1016/j.arabjc.2024.106018 Schulte U, Ei-Shagi H, Zenk MH (1984) Optimization of 19 Rubiaceae species in cell culture for the production of anthraquinones. Plant Cell Rep 3:51–54. https://doi.org/10.1007/BF00270970.PMID:24253392 Shetty P, Atallah MT, Shetty K (2003) Stimulation of total phenolics, L-DOPA and antioxidant activity through proline-linked pentose phosphate pathway in response to proline and its analogue in germinating fava beans ( Vicia faba ). Process Biochem 38:1707–1717. https://doi.org/10.1016/S0032-9592(02)00257-1 Soubeyrand E, Colombié S, Beauvoit B et al (2018) Constraint-based modeling highlights cell energy, redox status and α-ketoglutarate availability as metabolic drivers for anthocyanin accumulation in grape cells under nitrogen limitation. Front Plant Sci 9:1–14. https://doi.org/10.3389/fpls.2018.00421 Sreeranjini S, Siril EA (2019) Anthraquinone production in cell culture of Morinda citrifolia L. through cotreatment with elicitors and precursors. J Cytol Genet 20:13–18 Stalman M, Koskamp A-M, Luderer R et al (2003) Regulation of anthraquinone biosynthesis in cell cultures of Morinda citrifolia . J Plant Physiol 160:607–614. 10.1078/0176-1617-00773 Tian W, Wang C, Li D, Hou H (2020) Novel anthraquinone compounds as anticancer agents and their potential mechanism. Future Med Chem. 2020;12(7):627–644. 10.4155/fmc-2019-0322 Wu T, Kerbler SM, Fernie AR, Zhang Y (2021) Plant cell cultures as heterologous bio-factories for secondary metabolite production. Plant Commun 2. https://doi.org/10.1016/j.xplc.2021.100235 Zabalza A, Dongen JT, Van, Froehlich A et al (2009) Regulation of Respiration and Fermentation to Control the Plant Internal Oxygen Concentration 1 [ OA ]. 149:1087–1098. https://doi.org/10.1104/pp.108.129288 Zenk MH, El-Shagi H, Schulte U (1975) Anthraquinone production by cell suspension cultures of Morinda citrifolia . Planta Med 79–101. https://doi.org/10.1055/s-0028-1104768.PMID:1187876 Supplementary Files SupplemData.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 02 Apr, 2026 Reviewers invited by journal 02 Apr, 2026 Editor assigned by journal 23 Jan, 2026 First submitted to journal 22 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8650732","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":616370088,"identity":"b86fbe7e-dbbf-4b8c-bfa3-eb8aee5ea4fb","order_by":0,"name":"Yazmin Kalapuj","email":"","orcid":"","institution":"University of Buenos Aires: Universidad de Buenos Aires","correspondingAuthor":false,"prefix":"","firstName":"Yazmin","middleName":"","lastName":"Kalapuj","suffix":""},{"id":616370089,"identity":"1d4a2b63-b332-490a-b77a-784792e7bb25","order_by":1,"name":"María Perassolo","email":"","orcid":"https://orcid.org/0000-0002-3819-8150","institution":"University of Buenos Aires: Universidad de Buenos Aires","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"","lastName":"Perassolo","suffix":""},{"id":616370090,"identity":"d99223be-fa4a-42da-bd49-f28a23ce83b0","order_by":2,"name":"Julián Marcelo Rodrïguez","email":"","orcid":"","institution":"University of Buenos Aires: Universidad de Buenos Aires","correspondingAuthor":false,"prefix":"","firstName":"Julián","middleName":"Marcelo","lastName":"Rodrïguez","suffix":""},{"id":616370091,"identity":"fe7d7550-48b5-47d8-b1c5-21a287980648","order_by":3,"name":"Alejandra Beatriz Cardillo","email":"","orcid":"","institution":"University of Buenos Aires: Universidad de Buenos Aires","correspondingAuthor":false,"prefix":"","firstName":"Alejandra","middleName":"Beatriz","lastName":"Cardillo","suffix":""},{"id":616370092,"identity":"e8b7d3df-8f9e-4b7f-a803-0b97f154401f","order_by":4,"name":"Julián Rodríguez Talou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie2RvQrCMBhFvxBIHQJdM9lXaBGqIOKrpBTs5KqOASFT0AfwPVxVCk7+rA4dAkLnjo6miLo1HQVzhnCHHO4lAXA4fhAkzKEBut5yT0zct1EwAAfo0SNvqQC8lES1VvDmeqj4Is8UobFGsgjAO+vmYasUM37Kp0bph0iWkaBZ2KwoDCyR+XQXqJghmZv3mDQPMwp+GCWjZlitjIVfWhVSt/C3kghmb4kH/JRFipB5yC9lKpmlJVLofqsWw4ASvNXVrBitfUtLJD6xE9YfBKRZAAi+0dO2yw6Hw/GnPAFQvkJJREvl9AAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0006-9908","institution":"University of Buenos Aires Faculty of Pharmacy and Biochemistry: Universidad de Buenos Aires Facultad de Farmacia y Bioquimica","correspondingAuthor":true,"prefix":"","firstName":"Julián","middleName":"Rodríguez","lastName":"Talou","suffix":""}],"badges":[],"createdAt":"2026-01-20 15:40:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8650732/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8650732/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106724047,"identity":"74f38f4f-f1db-4478-8bca-1abe33e04c26","added_by":"auto","created_at":"2026-04-12 18:24:48","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":112133,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the Anthraquinone metabolic pathways. DHNA: 1,4-dihydroxy-2-naphthoic acid; DMAPP: 3,3-dimethylallylpyrophosphate; GAP: glyceraldehyde-3-phosphate; IPP: isopentenyl-5-pyrophosphate; OSB: o-succinylbenzoic acid; PEP: phosphoenolpyruvate; MEP: 2-C-methyl-D-erythritol 4-phosphate.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8650732/v1/056f0b0b9255c54df8620d0a.jpeg"},{"id":106724005,"identity":"21ab68ad-2e4c-4b3e-b2a0-183c898c3e6b","added_by":"auto","created_at":"2026-04-12 18:23:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8132458,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of MeJa elicitation and Pyr addition on biomass (g FW/L) and IC AQ production (µmol/g FW) in hairy root cultures of \u003cem\u003eR. tinctorum\u003c/em\u003e. Different letters represent significant differences among media according to Duncan’s test (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8650732/v1/3a386e7e955712b4db2ca819.png"},{"id":106724048,"identity":"4141feb5-1d1f-4a92-a6d9-9eef6acc8a35","added_by":"auto","created_at":"2026-04-12 18:24:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8258386,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MeJa elicitation and Pyr addition on total and EC AQ accumulation (µmol/L) in hairy root cultures of \u003cem\u003eR. tinctorum\u003c/em\u003e. Different letters represent significant differences among means according to Duncan’s test (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8650732/v1/a98572051a0a78fd2a67021b.png"},{"id":106435486,"identity":"cd45fe00-95b6-4bfd-981e-289f7d9ed2e6","added_by":"auto","created_at":"2026-04-08 13:52:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3590866,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MeJa elicitation and Pyr and KG addition on biomass production (expressed as gFW/L) and IC AQs (expressed as µmol/g FW) in hairy root cultures of \u003cem\u003eR. tinctorum\u003c/em\u003e. Different letters represent significant differences among means according to Duncan’s test (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8650732/v1/bbc0bdd1bdbfb0f8e8f1e3e6.png"},{"id":106435490,"identity":"96422603-0acf-42df-907c-6ec4feb7d809","added_by":"auto","created_at":"2026-04-08 13:52:03","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":519808,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MeJa elicitation and Pyr and KG addition on total and EC AQs(expressed as µmol/L) in hairy root cultures of \u003cem\u003eR. tinctorum\u003c/em\u003e. Different letters represent significant differences among media according to Duncan’s test (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8650732/v1/9ac93f3956e18750df67f379.jpeg"},{"id":106435487,"identity":"b1edadc6-cd85-43ea-8114-49e20ff18ee9","added_by":"auto","created_at":"2026-04-08 13:52:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3668063,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MeJa elicitation and Pyr and KG addition on specific (expressed as µmol/g FW) and volumetric (expressed as mg/L) TP production in hairy root cultures of \u003cem\u003eR. tinctorum\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8650732/v1/9ae7728f6ca7d03aa4cdede7.png"},{"id":106959638,"identity":"ea990e8e-1a54-481b-9f51-d586a897fa76","added_by":"auto","created_at":"2026-04-15 09:12:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3353127,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MeJa elicitation and Pyr and KG addition on specific (expressed as µmol/g FW) and volumetric (expressed as mg/L) TF production in hairy root cultures of \u003cem\u003eR. tinctorum\u003c/em\u003e. Different letters represent significant differences among media according to Duncan’s test (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8650732/v1/8a81734c60e6fd4cc5b336c0.png"},{"id":108180968,"identity":"f13fdaa9-a00f-450b-bcd2-1e9dcd7601ef","added_by":"auto","created_at":"2026-04-30 08:55:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27965672,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8650732/v1/e5a27999-af8e-4e92-b90c-5bf71536b390.pdf"},{"id":106435484,"identity":"2f8af4fc-9659-443e-a4dc-4ab89fb52022","added_by":"auto","created_at":"2026-04-08 13:52:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":53233,"visible":true,"origin":"","legend":"","description":"","filename":"SupplemData.docx","url":"https://assets-eu.researchsquare.com/files/rs-8650732/v1/8e0d9970c79d7f4f2a275f85.docx"}],"financialInterests":"","formattedTitle":"Pyruvate and α-ketoglutarate precursor feeding combined with methyl jasmonate elicitation increases anthraquinone accumulation in hairy root cultures of Rubia tinctorum","fulltext":[{"header":"Key message","content":"\u003cp\u003ePyruvate, α-ketoglutarate and elicitation increase anthraquinone productivity in hairy root cultures of . Pyruvate and α-ketoglutarate addition stimulate anthraquinone release to the culture medium.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eAnthraquinones (AQs) are specialized metabolites produced by plants of the Rubiaceae family that have been used as dyes since antiquity. In traditional medicine, crude extracts of \u003cem\u003eR. tinctorum\u003c/em\u003e have been used to treat and remove calcium oxalate and phosphate kidney stones. Additionally, interesting biological activities have been attributed to AQs, including antibacterial and antiviral properties, and they have also shown efficacy in photodynamic therapy against cancer cells (Duval et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tian 2020; Campora et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe three-ring base structure can be substituted in different ways, leading to a wide variety of compounds. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, rings A and B of AQs in Rubiaceae are derived from the condensation of α-ketoglutarate (KG) and isochorismate (Murthy et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The latter is formed by the action of isochorismate synthase (ICS) on chorismate, the final product of the shikimate pathway. The C ring is formed by incorporating a DMAPP unit that comes from the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The first step in this route is the conversion of glyceraldehyde 3-phosphate (GDP) and pyruvate (Pyr) into deoxyxylulose phosphate (DOXP), catalyzed by the deoxyxylulose phos phate synthase (DXS)(Eichinger et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Murthy et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePlant cell culture technology has emerged as an alternative platform for the production of specialized metabolites under controlled conditions (complying with GMP standards) and in shorter times. In addition, the cultivation of plant cells in bioreactors avoids unsustainable extractivism, reduces the use of land, herbicides, and insecticides, and decreases the production of gases responsible for the greenhouse effect (Gubser et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hasnain et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, these bioprocesses can be optimized through different strategies, including culture media design, elicitation, release and \u003cem\u003ein situ\u003c/em\u003e product removal (ISPR), precursor feeding, and metabolic engineering (Krasteva et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eElicitation with methyl jasmonate (MeJa) is one of the most effective strategies for increasing the production of specialized metabolites (Ho et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nallakaruppan and Thiagarajan \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This endogenous elicitor has been successfully applied to increase gingenosides, taxanes, podophyllotoxin, tropane alkaloids, rosmarinic acid, valerenic acid, and artemisinin, among others (Morey and Peebles \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). MeJa also triggers AQ production in suspension and hairy root cultures of \u003cem\u003eR. tinctorum\u003c/em\u003e, an effect that is mediated by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e signaling (Perassolo et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Perassolo et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrecursor feeding is another strategy to enhance the accumulation of specialized metabolites in plant cell cultures. The exogenous supply of pathway intermediates can also help to elucidate potential carbon flux limitations or regulatory bottlenecks within the metabolic pathway under study ; (Gerszberg and Wiktorek-Smagur, 2022; Rifqi et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)). Pyr and KG are key intermediates in the energy metabolism of plant cells and, apart from AQ biosynthesis, are also involved in the synthesis of many amino acids. KG has also been described as a signal molecule and has antioxidant activity. In addition, it participates in many enzymatic reactions mediated by 2-oxoglutarate-dependent dioxygenase (2-OD) in several plant metabolic pathways (Lei and Huang \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Soubeyrand et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, we evaluated the effects of the addition of precursors (Pyr and KG) and MeJa elicitation on the AQ production in hairy root cultures of \u003cem\u003eR. tinctorum\u003c/em\u003e. Effects on the phenylpropanoid and flavonoid pathways were also analyzed.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Plant cell culture\u003c/h2\u003e \u003cp\u003eHairy roots were obtained as described by Perassolo et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The cultures were maintained by subculturing the roots every 30 days in Lloyd and McCown\u0026rsquo;s Woody Plant Medium (WPM) contained in 100 mL Erlenmeyer flasks on a gyratory shaker at 100 rpm. The temperature was set at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C with a 16-h photoperiod using cool white LED lamps at an intensity of 1.8 W/m\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. MeJa elicitation and precursor addition\u003c/h2\u003e \u003cp\u003eElicitation and precursor feeding analysis followed two separate experiments with a full factorial design. In both cases, the experiments were initiated by inoculating approximately 0.25 g (fresh weight, FW) of \u003cem\u003eR. tinctorum\u003c/em\u003e hairy roots into 100 mL Erlenmeyer flasks that contained 25 mL of fresh medium, and cultures were harvested at 7 days post elicitation (dpe). The pH was kept constant by the addition of 50 mM MES buffer, pH 5.7, and all the treatments were performed in triplicate.\u003c/p\u003e \u003cp\u003eThe first experiment tested 2 variables, MeJa and Pyr, at two levels (2\u003csup\u003e2\u003c/sup\u003e): no addition of MeJa or Pyr was coded as 0, whereas MeJa (100 \u0026micro;M) and Pyr (20 mM) addition were coded as 1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After 14 days of culture, MeJa or its diluent (ethanol) was added to the Erlenmeyer flasks and hairy roots were cultured for 2 more days. At that moment Pyr or its diluent (water) was added, and the roots were cultured until harvest time (7dpe).\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\u003eFull factorial design (2\u003csup\u003e2\u003c/sup\u003e) for the experiment combining Pyr and MeJa\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExperimental Unit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eLevel Codification\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatment Codification\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMeJa\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003ePyr\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eMeJa (\u0026micro;M)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003ePyr (mM)\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\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eM0P0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eM0P1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eM1P0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eM1P1\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\u003eThe second experiment evaluated 3 variables, MeJa, Pyr and KG, at two levels (2\u003csup\u003e3\u003c/sup\u003e): no addition of MeJa, Pyr or KG was coded as 0, whereas MeJa (100 \u0026micro;M), Pyr (20 mM) and KG (20 mM) addition were coded as 1 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After 21 days of culture, MeJa or its diluent (ethanol) was added and hairy roots were cultured for 2 days. At that moment, Pyr, KG and/or their diluent (water) were added to the Erlenmeyer flasks and roots were cultured until harvest time (7dpe).\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\u003eFull factorial design (2\u003csup\u003e3\u003c/sup\u003e) for the experiment combining Pyr, KG and MeJa\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExperimental Unit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eLevel Codification\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatment Codification\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeJa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePyr\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMeJa (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePyr (mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eKG (mM)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eM0P0K0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eM0P1K0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eM0P0K1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eM0P1K1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eM1P0K0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eM1P1K0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eM1P0K1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eM1P1K1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Biomass determination\u003c/h2\u003e \u003cp\u003eFor fresh weight (FW) determination, hairy root cultures were harvested, washed twice with distilled water and vacuum-filtered for 2 minutes. Finally, the root biomass was placed on filter paper for 5 minutes to eliminate the excess of water and then weighed on an analytical balance. The final biomass was expressed as g FW/L, considering the FW determination and the volume of medium in each Erlenmeyer flask (25 mL). Root tissues were subsequently ground to a fine powder with liquid nitrogen and stored at -80\u0026deg;C for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Anthraquinone determination\u003c/h2\u003e \u003cp\u003eThe AQ content was determined according to Perassolo et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For intracellular (IC) AQs, approximately 100 mg of frozen biomass (FW) was extracted several times with 1.5 mL of 80% ethanol for 30 minutes at 80\u0026deg;C. The extracted fractions were collected together and the absorbance was measured spectrophotometrically at 434 nm. The extracellular (EC) AQ content was determined in sample medium, previously centrifuged at 13,000 rpm for 5 minutes. In both cases, the AQ content was estimated using the molar extinction coefficient of alizarin (5.5 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), the most abundant AQ in \u003cem\u003eR. tinctorum\u003c/em\u003e, according to Schulte et al. (Schulte et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). IC AQ content was expressed as \u0026micro;mol/g FW, whereas EC AQ content was expressed as \u0026micro;mol/L. Total AQs (\u0026micro;mol/L) were calculated as the addition of EC AQs\u0026thinsp;+\u0026thinsp;IC AQs, considering the final biomass achieved (g FW/L).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Total phenolic determination\u003c/h2\u003e \u003cp\u003eThe total phenolic (TP) content was determined by the Folin‒Ciocalteu method, according to Shetty et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Briefly, 100 mg of frozen roots were extracted with 100% methanol, and the samples were stored at -20\u0026deg;C for 48\u0026ndash;72 hours. Fifty \u0026micro;l of the extracts were mixed with 375 \u0026micro;l of a 0.5 N Folin-Ciocalteu solution and incubated for 5 minutes, and then 375 \u0026micro;l of a 1 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e solution were added. The samples were incubated in the dark for 1 hour, and the absorbance was measured spectrophotometrically at 725 nm (Shetty et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). A standard curve prepared with different concentrations of hydroquinone in 95% ethanol, and the absorbance values were converted to mg TP (hydroquinone equivalents) per gram of biomass (FW).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Total flavonoid quantification\u003c/h2\u003e \u003cp\u003eTotal flavonoid (TF) determination was performed according to Dewanto et al. (Dewanto et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). A total of 125 \u0026micro;l of the methanolic extracts (obtained as stated in subsection 2.5) were mixed with 630 \u0026micro;l of distilled water and 38 \u0026micro;l of a 5% NaNO\u003csub\u003e2\u003c/sub\u003e solution. The samples were incubated for 6 minutes, and 75 \u0026micro;l of a 10% AlCl\u003csub\u003e3\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO solution was added. After 6 min of incubation, 75 \u0026micro;l of a 1 M NaOH solution was added, the final volume was adjusted to 1.25 mL with distilled water, and the absorbance was recorded at 510 nm. A calibration curve was generated with rutin as a standard, and the absorbance values were converted to mg of flavonoids (as rutin equivalents) per gram of biomass (FW).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical analysis\u003c/h2\u003e \u003cp\u003eFor both factorial experiments, the significance of the treatment effects was evaluated using two-way ANOVA followed by post hoc analysis using the Duncan\u0026acute;s test. The software used for these analyses was InfoStat 2018 Version (Di Rienzo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). All experimental data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three independent replications.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Pyruvate addition and MeJa elicitation\u003c/h2\u003e \u003cp\u003eIn the first set of experiments (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), hairy root cultures of \u003cem\u003eR. tinctorum\u003c/em\u003e were elicited with MeJa on day 14. After two days of elicitation, Pyr was added, and the cultures were collected on day 7 post elicitation. In all the treatments, the pH levels at the end of the experiment were between 5.0 and 5.7.\u003c/p\u003e \u003cp\u003eThe effects of these treatments on IC AQs (\u0026micro;mol/g FW) are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. IC AQ content when only Pyr was added (M0P1) reached 10.62 \u0026micro;mol/g FW, whereas it was 8.65 \u0026micro;mol/g FW in M0P0 cultures. On the other hand, MeJa-treated cultures resulted in AQ contents of 16.35 \u0026micro;mol/g FW (M1P0) and 17.36 \u0026micro;mol/g FW (M1P1). As two-way ANOVA revealed, the interaction between factors was not significant, but both Pyr and MeJa had positive and significant effects on IC AQ accumulation (Table S1 of Supplementary Information). While Pyr addition accounted only for 3.8% of the total variance, MeJa accounted for almost 91% of the total variance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilar results were observed for total AQs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which increased up to 73% and 83% in M1P0 and M1P1 treatments, respectively, when compared to M0P0 treatment. However, no significant differences were found between M0P1 and M0P0 treatments, probably due to a slight decrease in hairy root growth after Pyr addition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe analysis of EC AQs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) showed that both variables positively affected the release of these specialized metabolites into the culture medium. Two-way ANOVA (Table S2) revealed a significant positive interaction between Pyr and MeJa, so the effects of one variable at each level of the other were analyzed.\u003c/p\u003e \u003cp\u003eWhen MeJa was 0, Pyr addition had a positive effect on EC AQs, increasing from ~\u0026thinsp;9.2 to ~\u0026thinsp;20.5 \u0026micro;mol/L, but in the presence of MeJa (level 1) EC AQs increased from ~\u0026thinsp;22.5 to ~\u0026thinsp;46 \u0026micro;mol/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Similar effects were observed for MeJa at each level of Pyr. These results were also plotted in an interaction graph, showing nonparallel lines (see Figure S1), which indicates that an interaction between the two factors occurred.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Effects of MeJa elicitation and Pyr and KG addition\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Effects on AQ production\u003c/h2\u003e \u003cp\u003eTo gain insight into the effects of feeding precursors on AQ accumulation, the addition of both Pyr and KG after elicitation with MeJa was assayed with a full factorial design (2\u003csup\u003e3\u003c/sup\u003e, see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Hairy root cultures were elicited on day 21, Pyr and KG were added two days after MeJa elicitation, and cultures were harvested at 7 days post-elicitation. For all the treatments, the pH values at the end of the experiment were between 5.0-5.7.\u003c/p\u003e \u003cp\u003eThe effects of this experiment on biomass production and IC AQs are plotted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. While MeJa was the only variable that had a significant negative effect on biomass production, the three variables affected AQ production, although double and triple interactions were not significant. MeJa represents 89% of the total variance in the model, whereas KG and Pyr account for 3.6% and 1.9%, respectively. In accordance with this, the increase in IC AQs attributed to Pyr and KG addition was 6.02 and 8.44%, respectively, whereas that of MeJa elicitation was 50.35%. ANOVA results are included as Supplementary Information (Table S3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA multiple comparison study revealed that M1P1K1 treatment resulted in the highest IC AQ production (18.5 \u0026micro;mol/g FW), followed by that of M1P0K1 treatment (17.3 \u0026micro;mol/g FW). Compared with M0P0K0, M1P0K0 and M1P1K0 treatments also resulted in higher IC AQ content (15.9 and 16.5 \u0026micro;mol/gFW, respectively, compared to 10.4 \u0026micro;mol/g FW of M0P0K0).\u003c/p\u003e \u003cp\u003eTotal and EC AQs are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The analysis of total AQs revealed that MeJa elicitation had the highest effect on total AQ accumulation (~\u0026thinsp;2020 \u0026micro;mol/L), when Pyr and KG were at the highest level (M1P1K1). KG and Pyr added individually also showed a positive effect on AQ production in the presence of MeJa, with AQ concentrations of ~\u0026thinsp;1860 (M1P0K1) and ~\u0026thinsp;1820 \u0026micro;mol/L (M1P1K0). When KG and Pyr were at level 0, the addition of MeJa resulted in an AQ content of ~\u0026thinsp;1790 \u0026micro;mol/L. At level 0 of MeJa, KG and Pyr alone or combined resulted in increases in total AQ production (~\u0026thinsp;1370, ~\u0026thinsp;1400, and ~\u0026thinsp;1410 \u0026micro;mol/L in M0P0K1, M0P1K0 and M0P1K1, respectively), which were significantly higher than that of M0P0K0 treatment (~\u0026thinsp;1240 \u0026micro;M). Triple interaction was significant, which means that the effect observed for any double interaction changes across the levels of the third variable. MeJa explained 87% of the variance of the model, whereas KG, Pyr, and the triple interaction represented 4.6%, 2.3%, and 1.4%, respectively (Table S4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe analysis of double interactions and simple effects revealed that KG-Pyr interaction was not significant at any level of MeJa, but KG showed significant differences at both levels of MeJa, while Pyr effect was significant only at MeJa\u0026thinsp;=\u0026thinsp;1. Regarding the interactions between MeJa and each precursor, they were significant only at level 1 of the other precursor. In other words, MeJa-KG interaction had a significant effect only at Pyr\u0026thinsp;=\u0026thinsp;1, whereas MeJa-Pyr interaction had a significant effect at KG\u0026thinsp;=\u0026thinsp;1. On the other hand, MeJa alone had a significant effect at any combination of KG and Pyr, whereas Pyr had a significant effect in the presence of MeJa, or both MeJa and KG. Regarding KG, it showed significant differences at almost any combination of MeJa and Pyr, except for the case where Pyr\u0026thinsp;=\u0026thinsp;1 and MeJa was 0. The effects of each factor and the interaction can be more easily perceived in Figure S2.\u003c/p\u003e \u003cp\u003eA slightly different response was observed for the accumulation of EC AQs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As shown in Table S5, the ANOVA reveals that although triple interaction was not significant, all the three double interactions (Pyr-KG, Pyr-MeJa and KG-MeJa) were significant. KG addition was the principal factor involved in the release of AQs into culture medium (58% of the total variance). On the other hand, MeJa accounted for 14% of the total variance, whereas Pyr accounted for only 4%. Further analysis of the simple effects revealed that the addition of KG provoked a significant increase in EC AQs at Pyr\u0026thinsp;=\u0026thinsp;0, and this effect was more pronounced at Pyr\u0026thinsp;=\u0026thinsp;1. The same behaviour was observed when analyzing simple effects of KG at each level of MeJa, although in this case the synergism resulted in a higher release of AQs. The analysis of Pyr interactions revealed that Pyr addition significantly increased AQ release when KG was also added (for Pyr-KG interaction), but it only increased AQ release when MeJa\u0026thinsp;=\u0026thinsp;0 (for Pyr-MeJa interaction). Similar results were observed for MeJa: it had a positive impact on AQ release only at KG\u0026thinsp;=\u0026thinsp;1 (for MeJa-KG interaction), and only when Pyr\u0026thinsp;=\u0026thinsp;0 (for Pyr-MeJa interaction). M1P0K1 treatment produced the highest EC AQs contents (56 \u0026micro;M), which represented a 6.1-fold increase compared with that in M0P0K0 treatment (9.2 \u0026micro;mol/L, see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Effects on phenylpropanoid and flavonoid pathways\u003c/h2\u003e \u003cp\u003eEffects of Pyr and KG addition together with MeJa elicitation on total phenolics (TP) and total flavonoids (TF) were also analyzed. The results of specific and volumetric TP production are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The ANOVA analysis revealed no significant differences between the main variables analyzed and their interactions, for both specific and volumetric TP content (Tables S6 and S7).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe study of specific and volumetric TF production revealed that only MeJa had a significant effect on TF content (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Tables S8 and S9). The treatments where MeJa\u0026thinsp;=\u0026thinsp;1 showed the highest levels of TF specific production, ranging from 6.9 to 7.1 mg/g FW, while those where MeJa\u0026thinsp;=\u0026thinsp;0 showed TF contents varying between 3.4 to 3.7 mg/g FW. The volumetric production showed a similar behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Effectively, volumetric TF in cultures where MeJa was added (MeJa\u0026thinsp;=\u0026thinsp;1) ranged from ~\u0026thinsp;720 to ~\u0026thinsp;780 mg/L, whereas their production was between ~\u0026thinsp;390 and 440 mg/L in cultures where MeJa\u0026thinsp;=\u0026thinsp;0.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe main focus of this research was to study the relationship between precursor addition and MeJa elicitation in hairy root cultures of \u003cem\u003eR. tinctorum\u003c/em\u003e. In the first set of experiments, we evaluated the combination of Pyr and MeJa following a 2\u003csup\u003e2\u003c/sup\u003e full factorial design. As mentioned in the introduction, Pyr is involved in the first step of the MEP pathway, and this route generates the DMAPP unit that gives rise to the C-ring of AQs. Our results showed that Pyr alone had a significant but little effect on IC AQ accumulation, whereas MeJa provoked a marked increase in both IC and total AQs. The most interesting finding was the synergism between Pyr and MeJa on EC AQ accumulation. Each factor alone increased AQ release, but the combination of both had a deep impact on EC AQ accumulation (greater than the sum of both effects).\u003c/p\u003e \u003cp\u003eThese promising results led us to incorporate another AQ precursor to our research. KG was chosen since its condensation with isochorismate is the first step in the biosynthesis of rings A and B of AQs. We tested Pyr and KG addition combined with MeJa by performing a 2\u003csup\u003e3\u003c/sup\u003e full factorial experiment. Our results showed that Pyr and KG in combination with MeJa increased IC and total AQ production. Although MeJa elicitation had the greatest impact on AQ accumulation in \u003cem\u003eR. tinctorum\u003c/em\u003e hairy roots, Pyr and KG also triggered AQ production. Effectively, MeJa elicitation and precursor feeding had a significant interaction effect, resulting in a total AQ production of ~\u0026thinsp;2020 \u0026micro;M. These effects revealed that Pyr and KG could be limiting substrates in the biosynthetic pathways of AQs in hairy root cultures of \u003cem\u003eR. tinctorum\u003c/em\u003e. These results are similar to those obtained with other AQ-producing plant cell cultures. Zenk et al. (Zenk et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1975\u003c/span\u003e) reported that the addition of the precursor ortho-succinylbenzoic acid (OSB) resulted in a 2-fold increase in the AQ content in \u003cem\u003eM. citrifolia\u003c/em\u003e cell cultures compared with that in untreated cell cultures. Stalman et al. (Stalman et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) reported that the addition of 20 mM Pyr resulted in a 4-fold increase in AQ accumulation in \u003cem\u003eMorinda citrifolia\u003c/em\u003e cell cultures. In related research, the addition of KG produced a 4.8-fold increase in AQ accumulation in \u003cem\u003eM. citrifolia\u003c/em\u003e cell suspensions compared with that in control cultures (Sreeranjini and Siril \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). KG addition (30 mg/L) also resulted in a significant increase in alizarin, purpurin and total AQ content in adventitious root cultures of \u003cem\u003eR. cordifolia\u003c/em\u003e (Gnanaraj et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Glutamate, an amino acid that can be converted into KG and proline in plants, was added to cell suspensions of \u003cem\u003eR. tinctorum\u003c/em\u003e (5 mM), and resulted in higher AQ content (Perassolo et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). On the other hand, cell suspensions of \u003cem\u003eCinchona\u003c/em\u003e \u0026acute;Robusta\u0026acute;, a Rubiaceae species, were fed with glyceraldehyde, tectoquinone, ferulic acid, and caffeic acid. The addition of glyceraldehyde, an earlier precursor of the MEP pathway, did not result in any increase in AQ accumulation. However, AQ levels were increased by 48% compared with those in control cultures when caffeic acid was fed to cell cultures, probably due to an indirect effect on the AQ biosynthetic pathway (Han et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The feeding of Pyr and KG to callus cultures of \u003cem\u003eCassia angustifolia\u003c/em\u003e, a non-Rubiaceae species, resulted in an approximately 30% increase in the Sennoside content (Chetri et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother interesting aspect we want to evaluate was the release of AQs to the culture medium. This is a highly relevant topic of research, since metabolite release improves product recovery and opens the opportunity to design strategies such as \u003cem\u003ein situ\u003c/em\u003e product removal. We found differences in the effects of Pyr on AQ release between our experiments: it had a synergic effect with MeJa in the first experiment but no synergism between them was detected in the second experiment. One possible explanation is the age of cultures: elicitation and precursor addition occurred between 14\u0026ndash;21 days of culture in the first experiment, whereas in the second experiment, they occurred between 21\u0026ndash;28 days of culture. Nevertheless, the incorporation of KG in the second experiment led to an interesting outcome: the increased AQ release produced by the addition of both precursors, Pyr and KG. Our research showed that KG resulted in the main factor responsible for the release of AQs into the culture medium. Its combination with MeJa elicitation resulted in the highest EC AQ content (56 \u0026micro;M). The presence of high ketoacid concentrations, such as Pyr, induced an increase in the oxygen consumption rate in pea roots, resulting in hypoxia in the plant roots, even in well-aerated nutrient solutions. Hypoxia has been described as an abiotic stress that could trigger specialized metabolite production in plants and might be another factor that explains the increase in AQ accumulation and release to the culture medium due to Pyr and KG addition (Zabalza et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince phenolic and flavonoid biosynthetic pathways compete for carbon precursors with the AQ pathways, we also evaluated the effect of elicitation and precursor addition on the production of these metabolites. In the present study, only MeJa elicitation enhanced total flavonoid accumulation (between 1.6- to 1.8-fold increase). These results are similar to those found in \u003cem\u003eM. citrifolia\u003c/em\u003e adventitious root cultures. Effectively, chitosan elicitation resulted in a 2-fold increase on AQ, total phenolic and flavonoid accumulation, compared to control cultures (Baque et al. 2012; Baque et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012b\u003c/span\u003e). MeJa also triggered AQs and phenolics production in adventitious roots of \u003cem\u003eR. tinctorm\u003c/em\u003e (Bi\u0026ccedil;er et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A metabolomic study in MeJa-elicited suspension cultures of \u003cem\u003eDamnacanthus major\u003c/em\u003e also showed higher accumulation of the AQ rubiadin and the flavonoid kaempferol, than those of unelicited cultures (Hyeon et al \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Furthermore, adventitious roots of \u003cem\u003eM. coreia\u003c/em\u003e elicited with chitosan showed higher AQ and total phenolic production than non-elicited cultures (Kannan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). On the contrary, external addition of KG and Pyr did not produce a positive effect on flavonoid accumulation in \u003cem\u003eR. tinctorum\u003c/em\u003e hairy roots. In \u003cem\u003eVitis vinifera\u003c/em\u003e cell suspensions, nitrogen limitation raised the intracellular levels of KG, resulting in higher flavonoid and anthocyanin accumulation. KG could be the metabolic signal responsible for this response, since it participates as a reducing agent in many enzymatic steps of the flavonoid pathway (Soubeyrand et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn summary, the present study reveals that both precursors of the AQ biosynthetic route, Pyr and KG, could be limiting the carbon flux to this specialized metabolic pathway, and this effect is more pronounced after MeJa elicitation. MeJa also stimulated the flavonoid pathway, whereas Pyr and KG did not affect flavonoid production. This paves the way for the development of other biochemical and metabolic engineering strategies to increase the carbon flux to AQ biosynthesis by raising Pyr and KG cellular levels and/or inhibiting the flavonoid pathway.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANOVA Analysis of variance\u003c/p\u003e\n\u003cp\u003eAQs \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;anthraquinones\u003c/p\u003e\n\u003cp\u003eKG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;α-ketoglutarate\u003c/p\u003e\n\u003cp\u003eDPE \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;days post elicitation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDW \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Dry weight\u003c/p\u003e\n\u003cp\u003eFW \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fresh weight\u003c/p\u003e\n\u003cp\u003eMeJa \u0026nbsp; \u0026nbsp; \u0026nbsp;methyl jasmonate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePyr \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;pyruvate\u003c/p\u003e\n\u003cp\u003eTF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; total flavonoids\u003c/p\u003e\n\u003cp\u003eTP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; total phenolics\u003c/p\u003e\n\u003cp\u003eWPM \u0026nbsp; \u0026nbsp; Lloyd and McCown’s Woody Plant Medium\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFundings:\u0026nbsp;\u003c/strong\u003eThis work was supported by Agencia Nacional de Promoción Científica y Tecnológica (PICT 2020-02033), Universidad de Buenos Aires (UBACyT 2023- 217BA) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP 2651). MP, ABC, VDB and JRT are researchers from CONICET. YRK is a fellow from Agencia Nacional de Promoción Científica y Tecnológica, and JMR is a fellow from Universidad de Buenos Aires.\u003c/p\u003e\n\u003cp\u003eCompeting interests:\u0026nbsp;The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eData availability statement: Data will be available on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompliance with Ethical Standards: Ethics declaration: not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaque MA, Elgirban A, Lee EJ, Paek KY (2012a) Sucrose regulated enhanced induction of anthraquinone, phenolics, flavonoids biosynthesis and activities of antioxidant enzymes in adventitious root suspension cultures of \u003cem\u003eMorinda citrifolia\u003c/em\u003e (L). 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Planta Med 79\u0026ndash;101. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1055/s-0028-1104768.PMID:1187876\u003c/span\u003e\u003cspan address=\"10.1055/s-0028-1104768.PMID:1187876\" 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":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Anthraquinones, Rubia tinctorum, Hairy roots, Elicitation, Methyl jasmonate, Precursor feeding","lastPublishedDoi":"10.21203/rs.3.rs-8650732/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8650732/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnthraquinones (AQs) are specialized metabolites produced by \u003cem\u003eRubia tinctorum\u003c/em\u003e that have traditionally been used as dyes, but also have important biological activities. The present research studied the effects of the addition of pyruvate (Pyr) and α-ketoglutarate (KG) precursors in combination with methyl jasmonate (MeJa) elicitation, via a full factorial design, on hairy root cultures of \u003cem\u003eR. tinctorum\u003c/em\u003e. MeJa was the main factor that triggered AQ accumulation, and the addition of Pyr and KG also significantly enhanced AQ production. Feeding of each precursor resulted in increased intracellular and total AQ production, which revealed that Pyr and KG are limiting precursors of AQ biosynthesis in hairy root cultures of \u003cem\u003eR. tinctorum\u003c/em\u003e. The three variables showed a significant interaction effect, resulting in total AQ production of ~\u0026thinsp;2022 \u0026micro;M. Moreover, the addition of Pyr and KG increased AQ release into the culture medium. KG was the main factor responsible for the secretion of AQs; combination with MeJa elicitation resulted in 56 \u0026micro;M of AQs in the culture medium. The phenolic and flavonoid contents were also evaluated, and the results showed that MeJa elicitation triggered only flavonoid production. At the same time, precursor addition had no significant effect on phenolic or flavonoid accumulation.\u003c/p\u003e","manuscriptTitle":"Pyruvate and α-ketoglutarate precursor feeding combined with methyl jasmonate elicitation increases anthraquinone accumulation in hairy root cultures of Rubia tinctorum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-08 13:51:58","doi":"10.21203/rs.3.rs-8650732/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-04-02T11:44:40+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-02T06:48:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-24T03:41:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2026-01-22T09:05:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"df5a2db2-7673-42ec-a3aa-f517feae4758","owner":[],"postedDate":"April 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-08T13:51:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-08 13:51:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8650732","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8650732","identity":"rs-8650732","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}