Integration of phenylalanine precursor feeding and elicitor treatment in concert with the cellulase-assisted extraction approach to improve stilbene compound production

Trans-resveratrol, trans-arachidin-1 (Ara-1), and trans-arachidin-3 (Ara-3) are major stilbene compounds found in elicited peanut hairy root culture and exert diverse potential biological and pharmaceutical activities. The aim of this study was to investigate the various approaches for enhancing the productivity of prenylated stilbene. The fast-growing peanut hairy root K2-K599-C line was elicited with chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD) (CHT + MeJA + CD) as combined elicitors together with phenylalanine (Phe) as a precursor to feeding at 24, 48, and 72 h. The accumulation of stilbene compounds was investigated in this study as well as stilbene biosynthesis-related genes and plant defense gene expression. Upon an exogenous supply of Phe, elevated amounts of Ara-1 and Ara-3 were detected through the entire 72 h elicitation period while the non-Phe precursor feeding conditions exhibited an increasing amount of Ara-1 and Ara-3 up to 48 h, decreasing at 72 h. In addition, Phe precursor may serve as an early regulator of gene expression for the stilbene biosynthesis pathway, transcription factors, and pathogenesis-related proteins while a delayed response in transcriptional regulation was observed in the elicitation without Phe precursor feeding. Cellulase-assisted extraction was performed prior to solvent extraction of the culture medium. The results indicated a significant 1.73- and 1.57-fold increase in Ara-1 and Ara-3, respectively, compared to the non-cellulase-assisted process. To the best of our knowledge, this is the first study to propose an integrated approach to improving stilbene compound production in peanut hairy root culture. An integrated approach of precursor feeding, elicitors, and enzyme-assisted extraction facilitates the improvement of stilbene compound production. Phenylalanine feeding induces early transcriptional regulation of plant response. Cellulase-assisted extraction improves the recovery of stilbene compounds.


Introduction
Stilbene compounds, such as trans-resveratrol (RES), pterostilbene, and trans-arachidin (Ara) are natural phenolic antioxidants and important to the plant defense mechanism.Stilbenes with fungicidal activity are produced in several unrelated plant species, such as the grapevine (Vitis vinifera), pine (Pinus sylvestris), and peanut (Arachis hypogaea).Several studies have indicated that stilbene compounds exert beneficial health properties.According to recent data, stilbene compounds represent potential antioxidant activity and are capable of suppressing oxidative stress-related disease.

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The most extensively studied natural stilbene is RES which exerts antimicrobial, anti-inflammatory, cardioprotective, and neurodegenerative disease prevention (Zhang et al. 2021).RES has been reported to exhibit anti-tumor properties with potent cytotoxicity while increasing the level of cytoplasmic nucleosomes and the DNA fragmentation mechanism in apoptotic HT-29 cells (Li et al. 2010).RES has also demonstrated the attenuation of oxidative damage in pancreatic tissue and protection of β cells in diabetes.Therefore, RES may be useful in diabetes prevention and treatment (Szkudelski and Szkudelska 2011).Besides, the prenylated forms of trans-arachidin-1 (Ara-1) and transarachidin-3 (Ara-3) are the most significant stilbene compounds found in peanut sprout and peanut hairy root culture.The attachment of the prenyl group to the stilbene backbone increases hydrophobicity and is reported to enhance the bioactivity of molecules compared to RES.Additionally, Ara-3 demonstrated strong antioxidant activity over RES by inhibiting the intracellular free radicals in PMA-induced HL-60 cells (Sobolev et al. 2011).The Ara-1 has attracted increased attention due to the inhibition of PGE2-or NO-mediated inflammation of RAW 264.7 macrophage cells (Djoko et al. 2007).Ara-1 has been reported to process anticancer activity in epithelial triple-negative breast cancer (TNBC) cell lines by promoting caspase-dependent cell apoptosis via the intrinsic pathway (Mohammadhosseinpour et al. 2022).
Peanut hairy root culture is a promising tool in the production of stilbene compounds due to its genetic stability and ability to promote rapid growth in hairy root tissue.Several strategies have been applied to improve metabolite production such as elicitation, precursor feeding, and gene manipulation.In this approach, elicitors are defined as substances that activate the plant physiological and morphological defense response to induce plant secondary metabolite production.The elicitor molecules can be abiotic elicitors such as chemical substances and physical stresses, while biotic elicitors can be derived from living organisms such as bacteria and fungi.The co-treatment of methyl jasmonate (MeJA) and methyl-β-cyclodextrin (CD) indicated the enhancement of RES, piceatannol, Ara-1, and Ara-3 in peanut hairy root culture (Yang et al. 2015).Different combinations of MeJA, CD, H 2 O 2, and MgCl 2 demonstrated differential improvement in Ara-1 and Ara-3 production in peanut hairy root culture (Sharma et al. 2022).The Kalasin2 peanut hairy root (K2-K599) culture established in our laboratory demonstrated high productivity in prenylated stilbenoids Ara-1 and Ara-3 by elicitation with various elicitor strategies such as a combination of paraquat (PQ), MeJA, and CD.The pre-exposure of PQ prior to the approach involving a combination of MeJA and CD in PQ_(MeJA + CD) induced different amounts of stilbene compounds compared to the post-application of PQ in the (MeJA + CD)_PQ, indicating a distinct response from the plant mechanism to elicitor treatment (Somboon et al. 2019).Due to the toxicity of PQ, an alternative elicitor substance was chosen, namely the environmentally friendly chitosan (CHT) in combination with MeJA and CD.The synergistic effect of CHT + MeJA + CD treated hairy root resulted in a higher yield of Ara-1 and Ara-3 compared to the PQ_ (MeJA + CD) approach (Chayjarung et al. 2021).
Precursor feeding is a well-established approach for inducing plant secondary metabolites.The addition of a precursor substrate probably increases the metabolic flux, thus enhancing the production of desired targets.Farnesyl pyrophosphate (FPP) was used as a precursor for artemisinin production in Artemisia annua L. hairy root culture through the acetate mevalonate (MVA) pathway.A 1.25-fold increase in artemisinin was observed compared to the control (Ahlawat et al. 2014).The feeding of 0.5 mM phenylalanine substrate into the phenylpropanoid pathway has been reported to enhance the production of wedelolactone, phenolics, and flavonoids in Sphagneticola calendulacea (L.) Pruski hairy root culture (Kundu et al. 2018).The production of β-caryophyllene, which exerts anti-inflammatory and anticancer activity, was investigated in Artemisia vulgaris L. callus and hairy root culture.Farnesyl diphosphate (FDP) at a concentration of 3 µM was used for precursor feeding through the MVA and MEP pathway, with the results demonstrating an improvement in β-caryophyllene production for both callus and hairy root culture (Balasubramani et al. 2021).Stilbene compounds are synthesized via the phenylpropanoid pathway starting with amino acid l-phenylalanine (Phe).The combination of tyrosine precursor feeding and the elicitation approach was employed to investigate the accumulation of stilbene compounds in Morus alba root culture.The results indicated that tyrosine feeding promoted stilbene synthesis during the early stage of growth while the elicitor treatment provided the highest content of mulberroside A, oxyresveratrol, and RES (Inyai et al. 2021).The phenylalanine ammonia lyase (PAL) is the first enzyme to catalyze Phe into cinnamic acid, with the diverse enzymes further catalyzing the various intermediates in the pathway.In the final step, the enzyme stilbene synthases (STS) or resveratrol synthases (RS) are responsible for catalyzing the formation of stilbene compound and obtaining the RES as a product.RES can be further transformed into high molecular-ordered stilbene oligomers in plants by specific enzymes as described by Jeandet et al. (2023); Jeandet et al. (2021).
Plant cell walls consist of multi-components of the polysaccharide structure such as cellulose, hemicellulose, and pectin, forming a rigid plant barrier.Cell wall-digesting enzymes such as cellulase, hemicellulase, and pectinase play a key role in the breakdown of plant cell walls.Numerous studies have recommended the use of cell walldigesting enzyme prior to conventional solvent extraction to improve the yield of plant bioactive compounds.
Pectinase was applied as an enzymatic pretreatment in Rosmarinus officinalis L. leaves for 1 h before solid-liquid extraction with 50% hydroethanolic solvent for 24 h.The obtained extract exhibited higher DPPH antioxidant activity (IC50 14.3 ± 0.8 µg/mL) than non-enzymatic pretreatment (IC50 20.6 ± 1.1 µg/mL) (Pontillo et al. 2021).The use of 2.5 U/mL cellulase prior to aqueous extraction has been reported to accelerate the breakdown of β-(1-4) glycosidic linkage in cell wall cellulose and enhance the extraction yield of phenolic compounds from pistachio green hull by 22% (Ghandahari Yazdi et al. 2019).Enzyme-assisted extraction allows the release of bioactive compounds adhered to the plant cell wall and improves the extraction yield in the following step of conventional extraction.
To date, various elicitor treatments have been elucidated to enhance the production of stilbene compounds in peanut hairy root culture.However, very few studies have reported a combination of the elicitation and precursor feeding approach in concert with the combination of enzymeassisted extraction to improve stilbene compound production.This study has a primary objective of investigating the number of stilbene compounds in peanut hairy root culture.To achieve this, a unique integration approach is employed, combining precursor feeding and elicitor treatment using CHT + MeJA + CD.Alongside the stilbene compound accumulation analysis, the expression of stilbene biosynthesisrelated genes and plant defense genes are thoroughly examined.Additionally, the study incorporates cellulase-assisted extraction prior to conventional solvent extraction.The findings from this research hold significant potential in advancing the production of bioactive stilbenes, marking a crucial step forward in this area of study.

Plant material
The new peanut hairy root line C (K2K599-C) was established from the peanut seedling cultivar Kalasin2, transformed with A. rhizogenes K599 (NCPPB 2659) according to the protocol described by Pilaisangsuree et al. (2018).The PCR analysis was used to confirm the hairy root line K2K599-C.The specific primers of rolB and rolC were used to detect the T-DNA integration region and virD primer was used to verify the non-contamination of the hairy root line.All primer sequences and PCR amplification conditions are demonstrated in Supplementary Table S1.This K2-K599-C peanut hairy root line was maintained by root multiplication subculture on antibiotic-free MS agar medium every 2 weeks.

Elicitor treatment and precursor feeding
Elicitation experiments were performed using co-treatment of 200 mg/L CHT, 100 µM MeJA, and 6.87 mM CD (CHT + MeJA + CD) as described by Chayjarung et al. (2021) Precursor feeding was performed by varying concentrations of phenylalanine (Phe) substrate of 5, 10, 15, 20, and 25 mM.In total, 4 g of K2-K599-C hairy roots were cut into 500-mL Erlenmeyer flasks containing 200 mL of ½ Murashige and Skoog (MS) medium and shaken with a rotary shaker at 150 rpm for 72 h.
The selected optimum concentration of Phe was added along with the combination of elicitors CHT + MeJA + CD as described above.Small scale hairy root culture was performed with 1 g of K2-K599-C hairy root in 50 mL of ½ MS medium.The elicitor treatment in combination with precursor feeding experiments were performed over time steps of 24, 48, and 72 h under dark condition at 25 °C with three biological replicates.

Culture medium extraction
Culture medium extraction was performed using two different procedures: standard solvent extraction and cellulase-assisted extraction.Standard solvent extraction was carried out by partitioning the culture medium with equal volumes of ethyl acetate and the process repeated 3 times.The evaporation of the ethyl acetate fraction was achieved using a rotary evaporator (Büchi) at 40 °C of dryness.The crude extract of hairy root culture medium was collected and weighed.EtOH was used to re-dissolve the crude extract to obtain a 10 mg/ml concentration for further analysis.
Cellulase-assisted extraction was performed by adding 0.5% (w/v) cellulase into the elicited hairy root culture before subjecting it to the standard solvent extraction process.The hairy root culture was incubated at 25 °C and shaken for 1 h, as previously described by Tothong et al. (2022).Following the incubation with cellulase, the hairy root tissues were isolated from the culture medium.The culture medium was further subjected to standard solvent extraction as described above.

High-performance liquid chromatography (HPLC)
The hairy root medium crude extract was analyzed using the HPLC method as described by Chayjarung et al. (2020).HPLC analysis was performed on a reversed-phase system with a C18 column (Luna 5 μm C18 (2) 100 A column, Phenomenex).Separation was achieved using mobile phase acetonitrile: water with 2% formic acid (30:70, v/v) at a constant flow rate of 1 mL/min.The quantitative analysis of RES was calculated according to the standard curve of commercial RES.The purified fractions of Ara-1 and Ara-3 were identified by the LC-MS/MS method and the content of each compound determined based on the standard curve of each purified fraction.The Ara-1 and Ara-3 amounts were expressed as mg/g dry weight.

RNA extraction and cDNA synthesis
The total RNA was isolated from the K2-K599-C hairy root tissue using the RNeasy plant mini kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions.The NanoDrop ND-2000 spectrophotometer (Thermo Scientific, USA) was used for quantifying the RNA samples.One mg of RNA was further reverse transcribed into cDNA using the ReverTra Ace™ qPCR RT master mix with gDNA remover (TOYOBO, Osaka, Japan).

Quantitative real-time PCR (qPCR)
The qPCR was performed using the CFX96 Touch real-time PCR detection system (Bio-Rad, California, USA).The specific primers in the phenylalanine ammonia lyase (PAL) family including PAL, PAL2; resveratrol synthase (RS) family including RS, RS3; WRKY transcription factor 31 (WRKY31); transcription factor MYB36-like (MYB36); ethylene response factor (ERF3); pathogenesis-related protein 2-like (PR2) and STH-2-like (STH2) were designed using the NCBI Primer Blast tool.All gene sequences were obtained from A. hypogaea, available in the nucleotide database (Gen-Bank, NCBI) as described in Supplementary Table S1.The specificity of the primer was confirmed by single melting peak analysis and the expected PCR product size visualized by agarose gel electrophoresis.All qPCR reactions were prepared following the instructions of iTaq™ universal SYBR® Green Supermix (Bio-Rad, California, USA).The amplification reactions were as follows: pre-denaturation step at 95 °C for 10 min, followed by 36 cycles with denaturation at 95 °C for 15 s, annealing at optimum temperature as described in Supplementary Table S1 for 10 s, and extension at 72 °C for 10 s.The relative quantification was reported as the fold change of target gene expression in the treated sample relative to the untreated sample, normalized to an elongation factor 1 alpha (ef1a) gene.

Scanning electron microscope (SEM)
The hairy root tissues were freeze-dried to dehydrate prior to SEM analysis.The longitudinal and transverse sections of hairy root samples were positioned on the sticky surface of the stubs and sputter coated.The SEM images were created using an SEM instrument (LEO Model 1450VP Variable Pressure, Carl Zeiss, Oberkochen, Germany) at a voltage of 1-30 kV.

Statistical analysis
Statistical analysis was performed by one-way analysis of variance (ANOVA) with a post-hoc test using SPSS software version 17.0.Statistically significant differences were considered at the P < 0.05 level.

PCR confirmation of hairy root line
The emerging roots from wounded sites were observed following 4 weeks of agrobacterium transformation.Individual transformed root clones were isolated and sub-cultured in MS medium containing antibiotic cefotaxime (100 mg/L) and carbenicillin (100 mg/L) until no contamination of agrobacterium could be observed.The selected K2-K599-C hairy root line was based on the rapid growth characteristic with high root biomass obtained after several subculture periods.The PCR identification of rolB, rolC, and virD2 genes was performed to confirm the K2-K599-C hairy root line compared to A. rhizogenes plasmid.According to the results in Fig. 1, the hairy root line K2-K599-C demonstrated the integration of agrobacterium rolB and rolC genes into the plant chromosomes with PCR product sizes of 495 and 480 bps, respectively.The virD2 gene with the 338 bps PCR product was not detected in K2-K599-C hairy root, indicating no contamination of the hairy root line with A. rhizogenes.

Optimum concentration of phenylalanine precursor feeding
The optimum concentration of Phe precursor feeding was verified in combination with the elicitor treatment.Various Phe substrate concentrations of 5, 10, 15, 20, and 25 mM were simultaneously added to the hairy root culture elicited with CHT + MeJA + CD and shaken for 72 h.The hairy root tissues and culture medium were separated after the elicitor and Phe precursor feeding procedures.The non-elicited hairy root control demonstrated a light-yellow color of hairy root tissue and culture medium as presented in Fig. 2a and  h.The elicitor CHT + MeJA + CD treated hairy root culture exhibited an intense yellow hairy root tissue as well as culture medium as shown in Fig. 2b and i.The elicitor CHT + MeJA + CD treatment in concert with Phe feeding exhibited a yellow to brown shade in the hairy root tissue and yellowish in the culture medium by increasing the range of the Phe concentration from 5 to 20 mM as demonstrated in Fig. 2c-f and j-m.Interestingly, at a higher concentration of 25 mM Phe feeding, the hairy root tissue appeared to be pale yellow in both the hairy root tissue and culture medium as illustrated in Fig. 2g and n.
The yellowish color of the hairy root tissue and culture medium indicated the production of stilbene compounds from hairy root tissue and these compounds were secreted into the culture medium as reported in our previous publication (Somboon et al. 2019).Therefore, the number of secreted stilbene compounds in the culture medium was detected using the HPLC method.The HPLC chromatogram of stilbene compounds including RES, Ara-1, and Ara-3 is demonstrated in Fig. 3.The amount of RES, Ara-1, and Ara-3 was compared between the elicitor CHT + MeJA + CD treatment sample and the combination of elicitor CHT + MeJA + CD and Phe precursor feeding.As the results in Fig. 4a show, significant increase in RES content to 1.54-and 1.43-fold were observed in the combined treatment of elicitor together with 15 mM Phe and 20 mM Phe precursor feeding, respectively, compared to elicitor treatment without Phe precursor feeding.Interestingly, the content of prenylated stilbene Ara-1 and Ara-3 was increased to 1.59-fold (Fig. 4b and 1.35-fold Fig. 4c), respectively, by adding 5 mM Phe precursor, indicating a significant difference from the treatment without Phe precursor feeding.However, no significant difference in Ara-1 and Ara-3 content was observed by increasing the Phe concentration to 10-25 mM Phe.Therefore, 5 mM Phe was chosen for the optimum concentration of precursor feeding for further experimentation.

Stilbene content at various time steps
A small scale hairy root culture treated with elicitor CHT + MeJA + CD in combination with 5 mM Phe precursor feeding (CHT + MeJA + CD + 5 mM Phe) was analyzed at time intervals of 24, 48, and 72 h.The stilbene compounds including RES, Ara-1, and Ara-3 were determined and compared between the un-elicited group (control), elicited with CHT + MeJA + CD group, and CHT + MeJA + CD + 5 mM Phe precursor feeding group.According to the results in Fig. 5, the un-elicited control group exhibited insignificant amounts of all the stilbene compounds, while the treatment with CHT + MeJA + CD demonstrated a significantly higher amount for all stilbene compounds than the control group (Fig. 5a-c).An increasing trend of RES, Ara-1, and Ara-3 was observed in the CHT + MeJA + CD treated group by increasing the elicitation time from 24 to 48 h while gradually decreasing at 72 h (Fig. 5a-c).The addition of 5 mM Phe as a precursor in combination with CHT + MeJA + CD, demonstrated a similar result to the CHT + MeJA + CD treated group but with an increasing trend in RES, Ara-1, and Ara-3 content compared to the non-Phe precursor feeding group.The highest RES, Ara-1, and Ara-3 content was detected in the Phe precursor feeding group at 48 h of treatment time.However, an insignificant difference was observed between CHT + MeJA + CD and CHT + MeJA + CD + 5 mM Phe precursor feeding.A significant difference in all stilbene content between CHT + MeJA + CD and CHT + MeJA + CD + 5 mM of Phe precursor feeding was observed only at the treatment period of 72 h (Fig. 5a-c).

Gene expression profiles
The gene expression profiles of PAL, PAL2, RS, RS3, WRKY31, MYB36, ERF3, PR2, and STH2 were generated in hairy root culture treated with CHT + MeJA + CD and CHT + MeJA + CD + 5 mM Phe precursor feeding at 24, 48, and 72 h.The fold change of gene expression was compared with the untreated hairy root control at each time point.According to the results in Fig. 6, all examined genes were upregulated with different patterns at various time points.Interestingly, at the early time point of 24 h, the PAL, PAL2, MYB36, ERF3, PR2, and STH2 genes significantly increased with the combination treatment of CHT + MeJA + CD + 5 mM Phe precursor feeding, at fold changes of 8.65-, 6.44-, 5.02-, 6.23-, 10.18-, and 93.82-, respectively, compared to the CHT + MeJA + CD treatment without the Phe precursor.At 48 h of treatment time, the PAL2, RS3, and STH2 gene expressions in the CHT + MeJA + CD treatment without the Phe precursor were upregulated with fold changes of 12.29-, 52.37, and 160.08, respectively, demonstrating higher expression

Gene expression patterns and stilbene compounds
The relationship between stilbene biosynthesis gene expression and stilbene content is demonstrated in Fig. 7.
Fig. 4 The quantification of stilbene compounds: a trans-resveratrol (RES), b trans-arachidin-1 (Ara-1), and c trans-arachidin-3 (Ara-3) in K2-K599-C hairy root culture medium elicited with a combination of elicitor CHT + MeJA + CD and various concentrations of Phe precursor feeding.Data represented as mean ± standard deviation.Different lowercase letters express the statistically significant differences (P < 0.05) Fig. 5 The quantification of stilbene compounds in culture medium: a trans-resveratrol (RES), b trans-arachidin-1 (Ara-1), and c trans-arachidin-3 (Ara-3) of non-elicited K2-K599-C hairy root control, elicited with a combination of CHT + MeJA + CD and CHT + MeJA + CD + 5 mM Phe precursor feeding at 24, 48, and 72 h.Data represented as mean ± standard deviation.Different lowercase letters express the statistically significant differences (P < 0.05) 1 3 Hairy root culture elicited with CHT + MeJA + CD exhibited the highest amount of Ara-1 and Ara-3 at 24 and 48 h while decreasing at 72 h (Fig. 7a and c), while the combination treatment of CHT + MeJA + CD + 5 mM Phe demonstrated a constant amount of Ara-1 and Ara-3 over the entire treatment period (Fig. 7b and d).At 24 h of elicitation, the supplement with the Phe precursor feeding upregulated the PAL, PAL2 gene expression to 8.65-and 6.44-fold changes (Fig. 7b), while the non-precursor feeding condition showed lower expression levels of 2.07-and 3.06-fold changes, respectively (Fig. 7a).A consistent PAL gene expression was observed in Phe precursor feeding conditions throughout the entire treatment period (Fig. 7b).However, a significant increase in PAL gene expression was detected from 24 to 72 h in non-precursor feeding conditions (Fig. 7a).The RS and RS3 genes from both treatment groups showed similar trends in the expression level, increasing from 24 h to the highest level at 48 h, while decreasing at 72 h, with the highest RS3 gene expression level observed at 48 h in non-precursor feeding conditions with a 52.37-fold change (Fig. 7a, b).
At the early 24 h elicitation period, the trend of the WRKY31, MYB36, and ERF3 transcription factor gene expression accords with the stilbene biosynthesis gene, demonstrating a higher gene expression level in Phe precursor feeding conditions with 8.60-, 5.02-, and 6.23-fold changes over the non-precursor feeding conditions which exhibited gene expression changes of 5.41-, 0.87-, and 2.34-fold, respectively (Fig. 7c, d).At 48 h, the WRKY31 gene expression tended to increase in both Phe precursor feeding and non-precursor feeding conditions, while decreasing afterward.In contrast, the MYB36 and ERF3 demonstrated lower gene expression at 48 h and a significant increase with a further late response at 72 h (Fig. 7c,  d).

Cellulase-assisted extraction and stilbene content
The cellulase-assisted extraction approach was applied to the hairy root culture elicited with CHT + MeJA + CD + 5 mM Phe precursor feeding to improve the number of stilbene compounds obtained from the extraction process.By adding 0.5% cellulase (w/v) to hairy root culture and shaking in the incubator for 1 h prior to standard solvent extraction of the culture medium, significant increases in Ara-1 and Ara-3 were observed of 1.73-and 1.57-fold, respectively, compared to standard solvent extraction without the cellulaseassisted process (Fig. 8).

SEM image of hairy root tissue
After cellulase-assisted extraction, the hairy root tissues were separated from the culture medium and subjected to SEM analysis.The SEM analysis was performed with the untreated hairy root control (Fig. 9a-c), the treated hairy root with CHT + MeJA + CD + 5 mM Phe precursor (Fig. 9d-f), and treated hairy root with a combination of CHT + MeJA + CD + 5 mM Phe precursor after cellulaseassisted extraction (Fig. 9g-i), The image of a smooth epidermal root surface on longitudinal section (Fig. 9a, b) and the transverse section with vascular tissue surrounded by undamaged cortex and outer epidermis were observed in the untreated hairy root control (Fig. 9c).The treatment of hairy root tissue with the elicitor and Phe precursor revealed a partial vertical fracture on the root surface (Fig. 9d) with irregular aggregates localized on the root exterior (Fig. 9e).The transverse section of the treated hairy root appeared to be slightly detached at the root cortex (Fig. 9f).The elicited hairy root treated with cellulase exhibited greater damage to the area along the root layer (Fig. 9g) with a dense irregular aggregate compacted on the root surface (Fig. 9h).Impairment of the root's epidermis was also observed on the transverse section of the hairy root treated with cellulase (Fig. 9i).

Discussion
This study emphasizes the improvement of stilbene compound production using diverse strategies.A new peanut hairy root line K2-K599-C was established in our laboratory to produce the stilbene compounds by elicitation.Stilbenes are a group of polyphenolic compounds and demonstrated to be defensive response to various exogenous stimuli (Chong et al. 2009).In the present study, the new K2-K599-C peanut hairy root line was elicited with a combination of elicitors CHT + MeJA + CD as previously described by Chayjarung et al. (2021).However, the highest content in each stilbene compound following the elicitation was observed at 48 h, differing from our previous K2-K599 line which showed the highest content at 72 h (Chayjarung et al. 2021).The distinct variation could be attributed to the integration of a random gene into the plant genome of the diverse hairy root line by A. rhizogenase (Butaye et al. 2005).
The exogenous supply of the precursor involved in the specific biosynthesis pathway is another approach for improving the desired secondary metabolite.The precursor feeding approach which uses tyrosine as a precursor has been successfully reported to enhance the production to 8.1-fold L-DOPA in Mucuna pruriens cell suspension culture (Rakesh and Praveen 2022).The growing of a Rhodiola rosea plant in MS agar, supplemented with cinnamyl alcohol as a precursor, induced the production of secondary metabolite rosin and rosavin to 92.7-and 8.0-fold, respectively (Javid et al. 2021).To improve the productivity of secondary metabolites, the integration of 5 mg/L phenylalanine precursor and combined elicitors of 50 mg/L methyl jasmonate and 1 mg/L dextran has been reported to increase the anthocyanin content and yield to 6.1-and 4.6-fold in Vitis vinifera cell suspension culture (Qu et al. 2011).It was initially reported that the combination of chitosan as an elicitor and squalene as a precursor induced the production of withanolides content in Withania somnifera cell suspension culture (Sivanandhan et al. 2014).The present study demonstrates the highest stilbene content including RES, Ara-1, and Ara-3 by elicitation with CHT + MeJA + CD at 48 h, and an increasing trend in these compounds was observed by integrating the elicitor treatment with Phe precursor feeding.The production of stilbene compounds with elicitor treatment was reduced by increasing the elicitation period to 72 h.Interestingly, the combination of elicitors and Phe precursor feeding prolonged the production of stilbene compounds through 72 h.The sufficiency of Phe as a starting precursor in the metabolic pathway could facilitate the improvement of secondary metabolite production for an extended period.
This study also investigated the gene transcript involved in the stilbene biosynthesis pathway, transcription factor, and pathogenesis-related protein.All genes were upregulated following the elicitor treatment with and without precursor feeding.However, the diverse gene expression pattern in both groups was observed at various time points.The PAL gene family: (PAL and PAL2), MYB36, ERF3, PR2, and STH2 revealed a greater gene expression level at 24 h after treatment with the CHT + MeJA + CD + 5 mM Phe precursor compared to CHT + MeJA + CD without precursor feeding.Phenylalanine ammonia lyase (PAL) is a key enzyme in the phenylpropanoid pathway, thus the PAL could be a major regulatory enzyme for responding to the supplementary Phe precursor in the stilbene biosynthesis pathway.It has been reported that different gene members in the family displayed a diverse gene expression pattern in response to developmental stages and distinct stresses (Rasool et al. 2021).The integration of Phe precursor feeding and elicitor treatment could probably accelerate the rapid response of stilbene biosynthesis and plant defense mechanism by rapidly inducing early response genes.Hence, the supplementation of Phe as a precursor feeding could induce the stilbene biosynthesis and prolong the constant number of Ara-1 and Ara-3 over the entire treatment period of 72 h, while the non-precursor feeding conditions resulted in a high number of Ara-1 and Ara-3 from 24 to 48 h, and decreasing at 72 h as illustrated by the combined graph in Fig. 7. Additionally, the increasing trend in the transcription factor WRKY31, MYB36, and ERF3 gene expression was also observed in Phe precursor feeding conditions over the non-precursor at the early time point.The MYB, ERF, and WRKY transcription factors have reportedly been involved in the regulation of diverse stresses in plants (Chen et al. 2014;Cui et al. 2021;Zhao et al. 2020).The MYB proteins also play a role as transcriptional activator or repressor to regulate the phenylpropanoid biosynthesis pathway (Liu et al. 2015).In addition, proteins such as STH2 and PR2 are also involved in the plant defense mechanism.The STH2 protein was rapidly induced in Solanum tuberosum after elicitation with Phytophthora infestans zoospores (Constabel and Brisson 1992).The PR2 protein has been reported to act as a plant defense response against Leptosphaeria maculans by inducing callose deposition in Arabidopsis (Constabel and Brisson 1992).Therefore, the Phe precursor could perform as an early metabolic regulator in cells, related to the biosynthesis of enzymes in the metabolic biosynthesis pathway, and may be involved in regulating the various signaling pathways encompassing the plant defense response.At the middle stage of 48 h, the PAL2 and STH2 from elicitors without precursor feeding were highly upregulated compared to elicitors with precursor feeding, while at the later stage of 72 h, the PAL, WRKY31, ERF3, and PR2 gene expressions last through the late response period.This suggests that in Based on the previous report, stilbene compounds produced from peanut hairy root were secreted into liquid culture medium after elicitation, although the secretion mechanism not yet fully understood (Pilaisangsuree et al. 2018).The liquid-liquid extraction method was used to facilitate the recovery of stilbenes from the culture medium since it can only extract the most obtainable stilbene compounds.Enzymes were applied to break down the plant cell wall prior to conventional solvent extraction, thus increasing the release of plant bioactive compounds from the intracellular part.In the present study, cellulase enzyme-assisted extraction was also applied in combination with CHT + MeJA + CD + 5 mM Phe precursor feeding to enhance stilbene compound recovery.According to the results, the optimum condition of cellulase enzyme-assisted extraction with 0.5% cellulase (w/v) and incubated shaking for 1 h (Tothong et al. 2022) before ethyl acetate extraction of the elicited hairy root culture medium showed a significant 1.73-and 1.57-fold increase in Ara-1 an Ara-3, respectively, compared to the non-enzymatic extraction process.The SEM image of cellulase-treated elicited hairy root revealed an impairment in the root epidermis with the destruction area along the root layer, indicating damage to the outer layer of the hairy root cell wall.A dense irregular aggregate substance attached along the root surface probably confirms the release of intracellular compounds.These results accord with those of Zu et al. (2009), who reported that the use of 1 mg/mL concentration cellulase can increase the extraction of paclitaxel yield from Taxus chinensis to 1.78-fold compared to the control.The combination of enzyme cellulase and pectinase was first utilized to assist in the hydrolyzing of a blueberry cell wall, releasing the anthocyanins and then extracting with citric acid-acidified 75% ethanol.The results indicated that enzymatic-assisted solvent extraction facilitated the maximum 25% extraction rate of anthocyanins from blueberry (Xu et al. 2016).

Conclusion
The transformed peanut hairy root culture line K2-K599-C was used as a source for stilbene compound production.The combination of CHT + MeJA + CD elicitation and Phe precursor feeding was applied to investigate the number of RES, Ara-1, and Ara-3 at various time points of 24, 48, and 72 h.The elicitor approach aimed to activate the plant defense response by various external substances which can induce plant secondary metabolites to alleviate the effect of various stresses.Supplementation of the Phe precursor may serve as an early metabolic regulator and accelerate the rapid response of stilbene biosynthesis throughout the entire 72 h elicitation period.In addition, several genes involved in the stilbene biosynthesis pathway (PAL and PAL2), transcription factors (MYB36, ERF3), and pathogenesis-related proteins (PR2, STH2) were highly upregulated at the early response stage of Phe precursor feeding compared to nonprecursor, while PAL2, RS3, WRKY31, MYB36, ERF3, PR2, and STH2 genes demonstrated high up-regulation in nonprecursor feeding at the middle to late stages of elicitation.This suggested that the defense-responsive genes may conduct a delayed response of transcriptional regulation in the elicitation without Phe precursor feeding.The cellulase was eventually employed as an enzyme-assisted process prior to ethyl acetate extraction to improve the recovery of stilbene compounds from the hairy root culture medium.The combination of various approaches, including elicitor treatment, precursor feeding, and enzyme-assisted extraction are promising strategies for improving stilbene compounds productivity from peanut hairy root culture.The scale-up for the mass production of peanut hairy root culture in combination with these elicitors, precursor feeding, and the enzyme-assisted extraction strategy should be further investigated for effectiveness in stilbene compound production.

Fig. 2
Fig. 2The characteristics of peanut hairy roots and culture medium after elicitation with CHT + MeJA + CD in combination with Phe precursor feeding at various concentrations: a and h non-elicited hairy root control, b and i elicitor

Fig. 3
Fig. 3 HPLC chromatograms of the hairy root culture medium treated with a CHT + MeJA + CD, b CHT + MeJA + CD + 5 mM Phe precursor feeding

Fig. 7
Fig. 7 Combination data represent the treatment of a and c CHT + MeJA + CD elicitor treatment, b and d CHT + MeJA + CD + 5 mM Phe precursor feeding treatment; with the effect of a and b stilbene content and stilbene biosynthesis gene

Fig. 9
Fig. 9 Scanning electron microscopy (SEM) of K2-K599-C peanut hairy roots: a-c untreated hairy root control, d-f hairy root treated with CHT + MeJA + CD + 5 mM Phe, and g-i hairy root treated with CHT + MeJA + CD + 5 mM Phe following cellulase-assisted extraction