Sodium lignosulfonate improves shoot growth of Oryza sativa via enhancement of photosynthetic activity and reduced accumulation of reactive oxygen species

Lignosulfonate (LS) is a by-product obtained during sulfite pulping process and is commonly used as a growth enhancer in plant growth. However, the underlying growth promoting mechanism of LS on shoot growth remains largely unknown. Hence, this study was undertaken to determine the potential application of eco-friendly ion-chelated LS complex [sodium LS (NaLS) and calcium LS (CaLS)] to enhance recalcitrant indica rice MR 219 shoot growth and to elucidate its underlying growth promoting mechanisms. In this study, the shoot apex of MR 219 rice was grown on Murashige and Skoog medium supplemented with different ion chelated LS complex (NaLS and CaLS) at 100, 200, 300 and 400 mg/L The NaLS was shown to be a better shoot growth enhancer as compared to CaLS, with optimum concentration of 300 mg/L. Subsequent comparative proteomic analysis revealed an increase of photosynthesis-related proteins [photosystem II (PSII) CP43 reaction center protein, photosystem I (PSI) iron-sulfur center, PSII CP47 reaction center protein, PSII protein D1], ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), carbohydrate metabolism-related proteins (glyceraldehyde-3-phosphate dehydrogenase 3, fructose-bisphosphate aldolase) and stress regulator proteins (peptide methionine sulfoxide reductase A4, delta-1-pyrroline-5-carboxylate synthase 1) abundance in NaLS-treated rice as compared to the control (MSO). Consistent with proteins detected, a significant increase in biochemical analyses involved in photosynthetic activities, carbohydrate metabolism and protein biosynthesis such as total chlorophyll, rubisco activity, total sugar and total protein contents were observed in NaLS-treated rice. This implies that NaLS plays a role in empowering photosynthesis activities that led to plant growth enhancement. In addition, the increased in abundance of stress regulator proteins were consistent with low levels of peroxidase activity, malondialdehyde content and phenylalanine ammonia lyase activity observed in NaLS-treated rice. These results suggest that NaLS plays a role in modulating cellular homeostasis to provide a conducive cellular environment for plant growth. Taken together, NaLS improved shoot growth of recalcitrant MR 219 rice by upregulation of photosynthetic activities and reduction of ROS accumulation leading to better plant growth.


Results
Physiological responses of MR 219 rice shoot towards CaLS and NaLS treatments. Utilization of ion chelated LS required details optimization in order to provide beneficial effects to the plant. In this study, different concentrations of NaLS and CaLS (0, 100, 200, 300 and 400 mg/L) were supplemented into MS medium in order to determine their growth promoting effects during shoot growth of MR 219 rice. Shoot apex was selected in this study as it contained shoot apical meristem cells that contributed to building block of special-Scientific Reports | (2021) 11:13226 | https://doi.org/10.1038/s41598-021-92401-x www.nature.com/scientificreports/ ized tissues in the aerial parts of the test plant. In general, high concentration of NaLS (200 mg/L ≥) was found to enhance shoot elongation of the MR 219 rice (Fig. 1a). When compared to control plantlet (MSO; Fig. 1b), no significant changes were recorded with all concentrations tested for CaLS (Fig. 1a,c). On the other hand, the optimum concentration of NaLS was recorded at 300 mg/L, with significant increment in shoot growth (28.15%; Fig. 1d) as compared to MSO (Fig. 1a,b). Hence, further experiments were not performed on CaLS-treated plants. To understand the underlying mechanisms of NaLS in stimulating the shoot growth of rice, comparative proteome profiling and biochemical assays were performed on MSO and 300 mg/L NaLS plantlet (NaLS-treated rice).

Comparative proteome analysis reveals an increased in photosynthesis-related proteins in
NaLS-treated rice. Comparative proteome analysis was carried out to elucidate the changes in MR 219 rice proteome in response to NaLS in plant growth. The proteome analysis was performed using Perseus Software v1.6.0.7 (Max Planck Institute of Biochemistry, Planegg, Germany) to execute the Pearson correlation and Principal Component Analysis (PCA). Pearson correlation revealed strong positive correlation (> 0.7) between the MSO and NaLS-treated rice ( Supplementary Fig. S1), which indicated that both MSO and NaLS-treated rice used were derived from the same organism with minimal contamination in the samples. In addition, the score plot of PCA showed grouping pattern of sample, wherein the MSO and NaLS-treated rice samples were totally separated because of significant change in proteome profile detected between treatment groups (Supplementary   Fig. S1) describes the relationship among all proteins identified. These proteins identified in loading plot correlates to the samples as shown in score plot of PCA ( Supplementary Fig. S1). Based on comparative proteome analysis, a total of 41 and 60 unique proteins were successfully identified in the MSO and NaLS-treated rice, respectively; wherein, a total of 371 proteins were shared between the two samples ( Fig. 2a). Through volcanic plot analysis, a total of 56 differentially expressed proteins were identified in response to NaLS. Among these proteins, a total of 15 proteins were upregulated, while a total of 41 proteins were downregulated in NaLS-treated rice as compared to the MSO plantlet ( Fig. 2a-c). In response towards NaLS, proteins with the greatest increase in abundance were photosystem II (PSII) CP43 reaction center protein, photosystem I (PSI) iron-sulfur center, PSII CP47 reaction center protein and PSII protein D1. Meanwhile, proteins with greatest decrease in abundance were chitinase 5, chitinase 1, ATP synthase epsilon chain, thioredoxin reductase NTRB (Table 1). Two upregulated proteins [PSI iron-sulfur center (PSAC), PSII CP47 reaction center protein (PSBC)] and two downregulated proteins [chitinase 5 (CHT5) and chitinase 1 (CHT1)] were selected for gene expression analysis. Gene expression of selected proteins was observed to be in agreement with the proteome profile ( Supplementary Fig. S2). The list of primers used in RT-qPCR can be found in Supplementary Table S2.
The proteins identified were subjected to gene ontology (GO) analysis, in which they were classified into three categories, namely, biological processes, cellular components and molecular functions (Fig. 2d-f). From GO analysis ( Fig. 2d-f) of biological processes, majority of the proteins were categorized under metabolic process (32.50%), followed by cellular process (34.58%), response to stimulus (8.33%) and biological regulation (7.92%). Meanwhile, when categorized under cellular components, majority of the proteins were involved in chloroplast (30.77%), cytoplasm (23.08%), nucleus (14.42%) and mitochondria (8.65%). In molecular functions, majority of the proteins identified were involved in binding (45.27%) and catalytic activity (40.54%). The changes in protein abundance in each category are shown in Fig. 2g. Subsequently, the identified proteins were subjected to KEGG pathway analysis 20 in order to determine the overall effects of NaLS on the proteome during shoot growth (Fig. 2h). Based on KEGG pathway analysis 20 , proteins involved in secondary metabolite biosynthesis were found to be the most affected pathway in response towards NaLS, followed by photosynthesis, carbohydrate metabolism, and lastly, amino acid biosynthesis pathway (Fig. 2h). A detailed categorization of the proteome profile can be found in Supplementary Table S2. www.nature.com/scientificreports/ NaLS enhances protein synthesis and photosynthetic activities during shoot growth. In addition to proteome analysis, biochemical assessments were performed to further elucidate the possible roles of NaLS during shoot growth. Our results in Fig. 3a recorded 3.54 mg/g fresh weight (FW) of total chlorophyll content in NaLS-treated rice as compared to the MSO (2.47 mg/g FW). The accumulation of chlorophyll content was coherent with the significant increase in rubisco activity, whereby significant increase in rubisco activity (1.39 µmol CO 2 /mg protein) was detected in NaLS-treated rice as compared to the MSO (0.54 µmol CO 2 / mg protein; Fig. 3b). Besides, significant increment of sugar content was also recorded in NaLS-treated rice (1.50 mg/g FW) as compared to the MSO (2.11 mg/g FW; Fig. 3c). Likewise, significant increment in protein contents were recorded in NaLS-treated rice (7.82 mg/g FW) as compared to the MSO (4.12 mg/g FW).
Regulation of cellular homeostasis and stress response in NaLS-treated rice. To investigate the possible involvement of secondary metabolites and stress response in NaLS-treated rice, peroxidase activity, malondialdehyde (MDA) content and phenyl ammonia lyase (PAL) activity were determined. The NaLStreated rice recorded a significant decrease in peroxidase activity (0.08 µmol/min mg protein) as compared to  Fig. 4b). As for PAL activity, a significant drop was also observed in NaLS-treated rice (16.84 nM/mg protein) as compared with the MSO (10.65 nM/mg protein; Fig. 4c).

Discussion
Previous study has shown that beneficial effects of LS ion chelated complex is very much dependent on the type of tissues, plant species and type of chelated ions used. For instance, Docquier et al. 11 reported the usage of CaLS (1000 mg/L) resulted in superior growth promoting effects compared to FeLS and potassium LS (KLS) in both Phalaenopsis and Sequoia sempervirens. In another study made by Kevers et al. 13 , a multiplication rate and vigor improvement of a shoot-proliferating poplar cluster were demonstrated after addition of FeLS and CaLS (10 mg/L). Meanwhile, in Vanilla planifolia, optimum shoot multiplication response was observed when supplemented with CaLS and NaLS (150 mg/L) 16 . Based on these studies, different concentrations and different types    15 has demonstrated that incorporation of CaLS promotes callus growth and root formation via enhanced endogenous auxin biosynthesis, nutrient uptake and regulation of cellular homeostasis 15 . However, in this study, NaLS was shown to be a better additive to improve the shoot growth of MR 219 rice when compared to CaLS. In this study, incorporation of NaLS were demonstrated to improve shoot development of MR 219 rice via stimulation of photosynthetic activities, carbohydrate metabolism and reduced accumulation of ROS. Based on these data, it is suggested that different ion-chelated LS may express different mode of action when introduced to different plant development stages 10 . Similar finding has also been reported by Low et al. 12 , whereby application of CaLS modulates different physiological response in different plant development stages of rice. In that study, application of CaLS observed an enhanced callus proliferation rate and adventitious root formation. However, application of CaLS was found to induce albino shoot formation during shoot induction study 12 . This indicate that a different mode of action of LS was expressed during shoot induction.
Incorporation of NaLS were observed to be the superior growth additive in enhancing shoot development when compared to CaLS. Although many studies have regarded that sodium ions (Na + ) as non-essential element in terrestrial plant, their benefits in stimulating plant growth should not be ignored. In optimum concentration, Na + could aids in regulating leaf turgor pressure and chlorophyll concentrations resulting in an enhancement of overall plant photosynthetic activity, which is essential for plant growth and development 21 . On top of that, Na + could also be used as a substitute for K + , reducing K + requirements for plant growth 22 . For instance, the germination rate, total dry weight and nutrient absorption of cotton plants were improved by Na supplementation 23 . Thus, application of NaLS increased the bioavailability of Na + in the medium and enhanced uptake of Na + in the plant, while reducing dependability towards K + for plant growth.
On the other hand, incorporation of CaLS did not promote any shoot development in MR 219 rice. Similar finding was reported by Docquier et al. 11 , whereby supplementation of CaLS did not exhibit any effects on shoot multiplication in S. sempervirens. However, supplementation of CaLS (1000 mg/L) were reported to improve root growth of S. sempervirens. According to Docquier et al. 11 , the effects of LS could be indirect or 'delayed' and they hypothesized that the CaLS may either induce changes in hormonal, mineral balance, numbers of auxin receptor and/or their affinity towards free auxin.
Based on our proteomic data, increased of proteins abundance related to photosynthesis (PSII CP47, CP43, D1 and D2 proteins) was observed suggesting the participation of NaLS in governing photosynthetic rate. These proteins play an important role in modulation of photosynthetic activities in plant, as photosynthesis involves in production of energy and sugar required for plant growth. Moreover, previous study reported that moderate concentration of sodium chloride (up to 50 mM) allowed optimum Na + accumulation on the leaves of Zygophyllum xanthoxylum which led to stimulation of photosynthesis activity and significantly increased relative growth rate 21 . Hence, NaLS may help in enhancing the photosynthetic rate to improve the rice growth as observed in this study.
Photosynthetic process is governed by a complex protein phosphorylation/dephosphorylation cascade 17 . The process is initiated by light-harvesting complexes, which absorb light and transfer excitation energy to the reaction centers of PSII and PSI. Subsequently, linear electron flow from PSII was initiated through a series of electron carrier intermediates producing and eventually reduced NADP to NADPH 24 . In present study, comparative proteome analysis reveals upregulation of photosynthesis-related proteins in response to NaLS ( Table 1). The PSII is a multiprotein-pigment complex, composed of major protein subunits such as reaction center subunits (D1 and D2 proteins) and PSII internal antenna subunits (CP43 and CP47 proteins). According to Table 1, upregulation of PSII major protein subunits (PSII CP47, CP43, D1 and D2 proteins) were observed in NaLS-treated rice, suggesting an enhancement of PSII biogenesis. The PSII is one of the key components involved in initiation of linear electron flow of photosynthesis generating reducing power required for CO 2 fixation to produce NADPH during photosynthesis 25 . Lacking in one of the major photosystem protein subunits may result in reduction of photosynthetic capacity and cellular metabolism. For instance, degradation of reaction center subunits (D1 and D2 proteins) was demonstrated to reduce photosynthetic performance in plants, which eventually reduced plant growth 18,25 .
In addition to proteome analysis, biochemical assessments were conducted to provide a further understanding on the possible roles of NaLS on shoot growth. Consistent with the upregulation of photosynthesis-related proteins in proteome profiling, the total chlorophyll content was significantly enhanced in response to NaLS treatment (Fig. 3a). The positive relationship between chlorophyll content and photosynthetic activity in plant has been widely observed 26 . In plants, chlorophyll is responsible for light-harvesting in photosynthesis, resulting in the excitation of electrons that are used to drive the linear electron flow in photosynthesis. Photosynthetic products produced will then be used for sugar production via Calvin cycle. In Calvin cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays an important role, whereby Rubisco catalyzes the assimilation of CO 2 with ribulose-1,5-bisphosphate (RuBP) producing 3-phosphoglycerate (PGA). The PGA produced will be integrated into the Calvin cycle producing sugars 27 . In present study, the upregulation of Rubisco protein (Table 1) and enhanced Rubisco activity (Fig. 3b) were observed in NaLS-treated rice. Therefore, the increased in chlorophyll content accompanied with enhanced Rubisco activity support the role of NaLS in improving rice growth via enhanced photosynthetic activity.
Photosynthetic activity was known to influence sugar accumulation in plant. The data obtained in Fig. 3c shows that NaLS-treated rice had recorded significant amount of sugar content compared to the MSO. These observations were consistent with previous studies, in which enhanced photosynthetic activity were accompanied with enhanced sugar production 28 www.nature.com/scientificreports/ aldolase (FBA); Table 1] in NaLS-treated rice. These two proteins were known to play a role in glycolysis pathway catalyzing the breakdown of glucose into pyruvate and energy, which will then be used for plant growth 30 . Likewise, NaLS-treated rice recorded a significant increase protein accumulation (Fig. 3d). The increased in protein content is consistent with the enhanced shoot growth and photosynthetic activity. Besides, studies have found positive correlation between protein content with nitrogen supply in plant 19,31 . Supporting this, it was demonstrated that deficiency in nitrogen supply in plant often reduces the growth of leaves by half. Nitrogen content is utilized as a source of building blocks for amino acid, which are prerequisite for protein biosynthesis in plant 19 . Besides, high protein content is crucial during plant growth and development as well. For instance, protein is involved as structural protein, transporter protein, and metabolic reaction in the cells 31 . These activities are important for proper functioning of the plant cells which will provide energy and food source to the plant and subsequently lead to improve plant growth. Aside from photosynthetic process, NaLS was observed to be involved in modulation of ROS and stress in plant ( Fig. 4 and Supplementary Table S1). During photosynthetic process, ROS were constantly generated. While ROS play an important role in plant signaling, excessive accumulation of ROS may cause oxidative damage towards plant cells, which leads metabolic homeostasis impairment. For instance, excessive ROS production caused by absorption of excessive sunlight by the light-harvesting complex resulted in photoinhibition of PSI 32 . Hence, it is vital to maintain cellular homeostasis in the plant during enhancement of photosynthetic activity. In present study, increased in protein abundance of peptide methionine sulfoxide reductase A4 (PMSRA4) and delta-1-pyrroline-5-carboxylate synthase 1 (P5CS1) that were found in NaLS-treated rice could act as stress regulator (Supplementary Table S1). Involvement of these proteins in regulating stress response in plant have been elucidated previously. As suggested by Romero et al. 33 , increased of PMSR4 activity could enhance tolerance level towards oxidative stresses. Similarly, P5CSI was shown to be vital as a stress regulator upon abiotic stress treatment.
Additionally, application of NaLS recorded a significant decrease in peroxidase activity (Fig. 4a). Low levels of peroxidase activity indicate low levels of ROS in plant cell. In line with the decrease in peroxidase activity, NaLS recorded a significant decrease in malondialdehyde (MDA) content (Fig. 4b). MDA is a major product of lipid peroxidation induced mainly by ROS and it reflects lipid peroxidation in plant cells in response towards stress 34 . Significant increase in MDA levels implies severe oxidative damage towards cell membrane. Studies have found that lipid peroxidation is a common phenomenon that occurs in plant cells when subjected to stress, and MDA are often used as a marker to determine the physiological status of the plant during plant growth 35 . On top of that, our study recorded a significant decrease of phenylalanine ammonia lyase (PAL) activity in NaLS-treated rice (Fig. 4c). The PAL is one of the rate-limiting enzymes, known to play a key role in secondary metabolite biosynthesis pathway. Secondary metabolites biosynthesis involves production of phenolic and flavonoid compounds which plays an important role in regulation of cellular homeostasis in plants in response towards stress 36,37 .
In general, regulation of cellular homeostasis is vital for enhancement of plant growth and development. During active acclimatization and adaptation process in response to stress environment, plant requires a bulk amount of energy for ATP biosynthesis and metabolism 38 . This could be seen during salt stress treatment of Hordeum marinum, whereby mitochondrial ATP synthase precursor and soluble inorganic pyrophosphatase 1 were upregulated to catalyze more energy to the plant indicating the urgent need for energy splurge in the form of ATP to establish homeostasis environment in the plant 39 . Therefore, the increase in abundance of stress regulator proteins, decrease in peroxidase activity, MDA content and PAL activity imply that incorporation of NaLS may play a role in reducing stress in plant cell. This allows the plant to be grown in a conducive environment, whereby energy produced by the plant could be used efficiently to promote plant growth and development rather than directed it for plant defense mechanism to maintain cellular homeostasis.

Conclusions
Our study demonstrated that the incorporation of 300 mg/L of NaLS in MS medium effectively enhanced shoot growth of MR 219 rice. Improvement of shoot growth was due to the stimulation of photosynthetic activities in the MR 219 rice, which could be evidenced by increased of PSI-and PSII-related proteins in proteome profiling as well as enhanced chlorophyll content and Rubisco activity in the plant (Fig. 5). In addition, it was found that NaLS successfully regulates cellular homeostasis in the plants, which was demonstrated by enhanced stress regulator proteins, reduced peroxidase activity, MDA content and PAL activity (Fig. 5). Hence, NaLS improved shoot growth of MR 219 rice via enhanced photosynthetic activities and ROS stabilization. Sigma-Aldrich, USA) were prepared in a stock solutions of 50 mg/mL. All the stock solutions were filtered sterilized using 0.22 μm cellulose acetate membrane before being kept at 4 °C.

Seed sterilization and LS treatment.
Surface sterilization were performed on mature MR 219 seeds according to previously described protocol 40 with slight modifications. Firstly, the seeds were de-husked and sterilized with 70% (v/v) ethanol for 1 min, followed by 50% (v/v) Clorox for 30 min. The seeds were rinsed with sterile distilled water before being dried on filter paper. The sterilized seeds were then transferred into our previ- Protein extraction and protein digestion. In proteomic analysis, plant samples were ground into fine powder using liquid nitrogen. The samples were then mixed with 500 μL protein extraction buffer containing 50 mM of ammonium bicarbonate and 10 mM phenylmethylsulfonyl fluoride (PMSF). The mixture was then vortexed, sonicated and centrifuged according to Yang et al. 43 and solubilized protein was collected. Desalting was carried out on total soluble protein obtained using acetone precipitation method 44 . Subsequently, the proteins content in the plant sample was determined at 595 nm through Bradford assay 45 . The proteins sample (100 μg) was digested with Trypsin Gold (Promega, USA) at a ratio of 1:200 parts of protein, according to previous study 43 .
Peptide separation and protein identification. Nano liquid chromatography tandem-mass spectrometry (nanoLC-MS/MS; Thermo Scientific, USA) analysis was performed according to Yang et al. 43 . Briefly, aliquot of 2 μL digested sample was injected into EASY-Spray Column Acclaim PepMap C18 100 (A0, 2 µm particle size, 50 µm id × 15 cm) at 35 °C. The sample elution process was performed similarly as described by Yang et al. 43 .
The eluents from the LC were directly introduced into a mass spectrometer (Orbitrap Fusion-Thermo Fisher Scientific, US). The instrument was operated in the data dependent acquisition. Full scan spectra were collected (OTMS1) using parameters defined by previous study 43 . Only precursors with an assigned monoisotopic m/z and a charge state up to 4 were further analyzed for MS2. All precursors were filtered using a 20 s dynamic exclusion window and intensity threshold of 5000. The MS2 spectra were analyses (ITMS2) following parameters reported by Yang et al. 43 . Precursors were fragmented by CID and HCD at normalized collision energy of 30% and 28%, respectively. Subsequently, raw data was analyzed using Thermo Scientific Proteome Discoverer Software 2.1 and SEQUEST HT was used as the database searching algorithm. The intensities of each MS ion were measured according to the accurate mass and time tag strategy 46 . Identification of the proteins was performed based on the searched against the UniprotKB database restricted to O. sativa (2020_01: 48,904 sequences) with a 1% strict FDR and 5% relax FDR criteria using Percolator. Search parameters were set up according to previous study 42 . Protein quantification and data analysis. The experiment was performed in triplicates with three biological replicates for MSO and NaLS-treated samples. The protein files (txt.format) obtained from Proteome Discoverer were uploaded to Perseus for comparative proteome analysis between MSO and NaLS-treated sam-   46 . Significant differences of protein abundance were determined based on the Student's t test (p < 0.05).
The p-values were also adjusted for multiple-testing using the permutation-based false discovery rate, with a randomization number of 250. Proteins were considered to be significantly differentially expressed between treatment groups with adjusted p-values of < 0.05.
Total photosynthetic pigments content. One hundred milligram of sample was ground into fine powder with the presence of liquid nitrogen. The ground powder was mixed with 2 mL of 80% (v/v) of acetone for 1 min in darkness. The homogenate was then centrifuged at 400×g for 5 min. The absorbance of the samples was then recorded at 470, 647 and 663 nm. The concentration of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll and carotenoids) were calculated according to calculations described in Lichtenthaler et al. 47 and expressed in mg/g fresh weight (FW).
Rubisco activity. Rubisco activity was measured spectrophotometrically according to Usuda 48  Total soluble sugar content. Total soluble sugar content was estimated according to Dubois et al. 49 with slight modifications. In brief, 0.1 g of sample was ground into powder in liquid nitrogen and extracted twice in 2 mL of 90% (v/v) ethanol at 60 °C for one hour. After each extraction, samples were centrifuged at 400×g for 5 min. One mL of the supernatant was mixed with 1 mL of 5% (v/v) phenol together with 5 mL of concentrated sulphuric acid. The mixture was allowed to cool before the readings was taken at 495 nm spectrophotometrically. The soluble sugar content was determined using glucose as a standard and expressed in mg/g FW.
Total protein content. Two hundred fifty milligram of plant sample were ground into powder with liquid nitrogen. Ice cold extraction buffer containing 1.8 mL of 50 mM ammonium bicarbonate (ABC) and 0.2 mL of 100 mM PMSF was added to the powdered sample. The homogenate was centrifuged at 10,000×g for 30 min and supernatant was collected as crude enzyme. All steps in enzyme extraction were performed at 4 °C. Bradford assay was performed to determine the protein concentration at 595 nm 45 . Total protein content in the sample was then determined by using bovine serum albumin as standard.
Peroxidase activity. The crude enzyme obtained during protein extraction was used in determination of peroxidase activity. One hundred microliters of crude enzyme were added with 950 µL of distilled water, 750 µL of 100 mM potassium phosphate buffer (pH 6.8), 600 µL of 100 mM pyrogallol and 600 µL of 100 mM H 2 O 2 . Peroxidase activity were measured at 420 nm between the second and fifth minute after addition of crude enzyme into the substrate. The peroxidase activity was expressed as micromoles of purpurogallin formed per minute per milligram of proteins 50 .
Malondialdehyde (MDA) content. MDA content was measured according to Luo et al. 51 with slight modifications. Approximate 0.2 g of frozen powdered samples were dissolved in 10 mL of 10% (w/v) trichloroacetic acid (TCA). The homogenate was then centrifuged at 12,000×g for 10 min. Subsequently, 2 mL of supernatant was mixed with 2 mL of 10% (w/v) TCA containing 0.6% (w/v) of thiobarbituric acid (TBA) and incubated at 100 °C for 20 min. Then, the homogenate was allowed to cool on ice followed by centrifugation at 12,000×g for 10 min. The supernatant was then measured at 532, 600 and 450 nm. The MDA content was calculated using the following formula; MDA content (μM) = 6.45 (OD 532 − OD 600 ) − 0.56 (OD 450 ).
Phenylalanine ammonia-lyases (PAL) activity. PAL activity was measured according to Schmidt et al. 52 with slight modifications. The crude enzyme obtained during protein extraction in total protein content determination was used to determine PAL activity in the sample. One hundred microliter of crude enzyme obtained were mixed with 1.15 mL containing 0.1 M sodium borate buffer (SBB; pH 8.8) and 10 mM l-phenylalanine. The mixture was then incubated at 37 °C for 1 h following which 250 µL of 5 N HCl was added to stop the reaction. PAL activity was then measured at 290 nm and the result was expressed in nanomoles of transcinnamic acid formed per milligram of proteins.
Real-time reverse transcription polymerase chain reaction (RT-qPCR) analysis. The RNA was isolated via RNeasy Plant Mini Kit (Qiagen, Germany) following the protocol described in Lai 53 . First strand cDNA was converted from 1 μg of isolated total RNA using QuantiNova Reverse Transcription Kit (Qiagen, Germany). Expression profile were assessed via RT-qPCR analysis. Real-time PCR was performed with Bio-Rad CFX96 system (Bio-Rad, US) with QuantiNova SYBR Green PCR (Qiagen, Germany) following the protocol as described in Lai et al. 54 . The real-time PCR reaction conditions used were as follows: 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 5 s. Three technical replicates on three biological replicates were performed