Cryptogein, a proteinaceous elicitor has been reported to affect growth and secondary metabolism in several plant species. We established two transgenic plant lines of Bacopa monnieri L., an indigenous medicinal species, following Agrobacterium tumefaciens mediated transformation. The transgenic plant lines obtained following transformation with strains LBA4404-nptII-crypt (plant line BmAt-ncrypt) and LBA4404-nptII (plant line BmAt-n), were maintained on selection media i.e. MS medium containing 100mg/l Kanamycin. To evaluate the effects of endogenous expression of cryptogein gene in crypt-transgenic plants of B. monnieri, in the present study, a proteomic approach was employed. Using 2D-PAGE followed by MALDI TOF MS/MS, we found 54 spots with reproducible differences in abundance (≥+2 or ≤-0.5-fold) between the plant lines. Bioinformatics analyses helped their identification, functional annotation and categorization.

Significantly upregulated gene expression (p≤0.5) of most of the key enzymes of triterpenoid saponin biosynthetic pathway (mevalonate kinase, mvk; mevalonate diphosphate decarboxylase, mdc; squalene synthase, sqs; β-amyrin synthase, bas and UDP-glycosyl transferase 2, ugt 2) in crypt-transgenic plant lines (BmAt-ncrypt) of B. Monnieri was observed as compared to non-transformed (Bm-NT) and empty vector control (BmAt-n) plant lines. Cryptogein-induced ROS production, a typical stress response of plant cells towards cryptogein, was significantly increased (p≤0.5) in specific activities and higher abundance of several anti-oxidant enzymes, such as SOD (3.0-5.0-fold), APX (2.3-3.0-fold) and CAT (2.6-fold) in BmAt-ncrypt. The upregulation of CaM (1.6-fold) and calcium-transporting ATPase-2 (2.0-fold) in BmAt-ncrypt indicate possible calcium-dependant cryptogein elicitation. A 3.0-fold increase in EIN3 levels with down-regulation of F-box protein, its regulator also indicate induction of JA-ET signalling pathway in crypt-transgenic plants. This study not only demonstrates involvement of multiple defence signalling pathways in crypt-transgenic plants of B. monnieri but also shows first evidence of the involvement of JA-ET signalling in cryptogein-induced elicitation.

Keywords: bacopa monnieri, cryptogein, triterpenoid saponin biosynthetic pathway, leaf proteome, defence signalling pathways

HR, hypersensitive response; SAR, systemic acquired resistance; WIPK, wound-induced protein kinase; SIPK, salicylic acid-induced protein kinase; TCA, trichloroacetic acid; 2-ME, 2-mercaptoethanol; SDS, sodium dodecyl sulphate; PMSF, phenyl methyl sulfonyl fluoride; 2DE, 2D gel electrophoresis; DTT, dithiothreitol; aact, acetyl-coa c-acetyl transferase; qRT-PCR, quantitative real time PCR; hmgr, hydroxyl methyl glutaryl-coa reductase, mvk, mevalonate kinase, mdc, mevalonate diphosphate decarboxylase; fpps, farnesyl diphosphate synthase; sqs, squalene synthase; bas, β-amyrin synthase; ugt 1, UDP-glycosyl transferase 1; fgt, flavonoid glucosyl transferase; pr 1, pathogenesis-related 1; NBT, nitro-blue tetrazolium; ROS, reactive oxygen species; HSPs, heat shock proteins

Cryptogein, a 10 kDa sterol scavenging protein secreted by the oomycete Phytophthora cryptogea,¹ belongs to 'elicitin' class of elicitors.² Cryptogein has been used to study the underlying molecular mechanisms of disease resistance in tobacco. Application of purified cryptogein protein, even in nanomolar quantity causes hypersensitive response (HR)-like necrosis in tobacco plants³ and also induces systemic acquired resistance (SAR) to P. parasitica var nicotianae .⁴

There are several reports on transformation with cryptogein (crypt) gene aimed to achieve the resistance against plant pathogens. Over expression of β-cryptogein induces HR in tobacco and triggers resistance against P. parasitica var nicotianae ^5,6 and other pathogens. Effective increase in biomass and/ or secondary metabolites accumulation in crypt-transgenic cultures in a number of plant species have also been observed,^7-9 where the crypt gene was either under the constitutive promoter⁷ or under the inducible promoter.⁸

The biochemical mode of action of this elicitor has been extensively studied. The cryptogein initiates a signal transduction possibly by binding cryptogein to an N-glycosylated plasma membrane receptor protein,^10,11 followed by the activation of several protein kinases, such as the wound-induced protein kinase (WIPK), the salicylic acid-induced protein kinase (SIPK).^12,13 Calcium influxes triggered by these kinases subsequently give rise to calcium-dependant transcriptional changes¹⁴ and activate a wide array of cellular responses, including an NADPH oxidase-dependent oxidative burst.^15,16 Upregulation of several chitinases, peroxidases, peptidyl-prolyl isomerases and proteinase inhibitors in intercellular fluid of cryptogein-treated tobacco leaves were observed using a proteomic approach.¹⁷

Bacopa monnieri(Scrophulariaceae), a herbaceous plant, has been used in Ayurvedic medicine in India for almost 3,000 years and is classified as medhya rasayana, a drug used to improve memory and intellect (medhya). Although different triterpenoid compounds are well distributed in plant kingdom, B. monnieri (popularly known as Brahmi) is the only herbal source of bacosides.¹⁸ We have been exploring various biotechnological approaches for improvement in production of active principles of B.monnieri.^19,20

Cryptogein gene either alone or in association with Ri T-DNA genes have been implicated for increased levels of principal bioactive compounds viz., bacoside A3, bacopasaponins (C, D, and F) and bacopasides (II and III) from B. Monnieri .¹⁹ The enhanced level of bacosides (1.66- to 2.05-fold as compared to non transformed plants) was stable for four years in plants maintained in vitro and the effects were sustained, even one year after transfer to greenhouse.²⁰ However, the biochemical pathways leading to the biosynthesis of bacosides have not been fully elucidated so far.

In the present study, a proteomic approach using 2D-PAGE followed by MALDI TOF MS/MS analysis was employed to evaluate the effects of endogenous expression of cryptogein gene in crypt-transgenic plants of B. monnieri.

In the present study leaf proteomes of B. monnieri plant line Bm-NT (non-transformed control) and three independent plant lines each of BmAt-n (empty vector controls) and Bm-At-ncrypt (crypt-transgenic plants) were analyzed and 54 spots with reproducible differential expression (≥ +2 or ≤ -0.5-fold) were identified in crypt-transgenic plants of B. monnieri. Among these 54 spots, gene expression of most of the key enzymes of triterpenoid saponin biosynthetic pathway in crypt-transgenic plant lines were significantly upregulated (p≤ 0.5) compared to non-transformed and empty vector control plant lines, as confirmed by qRT-PCR. In crypt-transgenic plants elevated ROS production in specific activities and higher abundance of several anti-oxidant enzymes, such as SOD (3.0-5.0-fold), APX (2.3-3.0-fold) and CAT (2.6-fold) was also noted along with up regulation of CaM (1.6-fold) and calcium-transporting ATPase-2 (2.0-fold), indicating possible calcium-dependant cryptogein elicitation.

In another interesting observation, 3.0-fold increase in EIN3 levels with down-regulation of F-box protein, its regulator, was noted which indicate induction of JA-ET signalling pathway in crypt-transgenic plants. In present study we demonstrate the first evidence of involvement of JA-ET signalling in cryptogein-induced elicitation and also hypothesize involvement of multiple defence signalling pathways in crypt-transgenic plants of B. monnieri.

Plant materials

For this study, transgenic B. monnieri plant lines were obtained via transformation with Agrobacterium tumefaciens strains LBA4404-crypt (harboring pBin19++crypt) and LBA4404-npt II (harboring pBin19++, empty vector). The synthetic β-cryptogein (crypt) gene²¹ was cloned under the 35S CaMV promoter and NOS terminator in a pBin19 vector.⁵

Three independent crypt-transgenic plant lines, BmAt-ncrypt (harboring npt II and crypt genes) were selected to study the effect of endogenous expression of crypt gene in crypt-transgenic plant lines of B. monnieri. However, B. monnieri plant lines Bm-NT (non-transformed) and three independent plant lines of BmAt-n (empty vector control, harboring only npt II gene) were considered as controls. Representative specimens of the three plant lines have been shown in Supplementary Figure 1. The transgenic plant lines were raised as described previously.²⁰ Southern hybridization analysis was performed to ensure the insertion of crypt gene into the genome of crypt-transgenic plant lines (Southern hybridisation details described in Supplementary Figure 2).

Supplementary Figure 1

1. Three representative six week old non-transformed wild type plants (Bm-NT); representative six week old plants of three selected plant lines each of B. monnieri transgenic lines
2. BmAt-n
3. BmAt-ncrypt. Bar= 1.8 cm

Supplementary Figure 2 Confirmation of integration of crypt in crypt-transgenic plant lines (BmAt-ncrypt) of B. monnieri by Southern hybridisation.

Lane M DNA Molecular Weight Marker VI, DIG-labeled (Roche), lane 1 EcoRI-digested Bm-NT genomic DNA, lane 2 EcoRI-digested genomic DNA of BmAt-n plant lines, lane 3, 4 and 5 EcoRI-digested genomic DNA of three independent transgenic plant lines of BmAt-ncrypt (arrow indicates the crypt-specific band), lane 6 EcoRI-digested pBin19++crypt plasmid DNA.

For Southern hybridisation, 20 μg of EcoRI-digested genomic DNA from three selected crypt-transgenic plant lines of B. monnieri was electrophoresed on a 0.8% agarose gel and transferred to a nylon membrane (Hybond-N, Amersham) by capillary transfer. DNA was fixed to the membrane by UV cross-linking. The pre-hybridizations and hybridizations were performed in DIG Easy Hyb solution (Roche, Germany) at 42°C. Double stranded probe for crypt gene was labelled with digoxigenin-11-dUTP by random primed labelling using DIG DNA Labelling and Detection Kit (Roche, Germany). After hybridization, membranes were washed twice for 5 min with 2X SSC (3M NaCl, 0.3 M sodium citrate, pH 7.0), containing 0.1% SDS at room temperature and then twice for 15 min with 0.5X SSC containing 0.1% SDS at 68°C. The digoxigenin-labelled hybridization products were detected according to the manufacturer’s instructions.

Southern hybridisation analysis confirmed integration of crypt into the genome of three selected BmAt-ncrypt plant lines. Detection of single crypt-specific band at three different positions in the blot suggests that the three crypt-transgenic plant lines were indeed arose from three independent transformation events and at the same time the crypt gene was integrated in single copy.

Leaf tissues were harvested from plantlets obtained after culturing 2 cm long shoot tip for 4 weeks on basal MS media with 3 % sucrose and 0.8 % agar to isolate total RNA or protein. Cultures were maintained at 24 ± 1 °C and 50-60 % relative humidity under 16-/8-h (light/dark) photoperiod.

Protein extraction from monnieri leaves

Total protein was extracted according to the method described by Wang et al.²¹ In brief, about 200 mg leaf tissue was ground to fine powder in liquid nitrogen and immediately suspended in 800 µl of ice cold 10% trichloroacetic acid (TCA) in acetone with 1% 2-mercaptoethanol (2-ME). The mixture was vigorously vortexed and incubated at -80°C for 30 min. After centrifugation (14000 rpm, 4°C, 10 min), the pellet was recovered and resuspended in ice cold 0.1M ammonium acetate in methanol. Subsequently, the mixture was vortexed and incubated at -80°C for 30min. Pellet was recovered by centrifugation and washed once each with ice cold 0.1M ammonium acetate in methanol, ice cold methanol and 80% acetone. After 5 min of brief air drying equal volume of tris-saturated phenol (pH 8.0) and SDS buffer [30% sucrose, 2% sodium dodecyl sulphate (SDS), 0.1M tris-HCl (pH 8.0), 5% 2-ME, 1mM PMSF (Phenyl methyl sulfonyl fluride)] were added to the pellet, vortexed and incubated on ice for 10 min. After centrifugation (14000 rpm, 4°C, 10 min) the upper phenolic phase was taken out in fresh eppendorf tube, 5 volumes of ice cold 0.1M ammonium acetate in methanol was added to it and mixed well. The mixture was incubated at -80°C for 1 h. The recovered pellet was washed once each with ice cold methanol and 80% acetone. Pellet was air dried and stored at -80°C for further 2DE analysis.

2D gel electrophoresis (2DE)

For 2DE the protein pellet was dissolved in resolubilization buffer (8 M urea, 2 M thiourea, 4 % CHAPS) mixed with 2% IPG buffer (pH 3-11) (GE Healthcare). Total protein content was determined following the method of Bradford²³ with bovine serum albumin (BSA) as standard. For first dimension of 2DE, IPG strips (Immobiline™ DryStrip, pH 3-11 NL, 11 cm, GE Healthcare) were passively rehydrated overnight with 200 μl of 2DE sample buffer containing about 150 μg protein. Isoelectric focusing was performed in a PROTEAN^® IEF Cell (Bio-Rad) at 4000 V for 2 h 30 min with linear voltage amplification and finally to 8,000 Vh with rapid amplification.

The focused strips were equilibrated once each for 15 min in equilibration buffer I [50 mM Tris-HCl, pH 8.8, 6 M urea, 20% [v/v] glycerol and 2% [w/v] (SDS) and 100 mg dithiothreitol (DTT)] and equilibrium buffer II (with 250 mg iodoacetamide). The equilibrated strips were then subjected to SDS-PAGE in a Criterion^TM Cell (BIO-RAD, USA) at constant 180 V for 2 h. After electrophoresis, gels were incubated for 30 min in the fixing solution (40% methanol; 10% acetic acid). Thereafter, the gels were stained overnight in 0.12% (w/v) Coomassie brilliant blue G-250 colloidal stain, destained and stored in 5% acetic acid for further analysis.

Image and data analysis

Coomassie stained 2D gels were visualized using Chemidoc™ XRS system (BIO-RAD, USA) and the gel images were analysed for spot detection, spot matching, background subtraction and spot intensity normalization using PD Quest Software, version 8.0 (Bio-Rad). Automated matching was performed and spot alignment was improved by manual spot detection and matching. All protein spots detected in gel were matched with the corresponding spots of master gel (reference gel) and we normalized each spot density against the whole gel densities. In order to minimize variations in sample loading, gel staining, and destaining, data were normalized by expressing protein abundance as a percentage of the total spot volume in the gel (relative volume %) to minimize gel-to-gel variation in spot intensity. The volume of the spot corresponded to the amount of protein expressed. The percentage volume of each spot was the average of the spot densities of nine different spots (three biological replicates of 3 plant lines BmAt-n=3x3=9; three biological replicates of 3 plant lines BmAt-ncrypt =3x3=9. For control BmNT plant lines, which are all non transformed three biological replicates n=3 were used).

Statistical analysis was performed to determine the significant differences between the groups (Bm-NT, BmAt-n and BmAt-ncrypt). Only the spots present in all the three biological replicates for each plant line were considered for comparison. Spots showing reproducible fold change patterns (above 2.0-fold for up-regulation and below 0.5-fold for down-regulation) and significant differences (P < 0.05) were designated as differentially expressed proteins.

Tryptic digestion and MALDI-TOF MS/MS analysis

Selected protein spots were excised manually from the gels and washed three times with ultra-pure water. In-gel trypsin digestion of the spots were performed according to the protocol described by Shevchenko et al.²⁴ Protein digests were finally dissolved in 0.1% TFA solution and digested samples were mixed (1:1, v/v) with the matrix solution [α-cyano-4-hydroxycinnamic acid (10 mg/ml) in 50 % (v/v) ACN and 0.1% (v/v) TFA], and 1 µl of this mixture was spotted on the MALDI target plate (Bruker Daltonics, Germany). MALDI-TOF MS/MS analysis was performed in MALDI-TOF MS/MS analyser (Bruker Daltonics, Germany); MS/MS spectra were collected and subsequently analysed in Flex Analysis v3.4 software. The Protein search was carried out using MASCOT (Matrix Science, London, England) and identified by NCBI and UniProt nrprotein sequence database using a MOWSE algorithm as implemented in the MASCOT search engine version 3.5.

For database searches following parameters were used: taxonomy: viridiplantae (green plants; 186963 sequences); cleavage specificity: trypsin with one missed cleavages allowed; mass tolerance of 100 ppm for precursor ions and a tolerance of 0.7 Da for the fragment ions; allowed modifications: carbamidomethyl (fixed), oxidation of methionine (variable); cleavage by trypsin: cuts C-terminal side of KR unless next residue is P. A Mascot score above 64 was considered as significant (P < 0.05).

Bioinformatics analysis

To evaluate the functional categories and hierarchies of identified proteins, Blast2Go (v. 3.0) program and UniProt database were used. Subcellular localizations of proteins were predicted by WoLF PSORT online software. The theoretical peptide mass and pI of the polypeptides were evaluated at EXPASy.

Relative gene expression (qRT-PCR) analysis of the enzymes involved in MVA, triterpenoids, MEP and flavanoid pathways

Total RNA, from the leaves of 4-weeks old plants, was extracted using RNAqueous Kit (Ambion, USA) and one µg total RNA was reverse transcribed using transcriptor first strand cDNA synthesis kit (Roche, Germany) following the manufacturer’s protocol. Three independent RNA extractions from three biological replicates were used for the analysis, and three technical replicates were analyzed for each biological test sample (n=3x3=9).

To investigate the effect of crypt on expression of the genes encoding secondary metabolite pathway enzymes, quantitative real time PCR (qRT-PCR) of 13 genes [five genes involved in MVA pathway, viz., acetyl-CoA C-acetyl transferase (aact), hydroxyl methyl glutaryl-CoA reductase (hmgr), mevalonate kinase (mvk), mevalonate diphosphate decarboxylase (mdc) and farnesyl diphosphate synthase (fpps); four genes involved in triterpenoid saponin biosynthesis, viz., squalene synthase (sqs), β-amyrin synthase (bas), UDP-glycosyl transferase 1 (ugt 1), UDP-glycosyl transferase 2 (ugt 2); one gene each of MEP (1-deoxy-D-xylulose-5-phosphate synthase, dxs), shikimic acid pathway (3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, dahs) and flavonoid biosynthesis (flavonoid glucosyl transferase, fgt); pathogenesis-related 1 (pr 1) was included as defence marker] was performed for the plant lines Bm-NT, BmAt-n and BmAt-ncrypt. For internal standard three reference genes were selected, viz, 18S rRNA, 26S rRNA and β-actin. Primer details of the genes are tabulated in Supplementary Table 1.

LightCycler 480 SYBR green I Master kit (Roche Diagnostics, Germany) was used to carry out the expression assays using LightCycler 480 II (Roche, Germany). The first strand cDNA was used as template. All the reactions were carried out in 10 µl reaction volume according to manufacturer’s protocol. The following amplification programme was used: pre incubation at 95°C for 5 min, followed by 45 cycles of amplification, each cycle comprised of 10 s denaturation at 95°C, 15 s annealing at respective temperatures as optimized for each set of primers and 30s extension at 72°C. This was followed by one cycle of melting curve analysis to check specificity of amplified product at 95°C for 5 s, 65°C for 1 min and 97°C for continuous acquisition. This was followed by one cycle of final cooling at 4°C.

The geNorm software was used to find out the most stable genes among the set of reference genes. In this study, the raw Cp values of all the reference genes are transformed into relative quantities using ΔΔCt method.²⁵ For relative quantification assays, the expression of the target gene is expressed as ratio of target-to-reference gene in the same sample. Melting curves for the genes are shown in Supplementary Figure 3.

Supplementary Figure 3 Melting curve of three reference genes and the 13 genes related to secondary metabolism and defence responses analysed in this study.

Anti-oxidant enzyme assay

Leaf tissues (0.5 g) were ground in a pre-chilled mortar and pestle in liquid nitrogen with 2 ml extraction buffer (50 mM potassium phosphate buffer pH 7.5, 1 mM EDTA, 1 mM PMSF). For analysis of APX, the extraction buffer also contained 5 mM ascorbate. The homogenate was centrifuged at 8000 rpm for 15 min at 4°C and the supernatant collected. Total protein content was determined following the method of Bradford²³ with BSA as standard.

Catalase (CAT, EC 1.11.1.6) activity was estimated according to Bergmeyer method,²⁶ which measures the initial rate of disappearance of H₂O₂ at 240 nm. The decrease in the absorption was followed for 3 min and 1 mmol H₂O₂ ml⁻¹ min⁻¹ was defined as 1 unit of CAT.

Ascorbate peroxidase (APX, EC 1.11.1.11) activity was measured as described by Nakano et al.²⁷ The assay depends on the decrease in absorbance at 290nm as ascorbate was oxidized. The concentration of oxidized ascorbate was calculated by using extinction coefficient of 2.8 mM⁻¹ cm⁻¹. One unit of APX was defined as 1 mmol ml⁻¹ ascorbate oxidized min⁻¹.

Guaiacol peroxidase (GPX, EC 1.11.1.7) activity was determined by measuring the increase of absorbance at 436 nm for 5 min relative to guaiacol oxidation to tetraguaiacol (ε = 25.5×10³ L mol^-1 cm^-1).²⁸ One unit of GPX was defined as 1 µM guaiacol oxidised min^-1.

Superoxide dismutase (SOD, EC 1.15.1.1) activity was assayed by measuring the inhibition of photo reduction of nitro-blue tetrazolium (NBT).²⁹ One unit of enzyme activity is defined as the amount of enzyme required to inhibit the NBT reduction by 50%.

Data from three independent transgenic lines for each of the transformed B. monnieri plant lines (BmAt-n and BmAt-ncrypt) were used for statistical analysis. All the experiments were randomized and repeated at least three times. Data were examined by a one-way analysis of variance (ANOVA) to detect significant differences (p≤ 0.05) in the mean. A post hoc mean separation was performed by the Tukey’s multiple comparison tests at the 5 % probability level using SPSS software (version 20.0). Variability in the data was expressed as the mean ± standard deviation (SD), if not mentioned otherwise.

Comparison of leaf proteomes of B. monnieri plant lines

The leaf proteome profiles of B. Monnieri plant line Bm-NT and three independent transgenic lines of BmAt-n and Bm-At-ncrypt were analyzed by 2D-PAGE followed by MALDI TOF MS/MS and spots with reproducible differences in abundance (≥ +2 or ≤ -0.5-fold) between the plants lines were targeted for further analysis (Figure 1). Following this criterion 54 spots (Table 1) were detected which were functionally annotated and grouped into nine different functional categories using different protein databases (Supplementary Figure 4). Between BmAt-ncrypt and Bm-NT plant lines 85% and 12% of the differentially abundant proteins were found to be up-regulated and downregulated respectively in BmAt-ncrypt plant lines. However, 90% of the differentially abundant proteins were up-regulated in BmAt-ncrypt in comparison with BmAt-n plant lines, whereas, 7% of differentially abundant proteins were downregulated. When the leaf proteome of BmAt-n plant lines were compared with that of Bm-NT, 15% and 54% of the differentially abundant proteins were found to be up-regulated and downregulated respectively in BmAt-n plant lines (Supplementary Table 2).

Figure 1 Representative 2DE gels of

1. Bm-NT
2. BmAt-n
3. BmAt-ncrypt

Arrow indicates the differentially abundant proteins which were subjected to MALDI-TOF MS/MS analysis.

Supplementary Figure 4: Functional categories of the differentially abundant proteins as predicted by using Blast2Go software (v. 3.0) and UniProt database.

The functional categories included metabolism and energy related (group A, n = 11), photosynthesis and chloroplast organization related (group B, n = 6), protein synthesis, folding and transport related (group C, n = 7), stress and defence related (group D, n = 10), transcription factor and signal transduction related (group E, n = 4), structural proteins (group F, n = 3), secondary metabolism related (group G, n = 2) and proteins of miscellaneous function (group H, n = 3). About 15% of the differentially abundant proteins could not be identified and hence categorized as unknown (group I, n = 8). The largest groups of the identified proteins were related to metabolism (group A, 20%) along with stress and defence related proteins (group D, 18%). The maximum number (37%) of differentially abundant proteins were found to be localized in chloroplast, followed by cytoplasm (29%), nucleus (15%) and mitochondria (9%) (Supplementary Figure 5). The functional categories as predicted by using Blast2Go software were similar to KEGG and UniProt databases. On the other hand, the subcellular localization as predicted by WoLF PSORT software had difference with UniProt database only in three cases (spot no. 10, 34 and 39). However, the subcellular localizations documented by UniProt database were finally considered. A heat map profile was generated to compare the differentially abundant protein among the plant lines of B. monnieri (Figure 2).

Supplementary Figure 5 Subcellular localisation of identified proteins as predicted by using WoLF PSORT online software. The findings were compared with the subcellular localisation information documented in different protein databases.

Figure 2 Heat map profile of differentially abundant proteins between Bm-NT, BmAt-n and BmAt-ncrypt plant lines.

Relative expression of key enzymes of representative secondary metabolite biosynthetic pathway and pr 1 in B. monnieri plant lines

To further validate the 2D-GE results, out of 54 spots the relative expression of 13 genes involved in secondary metabolism and defence responses in B. monnieri plant lines harboring crypt gene (BmAt-ncrypt) was compared with that of non-transformed (Bm-NT) as well as empty vector control (BmAt-n) plant lines by qRT-PCR analysis (Figure 3).

Figure 3 Relative expression analysis of 13 genes related to secondary metabolism and defence response among the non-transformed (Bm-NT), empty vector control (BmAt-n) and crypt-transgenic (BmAt-ncrypt) plant lines by qRT-PCR. In the Y-axis relative gene expression was plotted. Values represent mean± standard error (SEM) of three independent experiments (n= 9). Bars with the same letter are not significantly different according to ANOVA and Tuky’s multiple comparison test (p≤ 0.05) as calculated using SPSS (v. 20.0) software.

High to moderately variable expression pattern was observed for the MVA pathway enzymes. The expression of hmgr and fpps were downregulated (p≤ 0.05) by 2.1-fold and 1.4-fold respectively in BmAt-ncrypt plant lines with respect to Bm-NT. On the other hand, the expression of mvk and mdc was upregulated (p≤ 0.05) by 62.5-fold and 6.6-fold respectively in BmAt-ncrypt compared to Bm-NT. The expression of three key genes encoding the enzymes of triterpenoid saponin biosynthetic pathway viz., sqs, bas and ugt 2 were found to be significantly upregulated (p≤ 0.05) by 1.6, 1.8 and 7.0-fold respectively in crypt-transgenic plant lines BmAt-ncrypt than that of Bm-NT. However, dxs, encoding an enzyme of MEP pathway was downregulated (p≤ 0.05) in BmAt-ncrypt with respect to Bm-NT by 2.2-fold. However, the expression pattern of dahs (belonging to shikimic acid pathway) and fgt (involved in flavonoid biosynthesis) was similar between Bm-NT and BmAt-ncrypt plant lines. Strikingly, the expression of all the genes involved in triterpenoid saponin biosynthesis was significantly downregulated (p≤ 0.05) in BmAt-n in comparison with BmAt-ncrypt plant lines.

The expression of a defence marker gene pr 1 was also included in the present study to demonstrate whether over expression of crypt in crypt-transgenic plant lines of B. monnieri results in induction of defence responses through PR proteins. However, relative expression of pr 1 was found to be similar among different plant lines (Figure 3).

Effect of crypt on reactive oxygen species (ROS) scavenging enzymes in leaves of crypt-transgenic plants of B. monnieri

Antioxidant enzyme activity assay was performed in order to validate the findings of higher abundance of a number of ROS scavenging enzymes in the leaf proteome of crypt-transgenic plants (BmAt-ncrypt) of B. monnieri than that of the non-transformed (Bm-NT) and empty vector control (BmAt-n) plants.

The activity of CAT, APX and SOD were recorded significantly higher (p ≤ 0.05) in BmAt-ncrypt compared to Bm-NT (2.57-fold, 2.47-fold and 2.21-fold higher respectively). Interestingly, activities of APX and SOD were similar between Bm-NT and BmAt-n plant lines, whereas activities of CAT and GPX were significantly higher (p≤ 0.05) in Bm-NT than that of BmAt-n plant lines (Figure 4).

Figure 4 Specific activity of anti-oxidant enzymes in different plant lines of B. monnieri.

1. CAT activity (U mg^-1 protein)
2. APX activity (U mg^-1 protein)
3. GPX activity (U mg^-1 protein) and
4. SOD activity (U mg^-1 protein)

Bars with the same letter are not significantly different according to ANOVA and Tukey’s multiple comparison test (p≤ 0.05) as calculated using SPSS (v. 20.0) software.

Cryptogein (crypt)-induced elicitation, as established in transgenic plants expressing crypt gene showed enhanced accumulation of secondary metabolites in many plant species,^7,9,30 including B. monnieri ^19,20 In the present study, we compared the whole leaf proteome using 2DGE to understand the cross-talks between different plant defence systems that ultimately promoted bacosides (triterpenoid saponins) biosynthesis in crypt-transgenic plant lines as evident in our previous study.²⁰ Using a broad pH range IPG strip (pH 3-11) 54 differentially abundant proteins were identified and function of 85 % of these differentially abundant proteins could be identified in the present study. The relative expression of genes and their relevance was further examined for 13 out of 54 differentially abundant proteins.

The relative gene expression of key enzymes of triterpenoid saponin biosynthetic pathway viz., mvk, mdc, sqs, bas and ugt2 was found to be enhanced in crypt-transgenic plant lines of B. monnieri (BmAt-ncrypt) (Figure 3). Importantly, a 62.5-fold higher expression of mvk and 6.6-fold higher expression of mdc in BmAt-ncrypt relative to Bm-NT (non-transformed plants) was observed, implicating stimulation of MVA pathway in BmAt-ncrypt plant lines and possibly helping in overcoming marginal downregulation of hmgr and fpps. Triterpenoids and sesquiterpenoids are synthesized via the MVA pathway, whereas monoterpenoids, diterpenoid, and tetraterpenoids are synthesized via the MEP pathway³¹ The downregulation of dxs of MEP pathway and at the same time upregulation of mvk and mdc of MVA pathway suggest the inclination towards triterpenoids biosynthesis in crypt-transgenic plants. Significant upregulation (p≤ 0.05) of genes encoding branch point enzymes of tri-terpene biosynthesis (viz., sqs, bas and ugt 2) contributes positively towards higher bacosides accumulation in crypt-transgenic plant lines as reported in our earlier work.²⁰ In Withania somnifera crypt-co-transformed hairy roots, a metabolic shift from withasteroid (an end product of MVA pathway) formation to phenyl propanoid accumulation was observed.³² However, in our study, endogenous expression of crypt rather stimulated several genes of MVA and downstream triterpenoid saponin biosynthetic pathway, thus inducing higher bacosides accumulation in crypt-transgenic plant lines over non-transformed plants. So, it can be predicted that cryptogein-induced elicitation is probably species specific and may not elicit major end products of respective secondary metabolite biosynthetic pathways in the particular plant under study.

ROS are formed in many cellular reactions, including those involving cytochrome P450, peroxidase, lipoxygenase, and NADPH oxidase. ROS produced during the oxidative burst after the pathogen attack was implicated to induce defence responses.³³ The ROS scavenging enzymes include SOD, the first line of defence catalyzing the dismutation of superoxide anions (O₂^•–) to H₂O₂; APX, GPX and CAT, which are responsible for further detoxification of H₂O₂. Significantly higher abundance (2.0-2.5-fold) of a number of ATPases localized in vacuole, mitochondria and chloroplast (Figure 2) may contribute in elevated reactive oxygen species (ROS) production in crypt-transgenic plant lines over non-transformed plants. In cryptogein-induced HR the major enzymes responsible for a rapid generation of a huge amount of ROS are NADPH oxidases.¹⁵ Strikingly, upregulation of none of the NADPH oxidases could be detected in this study. However, elicitor induced ROS production, a typical response of the plant cells towards cryptogein could indirectly be confirmed by significantly higher (p ≤ 0.05) specific activity (Figure 4) as well as higher abundance of several ROS scavenging enzymes, such as SOD (3.0-5.0-fold), APX (2.3-3.0-fold) and CAT (2.6-fold) in crypt-transgenic plants over non-transformed and empty vector control plants of B. monnieri. Kumar et al.³⁰ reported similar findings where significantly higher activity of antioxidant enzymes along with lower abundance of ROS was observed in tobacco plantlets expressing β-cryptogein .³⁰

Interestingly, two proteins associated with protein folding in stress-free condition viz., cyclophylin 1 and TCP-1/cpn60 chaperonin family protein were downregulated in both BmAt-n and BmAt-ncrypt plant lines. In addition, two isoforms of heat shock proteins (HSPs) were expressed in higher abundance and BAG family molecular chaperone regulator 1 (BAG 1) was downregulated in BmAt-ncrypt in comparison with Bm-NT plant line. Down regulation of BAG 1, an inhibitor of HSPs³⁴ might be responsible for the higher abundance of HSP 70 family in crypt-transgenic plants. However, the Hsp 70 family proteins often act as molecular chaperons during various physico-chemical stresses, including oxidative stress.³⁵ Upregulation of glutathione gamma-glutamyl cysteinyl transferase 2 (PCS 2), aphytochelatin synthase involved in heavy metal detoxification³⁶ further signifies cryptogein-induced stress in BmAt-ncrypt plant lines. Undoubtedly, the crypt-transgenic plants survived the constitutive expression of cryptogein, most likely by the activity of ROS scavenging enzymes, HSPs, PCS among others that could relentlessly be preventing from cellular damage.

In addition, similar expression pattern of pr 1 among the plant lines (Figure 3) indicates that cryptogein may not induce defence responses through PR proteins-mediated defence pathway in crypt-transgenic plants of B. monnieri. This observation is in agreement with the findings of Amelot et al.² where treatment of tobacco BY-2 cells with purified cryptogein protein failed to induce significant upregulation (p ≤ 0.05) of pr 1, 2 and 3 genes.

 Understanding signal transduction pathways underlying cryptogein-induced production of secondary metabolites is very important in elucidating the highly complex events that occur during elicitation. The elicitor signal transduction involves several signal molecules and multiple defence pathways.^37,38 Cryptogein-mediated elicitation in B. monnieri is likely calcium-dependant as suggested by the higher abundance of calmodulin (CaM) (1.6-fold) along with calcium-transporting ATPase 2 (2.0-fold) in crypt-transgenic plants over non-transformed plants. In fact, Ca²⁺/CaM complex has been implicated in a variety of physiological processes including phytohormone signalling, transcriptional regulation, protein phosphorylation and dephosphorylation.³⁹

A three-fold higher abundance of EIN3 in crypt-transgenic plants (BmAt-ncrypt) over non-transformed plants (Bm-NT) suggested that cryptogein-induced elicitation in crypt-transgenic plants of B. monnieri might be mediated through JA-ET signalling pathway. Another interesting observation was the downregulation (-0.65-fold) of an F box protein in BmAt-ncrypt. However, EBF1 and EBF2, the two F box proteins regulate the EIN3 protein levels through a ubiquitin/proteosome pathway.⁴⁰ Therefore, downregulation of F box protein might provide the enhanced stability to EIN3. From these observations it can be assumed that in many aspects the defence responses elicited by endogenous expression of crypt gene were very similar to the exogenous treatment of purified cryptogein protein, regarding the induction of defence signalling pathways. An evidence of the involvement of JA-ET-mediated signalling in cryptogein-induced elicitation of B. monnieri crypt-transgenic plants is a novel finding of the present study.

Higher abundance of three vesicle-associated proteins, such as clathrin (1.5-fold), VAMP-like protein (1.8-fold) and Brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG 3) (2.1-fold) was observed in crypt-transgenic plants suggesting involvement of secretory pathway of protein trafficking in crypt-transgenic plant line. Important to note that rate of vesicular transport or secretion have not been measured in this study. Interestingly, Leborgne-Castel et al.⁴¹ reported ROS dependant increase of clathrin-mediated endocytosis after addition of cryptogein to tobacco Bright Yellow-2 (BY-2) cells followed by the elicitation of defence responses.

Unexpectedly robust effect on primary metabolism was also observed in crypt-transgenic plants over non-transformed plants, signified by the upregulation of two key glycolytic enzymes, viz., GAPDH (2.3-fold) and TPI (2.1-fold). Interestingly, carbohydrate metabolism was found to be altered in cryptogein-treated tobacco cells Ca²⁺-dependant NADPH oxidation caused by cryptogein activates the cytosolic pentose phosphate pathway and eventually provides the glycolytic intermediates thus, triggering glycolysis.⁴² A recent study showed that over expression of aldolase, an enzyme of Calvin cycle enhanced growth and promoted photosynthetic CO₂ fixation in the transgenic tobacco plants.⁴³ Therefore, higher expression of plastidial aldolase (5.8-fold) along with GAPDH and TPI in crypt-transgenic plants over non-transformed plants was likely a contributing factor behind the higher growth rate of crypt-transgenic plant lines as reported in our previous study.²⁰

In the present study a broad pH range IPG strip (pH 3-11) has been used, a narrower pH range (e.g., pH 4-7) for IEF may be used for better resolution and the issue of protein masking, if any, could be addressed. Several proteins (spot no. 2, 16, 20, 22, 30, 32, 36 and 40; tryptic fingerprint data were tabulated in Supplementary Table 3) could not be identified even with sufficient MS and/or MS/MS signals, probably due to the unavailability of sequence data of those specific proteins in the public databases considered in MASCOT search engine. These unknown, novel proteins need to be identified and further characterized in order to understand their functional relevance in present context.

 In the present study, a cross-talk between ROS scavenging system, plant defence and secondary metabolite biosynthetic pathways in cryptogein-induced elicitation is suggested in crypt-transgenic plants of B. monnieri (Figure 5). Higher abundance of a number of defence and stress related proteins in crypt-transgenic plants prove the hypothesis that endogenous expression of crypt mimics the pathogen attack caused by the exogenous treatment of cryptogein protein. However, higher accumulation of bacosides might be the consequences of cryptogein-induced defence responses leading to the stimulation of expression of genes encoding key enzymes of triterpenoid saponin biosynthetic pathway.

Figure 5 Alteration in abundance of proteins and biochemical changes associated with endogenous expression of cryptogein gene in crypt-transgenic plants of B. monnieri. Red arrow and green arrow indicate upregulation and downregulation respectively.

Pijush Paul gratefully acknowledges the University Grant Commission (UGC), Govt. of India for award of Senior Research Fellowship in the Department of Genetics, University of Calcutta. The authors thank Head, Department of Genetics; Head, Department of Botany, University of Calcutta and coordinator, DBT-IPLS-CU for the facilities provided.

All the authors declare that they have no conflict of interest.

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