Enhanced stress tolerance in transformed Ajuga bracteosa Wall. ex Benth. regenerants by upregulated gene expression of metabolic pathways

The rol oncogenes of Agrobacterium rhizogenes enhance the production of medicinally important compounds in plants and provide a first barrier against the overproduction of reactive oxygen species during biotic and abiotic stress. This study was designed to evaluate the expression of genes involved in biosynthetic pathways and their impact on metabolic contents and environmental stress tolerance in regenerated Ajuga bracteosa Wall. ex Benth. After successful transformation, real-time quantitative PCR confirmed the increased expression (1.94–6.59-fold) of HMGR, HDS, FDS, PAL, and TTG1 genes in transgenic lines. Furthermore, GC-MS coupled with principal component analysis revealed diverse concentrations of 97 metabolites in A. bracteosa. Transgenic lines showed greater survival under multiple stresses. This was revealed by significant chlorophyll content (8.13–21 μmoles/m2), higher quantum efficiency of PSII (Fv/Fm), and the performance index (PIabs) value. Similarly, catalase and peroxidase enzyme activities were enhanced during extreme drought (300–400 mM mannitol) and salinity (150–200 mM NaCl) conditions, compared to untransformed control. Wild type control plant leaves were completely necrotized by Aspergillus fumigatus (FCBP 66) and Fusarium solani (FCBP 0291), whereas transformed leaves had improved antifungal resistance. In conclusion, our data suggest that rolABC genes have a significant impact on the synthesis of metabolites involved in enhancing multistress tolerance in A. bracteosa.


Introduction
Plants are prone to a wide variety of environmental stresses due to their sessile and restricted nature. Overproduction of reactive oxygen species (ROS) under stress conditions can cause plant cell death. To deter this, plants use antioxidant systems which include different enzymes and primary and secondary metabolites (Czarnocka and Karpinski, 2018).
Traditionally, a considerable number of medicinal plants have been used as natural sources of antioxidants. Ajuga bracteosa Wall. ex Benth. (Lamiaceae) is an important medicinal plant that is broadly distributed in the Himalayan range (Park et al., 2017). A large number of pharmaceutically important compounds have been identified and characterized in this plant. Unfortunately, the yield of these phytochemicals in the wild plant is very low and is further compromised by environmental stress and lack of proper cultivation practices (Ahmad et al., 2012). Recent advances in genetic engineering offer a promising approach for instigating the production of secondary metabolites in hairy roots. However, tissue or organ specificity for the synthesis and translocation of certain groups of metabolites hinders the development of hairy root cultures (Isah, 2019).
To solve this problem, Kayani et al. (2016a) regenerated A. bracteosa plants from transgenic hairy roots and found improved production of phytoecdysteroids in regenerants. However, the molecular basis for the increase was not known.
Moreover, the correlation of rol genes to cope with stresses has not been observed in medicinal plants.

Transformation and regeneration
In vitro grown plants 2-3-months old ( Figure 2c) were used as explants for A. rhizogenes-mediated (LBA-9402 harboring pRi) genetic transformation. The transformation procedure and growth media for hairy root induction, proliferation, and stabilization were previously optimized by Kayani et al. (2016a) and followed accordingly. The hairy root lines obtained after infection were maintained in the growth room at 27 ± 2 °C in darkness ( Figure 2d). We selected three transformed hairy root lines, numbered R02, R04, and R06, based on their growth rate and metabolite production rate. Intact plants (ABRL1, ABRL2, and ABRL3) were regenerated from these hairy root lines according to the already optimized protocol of Tanaka and Matsumoto (1993). For multiple shooting, medium was supplemented with 2 µM BAP and plants were placed in the light while maintaining growth room conditions. When regenerants reached an optimum size (Figure 2e), they were micropropagated on plain MS medium. After 3-4 months rooted shoots were acclimatized into pots containing peat moss and sand ( Figure 2f).

Polymerase chain reaction
The presence of rolABC genes in putatively transformed and regenerated plants was confirmed by polymerase chain reaction (PCR). Already optimized conditions were used for genomic DNA isolation, plasmid DNA (positive control) extraction, and detection of rolA by PCR (Kiani et al., 2014). The sequences of the primers of rolA gene are given in Supplementary Table 1.

Total RNA extraction and cDNA synthesis
The TRIzol ® /ice based method (Luz et al., 2016) was used to extract RNA from transformed and wild type plant leaves, with slight modification. Briefly, plant samples (200 mg each) were freeze-dried, finely crushed, and homogenized with 1 mL of TRIzol ® . Then, 400 µL of chloroform was added to this homogenate and subjected to centrifugation at 12,000 rpm for 10 min at 4 °C. The supernatant was transferred to a new tube, placed on ice, and prechilled isopropanol was added in an equal ratio. Afterward, samples were centrifuged again under the abovementioned conditions. The pellet (containing RNA) was washed with

Gas chromatography and mass spectrometry (GC-MS)
Gas chromatography and mass spectrometry analysis was carried out with Thermo GC-TRACE ultra (v. 5.0) Thermo MS DSQ II (Thermo Fischer Scientific Inc.) fitted with a DB5-MS nonpolar capillary column (30 m length, 0.25 mm id, and 0.25 μm film thickness). Crude plant samples were completely dissolved in n-hexane and analyzed with GC-MS under the following experimental conditions: mobile phase (helium used as a carrier gas) with a flow rate of 1 mL/min and 1 μL injection volume. GC oven temperature was initially held at 70 °C and raised to 260 °C at 6 °C/min, and samples were fully run at 50-650 m/z. Identification of constituent compounds in crude samples was based on comparing their retention times and mass spectra with the known mass spectra stored in the National Institute of Standards and Technology (NIST) database search library. Principal component analysis (PCA) was carried out with the help of PAST 3.26 statistical software. The results of PCA are presented in terms of loading and scatter plots.

Abiotic stress and its effect on photosynthetic parameters
To assess the effect of stress on A. bracteosa regenerants, fresh green leaves from 3 independent transgenic lines (ABRL1, 2, and 3) and untransformed, in vitro grown wild type plants (WT) were propagated on MS media supplemented with 0, 100, 200, 300, and 400 mM mannitol (for drought stress) and 0, 50, 100, 150, and 200 mM NaCl (for salinity stress). For each treatment, leaves from WT and ABRL1, 2, and 3 were germinated in a growth room (21 days; 16/8 h photoperiod, 45 µE m 2 s 1 or 1000 lux light intensity, and 60% relative humidity). Each treatment was triplicated, and data were taken on the last day of the stress experiment.
The ratio of variable-to-maximum fluorescence (F v /F m ) for the quantum efficiency of photosystem II (PSII) and the performance index (PI abs ) from drought-and salt-stressed leaves were measured by chlorophyll fluorometer (pocket PEA-v04; Adsotech Instruments Ltd., Kings Lynn, UK). The fluorescence of fully extended leaves was measured after 30 min for dark adaptation at full intensity (3500 µm m -2 s -1 ). Total chlorophyll content was determined by SPAD 502 chlorophyll meter as µmoles/m 2 of leaf (Netto et al., 2005).

Estimation of relative water content
Relative water content (RWC) of leaves under drought stress was calculated by taking the initial weight after stress application as fresh weight (FW). Then, all of the tested leaves were soaked in distilled water for 12 h to find their turgid weight (TW). Finally, dry weight (DW) was recorded after drying turgid leaves at 80 °C for 24 h (Arndt et al., 2015). The following equation was used to calculate the RWC.

Enzyme extract preparation
Enzyme extracts of fresh leaves were prepared by following a previously reported method (Nayyar and Gupta, 2006) with some modifications. Briefly, 0.5 g of each leaf was freeze-dried and homogenized with 8 mL of enzyme extract solution containing 50 mM potassium phosphate buffer (pH 7.0) and 1% polyvinylpyrrolidone. The homogenate was centrifuged at 14,000 rpm for 30 min, and the supernatant was collected and stored at 4 °C for further enzyme assays.

Antioxidant enzyme assays
Catalase (EC 1.11.1.6) enzyme activity was determined following Aebi's (1984) method, with slight modifications. The enzyme mixture (3 mL) consisted of 0.1 mL of enzyme extract, 0.1 M of H 2 O 2 , and 50 mM of potassium phosphate buffer (pH 7.0). The decline in optical activity at 240 nm was measured as catalase activity/g FW. Peroxidase (EC 1.11.1.7) enzyme activity was determined using the guaiacol oxidation method by Chance and Machly (1955). The assay mixture (0.2 mL) contained 50 µL of enzyme extract, 50 mM of potassium phosphate buffer (pH 7.0), 50 mM of guaiacol, and 2% H 2 O 2 . Change in absorbance in 3 min at 470 nm due to the formation of tetraguaiacol was measured as peroxidase activity/g FW.

Fungal resistance assays
Fungal pathogens Aspergillus fumigatus (FCBP 66) and Fusarium solani (FCBP 0291) were propagated in Sabouraud dextrose agar medium. Fresh, green intact leaves were detached from A. bracteosa regenerated transgenic plants as well as wild plants, placed on the fungal cultures, and maintained at 28 °C and 16/8 h photoperiod for 6 days. Each petri plate contained two to three leaves with 5 replicates. Percentage of infection was measured using the following formula: Percentage of infection = (number of leaves infected-total number of leaves inoculated) × 100 The same assay was repeated with an additional modification, i.e. petri plates were provided with already moist filter paper (to maintain humidity). A 5 mm filter paper disc containing microsclerotia of A. fumigatus and F. solani was placed in the central area of the leaf. The experiment was conducted at 28 °C and 16/8 h photoperiod. The leaf area necrotized by fungal infection was measured at 24, 48, and 72 h postinfection (hpi) (Chowdhury et al., 2017).

Statistical analysis
All experiments were performed in triplicate, and values were represented as mean (n = 3) ± SD. PAST 3.26 statistical software was used for PCA of phytochemicals detected via GC-MS. For qPCR analysis two biological and three technical replicates were used for each sample. The significance of difference was calculated between wild type control and transgenic plants by two-way analysis of variance (ANOVA) using GraphPad Prism 5. Statistical significance was indicated by P ≤ 0.05.

Phenotype and molecular analysis of the transgenic plants
Nonchimeric A. bracteosa plants were regenerated from transgenic hairy roots in this study. These plants (harboring TL-DNA of pRi) presented morphological variability that was distinct from wild type A. bracteosa. The phenotype of regenerated plants was consistent with the previously reported data characteristic of A. bracteosa derived from transgenic hairy roots (Kayani et al., 2016a), i.e. they had a large number of curled leaves and short internodes with a bushy appearance. Plants regenerated from the transgenic hairy root lines were very different from the ones obtained from direct infection of A. tumefaciens harboring rolABC genes (Kayani et al., 2016b). Plants raised directly after the T-DNA transfer into tissue could be chimeric and had broad brittle leaves with ridges and furrows; the regenerants obtained from the hairy root lines were curled (like a bow) and not broad at all. PCR performed with rolA primers showed the presence of a 308 bp amplicon in regenerated plantlets and plasmid DNA (Figure 3a). This product size was similar to previous reports for rolA gene (Kiani et al., 2014).

Relative gene expression by quantitative real-time PCR
Among all the studied rol genes, rolB is considered the most powerful inducer of secondary metabolites, but it hampers the growth of the plant and its phenotype. However, when rolB gene is coupled with other rol genes such as rolA and rolC, the detrimental effect of rolB is overcome (Bulgakov, 2008). To assess the up-and downregulation of biosynthetic pathways, we analyzed the expression of HMGR, HDS, FDS, PAL, and TTG1 genes in wild type and transgenic regenerants of A. bracteosa by quantitative real-time PCR. We found upregulation of the studied genes involved in biosynthesis of secondary metabolites under the effect of rol genes, as compared to the wild type ( Figure 3b). Overall, these results implied that transgenic line ABRL3 had the highest expression of all genes studied. Data indicated that the expression of PAL was highest, whereas HDS had the lowest expression among all genes.

GC-MS analysis
A large number of volatile metabolites were identified in the aerial extracts of wild type and transformed A. bracteosa lines using GC-MS analysis. The 97 identified components classified into 7 distinct groups, along with their retention time (RT), molecular formula, molecular weight, and differential peak areas (%) are given in Table  1. Overall, ABRL3 line had the highest concentration of various metabolites compared to control (wild type). These inferences correspond to relatively higher gene expression in ABRL3.

Principal component analysis (PCA)
Principal component analysis (PCA) is a multivariate statistical technique that aims to explain the variability in a data set without losing important information (Kilimann et al., 2006). In this study, the extent of similarities or differences between wild type and three transgenic lines was evaluated by PCA based on the peak area of 26 selected metabolites (peak area ≥ 2). Principal component analysis identified three significant components; PC 1 explained 87.69% of the variance, while PC 2 had a variation of 7.94%.
The combination of PC 1 and PC 2 separated the four employed samples into distinct regions (Figure 4c), which indicated that significant discrimination of metabolites did exist in all samples. Samples with similar characteristics (ABRL1 and ABRL2) had nearby points on the graph, while WT and ABRL3 had the most distant relationship, confirming that metabolic composition is extremely variable between these two lines. Biplot pointed to a clear association among different lines of A. bracteosa and their metabolic profiles (Figure 4d). This interrelationship was further supported by hierarchical analysis (Figure 4e).

Abiotic stress tolerance and photosynthetic indices of the transgenic A. bracteosa lines
Although a plethora of salts are found in the soil, NaCl is considered the principal source of salinization (Shavrukov, 2013). Furthermore, mannitol, an effective osmotic agent, has been reported to mimic in vitro drought stress conditions in plants (Jolayemi and Opabode, 2018). Transgenic leaves fared better than the control, untransformed leaves under multiple stresses ( Supplementary Figures 1a and 1b). We observed that the transgenic leaves tolerated extreme drought and salt stress without visible signs of chlorosis. However, untransformed leaves showed signs of wilting at a drought stress of 100 mM mannitol and exhibited severe chlorosis with increased concentrations of NaCl (above 50 mM). These results are in agreement with previous findings that suggested salt and drought stress hamper plant growth and development (Ali and Ismail, 2014;Niazian et al., 2019). Nevertheless, the transgenic leaves survived extreme stress conditions due to the overexpression of rol genes and the production of stress-tolerant metabolites, as supported by previous reports (Bulgakov et al., 2012;Kayani et al., 2016aKayani et al., , 2017.

Total chlorophyll content
Salinity and drought can decrease the photosynthetic capacity of plants either due to stomatal closure or through direct damage to photosynthetic machinery (Mittler, 2006). In this study all leaves (control and transgenic) had the highest chlorophyll content under nonstress conditions. After 21 days, a significant difference (P < 0.001) was observed in total chlorophyll contents between control and transgenic leaves at higher concentrations of mannitol and NaCl. Under drought stress, control leaves had maximum chlorophyll (18.13 µmole/m²) at 100 mM mannitol and minimum (10.88 µmole/m²) at 400 mM mannitol. A similar pattern was observed under salt stress; control plant leaves had the highest chlorophyll (23.06 µmole/m²) at 50 mM NaCl and lowest (3.8 µmole/ m²) at 200 mM. Among the transgenic lines, maximum chlorophyll content (17.66 µmole/m²) was noted in ABRL2 under extreme drought stress (400 mM mannitol), followed by ABRL3 (15.75 µmole/m²), and ABRL1 (13.54 µmole/m²). ABRL3 leaves had the highest chlorophyll content (21 µmole/m²) at maximum salt stress (200 mM NaCl), while ABRL1 had the lowest (8.13 µmole/m²) chlorophyll content at this concentration.
These results showed that there was a clearer impact of drought and salt stress on the chlorophyll content of wild A. bracteosa than in the transformed plants (Figures 5a and  5b). These findings are supported by Bettini et al. (2016a) who reported that rolB is involved in the overexpression of 5 genes related to photosynthetic activity in transformed tomato plants. More recently, Bettini et al. (2020) further confirmed the role rolB plays in the photoprotection of transgenic tomato through improvement in chlorophyll a, b, and a/b ratios under white light, as compared with control plants.

Photosynthetic fluorescence parameters
The two photosynthetic parameters F v /F m and PI (abs) are considered sensitive indexes of photosystems I and II that can evaluate plant photosynthetic status under stressful conditions (Živčák et al., 2008). Previous studies have shown that when the F v /F m value is 0.79-0.83, the plant is healthy, and its photosynthetic apparatus is not damaged (Baker, 2008). Tables 2 and 3 show that both F v /F m and PI (abs) were influenced by stress in A. bracteosa. In this study, the F v /F m and PI (abs) values (0.80 ± 0.04 and 1.80 ± 0.5, respectively) were constant in all leaves (control and transgenic) when measured under nonstress conditions. However, both parameters decreased with an increase in stress, which is in line with results previously reported from different species (Maghsoudi et al., 2015). We found that transgenic lines had comparatively higher F v /F m and PI (abs) values which could be correlated to the expression of rolABC. Our observations are supported by different pieces of evidence that show the interaction of rolB with photosynthetic parameters. Many reports have suggested that rolB is involved in the photoprotection of PSII through overexpression of cytochrome b6/f complex and carbonic anhydrase in transgenic tomato, as illustrated by improved nonphotochemical quenching parameters, including F v /F m (Bettini et al., 2016a(Bettini et al., , 2020. While comparing the rate of change in F v /F m and PI (abs) values in 4 lines of A. bracteosa we found that PI (abs) changed greatly in WT under salt and drought stress. These results are consistent with Li et al. (2019) who also found PI (abs) to be more sensitive than F v /F m .

Relative water content (RWC) under drought stress
Relative water content is an important variable used to evaluate the metabolic activity, physiological water status, and survival of plants under water deficit (Hasheminasab et al., 2014). In the present study, transgenic leaves had significantly higher (P < 0.001) RWC compared to the wild leaves under drought (Figure 5c). Under extreme drought conditions (400 mM mannitol) ABRL3 had maximum RWC (41%), followed by ABRL2 (33.9%), ABRL1 (21.4%), and WT leaves (11.6%). It has been indicated by different researchers that drought stress reduces RWC in many Lamiaceae species (García-Caparrós et al., 2019). The retention of RWC in transgenic A. bracteosa could also be related to the expression rol genes. Pavlova et al. (2014) found that rol genes increased the proline synthesis that   Shahid et al. (2014) found that exogenous application of proline on stressed pea plants improved photosynthetic activity and RWC by regulating guard cells and stomatal closure. Therefore, plants with higher proline content are more resistant to stress conditions.

Increased level of ROS enzymes in transgenic lines
Plants have developed various antioxidant systems (both enzymatic and nonenzymatic) against ROS to prevent oxidative stress. The major elements of the nonenzymatic antioxidant systems include ascorbic acid, alkaloids, flavonoids, phenolics, GSH, α-tocopherol, and carotenoids. The enzymatic antioxidant system involves superoxide dismutase, ascorbate peroxidase, catalase, glutathione peroxidase, etc. (Sofo et al., 2015). In the current study, we found that catalase (Figures 6a and 6b) and peroxidase (Figures 6c and 6d) enzyme activities gradually decreased in the leaves of both transgenic and control plants. However, transgenic leaves followed the same pattern as chlorophyll content, photosynthetic parameters, and RWC, displaying higher catalase and peroxidase activities even in extreme stress conditions. This pattern of catalase and guaiacol peroxidase enzyme activity is consistent with wild and transformed Arabidopsis under salt and drought stress (Manuka et al., 2019). A similar trend was followed by ROS enzymes in tomato plants under multiple stresses (Waseem and Li, 2019).
In general, we found that catalase and peroxidase enzyme activities were at a maximum in ABRL3 under  extreme stresses and at a minimum in control leaves. Bulgakov et al. (2012) found that rolB increased the expression of genes encoding for ascorbate peroxidase, catalase, and superoxide dismutase which lead to stress tolerance and ROS scavenging in Arabidopsis thaliana, Panax ginseng, and Rubia cordifolia. Similarly, Dilshad et al. (2015Dilshad et al. ( , 2016 reported increased synthesis of flavonoids and antioxidant potential in Artemisia species expressing rolB and C genes. Flavonoids scavenge free radicals and remove superoxide and peroxide thus improving plant tolerance to salt and drought stress.
The results of the current study are supported by our previous reports indicating significantly increased production of phytoecdysteroids in rolABC-transformed hairy roots and intact plants of A. bracteosa (Kayani et al., 2016a(Kayani et al., , 2017 which could account for the increased antioxidant activities of this plant.

Fungal resistance in transgenic lines
In the current study, we also get an insight into the effect of rolABC on fungal resistance. We found that the expression of rolABC genes in A. bracteosa conferred resistance against two drug-resistant pathogens. Transgenic leaves had restricted necrotic zone symptoms, while control leaves were completely necrotized by A. fumigatus and F. solani (Figures 7a-7d). Moreover, the percentage of leaf area infected by A. fumigatus and F. solani was significantly lower in transgenic lines compared to control (Figures  7e and 7f). The aforementioned antifungal resistance can be ascribed to the increased levels of metabolites in transgenic lines, as reported by Özçelik et al. (2011). Likewise, Sánchez-Maldonado et al. (2016) reported the antifungal properties of phenolics and glycoalkaloids in potato plants. Similarly, Arshad et al. (2014) reported that rolB gene improved foliar tolerance of transgenic tomato leaves against two pathogenic fungal strains Alternaria solani and Fusarium oxysporum, compared to wild tomato plants. Bettini et al. (2016b) reported that rolA also enhanced the antifungal activities of tomato. Furthermore, Kiani et al. (2019) found higher antifungal resistance in rolABC-transformed Artemisia dubia, suggesting that rol genes modified the phytochemical constituents that are involved in microbial defense in this plant.
In conclusion, this study dissects the effects of rolABC genes on the metabolic pathways in A. bracteosa. The increased expression levels of all genes is in accordance with the accumulation of volatile metabolites as well as biotic and abiotic stress resistance in transformed plants.