Metal resistant PGPR lowered Cd uptake and expression of metal transporter genes with improved growth and photosynthetic pigments in Lycopersicon esculentum under metal toxicity

Plant growth promoting rhizobacteria (PGPRs) are very effective in immobilization of heavy metals and reducing their translocation in plants via precipitation, complex formation and adsorption. The present study was therefore designed to understand the role of Pseudomonas aeruginosa and Burkholderia gladioli in mitigation of Cd stress (0.4 mM) in 10-days old L. esculentum seedlings. The present work investigated growth characteristics, photosynthetic pigments, metal tolerance index, metal uptake and the contents of metal chelating compounds (protein bound and non-protein bound thiols, total thiols) in microbes inoculated Cd treated L. esculentum seedlings. The gene expression profiling of different metal transporters was conducted in order to investigate the quantitative analysis. Our results revealed Cd generated toxicity in seedlings in terms of reduced growth (root length, shoot length and fresh weight) and photosynthetic pigments (chlorophyll, carotenoid and xanthophyll) which enhanced upon inoculations of P. aeruginosa and B. gladioli. Further, the metal uptake along with levels of protein and non-protein bound thiols was also enhanced in Cd-treated seedlings. Gene expression studies suggested enhanced expression in the metal transporter genes which were further declined in the microbe supplemented seedlings. Therefore, micro-organisms possess growth promoting traits that enable them to reduce metal toxicity in plants.

Growth parameters. The effect of micro-organisms (P. aeruginosa (M1) and B. gladioli (M2)) on growth parameters of 10-day old L. esculentum seedlings under Cd stress was assessed in terms of root length, shoot length and fresh weight of the seedlings. It was observed that Cd stress resulted in reduction of the growth attributes of seedlings. Cd stress declined the root length and shoot length of seedlings by 32 and 31.61% in comparison to control plants. Furthermore, 35.64% decrease in the fresh was observed respectively. But supplementation of P. aeruginosa (M1) enhanced the root length, shoot length and fresh weight by 41, 39.8, 36.8%. Also, the application of B. gladioli (M2) improved the root length, shoot length and fresh weight by 48.2, 48.4, 52.7% respectively as compared to plants under Cd stress. Two-way ANOVA revealed the significant differences between root and shoot lengths and fresh weight (Table 1). photosynthetic pigments. The photosynthetic pigments were determined by analysing total chlorophyll, carotenoid and xanthophyll contents. Total chlorophyll content was lowered by 35.9% with 0.4 mM Cd concentration as compared to control seedlings. Seedlings treated with P. aeruginosa (M1) resulted in increased total chlorophyll content by 91.05%. Application of seedlings with B. gladioli (M2) also elevated the total chlorophyll content of the seedlings grown under Cd stress by 144.7%. The carotenoid content decreased by 54.7% in 0.4 mM Cd treated seedlings relative to untreated seedlings. Inoculation of P. aeruginosa (M1) led to elevation in carotenoid content by 213.6% in 0.4 mM Cd treated seedlings whereas seedlings amended with B. gladioli (M2) by Table 1. Effect of M1 (10 9 cells/ml) and M2 (10 9 cells/ml) on growth parameters. a Root length. b Shoot length and c Fresh weight in 10-days old L. esculentum seedlings under Cd stress (0.4 mM). Data is presented as means of 3 replicates ± S.D (standard deviation) and HSD values. F ratio values, * indicates significance at P ≤ 0.05 and ** indicates significance at P ≤ 0.01). Different letters on the graphs indicate that mean values of treatments are significantly different at p < 0.5 according to Tukey's multiple comparison test (CN-Control, Cd-Cadmium, M1-Pseudomonas aeruginosa, M2-Burkholderia gladioli). Data is presented as means of 3 replicates ± S.D (standard deviation) and HSD values. F ratio values, *indicates significance at P ≤ 0.05 and **indicates significance at P ≤ 0.01). Different letters on the graphs indicate that mean values of treatments are significantly different at p < 0.5 according to Tukey's multiple comparison test (CN-Control, Cd-Cadmium, M1-Pseudomonas aeruginosa, M2-Burkholderia gladioli).
Metal chelating compounds. An elevation in the accumulation of Cd in Lycopersicon seedlings triggers the synthesis of metal chelating compounds i.e protein bound thiols (PBT), non-protein bound thiols (NPBT) and total thiols. It was noticed that Cd led to significant elevation in protein bound thiols by 50.22% in Cd stressed seedlings as compared to control. With P. aeruginosa and B. gladioli application, an elevation in PBT was recorded by 77.7 and 96.2% respectively. Moreover, an increase in NBT and total thiols by 61.36 and 52.49% was recorded in Cd treated seedlings in comparison to untreated seedlings. Supplementation of both the strains in Cd exposed seedlings led to further elevation in the NBT by 36 (Figs 3 and 4).

Discussion
The present study revealed that Cd metal inhibited the growth of L. esculentum seedlings in terms of root and shoot length. This decline in plant growth in the presence of Cd might be due to lowered water potential and nutrient imbalance. It can also be due to obstruction in the proton pumps that further causes impairment in the cell division and elongation 32 . Our studies are in agreement with Dutta et al. and Ahmad et al. who suggested that Cd treated B. juncea showed reduced root and shoot lengths 33,34 . Moreover, reports of decline in the root and shoot lengths due to metal toxicity have been suggested in earlier studies conducted in Vigna mungo exposed to Cd 33 , Zea mays exposed to Cu and Pb 35 , O. sativa exposed to As 36 , and Pennisetum purpureum exposed to Pb 37 . Suppression in the root and shoot elongation is correlated to direct inhibition of root as well as shoot metabolism 38 . Supplementation of microbial strains (P. aeruginosa and B. gladioli) significantly improved the growth of seedlings in the present study. Micro-organisms are beneficial for the plant growth and development, and helps in phosphorous uptake in plants 39 . The mechanism by which micro-organisms enable phosphate uptake is mainly through extending branching pattern and root hair formation, through hormonal stimulation. This facilitates organic phosphates via microbial turnover that induces metabolic activities involved in solubilization and mineralization of organic phosphorous. The metabolic activity involves efflux of protons and different anions followed by the release of phosphatases enzymes that lead to phosphorus hydrolysis and mineralization respectively. Stimulation of root and shoot length by microbial inoculations was also observed by Liu et al. in Z. mays under Cd stress 40 . It was further reported that Enterobacter asburiae KE17 enhanced the growth and metabolism of soybeans under Cu and Zn toxicity 41 . The increase in root and shoot length was attributed to the plant growth promoting traits possessed by the inoculated micro-organisms.
The present study revealed that Cd toxicity resulted in declined fresh weight of the seedlings. Similar reports on reduced biomass under Cd stress in B. juncea have been observed by Verma et al. and Dutta et al. 33,42 . Reduction in the plant biomass during Cd stress is mainly due to its adverse affects upon mineral uptake, photosynthesis, chlorophyll synthesis, altered water and hormonal balance 43 . Cd is first exposed to roots which later on through apoplastic pathway may enter the system and can disrupt the complete metabolism of the plant 44 . Further, fresh weight of roots and shoots were found to be lowered in Cd-treated Russian knapweed 45 . Our results revealed that inoculations of P. aeruginosa and B. gladioli improved fresh weight of L. esculentum seedlings. There is a direct correlation of plant growth and biomass with plant growth promoting rhizobacteria (PGPRs) which was found to be increased after augmentation of PGPRs under Cd toxicity 46 . An elevation in the growth characteristics was also observed in Enterobacter inoculated O. sativa exposed to Cd 47 . Our studies were in the agreement with the previous studies conducted by Treesubsuntorn et al. who found that B. subtilis and B. cereus when inoculated to Cd exposed O. sativa plants resulted in the higher root and shoot biomass 48 . This is possibly due to plant growth hormone production (IAA) by assisted microbes that regulate the hormones within plant tissues and make them acclimatize towards stress conditions 48 . Moreover, Trichoderma sp. elevated the plant growth characteristics such as plantbiomass, yield, germination rate and many other plant growth promoting traits under arsenic (As) toxicity 49 . It was found that As tolerant strain improved the phosphate solubilisation from soil towards plant system via phosphate and As transporters that enhanced the metabolic and physiological activities of the Cicer arietinum plants 49 . Several other studies in Solanum nigrum L. 50 , Ocimum gratissimum L. 51   www.nature.com/scientificreports www.nature.com/scientificreports/ of P. aeruginosa and B. gladioli in growth promotion and phosphate solubilization in heavy metal toxicity have been reported [54][55][56] .
The results of the present study showed that Cd toxicity reduced the contents of photosynthetic pigments such as chlorophyll, carotenoid and xanthophylls in L. esculentum seedlings. It has been observed that Cd hinders photosynthetic machinery of rice plants by altering the levels of chlorophyll a, chlorophyll b, carotenoids and net  www.nature.com/scientificreports www.nature.com/scientificreports/ photosynthetic activity 57 . It also causes the structural changes in leaves leading to damage in their photosynthetic apparatus. It was investigated by Amirjani. that Cd exposure reduced the levels of carotenoids, total chlorophyll, chlorophyll a and chlorophyll b in Triticum aestivum 58 . The decline in the levels of photosynthetic pigments is mainly due to loss of cell wall and membrane integrity of thylakoid membrane. Many other heavy metals such as Zn, Fe, Cu, Hg, Cr and Pb leads to impediment of enzymes such as rubisco, chlorophyll synthase, protochlorophyllide reductase and δ-aminolevulinic acid dehydratase involved in the synthesis of chlorophyll 59 , that result in the breakdown of chlorophyll pigments 60 . Moreover, Cd-mediated breakdown of chlorophyll might be due to the activation of enzymes involved in hydrolysis of chlorophyll such as chlorophyllase 61 . They also lead to the activation of xanthophyll cycle in order to protect the photosynthetic apparatus from the metal exposure 62 . Moreover, carotenoids protect the plant's photosynthetic machinery from photo-oxidative disruptions through ROS scavenging. Reduction in carotenoids lead to PSII damage by retrogression of D1 protein that inhibits chlorophyll synthesis 63 . The observations made in our study are in accordance with Chen et al. who reported decline in the chlorophyll and carotenoid levels in Cd treated B. campestris and B. juncea 44 . Enhancement in the photosynthetic pigments could occur in the presence of plant growth promoting bacteria that increases nutrient uptake in plants through phosphate solubilization and exudating essential substances that play crucial role in synthesis of photosynthetic pigments required for light harvesting complex and its photo assimilation 64 . Inoculation of Klebsiella pneumoniae in V. mungo enhanced the levels of chlorophyll under Cd stress 33 . It was suggested by Rizvi et al. that Azotobacter chrococcum when supplemented with Cu and Pb exposed Zea mays plants enhanced the chlorophyll contents 35 .
Furthermore, elevation in chlorophyll as well as carotenoid contents in O. sativa was observed upon inoculations with Pseudomonas putida and C. vulgaris upon As treatment which is attributed to lessened accumulation of As that prevented the toxicity symptoms of As. We can speculate that it is most likely due to the growth promoting effects of PGPRs as well as enhanced protein levels that in turn stimulated the pigment levels. Several studies of PGPRs in enhancing the levels of chlorophyll and carotenoids under Cd stress have been reported such as Proteus vulgaris in pigeon pea 65 , Enterobacter in rice 47 , Azotobacter in Plantago ovata 66 , and K. pneumoniae in rice 53 .
In the current study, Cd stress lowered the heavy metal tolerance index and enhanced Cd-uptake in L. esculentum seedlings. It was demonstrated that inhibition of seed germination in various cereal crops such as rice, wheat and barley in response to different heavy metals is probably due to the morphological and physiological changes in roots that results in reducing heavy metal tolerance 67 . The results also signifies that treatment of seedlings with microbial strains (P. aeruginosa and B. gladioli) individually mitigates Cd induced reduction in heavy metal tolerance index. Heavy metal tolerance index in Ni and Cd treated tomato seedlings was reduced upon increased concentrations of Ni and Cd which upon supplementation of Methylobacterium oryzae and Burkholderia sp. was enhanced 68 . According to them, these metals raised stress marker ethylene levels which was reduced upon microbial inoculations. Along with this, they also observed that microbes precipitated heavy metals and lowered their availability by binding to functional groups such as hydroxyl, carboxyl, amide group etc. and chelating them via extracellular produced polymers such as humic substances and polysaccarides 68 . The results of the present study showed that Cd content was more in roots as compared to shoots. Similarly, studies with enhanced accumulation of different metal ions in roots were reported in Z. mays under Cu stress 35 , O. sativa under As stress 36 , and rapeseed under Cd presence 69 . The reduction in the uptake and translocation of heavy metals towards shoots is the adaptive measure adopted by the plants under stressful conditions 70 . Augmentation of micro-organisms in the present study reduced the Cd accumulation in roots as well as shoots. Previous studies conducted by Rizvi et al. suggested that Z. mays when inoculated with A. chroococcum reduced the Cu and Pb accumulation in plant organs which is most likely due to the secretion of different metabolites, protons and exudates that act as metal chelators and immobilise Pb 35 . Moreover, it was reported that metal tolerant strain Bacillus megaterium reduced the Ni translocation 20 , and As-tolerant Exiguobacterium reduced As translocation in Vigna radiata plants by colonization at the root surfaces 71 . It has been found that Acinetobacter lwofii promotes the growth and reduces As uptake in V. radiata 72 They speculated that A. lwofii produced growth promoting substances such as indole-3 acetic acid and siderophores that help them to resist the metal contamination. In addition, they also form biofilms which restricted the As uptake in these plants 72 .
Molecular studies revealed that PGPRs led to As biotransformation in wheat plants where up regulation of arsC, aioA and arsM genes reduced As levels in roots as well as shoots 10,73 . Furthermore, metal homeostasis within plants is mainly regulated by metal transporters and transport proteins depending upon the bioavailability of these metals 74 . In present study, gene expression profiling of metal transporter genes in seedlings under Cd metal stress have been studied through qRT-PCR. It was observed that expression of metal transporters were enhanced that resulted in the accumulation of Cd in roots and shoots which were lowered in the presence of microbes. A study conducted by Chen et al. showed that cloning of As-antiporter ACR3 Pteris vittata, PvACR3;1 was done and expressed in Arabidopsis thaliana and Nicotiana tabacum and yeast 75 . The results observed by them in yeast indicated that PvACR3;1 is a well developed antiporter that efflux As into the medium. Moreover, they also found that Arabidopsis thaliana and Nicotiana tabacum accumulated higher levels of As and reduced their accumulation in shoots 75 .
It was further reported that Pseudomonas putida possesses very well developed CzcCBA efflux system which involved three main genes CzcA, CzcB and CzcC and effect of these genes were studied in tobacco plants under Cd exposure 76 . They postulated that CzcB and CzcC genes led to lowered accumulation of Cd in shoots thereby reducing the Cd toxicity in contaminated soils. Similar studies revealed by Manzoor et al.depicted that microbes such as Pseudomonas possess metal resistance genes such as CzcR, Pbr A, CadA2 and ZntA that encodes different efflux proteins involved in metal detoxification processess. These genes belongs to P IB -type ATPases that transport metal ions from cytoplasm towards periplasm and precipitated to prevent the entry into the nearby cells 77 . Moreover, CzcA gene (encoded by bacteria) is a key antiporter and it has been found to be involved in controlling metal ion bioavailability at contaminated sites 78 . Therefore, the reduction of metal uptake via inoculation of plant (2019) 9:5855 | https://doi.org/10.1038/s41598-019-41899-3 www.nature.com/scientificreports www.nature.com/scientificreports/ growth promoting microbes enabled the Lycopersicon plants to survive better under metal exposure by detoxifying them that would otherwise affected the overall plant health.
The present study also revealed that metal chelating compounds such as total thiols, protein bound thiols and non-protein bound thiols were enhanced in seedlings under Cd stress. These metal chelators usually consist of sulfhydrl (-SH) groups that effectively bind to metals in order to immobilise them 1 . Our studies are in accordance with the studies of Aly and Mohamed. in Scenedemus bijugatus and maize under Cu stress 79 . Moreover, Cu accumulation can modulate glutathione levels which further stimulates the metal binding properties and sequester the metals 80 . It has been studied that thiol metabolism is the most crucial pathway in regulation of heavy metal stress tolerance. It mainly consists of non-protein thiols (NBTs), phytochelatins, protein thiols and glutathione that form complexes with metals and mediate their transport and sequestration into the vacuoles 81 . Moreover, NBT imparts antioxidant properties to the plants. The most important metabolite of thiol metabolism, cysteine produced during sulphur assimilation leads to the synthesis of phytochelatins and glutathione. The supplementation of P. putida in rice plants under As toxicity enhanced the levels of NBTs, phytochelatins and glutathione. According to their study, it is most probably due to complexation of As in shoots and roots 36 . This elevation in the levels of thiols might be due to increased nutrient uptake of different nutrients such as nitrogen, phosphorous, magnesium, potassium and sulphur in the presence of micro-organisms that stimulate the synthesis of these metal chelating compounds.

Conclusions
The results of the present study concluded that presence of plant growth promoting rhizobacterial strains P. aeruginosa and B. gladioli in soils promoted growth of plants in terms of root length, shoot length and fresh weight in L. esculentum seedlings subjected to Cd stress. A significant increase in the photosynthetic efficiency was also observed in Cd exposed microbe inoculated seedlings by elevating the contents of photosynthetic pigments such as chlorophyll, carotenoid and xanthophylls. Further, it was found that presence of these microbes reduced the Cd metal accumulation in the seedlings. The decreased levels of Cd resulted in alleviation of Cd toxicity through decreasing its bioavailability. The effective role played by microbes in reduction of metal uptake is mainly due to immobilization that results in binding the metal to root by complex formation which further prevent its translocation towards shoot. The role of metal chelating compounds such as non-protein thiols and protein thiols have also been elucidated in binding actions of heavy metals by microbes. Moreover, PGPRs also promote plant growth by enhancing mineral uptake, phytohormone production and nitrogen fixation. Therefore, all these traits act as driving force in enhancing the growth of plants under metal polluted environment. The present study therefore projects the contribution of micro-organisms in decreasing Cd toxicity and accumulation, implicating their roles for achieving the goal of lower Cd concentrations in tomato plants with better growth conditions.

Materials and Methods
Inoculation of microbial strains. Selected microbial strains (Pseudomonas aeruginosa strain no.

MTCC7195) and
Burkholderia gladioli strain no. MTCC10242) were obtained from IMTECH, Mohali, Punjab (India). These strains were cultured individually by addition of bacterial strains in 50 mL of nutrient broth (13 gL −1 ) medium. The flasks were later placed in the BOD incubator (Caltan (Deluxe Automatic), New Delhi, India) for 24-48 h at 28 °C for their proliferation. After their growth sub-culturing was done so as to maintain them for future. The culture was grown by adding 1 mL of above grown pure culture of bacteria (10 9 cells/mL) in 50 mL nutrient broth at 28 °C for 24-48 hrs. It was then centrifuged at 8000 rpm, 4 °C for 15 minutes to obtain the pellet. Pellet was washed twice using distilled water and resuspended in distilled water. The concentration of 10 9 cells/mL was adjusted for the experimental purpose. plant material and treatments. The certified seeds of L. esculentum (tomato) var. Pusa Ruby were sterilized by using 0.01% mercuric chloride (HgCl 2 ) solution. The seeds were dipped for a minute and rinsed thoroughly for 5-6 times using distilled water. Petriplates were lined with Whatman filter paper (grade 1 with diameter 10 cm) onto which thirty seeds were raised by soaking the seeds with microbial suspensions (having diameter approx. 10 cm) at 10 9 cells/mL concentration and CdCl 2 solution containing Cd (0.4 mM). The Petri plates were tracked in seed germinator for 10 days at controlled conditions of light (16-h photoperiod with white fluorescent light intensity 175 μmol m −2 s −1 ), temperature (22-25 °C), and relative humidity (80-90%) in seed germinator. Cd concentration was selected on the basis of IC50 (0.4 mM) value.
Growth parameters. Growth parameters were assessed after harvesting the seedlings by examining their root length and shoot length. Fresh weight was also recorded from the harvested seedlings after 10-days. evaluation of Heavy metal tolerance index. Heavy metal tolerance index was calculated using method of Balint et al. 82 . For this, dry weights of seedling samples were taken and tolerance index was calculated using the formula given below: Metal accumulation. Cd-metal accumulation in plant tissues (roots and shoots) was estimated by using Atomic Absorption Spectrophotometer (Shimadzu 6200, Agilent technologies GTA 120). The seedlings were harvested and their roots and shoots were separated and allowed to dry in oven at 65 °C for 48 hrs. The dried plant samples were further digested using the method proposed by Allen et al. 83 . For digestion, 200 mg of powdered www.nature.com/scientificreports www.nature.com/scientificreports/ sample was taken which was then digested in aqua regia (H 2 SO 4 : HNO 3 : HClO 4 , v/v) in ratio 1:3:1 using glass beakers on a hot induction plate. After that, digested samples were cooled and filtered using 0.22-μm nylon syringe filters. The samples were further diluted using double distilled water to make up the final volume upto 50 ml. These digested samples were stored at room temperature and used for further analysis. photosynthetic pigments. Determination of chlorophyll and carotenoid content. Estimation of chlorophyll and carotenoid content was done by the method given by Arnon 84 and Maclachlan and Zalik 85 , respectively. For this purpose, 1 g of fresh seedlings were macerated in 4 mL of 80% acetone. It was further centrifuged at 12,000 rpm for 15-20 min at 4 °C. The supernatant was taken for estimation of chlorophyll and carotenoid  www.nature.com/scientificreports www.nature.com/scientificreports/ contents. Absorbance at 645 nm and 663 nm was taken for chlorophyll analysis, whereas absorbance at 480 and 510 nm was measured for carotenoids using spectrophotometer (Thermo electron corporation, Genesys 10 UV).
Determination of xanthophyll content. Estimation of xanthophyll content was done by method proposed by Lawrence 86 . For this purpose, 50 mg of oven dried powdered seedling sample was kept in a 100-mL volumetric flask. To this, addition of 30 mL of combined extract (hexane (10 mL):acetone (7 mL):absolute alcohol (6 mL):toluene (7 mL)) was done and flask was shaken for 15-20 min. It was then followed by mixing 40% methanolic KOH (2 mL) to the flask. The flask was then kept in the water bath (58 °C) for 20-25 min after which the samples were placed under dark conditions for an hour. 30 mL of hexane along with 10% sodium sulfate was mixed to a volume of 100 mL followed by vigorous shaking for a minute. Again the flask was incubated under dark conditions. The upper layer was shifted into a 50-mL volumetric flask and volume was adjusted using hexane. Absorbance was taken at 474 nm.
Metal chelating compounds. Total thiols. Determination of total thiols was done by homogenising 0.5 g of seedling sample in 20 mM ascorbate buffer prepared using 20 mM EDTA (Ethylene diamine tetra acetic acid). Centrifugation was carried out at 12,000 g for 15-20 min, at 4 °C. To 0.5 mL of above obtained supernatant 2.4 mL of 200 mM Tris HCl and 10 mM of DTNB (5,5-dithio-bis-[2-nitrobenzoic acid]) was mixed. It was then allowed to stand for 20 min after which absorbance was recorded at 412 nm 87 .
Non-protein thiols. 0.5 g of fresh seedlings were macerated in 3 mL of ice cold 5% sulfosalicylic acid. The sample was then centrifuged at 12,000 g for 15-20 min at 4 °C. To 100 µL of extract, PPB (potassium phosphate buffer, 0.1 M; pH-7), 0.5 mL of 1 mM DTNB and 0.5 M EDTA was mixed. It was then placed for 15 minutes and absorbance was noted at 412 nm 88 .
Protein bound thiols. Estimation of protein bound thiols was done by subtraction of the non protein thiols (NPT) from total thiols (TT).
Gene expression analysis of metal transporters. RNA was isolated from tomato seedlings using Trizol method (Invitrogen, Life Technologies, USA). Analysis of RNA was done qualitatively as well as quantitatively through agarose gel electrophoresis (2%) and Nano Drop spectrophotometer (By Thermo Scientific, USA). The RNA isolated was treated with DNase (DNA-free TM kit; Ambion TURBO DNA-freeTM, From Life Technologies, USA) in order to restrict DNA contamination. Further, cDNA was synthesised with ImProm-IITM Reverse Transcription System (Promega, Madison, USA) using 1 μg of RNA (treated by DNase) as template and oli-godT12 primer (First Choice RLM-RACE Kit, Ambion, Life Technologies, Carlsbad, USA) 89 . The primers used in the current study are listed in the Table 3 (Integrated DNA technologies, USA).
Gene expression studies using qRT-PCR. Molecular studies in 10-day old tomato seedlings subjected to different treatments were done by expression studies of different metal transporter genes mentioned in Table 1. using quantitative real time RT-PCR (qRT-PCR) 90 . IM-Prom-IITM Reverse Transcription System (Promega, USA) was used for cDNA preparation. The primers designed for qRT-PCR profiling was carried out using Primer3 software. Expression study was preceded according to the manual instructions given along with the instrument Light Cycler 96 Real Time PCR System (Hoffmann-La Roche, Switzerland). PCR reaction (20 μL) preparation was done by combining the following reaction mixtures: cDNA (appropriately diluted), 1× Light Cycler 480 SYBR Green I Master (Hoffmann-La Roche, Switzerland) and 1 μM primers (Integrated DNA Technologies, USA, refer Table 1 for sequences). The prerequisite for qRT-PCR (thermal cycler) were: incubation period at 95 °C for 10-15 min, followed by 45 cycles of 3 step amplification (95 °C-10 s, 60 °C-15 s and 72 °C-25 s). Data interpretation was done through dissociation curve (heating upto 95 °C-10 s under normal conditions and cooling to 65 °C-60 s). Later, heat was given slowly upto 97 °C (1 s) at lowered ramping rate of 0.2 °C/s to check distinct qPCR reaction rate. Data was assessed in triplicates that also includes the negative control (non-template). Ubq (Ubiquitin) gene was used as house-keeping control gene for normalization purposes. The data was calculated using threshold cycle (Ct) of the amplification curve. The relative gene expression level was assessed using the 2 −ΔΔct method 91,92 (where Ct = (Ct, target − Ct, Ubiquitin)time × − (Ct, target −Ct, Ubiquitin time 0). statistical Analysis. The results of the present study were interpreted statistically through two-way analysis of variance (ANOVA) and Tukey's multiple comparison test to find the HSD (Honestly Significant Difference) among means. The values are mentioned as means ± standard deviation (S.D) and significant differences were checked at p ≤ 0.05 and 0.01. The data was examined in well built software in Microsoft excel.