Seeds of B. juncea (cv. RLC-1) were given pre-sowing treatment with 24-epibrassinolide (EBR) solutions (0 and 100 nM EBR/L) for 8 h. Petri-plates were lined with Whatman1 filter paper and were supplemented with different imidacloprid (IMI) concentrations (0, 150, 200, and 250 mg IMI/L). The EBR treated seeds were rinsed with distilled water and grown in Petri-plates supplemented with IMI solutions (three petri-plates for each treatment). The Petri-plates were kept in seed germinator (temperature = 25 ℃ , photoperiod = 16 h, light intensity = 175 µmol m ^-2 s^-1) and the seedlings were harvested 10 days after sowing for further analysis.

Seed soaking with 24-epibrassinolide recovers the impaired growth of B. juncea seedlings under imidacloprid stress by modulating the expression of genes encoding key enzymes including chlorophyllase, citrate synthase, succinyl Co-A ligase, succinate dehydrogenase, fumarate hydratase, malate synthase, phytoene synthase, chalcone synthase, and phenylalanine ammonialyase.

In the present study, as compared to control seedlings, the expression of gene CHLASE (encoding chlorophyllase) was observed to increase by 2.66-fold under IMI toxicity, but seed soaking with EBR significantly reduced the expression of CHLASE to 1.07-fold in the seedlings under IMI toxicity . Data analysis using two-way ANOVA and Tukey's HSD showed significant difference for CHLASE expression in B. juncea seedlings (FIMI p < 0.01, FEBR p < 0.01, FIMI * EBR p < 0.001). MLR analysis of the fold change in CHLASE expression also revealed the increased expression of gene with IMI toxicity and EBR application (positive betaIMI-value), whereas interaction between IMI and EBR was observed to be negative .Further, in comparison to control seedlings, the expression of PSY (encoding phytoene synthase) and CHS (encoding chalcone synthase) was significantly enhanced by 5.22 and 4.54-folds respectively in the seedlings raised from EBR treated as well as untreated seeds grown under IMI stress . Significant differences in expression PSY (FIMI p < 0.001, FEBR P<0.05) and CHS (FIMI * EBR p < 0.001) were observed after analyzing the data using two-way ANOVA and Tukey's HSD. MLR analysis of fold change in gene expression also revealed the role of EBR in modulation of gene expression of PSY and CHS. Concentrations of IMI as well as EBR were regressed positively on the fold change in gene expression of PSY and CHS, thus revealing enhanced expressions of these genes under both the treatments. Moreover, interaction between IMI and EBR was positive for PSY expression, whereas negative interaction was observed for the expression of CHS .In the present study, the expression of PAL was also observed to enhance significantly by 6.68-fold in the seedlings raised from EBR treated seeds and grown under IMI stress . After analyzing the data using two-way ANOVA and Tukey's HSD, significant difference in the expression of PAL was observed (FIMI p < 0.01, FEBR p < 0.01, FIMI * EBR P<0.05). MLR analysis of the fold change in gene expression also confirmed the role of EBR in increasing the PAL gene expression under IMI pesticide stress. Positive beta-regression coefficients were observed for IMI, EBR, and IMI * EBR .The expression of genes encoding the key enzymes involved in organic acid metabolism was also studied to understand the role of EBR in organic acid metabolism under IMI pesticide stress. It was observed that as compared to control seedlings, the expression of CS (encoding citrate synthase, 2.35-fold), SUCLG1 (encoding succinyl-Co-A ligase, 1.57-fold), SDH (encoding succinate dehydrogenase, 2.01-fold), FH (encoding fumarate hydratase, 1.57-fold), and MS (encoding malate synthase, 1.91-fold) were increased in B. juncea seedlings raised from untreated seeds and grown under IMI pesticide toxicity . However, seed soaking with 100 nM EBR and germinating them under IMI toxicity resulted in further enhancement in expression of CS (2.61-fold), SUCLGD1 (4.18-fold), SDH (2.55-fold), FH (3.73-fold), and MS (4.03-fold). Data analysis using two-way ANOVA and Tukey's HSD showed significant differences in the expression of CS (FEBR p < 0.01, FIMI * EBR p < 0.01), SUCLG1 (FEBR p < 0.001, FIMI * EBR P<0.05), SDH (FEBR p < 0.01), FH (FEBR p < 0.001), and MS (FEBR p < 0.001). MLR analysis showed that gene expression in seedlings under IMI stress as well as after the EBR seed treatment was increased as indicated by positive beta-regression coefficients. Whereas, negative interactions were noticed between IMI and EBR treatments for the expression of all genes studied related to organic acid metabolism.