Given the molecular results, inoculation of two bacteria treatments individually enhanced the expression of the interested genes in the roots of C. roseus. Among them, P. fluorescens had a positive and significant effect on the expression of genes involved in first step (G10H) and the last step (T16H, DAT and CrPRX) of the biosynthetic pathway. Previously, up-regulation of TDC genes had been reported in the roots of C. roseus treated with this elicitor (Ahmadzadeh et al., in press). Li et al. (2013) found the significant changes in the expression level of genes involved in the TIA biosynthesis, such as Tdc and G10H, by overexpressing the transcription factor ORCA2 in the hairy roots of C. roseus. In other words, these gene have a common transcription factor. Liu et al. (2017) showed that the transcription factors ORCA2 and ORCA3 regulate the expression of G10H and TDC genes. These transcriptional factors are activated in response to methyl jasmonate and jasmonic acid. However, in the present study, they probably were activated under the bacterial treatments, especially P. fluorescens, in the roots. Researches revealed that the G10H promoter contains unique binding sites of several transcriptional factors, suggesting that the G10H promoter may be regulated by a different transcriptional cascade (Suttipanta et al. 2007).
In the vindoline biosynthesis pathway, the two genes T16H and DAT involved in first and last steps respectively (Shabani et al., 2014) were elected for investigation. Owing to the production of vindoline with organ-dependent manner in green tissues, the expression of genes is not predicted through this branch of TIA biosynthesis pathway in root tissue under normal conditions (Dutta et al., 2005). However, molecular analysis demonstrated the expression of both genes in the roots under all treatments. The expression of some genes in this pathway, including the D4h in root tissue, has already been reported by Dutta et al. (2005). In order to prove the correspondence of observed hybridization signals with the real expression of the D4H gene in the root, they sequenced RT-PCR products and finally obtained more than 99% homology by blasting with sequences in the data bank (GenBank Acc. No. O04847).
In this study, expression of T16H and DAT genes in the root was affected by P. fluorescens and combine inoculation treatments. Also, the response of these two genes to all used treatments was almost the same. Thus, the treatments that increased the expression of T16H gene also increased the expression of DAT gene in the root. This result was not unexpected. This is because these two genes are in a branch of the biosynthetic pathway and it is possible that they have a common promoter to regulate expression or are activated by a common transcription factor. The possibility of co-response of some biosynthetic pathway genes of TIA to elicitors has also been mentioned in other reports. The expression of several genes involved in the biosynthetic pathway of indole alkaloids, including Str and Tdc, is coordinately induced by fungal elicitors such as yeast extract (Pauw et al. 2004).
The CrPRX gene is the final gene of the TIA biosynthetic pathway and encodes the enzyme anhydrovinblastine synthase. The product of this gene combines the two substances catharanthine and vindoline and it causes the formation of vinblastine and eventually vincristine (Goklani et al., 2009). CrPRX gene expression in root in response to all treatments was significantly increased compared to the control, but the most effective treatment was P. fluorescens. In the study of Wang et al., 2016, the transcript level of CrPRX in C. roseus increased in response to different concentrations of ethephon. Maximal amounts of CrPRX transcripts were detected in seedlings treated by 100 𝜇M ethephon. Since the studied rhizobacteria increase the susceptibility of the plant to ethylene (Beneduzi et al., 2012), it may be possible to influence the expression of TIA biosynthetic pathway genes in this way.
Overall, P. fluorescens was effective than other treatments in root and caused the significant up-regulation of studied genes, particularly last step gene, CrPRX, compared to the control plant. In sequent, A. brasilense treatment was able to increase the expression of G10H and CrPRX, significantly.
van der Fits and Memelinc, (2000) showed that methyl jasmonate (MJ) treatment stimulated TIA metabolism in C. roseus cell suspension and increased the expression of all genes implicated in the TIA biosynthetic pathway. In addition, the transcription factor ORCA3 was activated in response to MJ, which in turn regulated the expression of some other genes in this pathway, including the STR. Suttipanta (2011) studied the transcription factor WRKY in the C. roseus plant, which is expressed predominantly in roots and also in response to phytohormones such as jasmonate, gibberellin, and ethylene. They demonstrated that a high expression of transcription factor CrWRKY2 in response to methyl jasmonate in the hair roots culture of C. roseus up-regulates several TIA pathway genes.َBesides, it promoted the expression level of the transcription factor activating ORCA3 and the inhibitor of ZCT. Simultaneous induction of activators and inhibitors may be necessary to activate or repress some genes in response to elicitors (Shabani et al., 2014). It is noteworthy that some transcription factors are tissue specific, and some treatments are involved in the simultaneous induction of transcription factors of either activators or inhibitors. According to Pattra et al. (2018), inhibitors such as JAZs and RMT1 mediate the interaction of CrMYC2 and BIS regulators, as well as balance the metabolic flux of TIA. Zhang et al. (2011) showed that application of methyl jasmonate increases the expression of Tdc, G10H, Str, etc., genes in the biosynthetic pathway of TIA in the C. roseus. Another study revealed that ethylene treatment has a positive effect on C. roseus alkaloids at transcriptional and metabolic levels (Wang et al., 2016). Also, Shabani et al. (2014) found that some genes in the TIA pathway, such as the G10H gene, play critical role in responses to ethylene. Papon et al. (2005) reported high expression of this gene in response to cytokine and ethylene. Similarly, in this study, inoculation treatments individually had positive and significant effects on G10H in roots. In agreement with these results and previous research, it can be suggested that bacterial inoculation treatments by producing cytokinin hormone or increasing plant susceptibility to ethylene in the root, have up-regulated the G10H gene in conjunction with other genes studied.
Given the results, it could be inferred that exposure of C. roseus to bacterial inoculation treatment probably induced the production of hormones such as gibberellin and cytokinin, as well as activated the jasmonic acid and ethylene responses. These increased the expression level of transcription factor CrWRKT2, subsequently activators ORCA3 and ORCA2 in conjunction with some inhibitors, which in turn influenced the expression level of genes involved in the TIA biosynthetic pathway. Considering the positive correlation between relative expression level of these gene and the accumulation of corresponding alkaloids in C. roseus (Dutta et al., 2005; Goklani et al., 2009; Jaggi et al., 2011 and Wang et al. 2016), it can be concluded that up-regulation of G10H, DAT, T16H and CrPRX genes under these treatments, especially by P. fluorescens, can result in a higher production rate of vinblastine and vincristine alkaloids as final products in the TIA biosynthetic pathway. As an example, it is shown that an increase in the expression level of DAT has been found to result in the accumulation of vinblastine and vincristine alkaloids (Wang et al., 2012 and Khataee et al., 2019). In this case, metabolite studies were performed in the plant.
Root inoculation with plant probiotic bacteria promoted significant increase in growth and alkaloid content (Karthikeyan et al., 2010). Some studies have shown that different types of probiotic bacteria have a positive effect on alkaloid content in the root of C. roseus (Jaleel et al., 2007a; Jaleel et al., 2009; Karthikeyan et al., 2010). Jaleel et al. (2007b) studied the effect of P. fluorescens along with drought stress on vegetative traits, and ajmalicine content in roots of C. roseus plant. They suggested that ajmalicine content increased significantly due to exposure of drought-treated plants with P. fluorescens compared to non-treated plants and control under drought stress. Experimentally, Jaleel et al. (2009) showed that supplementing plant growth regulators as the same as P. fluorescens elicitor significantly changed the constituents of metabolites (ajmalycin, serpentine, catarantine, and vindoline) in the roots of C. roseus. Karthikeyan et al. (2009) also found, by inoculating C. roseus seeds and seedlings with P. fluorescens and A. brasilense separately or in combination that bacteria can be used as a suitable agent to increase alkaloids in C. roseus roots. Also, the effect of these beneficial bacterial strains has been demonstrated on alkaloid contents of Hyoscymus niger L. and increasing the hyoscyamine and scopolamine yield in roots and shoots (Ghorbanpour et al., 2013).
To comparative analysis of molecular and metabolite results, all results were presented in Table 4. As seen in this table, P. fluorescens by increasing the expression of all studied genes in the biosynthetic pathway and A. brasilense by increasing the expression of gene at the beginning (G10H) and end of the pathway (CrPRX) were able to significantly increase the levels of both vinblastine and vincristine alkaloids compared to the control.
Table 4
Comparative analysis of molecular and metabolite evaluations in the roots of C. roseus inoculated with bacteria relative to control
Bacterial inoculation treatments | Genes expression relative to control | Alkaloids amount relative to control |
Upstream TIA pathway gene | Downstream TIA pathway genes | Vinblastine (µg/g) | Vincristine (µg/g) |
Vindoline pathway | Terminal gene |
G10H | T16H | DAT | CrPRX |
Combined inoculation | IncreaseNs | Increase* | Increase* | Increase* | Decrease* | Unchanged |
P. fluorescens | Increase** | Increase ** | Increase ** | Increase ** | Increase* | Increase* |
A. brasilense | Increase ** | IncreaseNs | IncreaseNs | Increase ** | Increase* | Increase* |
Gene expression was measured relative to the control sample (no bacterial inoculation). Ns,*, **: Non-significant, significant at the level of 5% and 1% probability, respectively. P.F: P. fluorescens and A.b: A. brasilense The means with different letters have a significant difference with each other at the level of 5% probability. |