Effect of the Plant Probiotic Bacteria on TIA Biosynthesis Pathway Gene Expression Proling, Vinblastine and Vincristine Content in the Root of Catharanthus Roseus

Catharanthus roseus is the sole resource of vinblastine and vincristine, which are two of the biggest concerns of TIAs because of their powerful anticancer activities. Increasing the concentration of these alkaloids in various organs of the plant is one of the important goals in C. roseus breeding programs. Plant probiotic bacteria (PBB) act as biotic elicitors and can induce the synthesis of secondary products in plants. The purpose of this research is to study the individual and combined effects of P. uorescens and A. brasilense on expression of the TIA biosynthetic pathway genes (G10H, DAT, T16H and CrPRX) using qRT-PCR and the content of vinblastine and vincristine alkaloids using HPLC method in roots of C. roseus. P. uorescens drastically increased the content of vinblastine and vincristine alkaloids, compared to the control in the roots, up to 174 and 589 (µg/g), respectively. According to the molecular analysis, bacterium signicantly increased the expression of more genes in the TIA biosynthetic pathway compared to the control. P. uorescens increased the expression of the nal gene of the biosynthetic pathway (CrPRX) 47.9 times compared to the control. Therefore, the ndings indicate the coordination of transcriptional and metabolic outcomes. The same result was also observed for A. brasilense. According to the results, it can be concluded that, the seed priming and root of the seedling treatments of probiotic bacteria can be used as a good tool in the enhancement of alkaloid contents in medicinal plants, as it provides an eco-friendly approach.


Introduction
Medicinal plants are recognized as an important therapeutic tool to alleviate the ailments of human's kind (Sain and Sharma, 2013). Catharanthus roseus is one of the medicinal plant belonging to the family Apocynaceae (Almagro et al., 2015). C. roseus produces more than 130 types of terpenoid indole alkaloids (TIAs) (Gupta et al., 2007). This plant is the only source of vinblastine and vincristine, which are among the largest types of TIA due to their powerful anti-cancer activities (Zhu et al., 2014 andSun et al., 2016). However, the production of these bene cial drugs has been limited due to their small amount in the plant (Pan, et al., 2010 andAlmagro et al., 2015) and the major challenge in the pharmaceutical industry is the low production rate of these alkaloids (Soltani et al., 2020). Due to the pharmaceutical importance and the low content of vinblastine and the related alkaloid vincristine in plants. C. roseus, became one of the best-studied medicinal plants ( Van der Heijden et al., 2004), and increasing the concentration of important medicinal alkaloids in its various organs is one of the most important goals in C. roseus breeding programs (Gupta et al., 2007).
All parts of the plant have alkaloid, but, maximum concentrations are found in the root organ, particularly during the owering stage (Jaleel, et al., 2008). Therefore, this plant is considered as a very important medicinal plant in most pharmacopoeias due to the presence of valuable alkaloids in the shoots and roots.
The multi-step TIA biosynthetic pathway is quite complex and is under strict molecular regulation (Dutta et al., 2005). Many of the genes involved in this pathway of C. roseus have been cloned and sequenced for the analysis of their expression in various plant organs (Gupta et al., 2007). Critical early steps in the biosynthesis of TIAs include the reactions catalyzed by tryptophan decarboxylase (TDC), geraniol 10-hydroxylase (G10H), and strictosidine synthase (STR). TDC and G10H catalyze the rst committed steps towards TIA biosynthesis in the indole and terpenoid precursor branches, respectively (Goklany et al., 2009). Strictosidine is the central intermediate in the biosynthesis of different TIAs, which is formed by the condensation of secologanin and tryptamine. Secologanin is derived from terpenoid (isoprenoid) biosynthetic pathway, while tryptamine is derived from indole biosynthetic pathway. Then various speci c end products are produced by different routes during downstream process (Zhu et al., 2014). The downstream TIA pathway genes include deacetylvindoline-4-Oacetyltransferase (DAT), Tabersonine 16-hydroxylase (T16H), and Catharanthus roseus peroxidase (CrPRX) (Wang et al., 2016) (Fig. 1). TIA pathway is affected by biotic and abiotic factors (Favali, et al., 2004). According to some researchers, rhizosphere microorganisms act as biotic elicitors and can induce the synthesis of secondary products in plants (Sekar and Kandavel, 2010). Among these microorganisms, some have positive effects on plant growth promotion constituting the plant growth promoting rhizobacteria (PGPR) such as Azospirillum, Azotobacter, Pseudomonas uorescens and several gram positive Bacillus sp (Jaleel et al., 2007b). These rhizobacterias induce the jasmonic acid and ethylene responses in plants (Pieterse et al., 2001 andBeneduzi et al. 2012). Tissue culture studies have also shown that the external application of these compounds has induced the production of secondary metabolites in some plant species (Sekar and Kandavel, 2010). Generally, the phytohormone jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are major elicitors of TIA biosynthesis in C. roseus (Shen et al., 2017). The Naeem et al., (2017) review provides information regarding the role of potent PGRs such as gibberellic acid (GA3), in boosting the growth, metabolism, and other plant processes, particularly the production of anticancer alkaloids (vinblastine and vincristine) in C. roseus plants. Therefore, the mentioned bacteria can induce the synthesis of secondary metabolites in plants, through the production of phytohormones (Sekar and Kandavel, 2010). Singh et al., (2021) identi es the best combination of endophytes consisting of plant growth-promoting and alkaloid enhancing endophytes that can maximize the plant growth and TIAs yield in various C. roseus plant cultivars under eld conditions.
It should be noted that the uorescent strains of Pseudomonas and A. brasilense are both bene cial bacteria and have been mentioned in various sources as the most important and largest group of probiotic bacteria (Ahmadzadeh and Shari Tehrani 2021). A review of the resources shows that the only effect of these bacteria on growth parameters and metabolite levels has been studied in C. roseus (Jaleel et al., 2007b andKarthikeyan et al., 2009), while, in the present study, the effect of rhizobacteria on the expression of TIA biosynthetic pathway genes in C. roseus was evaluated for the rst time. The purpose of the research is investigation of the individual and combined effect of two species of rhizobacteria as a biotic treatment on expression some of the upstream and downstream TIA biosynthetic pathway genes, vinblastine and vincristine alkaloids content in roots of C. roseus.

Bacterial strains and growth conditions
The bacterial strains used in this study were Pseudomonas uorescens and Azospirillum brasilense. The isolates provided from the Soil and Water Research Institute (Soil Biology Research Department), Karaj, Iran. The both of the bacteria were grown on nutrient agar (NA) for routine use. A single colony was transferred to 500 mL asks containing NB grown aerobically in asks on a rotating shaker (150 rpm) for 48 h (Karthikeyan et al., 2010). The bacterial suspensions were then diluted in distilled water to a nal concentration of 10 8 colony forming units (CFU) per milliliter, and the resulting suspensions were treated with C. roseus plants. Bacterial inoculation treatment was applied to the plant in three stages including seed, root inoculation and inoculation of soil around the roots.

Plant test
The periwinkle seeds were provided by Pan American Seed Company (www.panamseed.com) and were surface sterilized in 0.1% NaClO for 15 min with frequent shaking and rinsed thoroughly in distilled water to remove NaClO. Surface-sterilized seeds were then soaked in a bacterial inoculum for 30 minutes, with shaking, and sown in culture trays, super cially. After irrigation, the culture trays were kept under plastic cover for complete absorption of moisture and natural light at a temperature of 20 to 25°C. Seedlings after emergence and in the six-leaf stage were removed from the culture trays and immersed in a bacterial inoculum and then planted in separate pots (15 cm diameter × 15 cm height), lled with a 2:1:1 mixture of farm soil, sand and peat, with the same weight (500 g).
Inoculation of root and soil around the roots were performed at the time of seedling transfer to the pot. For root inoculation, the root of the seedlings were washed with distilled water and then immersed in each of the inoculants for 20 minutes, in the six-leaf stage. Distilled water was used in equal volume with bacterial inoculation treatment as a control (no bacterial inoculation). The plants were uprooted at owering stage the day 30 th after transferring from culture tray to pots for analyzing gene expression and estimation of vinblastine and vincristine content in the root.
In uence of bacterial treatments on expression of G10H, T16H, DAT and CrPRX genes RNA extraction, cDNA synthesis, and primer designing Root samples were ash-frozen in liquid nitrogen upon harvesting at the owering stage and ground in liquid nitrogen using a mortar and pestle. Total RNA was extracted from 100 mg of roots using the RibospinTM Seed/Fruit according to the manufacturer's instructions (GeneAll, South Korea). RNA concentration was quanti ed using the NanoDrop 2000c Spectrophotometer (Thermo Scienti c NanoDrop 2000, USA) and were quali ed by 1% agarose gel electrophoresis. The rst strand cDNA was synthesized from 1µg of total RNA using the Hyperscript RT-PCR master mix® according to the manufacturer's instructions in the nal volume of 10 μl (GeneAll, South Korea). The cDNA was diluted to 100 ng/μL as the template for the real-time PCR analysis (Soltani et al., 2020).
The genes monitored in this study were G10H (gene at the beginning of the pathway biosynthesis of TIAs, in the terpenoid pathway), DAT, T16H (vindoline pathway genes) and CrPRX (Terminal gene of the pathway). Primer sequences for target genes and the 40s ribosomal protein S9 (Rps9) reference gene were obtained from various resources ( Table 1) and blasted in the National Center for Biotechnology Information (NCBI) genomic database (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) ( Table 1). Rps9 was validated as an appropriate housekeeping gene for the C. roseus by verifying the Ct pro le for Rps9 remained nearly constant for all treatments (Goklany et al., 2009). To ensure the speci c ampli cation of designed primers for these genes the PCR reaction was performed using cDNA.  To prepare a standard sample, 1 g of vinblastine sulfate and 10 g of vincristine sulfate were dissolved in 10 ml of distilled water. Then, the standard solution of vincristine and vinblastine (1000 μg/ml) was prepared by dissolving a certain amount of vinblastine sulfate and vincristine sulfate solution in methanol. The standard calibration curve was drawn. Finally, the amount of these two alkaloids in the samples was estimated by matching the standard curve.
The yield of alkaloids is calculated by multiplying the amount of alkaloids (µg/g of dry root weight) by the dry weight of the roots (g).

Statistical analysis
In this research, the effect of two bacterial strains at four levels is studied. A control (no bacteria), P. uorescens, A. brasilense and combined inoculation are investigated on C. roseus plants in a factorial experiment based on a randomized complete block design (RCBD) with three replications. The relative gene transcription was quanti ed using the comparative threshold cycle (CT) method and the data were analyzed using the REST® software [Pfa et al., 2002] according to the ΔΔCT method [Livak and Schmittgen, 2001]. Analysis of metabolite data was performed using SAS Ver. 9.2. LSD (the least signi cant difference) procedure was used to compare the means.

Results
Monitoring TIA gene expression in roots of C. roseus The transcript level of G10H gene in the terpenoid pathway (Upstream TIA pathway gene) was signi cantly upregulated. Figure 2-A shows the effect of different treatments on the relative expression of G10H gene compared to the control in the owering stage of C. roseus. Individual inoculation treatments of two bacteria had a signi cant effect on G10H gene expression. The highest transcript level was observed in P. uorescens. In combined inoculation, no signi cant increase was observed in gene expression.
Among the studied treatments, P. uorescens inoculation treatment increased the expression of T16H gene up to 22 times compared to the control. Combined inoculation resulted in increased gene expression, which was signi cant at the 5% probability level (Fig. 2-B).
According to Fig. 2-C, treatment with P. uorescens had a signi cant effect on DAT gene expression compared to the control (34-fold). Combined inoculation showed a positive and signi cant effect at the level of 5% probability on the expression of the gene.
In the case of CrPRX gene, the nal gene of TIA biosynthetic pathway, P. uorescens and A. brasilense had a positive and signi cant effect on the 1% probability level and combined inoculation had a positive and signi cant effect on the 5% probability level (Fig. 2-D). P. uorescens increased the expression of CrPRX gene 47.9 times compared to the control.
Metabolite analysis in roots of C. roseus Based on different concentrations of standard solutions injected into the device, the retention time (minutes) is speci ed for the vinblastine and vincristine alkaloids. The graphs of the other samples were interpreted based on the retention time of the alkaloids.
HPLC analyzing the extracts of C. roseus root inoculated with bacterial treatments showed a signi cant difference for both alkaloids and vincristine yield per plant (Table 2). According to mean comparison (Table 3 and Fig. 3), P. uorescens and A. brasilense, respectively, were characterized as the best treatments to induce signi cant production of vincristine and vinblastine alkaloids. In addition, these treatments individually caused increase of vincristine yield per plants root in the same manner as amount per gram of dry root weight.
Comparatively, the vincristine content was estimated at approximately 589 µg/g of dry root weight in plant roots treated with P. uorescens indicating remarkable value, which was signi cantly more than the control. Also, applying this treatment increased the vinblastine contents by up to 175 µg /g of root dry weight compared to the control. However, combined inoculation elicited the minimum production of vinblastine alkaloid, which is not signi cant content compared to the control sample.   in the present study, they probably were activated under the bacterial treatments, especially P. uorescens, 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 rst 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 nally 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. uorescens 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. Overall, P. uorescens was effective than other treatments in root and caused the signi cant 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, signi cantly.
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 roseus seeds and seedlings with P. uorescens 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 bene cial 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. uorescens 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 signi cantly increase the levels of both vinblastine and vincristine alkaloids compared to the control.

Conclusions
In the present study, P. uorescens drastically increased the content of two alkaloids vinblastine and vincristine, compared to the control and other bacterial treatments in the roots of C. roseus. A review of the results of molecular analysis showed that the bacterium signi cantly increased the expression of more genes in the TIA biosynthetic pathway compared to the control. Therefore, the positive effect of this treatment on the amount of evaluated alkaloids indicates the compatibility of the results of transcription and metabolic. The same result was observed for A. brasilense (Table 5). From the results of this investigation, it can be concluded that, the seed priming and seedling treatments of plant probiotic bacteria can be used as a good tool in the enhancement of alkaloid contents in medicinal plants, as it provides an eco-friendly approach.
Considering the positive effect of bacteria on two important alkaloids of C. roseus, the results of this research can be considered an important step in the pharmaceutical industry.