Enhancement of diterpenoid steviol glycosides by co-overexpressing SrKO and SrUGT76G1 genes in Stevia rebaudiana Bertoni.

Stevia rebaudiana (stevia) contains commercially important steviol glycosides, stevioside and rebaudioside A, these compounds have insulinotropic and anti-hyperglycemic effect. Steviol, stevioside and rebaudioside-A have taste modulation and insulin potentiation activity. Stevia leaves are composed of steviol (2-5%), stevioside (4-13%) and rebaudioside-A (1-6%). Stevioside has after-taste bitterness, rebaudioside-A is sweetest in taste among all the glycosides present. Therefore, lower ratio of rebaudioside-A to stevioside has bitter after-taste, which makes stevia plants unpalatable. By over-expressing the genes, SrUGT76G1 and SrKO, we propose to increase the ratio of RebA to stevioside in stevia. Various lines were generated and amongst them, seven lines had both the transgenes present. Co-overxpresion of SrUGT76G1 and SrKO led to the increased concentration of RebA in all the seven transgenic lines (KU1-KU7) than control plant and RebA to stevioside ratio also increased significantly. Steviol, stevioside and RebA showed a differential concentration in all the seven lines, but the pattern was the same in all of them and the ratio of RebA to stevioside increased dramatically. In transgenic line 2 (KU2), RebA showed a steep increase in concentration 52% the rebaudioside-A to stevioside ratio increased from 0.74 (control) to 2.83. In overall all the lines, RebA showed a positive correlation with steviol and stevioside. Overexpression of SrKO led to an increase in steviol which increased the stevioside, overexpression of SrUGT76G1 ultimately increased RebA concentration. In conclusion, concentration of RebA increased significantly with co- overexpression of SrUGT6G1 and SrKO genes. Lines with increased RebA are more palatable and commercially viable.

Integrated miRNA, target mRNA, and metabolome profiling of Tinospora cordifolia with reference to berberine biosynthesis.

MicroRNAs play a central role in gene regulation and emerge as novel targets for secondary metabolites improvement in plants. The crops thus can be improved through knowledge obtained by the study of miRNAs because of their conserved nature in gene regulation. The present study has been carried out on Tinospora cordifolia (T. cordifolia), because of its illimitable application for the treatment of various diseases. This plant has tremendous medicinal properties, yet unexplored at the molecular level, and has not received much recognition in the scientific field. Thus, here computational analysis was performed to identify T. cordifolia miRNAs using EST database. Using these miRNAs, we predicted their targets which were found to be associated with the regulation of diverse gene networks including 433 berberine biosynthesis genes in T. cordifolia. Further, selected miRNAs were validated and their expression was detected in different T. cordifolia tissues followed by expression analysis of their target mRNAs. These data were then compared with the metabolic profile of T. cordifolia with an emphasis on therapeutically important compound berberine. In this study, we did simultaneous miRNA/target gene expression and metabolome analysis which opens a new way for initiating new proposition and prioritization of miRNAs/genes/metabolites for targeted follow­up metabolic engineering experimentations. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03342-9.

Salivary metabolite signatures of oral cancer and leukoplakia through gas chromatography-mass spectrometry.

Background: Saliva contains a large array of metabolites, many of which can be informative for the detection of diseases. Gas chromatography-mass spectrometry (GC-MS) is a system that has long been used for metabolite profiling owing to its sensitivity, specificity, reproducibility and synchronized analysis; it has relatively broad coverage of compound classes including sugars, sugar alcohols, glycosides and lipophilic compounds. Aim and Objectives: The present study was conducted to explore the use of GC-MS in assessing variation in salivary metabolites and to recognize the metabolites which can be used as disease diagnostic tools and metabolite markers for detection of oral squamous cell carcinoma. Materials and Methods: The present study included clinically and histopathologically confirmed oral squamous cell carcinoma (OSCC) and oral leukoplakia patients (OLK) and the control group. Patients were divided into three groups: OSCC (n = 30), OLK (n = 30) and healthy individuals as controls (n = 30). Patients were refrained from eating, drinking, smoking or oral hygiene procedures for at least 1.5 h before the collection. Saliva was collected between 9.00 and 10.00 am. Samples were stored at -80°C. Filtered samples were used for GC-MS. Results: Fifteen compounds differed significantly between control, OLK and OSCC. These metabolites were decanedioic acid, 2-methyloctacosane, eicosane, octane, 3,5-dimethyl, pentadecane, hentriacontane, 5, 5-diethylpentadecane, nonadecane, oxalic acid, 6-phenylundecanea, l-proline, 2-furancarboxamide, 2-isopropyl-5-methyl-1-heptanol, pentanoic acid, Docosane. Conclusion: The findings of the study suggest the application of salivary metabolomics as a promising tool in the identification of tumor-specific biomarkers in early diagnosis and prediction of OSCC and oral leukoplakia. In future, standardizing the protocol for salivary analysis and overcoming some of the limitations will be helpful to establish salivary metabolomics as a reliable, the highly sensitive and specific method for clinical use as an independent diagnostic aid.

Set of miRNAs Involved in Sulfur Uptake and the Assimilation Pathway of Indian Mustard (B. juncea) in Response to Sulfur Treatments.

MicroRNAs (miRNAs) play an important role in the regulation of gene expression. They play a regulatory role in various nutrient assimilatory pathways of plants; however, their role in the regulation of sulfur uptake and assimilatory pathways in mustard cultivars under high/low sulfur conditions is not elucidated. Sulfur is essential for plant growth and development, and its deficiency can cause a decline in oil seed content and thus lower the economic yield in Brassica juncea. In this study, different miRNAs involved in the regulation of sulfur uptake and assimilation pathways in B. juncea were identified using a psRNA target analyzer and miRanda database tools. The predicted miRNAs that belong to 10 highly conserved families were validated using stem-loop RT-PCR. The B. juncea cultivars Pusa Jaikisan, Pusa Bold, and Varuna were kept in sulfur-excessive (high) and -deficient (insufficient) conditions, and expression studies of miRNAs and their target mRNAs were carried out using qRT-PCR. The correlation between the expression pattern of miRNAs and their target genes showed their potential role in sulfur uptake and assimilation. Analysis with 5' RACE revealed the authentic target of miRNAs. The influence of S treatments on metabolites and sulfur content was also studied using GC-MS and a CHNS analyzer. Our study showed the potential role of miRNAs in the regulation of sulfur uptake and assimilation and put forward the implications of these molecules to enhance the sulfur content of B. juncea.

Co-expression of anti-miR319g and miRStv_11 lead to enhanced steviol glycosides content in Stevia rebaudiana.

BACKGROUND: miRNAs are major regulators of gene expression and have proven their role in understanding the genetic regulation of biosynthetic pathways. Stevioside and rebaudioside-A, the two most abundant and sweetest compounds found in leaf extract of Stevia rebaudiana, have been used for many years in treatment of diabetes. It has been found that the crude extract is more potent than the purified extract. Stevioside, being accumulated in higher concentration, imparts licorice like aftertaste. Thus, in order to make the sweetener more potent and palatable, there is a need to increase the intrinsic concentration of steviol glycosides and to alter the ratio of rebaudioside-A to stevioside. Doing so would significantly increase the quality of the sweeteners, and the potential to be used on a wider scale. To do so, in previous report, miRNAs associated with genes of steviol glycosides biosynthetic pathway were identified in S. rebaudiana. In continuation to that in this study, the two miRNAs (miR319g and miRStv_11) targeting key genes of steviol glycosides biosynthetic pathway were modulated and their impact was evaluated on steviol glycosides contents. RESULTS: The over-expression results showed that miRStv_11 induced, while miR319g had repressive action on its target genes. The knock-down constructs for miR319g and miRStv_11 were then prepared and it was demonstrated that the expression of anti-miR319g produced inhibitory effect on its target miRNA, resulting in enhanced expression of its target genes. On the other hand, anti-miRStv_11 resulted in down-regulation of miRStv_11 and its target gene. Further miRStv_11 and anti-miR319gwere co-expressed which resulted in significant increase in stevioside (24.5%) and rebaudioside-A (51%) contents. CONCLUSION: In conclusion, the role of miR319g and miRStv_11 was successfully validated in steviol gycosides biosynthetic pathway gene regulation and their effect on steviol gycosides contents. In this study, we found the positively correlated miRNA-mRNA interaction network in plants, where miRStv_11 enhanced the expression of KAH gene. miRNAs knock-down was also successfully achieved using antisense precursors. Overall, this study thus reveals more complex nature and fundamental importance of miRNAs in biosynthetic pathway related gene networks and hence, these miRNAs can be successfully employed to enhance the ratio of rebaudioside-A to stevioside, thus enhancing the sweetening indices of this plant and making it more palatable.

In silico analysis and expression profiling of miRNAs targeting genes of steviol glycosides biosynthetic pathway and their relationship with steviol glycosides content in different tissues of Stevia rebaudiana.

miRNAs are emerging as potential regulators of the gene expression. Their proven promising role in regulating biosynthetic pathways related gene networks may hold the key to understand the genetic regulation of these pathways which may assist in selection and manipulation to get high performing plant genotypes with better secondary metabolites yields and increased biomass. miRNAs associated with genes of steviol glycosides biosynthetic pathway, however, have not been identified so far. In this study miRNAs targeting genes of steviol glycosides biosynthetic pathway were identified for the first time whose precursors were potentially generated from ESTs and nucleotide sequences of Stevia rebaudiana. Thereafter, stem-loop coupled real time PCR based expressions of these miRNAs in different tissues of Stevia rebaudiana were investigated and their relationship pattern was analysed with the expression levels of their target mRNAs as well as steviol glycoside contents. All the miRNAs investigated showed differential expressions in all the three tissues studied, viz. leaves, flowers and stems. Out of the eleven miRNAs validated, the expression levels of nine miRNAs (miR319a, miR319b, miR319c, miR319d, miR319e, miR319f, miR319h, miRstv_7, miRstv_9) were found to be inversely related, while expression levels of the two, i.e. miR319g and miRstv_11 on the contrary, showed direct relation with the expression levels of their target mRNAs and steviol glycoside contents in the leaves, flowers and stems. This study provides a platform for better understanding of the steviol glycosides biosynthetic pathway and these miRNAs can further be employed to manipulate the biosynthesis of these metabolites to enhance their contents and yield in S. rebaudiana.