Improving organoleptic and antioxidant properties by inhibition of novel miRstv_7 to target key genes of steviol glycosides biosynthetic pathway in Stevia rebaudiana Bertoni.

Stevioside (5-10%) and rebaudioside-A (2-4%) are well-characterized diterpene glycosides found in leaves of Stevia rebaudiana known to have natural sweetening properties with zero glycaemic index. Stevioside has after-taste bitterness, whereas rebaudioside-A is sweet in taste. The ratio of rebaudioside-A to stevioside needs to be changed in order to increase the effectiveness and palatability of this natural sweetener. Plant-specific miRNAs play a significant role in the regulation of metabolic pathways for the biosynthesis of economically important secondary metabolites. In this study inhibition of miRNA through antisense technology was employed to antagonize the repressive action of miRstv_7 on its target mRNAs involved in the steviol glycosides (SGs) biosynthesis pathway. In transgenic plants expressing anti-miRstv_7, reduced expression level of endogenous miRstv_7 was observed than the non-transformed plants. As a result, enhanced expression of target genes, viz. KO (Kaurene oxidase), KAH (Kaurenoic acid-13-hydroxylase), and UGT76G1 (UDP-glycosyltransferase 76G1) led to a significant increase in the rebaudioside-A to stevioside ratio. Furthermore, metabolome analysis revealed a significant increase in total steviol glycosides content as well as total flavonoids content. Thus, our study can be utilized to generate more palatable varieties of Stevia with improved nutraceutical values including better organoleptic and antioxidant properties.

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.

Regulatory trends in engineering bioactive-phytocompounds.

The secondary plant metabolites are of enormous importance because of their extensive medicinal, nutraceutical, and industrial applications. In plants, these secondary metabolites are often found in extremely small amounts, therefore, following the discovery of any prospective metabolite, the main constraining element is the ability to generate enough material for use in both industrial and therapeutic settings. In order to satisfy the rising demand for value-added metabolites, researchers prefer to use different molecular approaches for scalable and sustainable production of these phytocompounds. Here, we discuss the emerging regulatory trends in engineering these bioactive-phytocompounds and provide recommendation on successful employment of these state-of-the-art technologies for translation of these academic researches into novel process and products.

Increased artemisinin production in Artemisia annua L. by co-overexpression of six key biosynthetic enzymes.

Malaria remains a global health issue, especially in resource-limited regions. Artemisinin, a key antimalarial compound from Artemisia annua, is crucial for treatment, but low natural yields hinder large-scale production. In this study, we employed advanced transgenic technology to co-overexpress six key biosynthetic enzymes-Isopentenyl Diphosphate Isomerase (IDI), Farnesyl Pyrophosphate Synthase (FPS), Amorpha 4,11-diene Synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), cytochrome P450 oxidoreductase (AACPR) and artemisinic aldehyde D11 reductase (DBR2)-in A. annua to significantly enhance artemisinin production. Our innovative approach utilized a co-expression strategy to optimize the artemisinin biosynthetic pathway, leading to a remarkable up to 200 % increase in artemisinin content in T1 transgenic plants compared to non-transgenic controls. The stability and efficacy of this transformation were confirmed in subsequent generations (T2), achieving a potential 232 % increase in artemisinin levels. Additionally, we optimized transgene expression to maintain plant growth and development, and performed untargeted metabolite analysis using GC-MS, which revealed significant changes in metabolite composition among T2 lines, indicating effective diversion of farnesyl diphosphate into the artemisinin pathway. This metabolic engineering breakthrough offers a promising and scalable solution for enhancing artemisinin production, representing a major advancement in the field of plant biotechnology and a potential strategy for more cost-effective malaria treatment.

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.

Natural inhibitors: A sustainable way to combat aflatoxins.

Among a few hundred mycotoxins, aflatoxins had always posed a major threat to the world. Apart from A. flavus, A. parasiticus, and A. nomius of Aspergillus genus, which are most toxin-producing strains, several fungal bodies including Fusarium, Penicillium, and Alternaria that can biosynthesis aflatoxins. Basically, there are four different types of aflatoxins (Aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2), Aflatoxin G1 (AFG1), Aflatoxin G2 (AFG2)) are produced as secondary metabolites. There are certainly other types of aflatoxins found but they are the by-products of these toxins. The fungal agents generally infect the food crops during harvesting, storing, and/or transporting; making a heavy post-harvest as well as economic loss in both developed and developing countries. And while ingesting the crop products, these toxins get into the dietary system causing aflatoxicosis, liver cirrhosis, etc. Therefore, it is imperative to search for certain ways to control the spread of infections and/or production of these toxins which may also not harm the crop harvest. In this review, we are going to discuss some sustainable methods that can effectively control the spread of infection and inhibit the biosynthesis of aflatoxins.

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.

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.

Germline transformation of Artemisia annuaL. plant via in planta transformation technology "Floral dip".

The therapeutic efficacy of Artemisia annua L. is governed by artemisinin (ART), prevalently produced by A. annua extraction. Due to the modest amount of ART (0.01-1 %dw) in this plant, commercialization of ACTs is difficult. In this study, the floral-dip based transformation protocol for A. annua was developed to enhance expression of artemisinin biosynthesis genes and ART content. For dipping, the effective infiltration media components were optimized, and to obtain high transformation (26.9%) partially open bud stage capitulum of floral development was used. Hygromycin phospho-transferase (hptII) selection marker was used to validate the transformed T1 progenies. The copy numbers of the transgene (hptII) in T1 progenies were determined using a sensitive, high-throughput SYBR Green based quantitative RT-PCR. The results of the hptII transgene were compared with those of the low copy number, internal standard (hmgr). Using optimised PCR conditions, one, two and three transgene copies in T1 transformants were achieved.

Influence of Plant Growth Regulators on Glandular Trichome Density and Steviol Glycosides Accumulation in Stevia rebaudiana.

With the advent of modern lifestyles, diabetes-related comorbidities attributed the importance of low-caloric natural sweetener plants such as Stevia rebaudiana. This plant is the viable source of steviol glycosides (SGs) and other economically important secondary metabolites. Glandular trichomes (GTs) play the role as a reservoir for all secondary products present in the plant species. Therefore, the present study was carried out to evaluate the influence of different plant growth regulators (PGRs) on GT density and its impact on the SG content. The direct shoot regeneration system was developed on Murashige and Skoog (MS) + benzyl aminopurine (BAP) (1.0 mg/L) + naphthaleneacetic acid (NAA) (0.5 mg/L), and MS + BAP (1.5 mg/L) + NAA (0.5 mg/L) from nodal and leaf explants, respectively. Among the combination of PGRs used, MS medium fortified with BAP (1.0 mg/L) and 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5 mg/L) played a significant role in increasing the GT density on leaf and stem tissues of S. rebaudiana. Furthermore, high-performance thin-layer chromatography and gas chromatography-mass spectrophotometry data confirmed a notable rise in SGs and other valuable secondary metabolites. Thus, the protocol developed can be used for the propagation of stevia with an improved metabolic profile at a large scale.

Identification and the potential involvement of miRNAs in the regulation of artemisinin biosynthesis in A. annua.

Micro RNAs (miRNAs) play crucial regulatory roles in multiple biological processes. Recently they have garnered the attention for their strong influence on the secondary metabolite production in plants. Their role in the regulation of artemisinin (ART) biosynthesis is, however, not fully elucidated. ART is a potent anti-malarial compound recommended by WHO for the treatment of drug-resistant malaria. It is produced by Artemisia annua (A. annua). The lower in planta content of ART necessitates a deep understanding of regulatory mechanisms involved in the biosynthesis of this metabolite. In this study, using modern high throughput small RNA-sequencing by Illumina Nextseq 500 platform for identification and stem-loop RT PCR for validation, miRNAs were identified in the leaf sample of A. annua plant. Here, we report a total of 121 miRNAs from A. annua that target several important genes and transcription factors involved in the biosynthesis of ART. This study revealed the presence of some important conserved miRNA families, miR396, miR319, miR399, miR858, miR5083 and miR6111 not identified so far in A. annua. The expression patterns and correlation between miRNAs and their corresponding targets at different developmental stages of the plant using real-time PCR indicate that they may influence ART accumulation. These findings thus, open new possibilities for the rational engineering of the secondary metabolite pathways in general and ART biosynthesis in particular.

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.