Steviol Glycosides Content Research Articles

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

BackgroundmiRNAs 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.ResultsThe 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.ConclusionIn 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.

Impact of cultivation factors in vitro on the growth and the biosynthesis of steviol glycosides in Stevia rebaudiana cell cultures.

The growth and the biosynthesis of steviol glycosides (SGs) in Stevia rebaudiana cell cultures subjected to the impact of different cultivation factors were investigated under in vitro conditions. The changes in composition and concentration of constituted components of the nutrient medium was found to exert a significant influence on the of cell culture growth and the biosynthesis of SGs. It was found that combination of growth regulators in the nutrient medium is more important factor for proliferation of isolated cells of S. rebaudiana while the level of SGs accumulation depends to greater extent on genotypical features of cell strain. Light was shown to activate the biosynthesis of SGs, providing evidence for the suggestion about chloroplasts participation in this activation. In morphogenic callus cultures, the content of SGs was about ten times higher, indicating that the level of the accumulation of the compounds in question depends on cell differentiation and specialization.

Growth and Steviol Glycoside Content of Stevia rebaudiana Bertoni in the Thin-Layer Liquid Culture Treated with Late-Stage Gibberellin Biosynthesis Inhibitors

Stevia (Stevia rebaudiana Bertoni) is a source of steviol glycoside, a natural sweetener which has almost zero calorie and glycemic index. Steviol glycoside shares the same precursor with gibberellin (GA), ent-kaurenoic acid. Therefore, to manipulate the biosynthesis pathway of steviol glycoside, a gradient of late-stage GA biosynthesis inhibitors, Daminozide and Prohexadione-Ca, was applied to the thin-layer liquid culture of stevia, which was 10 mL of liquid medium in the 100-mL Erlenmeyer flask. The culture was incubated for 3 weeks, followed by the observation of growth parameters and steviol glycoside content. Results showed that both Daminozide and Prohexadione-Ca significantly increased the steviol glycoside content, indicating that the disruption of GA biosynthesis could divert the metabolic pathway toward steviol glycoside production. However, the inhibition effect of Prohexadione-Ca was stronger than Daminozide, and caused poor growth of stevia plantlets. Therefore, Daminozide at the concentrations of 10 ppm and 20 ppm was potential to modulate steviol glycoside production from the stevia culture.

Pertumbuhan, Produksi Biomassa, dan Kandungan Glikosida Steviol pada Lima Klon Stevia Introduksi di Bogor, Indonesia

Stevia rebaudiana Bertoni, a sweetener plant, has been cultivated in Indonesia since the early 1980s, but the yield is lower than in other countries. Five selected introduced stevia clones were planted at Megamendung, Bogor, Indonesia (6°39’ S, 106°56’ E, 800 m above sea level) on December 2014 until November 2015 to evaluate their growth and biomass yield. The growth and yield were observed from 6 to 12 months after planting. In addition, stevioside, rebaudioside A (reb A), and total steviol glycoside (TSG) contents of leaves were measured using high performance liquid chromatography (HPLC). At six months after planting, stem diameter was 8-11 mm, plant canopy diameter was 23-30 cm, plant height was 24-35 cm, and plant mortality was less than 5% except for clone BU at 30.7% due to Sclerotium attack. The plants flowered at different times, therefore had different harvest durations, from 4 weeks (clone BM) to 8 weeks (clone BP). The highest leaf yield was clone BM (6.04 ton ha-1 per year), followed by BX (4.91 ton ha-1 per year), and BP (4.38 ton ha-1 per year). From the five clones tested, clone BM was the best for leaf, TSG, and stevioside yields; whereas clone BS and BP were the best for reb A yield. Keywords: rebaudioside A, Stevia rebaudiana, stevioside, total steviol glycoside

Mutations in the uridine diphosphate glucosyltransferase 76G1 gene result in different contents of the major steviol glycosides in Stevia rebaudiana

In the metabolic glycosylation grid of steviol glycosides, UGT76G1 was shown to catalyze at least eight different glucosylation steps, including the formation of rebaudioside B (Reb B) and rebaudioside A (Reb A) (Olsson et al., 2016). In this study, the accumulation of steviolbioside, Reb B, stevioside (ST) and Reb A in more than 140 samples of stevia leaves collected from different regions in China were analyzed by high-performance liquid chromatography (HPLC), and five genotypes, ‘N01-N05’, with significantly different levels of the abovementioned glycosides were discovered. Mutations in the UGT76G1 gene cloned from cDNAs from these five genotypes were identified, and the functions of the recombinant UGT76G1 variants were ascertained by adding steviolbioside and ST substrates. In addition, homology modeling and molecular docking were used to elucidate the functional differences between variants and UGT76G1. Comparing the sequences of the five variants ‘N01-N05’ with UGT76G1 (AY345974.1) revealed that base substitutions were not observed in ‘N01’. By contrast, ‘N02’ exhibited 9 single nucleotide polymorphisms (SNPs) and 9 associated amino acid substitutions or insertions with notable variations in the protein structure; however, an enzyme assay showed similar functionalities between the variant and UGT76G1. In ‘N03’, 49 SNPs and 29 associated amino acid substitutions or insertions were identified and shown to induce significant variations in the protein structure, especially in the binding pocket, resulting in the lack of functionality of this variant in the enzyme assay. These results were in agreement with the docking profiles. Moreover, a nonsense mutation of p.1090T > G in ‘N04’ and an insertion of a 68 base fragment in ‘N05’ were found, and both produced a premature protein without any catalytic activity. Therefore, UGT76G1, which is vital to the content of main steviol glycosides, should be a key gene marker for the molecular breeding of Stevia rebaudiana. Our investigations also revealed the location and orientation of active groups of the receptors and donors in the UGT76G1 enzyme, which play key roles in determining whether the enzyme has any enzymatic activity.

Salt Stress-Induced Changes in In Vitro Cultured Stevia rebaudiana Bertoni: Effect on Metabolite Contents, Antioxidant Capacity and Expression of Steviol Glycosides-Related Biosynthetic Genes

Stevia rebaudiana is an industrially and medicinally important herb, mainly due to its steviol glycoside content, which is a calorie-free natural sweetener. Moreover, it contains other phytochemicals that make this species potentially beneficial for health. Salt stress affects plant growth, development and plant metabolism. The present study was executed with the objective of evaluating the effect of different NaCl concentrations (0.0, 0.5, 1.0 and 1.5 g L−1) on growth parameters, total soluble sugars, steviol glycosides and phenolic compounds (total soluble phenols, flavonoids and hydroxycinnamic acids), as well as antioxidant capacity (with a FRAP and DPPH assay). We also analysed in this study the expression levels of genes involved in the three stages of the steviol glycosides biosynthesis pathway in S. rebaudiana. Treatment with NaCl decreased root numbers and weights (dry and fresh), while increasing antioxidant capacity (according to the FRAP assay), hydroxycinnamic acid and total soluble sugar content. Furthermore, the up-regulation of several genes (CMS, CMK, HDR and UGT76G1) encoding key enzymes of the steviol glycoside biosynthetic pathways was observed after NaCl treatments. The results provide new insights into the physiological, biochemical and molecular response mechanisms of in vitro-grown stevia plants to NaCl.

Effects of cytokinins, gibberellic acid 3, and gibberellic acid 4/7 on in vitro growth, morphological traits, and content of steviol glycosides in Stevia rebaudiana

Steviol glycosides (SGs) and gibberellic acids share a part of their biosynthesis pathways. Despite the widespread studies on the effect of gibberellic acid 3 (GA3), the effect of gibberellic acid 4 and 7 (GA4/7) on Stevia rebaudiana has never been studied. This study aimed at a comparative evaluation of different hormone effects, i.e., 1 mg L−1 GA4/7, 1 mg L−1 GA3, or 0.5 mg L−1 kinetin and 0.5 mg L−1 6-benzylaminopurine (BAP) (KB 0.5), on in vitro propagation, growth, morphological properties, and content of SGs in leaf samples of stevia. In comparison with the control group (hormone-free), the treatments of KB 0.5 or GA3 produced the highest biomasses and largest leaf areas. The three hormonal treatments produced a similar number of leaves, the ratio of fresh to dry weight, and leaf length. GA4/7-treated explants produced the highest ratio of leaf area to leaf length. The effect of GA4/7 on shoot elongation was greater than that of the control or even GA3. While the effect of GA3 on rebaudioside-A (Reb-A) production was similar to that of the control (16.2 and 18.04 mg g−1, respectively), GA4/7 resulted in a lower amount of it (13.31 mg g−1). Except for GA4/7, which induced more stevioside accumulation, the treatments’ effects were comparable to that of the control. The ratio of stevioside to Reb-A was the highest for GA4/7 (2.62), followed by GA3 (1.93), and then the two others. Sum of Reb-A and stevioside content was not changed by the use of any of the treatments.

Acute gamma irradiated Stevia rebaudiana Bertoni enhanced particular types of steviol glycosides

Stevia rebaudiana Bertoni from Asteraceae family is commercially valuable for its steviol glycosides (SGs) contents, which is 300 times sweeter than commercial sugar. The bottleneck in Malaysia is the lack of suitable stevia varieties that are able to thrive well under her climatic conditions and still produce high SGs. Mutation induction including gamma irradiation is effective in generating genetic variations and developing new plant varieties with desired traits. This study was aimed to determine the effects of acute gamma irradiation on phenotypic changes and enhancement of SGs contents of Stevia rebaudiana Bertoni variety AKH L1 (herein after will be designated as AKH L1). In vitro shoot tip explants of AKH L1 were subjected to a gamma doses regime of 10Gy to 50Gy, following which phenotypic changes of the irradiated explants and subsequent regenerated plantlets were observed. All irradiated explants exhibited different survival rates, with the lowest at 9.33±8.33% when subjected to 50Gy, while all the control (non-irradiated explants) survived. The LD50 was found to be at 23Gy. Subsequent irradiation of 900 shoot tip explants at 23Gy, produced 468 surviving shoot tips, which were all capable to develop and successfully sub-cultured until the fourth generation, M4. These M4 in vitro mutant plantlets exhibited significant increase in the numbers of leaf (16.07±5.19) and average leaf size (1.12±0.26cm x 0.54±0.15cm). HPLC analysis performed in parallel further revealed the mutant plants contained higher concentrations of stevioside (387.04ppm), rebaudioside A (670.18ppm) and rebaudioside D (106.26ppm) compared to the non-irradiated plantlets, which exhibited 96.87, 194.42 and 28.25ppm, respectively.

Plant growth retardants (PGRs) affect growth and secondary metabolite biosynthesis in Stevia rebaudiana Bertoni under drought stress

Beyond the inhibitory action against the gibberellin biosynthesis, some plant growth retardants (PGRs) can play an important role in regulating plant responses to abiotic stress through the induction of different tolerance mechanisms. The aim of the present study was the exploitation of the potential of PGRs in enhancing the resistance to drought stress in Stevia rebaudiana Bert. Therefore, the effects of three PGRs on stevia plants grown under drought stress condition were investigated. Stevia plants were first subjected to water stress and, second, treated with PGRs to detect PGRs effect on biometric, productive and phytochemical characteristics of drought stressed-plants. The control plants were uniformly irrigated at 3-day intervals, while water-stress conditions were imposed by watering the plants at 12-day intervals. Subsequently, the Chlorocholine chloride (CCC, as Copalyl diphosphate synthase inhibitor and Kaurene synthase inhibitor), Paclobutrazol (PBZ, as Kaurene oxidase inhibitor) and Daminozide (DAM, as anti-gibberellins) were applied in drought stressed-plants. The CCC and DAM were sprayed on stevia shoots, while PBZ was drenched. The obtained results showed that leaf dry weight of stevia plants was significantly reduced by drought stress, but this parameter increased as a consequence of CCC and PBZ treatments. Drought stress also caused a significant reduction in total steviol glycoside (SVglys) content. This reduction was more pronounced in drought stressed-plants treated with CCC, while PBZ was able to counteract the SVglys reduction, with SVgly content similar to that observed in the control. Similarly, PBZ was able to increase the soluble sugar production and total antioxidant capacity in the leaves of stressed-stevia plants. These findings suggested that CCC and, in particular, PBZ had a protective effect on stevia growth under drought stress by induction of antioxidant defenses and soluble sugar production. CCC seems to inhibit gibberellin biosynthesis, preventing the SVglys production, while DAM and PBZ, as gibberellin inhibitors, didn't have a negative effect on SVglys production in drought stressed-plants. This observation seems to emphasize their role in limiting the rate of target enzymes of CCC in SVglys biosynthetic pathway. Moreover, the induction of glucose production, as a substrate for SVglys biosynthesis, could be a convincing evidence for SVglys promotion in PBZ treated-plants.

Study of gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni under various mannitol concentrations.

Stevia rebaudiana produces sweet steviol glycosides that are 300 times sweeter than sugar and have the beneficial effects on human health including anti-hyperglycaemic. Tissue culture is the best method with high efficacy to propagate stevia. Abiotic stress has an impact on steviol glycoside contents in stevia. Therefore, we investigated the effect of mannitol on the expression of four genes involved in the biosynthesis of stevia including UGT74G1, UGT76G1, kaurene oxidase and kaurene synthase genes and steviol glycosides accumulation in stevia under in vitro conditions. The highest expression of UGT76G1 gene occurred in the plants grown under 20g/l mannitol. While for the kaurene synthase gene, the highest amount of gene expression was observed at 40g/l mannitol. The results were different about kaurene oxidase gene. As the highest and lowest gene expression were seen in 50 and 30g/l mannitol conditions respectively. There were the same results for UGT74G1 that means the most appropriate and also the most inopportune treatment for the gene expression were same as the condition for the kaurene oxidase gene. Compared with control, adding mannitol to media in all concentrations increases the expression of UGT76G1 gene. Estimation of steviol glycosides contents under different treatments of mannitol carried out by HPLC. According to the results, the highest amount of stevioside was produced under 20g/l mannitol treatment. However, rebaudioside A was accumulated in its maximum amounts under 30g/l mannitol. It can be concluded that adding mannitol to media in the certain concentration increases steviol glycoside contents in the stevia.

Deciphering performances of fifteen genotypes of Stevia rebaudiana in southwestern France through dry biomass and steviol glycoside evaluation

Stevia rebaudiana (Bertoni) is a perennial shrub native of Paraguay whose leaves naturally accumulate steviol glycoside (SG) sweeteners. Optimization of S. rebaudiana semi-perennial cultivation in Europe is based upon breeding adapted high-value genotypes. The basis of a pre-breeding program is to evaluate in field conditions the performances of a large set of genotypes that could then be used as genitors for new varieties. This study investigates the performances of fifteen clones gathered from different origins in the second year of production under the environmental conditions of southwestern France. The performances were evaluated in terms of SG yield on the basis of dry leaf biomass yield, SG content and composition. The fifteen genotypes showed a very wide range of performances, expressing ratios of 15, 3.5 and 4 between the worst and best genotypes for SG yield, dry leaf biomass yield and SG content respectively. The sweetest SG (RebA) could represent 72% of total SG content in the best genotype. We confirmed the major role of dry leaf weight in SG yield. Five canopy descriptors out of ten, number of principal stems, stem density (number of stems/m² of canopy area), canopy volume (m3), Harvest Index (HI) and Specific Stem Mass (g/m2), were relevant to describe aerial biomass variability and classify the genotypes. This study thus provides biomass descriptors as tools for future breeding purposes and highlights the urgent need to evaluate the great genetic diversity of S. rebaudiana as a prerequisite for breeding purposes.

Plant growth and steviol glycosides as affected by foliar application of selenium, boron, and iron under NaCl stress in Stevia rebaudiana Bertoni

Stevia (Stevia rebaudiana Bert.) is well known for its high content of sweet steviol glycosides (SVglys) in its leaves (about 4–20% of dry weight) depending on the environmental, climatic, and growth conditions. The present study aimed to investigate the effects of foliar iron, selenium, and boron on stevia, irrigated with different concentrations of saline water. The experiment was conducted during two consecutive years in Firuzabad and Anzali, Iran. Results indicated that plant height, number of branches, leaf and biological yield and leaf mass ratio decreased by increasing the NaCl concentration of irrigation water. In both places, total SVglys content, and SVglys yield increased when the plants were irrigated with a 30 mM of saline water while higher salinities (90 mM) resulted in decreasing the traits. In addition, the foliar application of selenium alone and in combination with boron, and iron played a significant role in growth, yield parameters and SVglys accumulation including rebaudioside-A,-B and -C, Stevioside, and Dulcoside-A. Firuzabad had the highest mean values of vegetative traits, leaf yield, and SVglys yield in the second year of the study. Regarding Firuzabad, leaf yield and SVglys percentages had a compensatory relationship, which can play a role in the observed increase in the SVglys percentage under a moderate level of NaCl (30 mM) stress. Finally, the foliar application of selenium either alone or in combination with iron could alleviate the adverse effects of NaCl stress on the studied traits. However, further studies are required to recognize the roles of selenium in SVglys accumulation and growth of stevia.

Antioxidant Capacity and UPLC-PDA ESI-MS Phenolic Profile of Stevia rebaudiana Dry Powder Extracts Obtained by Ultrasound Assisted Extraction

Stevia leaves, which are commonly used as a natural sweetener in food products, have increased in importance for antioxidant delivery due to their high content of phenolic compounds. In this study, the influence of the drying process on stevia leaves, with regards to phenolic content and antioxidant activity during drying kinetics 40 °C for 7 h, was studied. The effect of solvent concentration and extraction time using a 32 factorial design on total phenol content (TPC), and on antioxidant activity of extracts obtained from dried stevia leaves, by ultrasound assisted extraction (UAE) as alternative method was evaluated. Steviol glycosides contents were also evaluated by a conventional and UAE method. Phenols identification, quantification and purification were performed by Ultra Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (UPLC-ESI-MS), Ultra Performance Liquid Chromatography-Photodiode Array (UPLC-PDA) and advanced automated flash purification, respectively. Drying time affected the moisture content of stevia leaves. A constant weight was reached after six hours of drying, and higher antioxidant activity was observed, while the highest TPC was obtained after seven hours of drying. The highest TPC (91.57 ± 8.8 mg GAE/g dw) and antioxidant activity (603.24 ± 3.5 μmol TE/g dw) in UAE method was obtained when ethanol 50% at 5 min was used. Steviol glycosides extracted by UAE were recorded with a content of 93.18 ± 1.36 mg/g dw and 98.97 ± 1.75 mg/g dw for stevioside and rebaudioside A respectively. Six phenolic compounds including four phenolic acids and two flavonoids were identified and quantified by UPLC-PDA, and confirmed by ESI-MS reporting its fragmentation pattern. Diosmin and chlorogenic acid were the most abundant compounds with values of 2032.36 μg/mL and 434.95 μg/mL respectively. As a novelty we found that the antioxidant activity evaluated in partially purified fractions suggested that biological activity might be attributed to the synergistic effect of the six phenols present in the stevia leaves extract. In addition to its sweeting properties, stevia leaves constitute a potential source of polyphenolic compounds, with antioxidant activity that could be used as a food additive.

The response of Stevia (Stevia rebaudiana Bertoni) to nitrogen supply under greenhouse condition

ABSTRACTStevia (Stevia rebaudiana Bertoni) is a perennial plant producing some natural sweeteners. Stevia is considered as a new crop in some countries. This study was conducted to find the stevia response to nitrogen fertilizer supply. Different levels of nitrogen (0, 30, 60, 90, 120, and 150 kg/ha from urea source) were used in a greenhouse condition and then the stevia growth and metabolites were assessed under different availability of nitrogen. Results showed that the optimum growth of stevia was obtained by 60 kg/ha nitrogen and more nitrogen supply did not enhance the stevia growth. It was observed that the total steviol glycosides (SVglys) content of Stevia was significantly increased by nitrogen fertilizer application just up to 30 kg/ha, while it decreased by more rates of nitrogen fertilizer. Our result clearly showed that SVglys yield reached to maximum value by application of 60 kg/ha of nitrogen fertilizer. Since the variation of SVglys content and shoot growth of the stevia were compromised by 60 kg/ha nitrogen, it can be concluded that 60 kg/ha of nitrogen fertilizer could be considered as an optimum rate of nitrogen for stevia and could also be recommended for greenhouse conditions.

Growth, development and steviol glycosides content in the relation to the photosynthetic activity of several Stevia rebaudiana Bertoni strains cultivated under temperate climate conditions

Abstract The objective of this study was to evaluate the development and the quality of leaves from several S. rebaudiana Bertoni strains cultivated under temperate climate conditions in the tunnel and in the open field in the relation to the photosynthetic activity. Investigated stevia strains differed significantly in terms of whole plant and leaf morphology, photosynthetic efficiency as well as in the content of steviol glycosides, and antioxidant capacity, independently of cultivation place. On the basis of several chlorophyll a fluorescence parameters analysis it was stated that plants of all examined strains, did not exhibit photosynthesis inhibition. The studies of the relationship between photosynthetic activity and the level of secondary metabolites indicated that the highest content of steviol glycosides and phenolic compounds had plants showing also the highest value of the energy fraction photochemically converted in PS II (YII), while the highest ratio of rebaudioside A to stevioside content corresponded with low amount of energy dissipated as a heat (YNPQ). This results might indicate that steviol glycosides play a role in the better adaptation to environmental conditions. Karyology and cytometric studies revealed that all investigated stevia strains were diploids without significant differences in 2C DNA amount. Therefore, observed diversity in the content of steviol glycosides and phenolic compounds between investigated S. rebaudiana strains was not the effect of polyploidization but probably resulted from the differences in the metabolic activity of the examined plants.

Steviol Glycoside Content Dynamics during the Growth Cycle of <i>Stevia rebaudiana</i> Bert

The sweetener compounds in Stevia, steviol glycosides (SG), are mainly found in the leaves. The SG content depends on the plant’s growth stage and is usually highest just before flowering. In temperate areas, Stevia is a polyannual crop (5 - 7 years) with a vegetative period lasting from April-May until October, during which time the crop can be harvested two or three times. This research focuses on the need for knowledge about Stevia’s response to temperate climates in Southern Europe. Two field assays were established from June to October 2013 at two sites in Navarra (Spain). The biomass and content of the two major SG, stevioside (ST) and rebaudioside A (RA), were measured using High Performance Liquid Chromatography (HPLC) in 66 cloned plants, at different developmental stages. Although the results from the two sites showed different SG leaf content dynamics during the plant growth, the optimum harvest date at both sites coincided with the bud-flowering stage at the beginning of September (around 96 days after planting), when a ST yield of 27 g·m-2 was reached. These results show Stevia’s potential as a commercial crop for stevioside production in northern Spain.

Effect of Treatment with Gibberellin, Gibberellin Biosynthesis Inhibitor, and Auxin on Steviol Glycoside Content in Stevia rebaudiana Bertoni

Steviol glycosides (SGs) are natural compounds present in Stevia rebaudiana (Bertoni) Bertoni, which are approximately 200–400 times sweeter than sucrose. A biosynthetic pathway of SGs shares a biosynthetic pathway of gibberellin (GA) through a formation of ent-kaurenoic acid. Previous studies have demonstrated that SG-related genes are upregulated by exogenous GA treatment. However, the transcription level of UGT85C2, which is involved in a glycosylation step to steviol from steviolmonoside and suggests a rate-limiting step in the SGs biosynthesis, remains controversial in GA treatments. To have a detailed understanding of the relationship between SGs and GAs, a concentration gradient of exogenous GA3, GA inhibitor daminozide (DAM), and naphthaleneacetic acid (NAA) was used for treatment by the leaf infiltration method. Results showed that transcription level of UGT85C2 and the total number of stevioside and rebaudioside-A induced by GA3, DAM, or NAA treatment were significantly upregulated compared to an untreated group. These results suggest that the GA biosynthetic pathway affects the number of SGs and that hormonal crosstalk occurs between GA and auxin, which changes the number of SGs in S. rebaudiana. Controlling the GA biosynthesis could be considered to modulate the yield of SGs.

Differential effects of plant growth regulators on physiology, steviol glycosides content, and antioxidant capacity in micropropagated tissues of Stevia rebaudiana

AbstractPlants naturally produce secondary metabolites on exposure to abiotic/biotic stress. Production of secondary metabolites, like phenols or flavonoids, is a defence mechanism against different stresses. This study explores the influence of plant growth regulators (PGRs) on the growth and secondary metabolites of

Molecular dissection of transcriptional reprogramming of steviol glycosides synthesis in leaf tissue during developmental phase transitions in Stevia rebaudiana Bert

Stevia is a natural source of commercially important steviol glycosides (SGs), which share biosynthesis route with gibberellic acids (GAs) through plastidal MEP and cytosolic MVA pathways. Ontogeny-dependent deviation in SGs biosynthesis is one of the key factor for global cultivation of Stevia, has not been studied at transcriptional level. To dissect underlying molecular mechanism, we followed a global transcriptome sequencing approach and generated more than 100 million reads. Annotation of 41,262 de novo assembled transcripts identified all the genes required for SGs and GAs biosynthesis. Differential gene expression and quantitative analysis of important pathway genes (DXS, HMGR, KA13H) and gene regulators (WRKY, MYB, NAC TFs) indicated developmental phase dependent utilization of metabolic flux between SGs and GAs synthesis. Further, identification of 124 CYPs and 45 UGTs enrich the genomic resources, and their PPI network analysis with SGs/GAs biosynthesis proteins identifies putative candidates involved in metabolic changes, as supported by their developmental phase-dependent expression. These putative targets can expedite molecular breeding and genetic engineering efforts to enhance SGs content, biomass and yield. Futuristically, the generated dataset will be a useful resource for development of functional molecular markers for diversity characterization, genome mapping and evolutionary studies in Stevia.

Effect of nutrient omission and pH on the biomass and concentration and content of steviol glycosides in stevia (Stevia rebaudiana (Bertoni) Bertoni) under hydroponic conditions

Steviol glycosides (SGs), have recently been approved in western countries as sources of intense natural sweeteners. SGs are found in Stevia rebaudiana, mainly in the leaves, and industry prefers rebaudioside A (Reb-A) over other steviol glycosides for its superior flavour profile. Hence leaf biomass and concentration of SGs (and their product, SG content) are of primary agronomic interest. Experiments were conducted under controlled conditions in nutrient solution to assess the effects of nutrient deficiencies and pH on biomass production, and concentration and plant content of SGs. Total SG content was low in plants deficient in the macronutrients N, P, S, Mg or Ca because of reduced photosynthesis and because of the decreased leaf yield, even though lack of N resulted in greater concentration of stevioside in the leaves. Lack of N or P reduced the proportion of Reb-A to total SGs. Plants deficient in K had less yield than in the nutrient-complete control, but not significantly so and SG concentration in the leaves was similar to that of the control. Deficiency of the micronutrients Cu and Fe led to low SG yield, because of reduced SG concentration in leaves, and because of reduced leaf yield, respectively. Lack of other micronutrients did not influence SG content. Neutral to alkali conditions reduced plant growth and leaf yield, most likely due to deficit of P, but pH had no effect on SG concentration. Our results are indicative, but preliminary, and require confirmation in open field trials over several years.