Accumulation Of Steviol Glycosides Research Articles

Effect of UV Stress on the Antioxidant Capacity, Photosynthetic Activity, Flavonoid and Steviol Glycoside Accumulation of Stevia rebaudiana Bertoni

Lighting conditions are an important controller of plant growth and development, and they affect secondary metabolite synthesis. In this research, we explored the effect of additional UV irradiation of various ranges in addition to the main one at PPFD 160 µmol m−2 s−1 on the accumulation of some secondary metabolites of stevia (Stevia rebaudiana Bertoni). The fresh weight of leaves was slightly higher under additional UV-A and UV-B irradiation compared with the control variant, and the leaf surface area was significantly larger, respectively, by 23.3 and 20.7% than in the control variant, while the rate of photosynthesis did not decrease. Plants under additional UV-B and UV-C irradiation were under the greatest light stress, as evidenced by a decrease in antioxidant capacity by an average of 30% compared to the control and UV-A. The total flavonoid content was significantly higher (by 74%) under UV-B irradiation. The highest concentration of steviol glycoside was observed during budding and flowering under UV-B and UV-C irradiation (by 13.2 and 11.3%, respectively). Analysis of hyperspectral images, chlorophyll fluorescence, and vegetation indices showed light stress increasing under UV-C irradiation, which caused an increase in the relative chlorophyll content, scorches, leaf morphology changes, a CO2 absorption rate decrease, and plant growth inhibition. UV-B irradiation can be used as an optimal type of irradiation based on a set of indicators.

Correction to: The Influence of Ex-Vitro Acclimatization of Elicitor-Treated Stevia rebaudiana (Bertoni), on Growth Biomass, Physiological Traits, Steviol Glycosides Accumulation, and Biosynthesis Pathway Gene Expression Pattern

Correction to: The Influence of Ex-Vitro Acclimatization of Elicitor-Treated Stevia rebaudiana (Bertoni), on Growth Biomass, Physiological Traits, Steviol Glycosides Accumulation, and Biosynthesis Pathway Gene Expression Pattern

The Influence of Ex-Vitro Acclimatization of Elicitor-Treated Stevia rebaudiana (Bertoni), on Growth Biomass, Physiological Traits, Steviol Glycosides Accumulation, and Biosynthesis Pathway Gene Expression Pattern

The Influence of Ex-Vitro Acclimatization of Elicitor-Treated Stevia rebaudiana (Bertoni), on Growth Biomass, Physiological Traits, Steviol Glycosides Accumulation, and Biosynthesis Pathway Gene Expression Pattern

The effect of alginate as an elicitor on transcription of steviol glycosides biosynthesis pathway related key genes and sweeteners content in in vitro cultured Stevia rebaudiana.

Stevia rebaudiana is a medicinal herb that accumulates non-caloric sweeteners called steviol glycosides (SGs) which are approximately 300 times sweeter than sucrose. This study used alginate (ALG) as an elicitor to increase steviol glycosides accumulation and elucidate gene transcription in the steviol glycosides biosynthesis pathway. To minimize the grassy taste associated with stevia sweeteners, plantlets were grown in complete darkness. ALG was applied to stevia plants grown in suspension culture with a Murashige and Skoog (MS) medium to determine its effect on SGs' content and the transcription profile of SG-related genes using the HPLC and RT-qPCR methods, respectively. Treatment with alginate did not significantly affect plantlet growth parameters such as shoot number, dry and fresh weight. Rebaudioside A (Reb A) content increased approximately sixfold in the presence of 1g L-1 alginate and KS, KAH, and UGT74G1 genes showed significant up-regulation. When the concentration was increased to 2g L-1, the transcription of KO and UGT76G1, responsible for the conversion of stevioside to Reb A, was increased about twofold. The current study proposes that adding alginate to the MS suspension medium can increase Reb A levels by altering the SG biosynthesize pathway's transcription profile. The present experiment provides new insights into the biochemical and transcriptional response mechanisms of suspension-cultured stevia plants to alginate.

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.

A Novel Bacillus safensis-Based Formulation along with Mycorrhiza Inoculation for Controlling Alternaria alternata and Simultaneously Improving Growth, Nutrient Uptake, and Steviol Glycosides in Stevia rebaudiana under Field Conditions.

The excess use of chemicals by farmers in the agroecosystems degrades soil quality, disturbs soil ecology, and increases soil salinity and health hazards in humans. Stevia rebaudiana is an important medicinal and aromatic crop whose leaves contain steviol glycosides (SGs). The Bacillus safensis NAIMCC-B-02323 strain STJP from the rhizosphere of S. rebaudiana producing salicylic acid (16.80 µg/mL), chitinase (75.58 U/mL), β-1,3-glucanase (220.36 U/mL), and cellulase (170 U/mL) was taken as a plant growth-promoting rhizobacteria (PGPR). The cell-free supernatant (CFS) from strain STJP showed significant biocontrol activity against Alternaria alternata (80%), suggesting the protective role of extracellular metabolite(s) against phytopathogens. Paneer whey-based bioformulation (P-WBF) was developed to exploit B. safensis STJP to enhance the growth, nutrient uptake, soil properties, stevioside content, and SGs biosynthesis in S. rebaudiana under an A. alternata-infested field. The combined treatment of P-WBF and mycorrhiza (Glomus fasciculatum ABTEC) significantly enhanced plant growth parameters after 90 days, in comparison with control. The symbiotic action (P-WBF and mycorrhiza) displayed much better results in terms of chlorophyll a and b (improved by 132.85% and 39.80%, respectively), protein (by 278.75%), flavonoid (by 86.99%), carbohydrate (by 103.84%), antioxidant (by 75.11%), and stevioside (by 120.62%) contents in plants as compared to the untreated set. Further, the augmentation of potassium (by 132.39%), phosphorous (by 94.22%), and zinc (by 111.11%) uptake in plant tissues and soil was also observed by the application of P-WBF and mycorrhiza. The expression of UGT74G1 and UGT85C2 genes related to SG biosynthesis was upregulated (2.7- and 3.2-fold, respectively) in plants treated with P-WBF and mycorrhiza as further confirmed by the accumulation of SGs. The results suggest that the application of P-WBF and mycorrhiza not only provides an ecofriendly and sustainable solution to improve stevioside content in S. rebaudiana by a nutrient-linked mechanism but also paves the way to enhanced production of stevioside.

The Potential of Cold Plasma and Electromagnetic Field as Stimulators of Natural Sweeteners Biosynthesis in Stevia rebaudiana Bertoni.

Stevioside (Stev) and rebaudioside A (RebA) are the most abundant steviol glycosides (SGs) responsible for the sweetness of Stevia rabaudiana Bertoni. As compared to Stev, RebA has a higher sweetening potency, better taste and therefore is the most preferred component of the stevia leaf extracts. The aim of this study was to determine the effect of pre-sowing seed treatment with abiotic stressors cold plasma (CP) and electromagnetic field (EMF) on the amount and ratio of RebA and Stev in the leaves of stevia. Additionally, the effect on total phenolic content, flavonoid content and antioxidant activity was investigated. Seeds were treated 5 and 7 min with cold plasma (CP5 and CP7 groups) and 10 min with electromagnetic field (EMF10 group) six days before sowing. The germination tests in vitro demonstrated that all treatments slightly increased germination rate and percentage. HPLC analysis revealed that CP and EMF had strong stimulating effect on SGs accumulation. All treatments increased RebA concentration approximately 1.6-fold; however, the ratio of RebA/Stev decreased from 8.5 in the control to 1.9, 2.5 and 1.1 in CP5, CP7 and EMF10 groups respectively, since the concentration of Stev increased more than RebA, 7.1, 4.6 and 11.0-fold, respectively, compared to control. However, treatments had opposite effect on total phenolic content, flavonoid content, and antioxidant activity. We have demonstrated for the first time that short time pre-sowing treatment of stevia seeds with CP and EMF can be a powerful tool for the enhancement of biosynthesis of RebA and Stev, however it can have negative impact on the content of other secondary metabolites.

The productivity of Stevia rebaudiana (Bertoni) on dry leaves and steviol glycosides of four varieties grown in six regions of Morocco

Stevia rebaudiana Bertoni, is a perennial herbaceous plant native to Paraguay. This plant produces diterpene glycosides that are powerful natural low calorie sweeteners , about 300 times sweeter than saccharose. Stevia has now become an important agricultural crop for the production of a high-potency natural sweetener. Cultural practices are still in development and the productivity potential of desirable steviol glycosides in more or less stable proportions and under different agriculture conditions are largely unknown. Currently, commercial growers and processors all over the world are seeking to maximize the productivity of rebaudioside A , a steviol glycoside . The present study aimed to characterize the effects of region and varieties on stevia dry leaf yield, steviol glycoside content, and steviol glycoside yield under Moroccan conditions. The content of steviol glycosides obtained from dry leaves of four varieties of Stevia rebaudiana , compared to the edaphoclimatic conditions in six different areas in Morocco, was determined using High-Performance Liquid Chromatography (HPLC). Dry leaf yield, leaf steviol glycoside content, and leaf steviol glycoside yield varied according to varieties and regions, with significant interaction of varieties by regions. Dry leaf yield varied from 1.57 to 7.86 t/ha. Stevioside yield ranged from 57.04 to 739.77 kg/ha and rebaudioside A yield varied between 60.8 and 614.04 kg/ha, with Reb A/STV ratios that reached up to 2.62. The total of steviol glycosides yield ranged from 163.21 to 1353.2 kg/ha with significant interactions between regions and varieties concerning sweet glycoside contents. Stevia offers great potential for introduction as a commercial crop producing a biological sweetener under Moroccan conditions. • Rebaudiana crops have proved to be very well adapted to the Moroccan conditions. • High dried leaf yields of stevia were detected with significant variability. • The accumulation of steviol glycosides was very important in the 4 varieties. • Biomass and steviol glycosides are influenced by Variety × environment interactions.

UHPLC coupled with charged aerosol detector for rapid separation of steviol glycosides in commercial sweeteners and extract of Stevia rebaudiana

Natural sweeteners are in high demand as a part of a healthy lifestyle. Among them, sweeteners with decreased caloric value and suitability for diabetes patients are most requested. Extension in their consumption extends the need for their quality control. A fast gradient UHPLC coupled with charged aerosol detection enabling quantitation of stevioside, rebaudioside A-D, and steviolbioside in commercial sweeteners and Stevia rebaudiana plant extracts has been developed. The method was developed to achieve high efficiency, simplicity, versatility, and low solvent consumption. All steviol glycosides were baseline-separated in less than 4 min with a total run time of 7 min. Buffer-free eluents were used in the separations and only 2.45 mL solvent were needed per analysis. The Luna Omega Polar column featuring polar modification of the C18 stationary phase was employed with mobile phases composed of water and acetonitrile for the excellent separation of polar steviol glycosides. The flow rate of the mobile phase 0.35 mL/min, column temperature 50 °C and injection volume 2 µL were used. Critical pair of glycosides, stevioside and rebaudioside A, were baseline separated with a resolution of 2.41. The universal charged aerosol detector allowed quantitation of steviol glycosides with a limit of detection and quantitation 0.15 and 0.5 µg/mL, respectively. Method intra-day precision was less than 2% (RSD), and the recovery was 89.6–105.0% and 93.8–111.4% for plant material and sweetener tablets, respectively. The quantity of steviol glycosides in three out of four commercial sweeteners was 3.0–12.3% higher than declared. The content was about 12.4% less than declared in one sample. But the difference from the labeled content corresponded to trueness and precision of the developed method together with variability of sweeteners production. The most abundant glycoside detected in sweeteners was stevioside followed by rebaudioside A. A leaf-to-stem ratio describing the dominant accumulation of steviol glycosides in leaves affected the differences in the amount of steviol glycosides among plant samples.

Attaining higher biomass and steviol glycosides yields of Stevia rebaudiana through adjustment of plant population and nitrogen rate

Stevia rebaudiana is being cultivated in many parts of the world for its industrial uses as a low-caloric natural sweetener. However, the effects of plant population and nitrogen (N) levels and their interactions on biomass yield and steviol glycosides accumulation are not properly known in the mild-temperate conditions. Therefore, an experiment was laid out with ten treatment combinations having two plant densities (50,000 and 100,000 plants ha−1) and five N levels (0 to 160 kg ha−1). The overall dry leaf yield of stevia ranged from 1.74 to 3.35 Mg ha−1, while total steviol glycosides (SGs) concentration of the stevia leaf varied from 7.6 to 9.3%. The dense population (100,000 plants ha−1) registered about 64.9 and 71.8% higher dry leaf yields compared with thin population (50,000 plants ha−1) during 2014 and 2015, respectively. Irrespective of plant density, N at 120 kg ha-1 recorded significantly (P ≤ 0.05) higher dry leaf yields (3.35 and 2.83 Mg ha−1) compared with control during both the experimental years. However, substantially higher apparent N-recovery and agronomic N-efficiency were registered with lower doses (40−80 kg ha−1) of N. The total SGs yield (g plant−1) was not reduced with dense plantation. Similarly, the significantly higher total SGs yield was recorded with the application of N at 120 kg ha−1. The response curve suggests that the dense plantation and N up to 143 kg ha−1 can be practiced to increase dry leaf yield of stevia without sacrificing the production of total SGs under mild temperate conditions.

Identification of the Key Residues of the Uridine Diphosphate Glycosyltransferase 91D2 and its Effect on the Accumulation of Steviol Glycosides in Stevia rebaudiana

Stevia (Stevia rebaudiana Bertoni) possesses substantial value for its unique sweet compounds-steviol glycosides (SGs). In the metabolic glycosylation grid of SGs, SrUGT91D2 has been shown to catalyze formation of 1,2-β-d-glucoside linkages at the C13- and C19-positions and play a crucial role in the synthesis of SGs, including the formation of stevioside (ST), rebaudioside E (RE), and rebaudioside D (RD). However, the key residues of the SrUGT91D2 enzyme and how SrUGT91D2 affects the accumulation of SGs in S. rebaudiana remain unclear. In the present study, cloning and functional analysis of full-length SrUGT91D2 gene sequences were performed in 10 different S. rebaudiana genotypes with divergent SG compositions. After sequence analysis, it was found that most of the sequences of this gene (more than 50%) in each genotype were consistent with the UGT91D2e_No.5 allele, which has been reported to exert catalytic activity on 1,2-β-d-glucoside. Moreover, six variants (UGT91D2e_No.5, SrUGT91D2-11-14, SrUGT91D2-110, SrUGT91D2-023, SrUGT91D2-N01, and SrUGT91D2-N04) of this gene were obtained, and their activities were identified. Although there were some differences among these variants, the only type of mutation was partial base substitution at a very low level. In addition, the expression analysis of SrUGT91D2 in each genotype showed that the expression level of the gene was significantly different among genotypes, and a significant positive correlation was found between the content of RD (which was closely influenced by SrUGT91D2) and the expression level of SrUGT91D2 in each genotype (correlation coefficient = 0.91). Thus, it was indicated that SrUGT91D2 was relatively conserved in S. rebaudiana, and the differential effect of SrUGT91D2 on the accumulation of related SGs mainly derived from its expression level. Furthermore, based on homologous modeling and molecular docking analysis, T84, T144, A194, S284, E285, V286, G365, E369, R404, and G409 were predicted to be key residues in the glucosylation of SGs by SrUGT91D2. After site-mutation and enzyme assays, it was confirmed that T84, T144, R404, A194, and G409 are the key residues in the SrUGT91D2 protein, especially T144 and G409. This work provided valuable information for understanding the structure-activity relationship of the SrUGT91D2 protein and the molecular mechanism of SG accumulation in stevia.

Steviol glycoside accumulation and expression profiling of biosynthetic pathway genes in elicited in vitro cultures of Stevia rebaudiana

In the present study, different elicitors, viz., polyethylene glycol (PEG), alginate (ALG), chitosan (CHI), salicylic acid (SA), and yeast extract (YE), were used in in vitro shoot cultures of Stevia rebaudiana for 4 wk to decipher their effect on growth, biomass yield, and accumulation of steviol glycosides (SG), especially rebaudiosides. The highest leaf number (16.33), root number (4.67), and shoot length (3.80 cm) were observed in media supplemented with 0.5 mg/L YE, whereas maximum shoot number (2.44) and root length (1.89 cm) were recorded in 1.5 mg/L CHI and 1.0 mg/L PEG respectively. Elicitation by 1.0 mg/L YE resulted in enhanced biomass production with a maximum fresh weight of leaves (114.01 mg/plantlet), stem (90.27 mg/plantlet), and shoots (204.28 mg/plantlet). SG content was quantified through ultra-high-performance liquid chromatography (UHPLC). Stevioside (ST) content was increased by 5-fold (0.77 mg/g leaf DW) in 2.0 mg/L ALG and rebaudioside A (RA) by 7-fold (1.9 mg/g leaf DW) in 0.5 mg/L ALG. Enhancement of SG is mainly regulated by a key gene, UGT85C2, which was further validated through qRT-PCR and in silico promoter analysis. Also, cis-regulatory elements governing transcriptional regulation linked with the biosynthesis of SG were identified in key genes. The study unveils ALG as a promising elicitor to produce quality biomass with enhanced metabolite profile in S. rebaudiana.

UHPLC Coupled with Charged Aerosol Detector for Rapid Separation of Steviol Glycosides in Commercial Sweeteners and Extract of Stevia Rebaudiana

Natural sweeteners are in high demand as a part of a healthy lifestyle. Among them, sweeteners with decreased caloric value and suitability for diabetes patients are most requested. Extension in their consumption extends the need for their quality control. A fast gradient UHPLC coupled with charged aerosol detection enabling quantitation of stevioside, rebaudioside A-D, and steviolbioside in commercial sweeteners and Stevia rebaudiana plant extracts has been developed. The method was developed to achieve high efficiency, simplicity, versatility, and low solvent consumption. All steviol glycosides were baseline-separated in less than 4 min with a total run time of 7 min. Buffer-free eluents were used in the separations and only 2.45 mL solvent were needed per analysis. The state of the art column Luna Omega Polar featuring polar modification of C18 stationary phase was used for the excellent separation of polar steviol glycosides. Critical pair of glycosides, stevioside and rebaudioside A, were baseline separated with a resolution of 2.41. The universal charged aerosol detector allowed quantitation of steviol glycosides with a limit of detection and quantitation 0.15 and 0.5 µg/mL, respectively. Method intra-day precision was less than 2% (RSD), and the recovery was 89.6-105.0% and 93.8-111.4% for plant material and sweetener tablets, respectively. The quantity of steviol glycosides in three out of four commercial sweeteners was 3.0-12.3% higher than declared. The content was about 12.4% less than declared in one sample. The most abundant glycoside detected in sweeteners was stevioside followed by rebaudioside A. A leaf-to-stem ratio describing the dominant accumulation of steviol glycosides in leaves affected the differences in the amount of steviol glycosides among plant samples.

Genotypic characterization and steviol glycoside quantification in a population of Stevia rebaudiana Bertoni from Paraguay

Stevia rebaudiana (Bertoni) Bertoni is a widely grown species in various regions of the world, mainly due to its sweetening properties attributed to steviol glycosides. Despite the fact that Paraguay is the center of origin of S. rebaudiana, genetic characterization of local genotypes has not been studied. Considering that accumulation of steviol glycosides is a complex trait, molecular breeding could be an effective method for its improvement. Therefore, the identification of molecular markers associated with steviol glycoside accumulation is important. The aim of this study was to assess the genetic relationship among eight advanced lines of S. rebaudiana and two varieties selected from local germoplasm and quantify their steviol glycoside content. The stevioside and rebaudioside A content in methanolic extract was determined by high-performance liquid chromatography (HPLC), and the molecular characterization of the ten genotypes of S. rebaudiana was performed using SSR and ISSR markers. A wide range of variability was observed for the metabolites of interest under greenhouse conditions. The variety Eirete presented highest stevioside and rebausiode A content, followed by the variety Katupyry. One of the advanced line presented both metabolite content similar to that of Katupyry. Genotyping of these lines showed two main clusters. Cluster I contained the both varieties and advanced lines with higher stevioside and rebaudioside A content. Our results suggest that S. rebaudiana lines with genetic profile similar to that of cluster I, determined mainly by genotype at the markers SUGMS 35 and ISSR 47, might present higher stevioside and rebaudioside A content.

Photosynthetic performance and stevioside concentration are improved by the arbuscular mycorrhizal symbiosis in Stevia rebaudiana under different phosphate concentrations.

In plants, phosphorus (P) uptake occurs via arbuscular mycorrhizal (AM) symbiosis and through plant roots. The phosphate concentration is known to affect colonization by AM fungi, and the effect depends on the plant species. Stevia rebaudiana plants are valuable sources of sweetener compounds called steviol glycosides (SGs), and the principal components of SGs are stevioside and rebaudioside A. However, a detailed analysis describing the effect of the phosphate concentration on the colonization of AM fungi in the roots and the relationship of these factors to the accumulation of SGs and photochemical performance has not been performed; such an analysis was the aim of this study. The results indicated that low phosphate concentrations (20 and 200 µM KH2PO4) induced a high percentage of colonization by Rhizophagus irregularis in the roots of S. rebaudiana, while high phosphate concentrations (500 and 1,000 µM KH2PO4) reduced colonization. The morphology of the colonization structure is a typical Arum-type mycorrhiza, and a mycorrhiza-specific phosphate transporter was identified. Colonization with low phosphate concentrations improved plant growth, chlorophyll and carotenoid concentration, and photochemical performance. The transcription of the genes that encode kaurene oxidase and glucosyltransferase (UGT74G1) was upregulated in colonized plants at 200 µM KH2PO4, which was consistent with the observed patterns of stevioside accumulation. In contrast, at 200 µM KH2PO4, the transcription of UGT76G1 and the accumulation of rebaudioside A were higher in noncolonized plants than in colonized plants. These results indicate that a low phosphate concentration improves mycorrhizal colonization and modulates the stevioside and rebaudioside A concentration by regulating the transcription of the genes that encode kaurene oxidase and glucosyltransferases, which are involved in stevioside and rebaudioside A synthesis in S. rebaudiana.

Factors affecting organogenesis of Stevia rebaudiana and in vitro accumulation of steviol glycosides

Factors affecting organogenesis of Stevia rebaudiana and in vitro accumulation of steviol glycosides

Melatonin promotes seed germination under salinity and enhances the biosynthesis of steviol glycosides in Stevia rebaudiana Bertoni leaves.

Melatonin (MEL) can act as a plant growth regulator and biostimulator in stressful situations. Using MEL in seed pretreatment also affects the future growth of plants. Therefore, this research investigated the effects of MEL on seed germination and seedling growth under NaCl in in vitro conditions. The additional effects of MEL on the accumulation of steviol glycosides (SGs) and on the expression of appropriate genes were also studied. Five μM of MEL was the best concentration for seed germination, while 20 μM exerted a positive impact on the biomass of stevia plantlets. NaCl significantly decreased seed germination, but MEL alleviated this effect when seeds were germinated in 50 mM of NaCl. Under salinity, the values of almost all morphological traits decreased as MEL concentration increased. The highest amounts of stevioside and rebaudioside A (Reb A) were observed as a result of treating seeds with 5 and 20 μM of MEL, respectively. When adding NaCl, positive impacts of MEL on the accumulation of both SGs were also observed. Expression analyses of the genes involved in SGs biosynthesis was explored in seeds and leaves, and the transcripts of key enzymes occurred in both the tissues. However, quantitative polymerase chain reaction (qPCR) analysis showed that all tested genes were upregulated in younger leaves, contrary to older ones. Also in younger, rather than older, leaves SG gene expression varied according to MEL concentration. This study, therefore, presents the promising potential of MEL for improving stevia seed germination under salinity conditions and for enhancing the production of SGs in stevia plants.

Optimized Nitrogen Topdressing Strategies Enhance Steviol Glycoside Productivity in Stevia (Stevia rebaudiana Bertoni) Plants

The nitrogen (N) management is crucial to stevia growth and steviol glycoside (SG) production. Although previous studies have concentrated on the impacts caused by N fertilization rates, very few studies are concerning N topdressing strategies. We hypothesized that stevia biomass and SG productivity can be effectively increased by altering the ratio of base fertilization:first topdressing at the seedling stage:second topdressing at the branching stage. The current plot experiment was undertaken to evaluate such effects. Compared with one-time basal fertilization, optimized N topdressing ratios (especially the 5:3:2 and 4:3:3 ratios) significantly enhanced the leaf biomass formation and aboveground N accumulation. Different results were observed for the leaf SG concentrations, and increasing the topdressing proportions critically reduced the total leaf SG concentrations (3.3–24.7%) at the harvest stage. The highest values for total SG accumulation were exhibited under the 6:4:0 and 5:3:2 topdressing strategies, which depend on the changes in biomass and SG concentration. Moreover, a significant negative correlation between leaf SGs and the N concentration was observed at all growth stages, but a positive correlation was observed with the leaf C/N ratio and soluble sugar concentrations. Our results suggest the potential for increasing SG productivity by optimizing N fertilization regimes, especially with an appropriate reduction in the topdressing ratio after the vegetative growth stage. Our findings further expanded the effect of topdressing strategies on SG production based on the N fertilization levels.

Comparative transcriptome analysis revealed gamma-irradiation mediated disruption of floral integrator gene(s) leading to prolonged vegetative phase in Stevia rebaudiana Bertoni

Stevia rebaudiana Bert. is getting global attention because of its ability to synthesize commercially important low/no calorie natural sweeteners (LNCSs) steviol glycosides (SGs). Considering, higher accumulation of SGs in vegetative phase followed by decrement during reproductive phase necessitate the understanding of different molecular components of floral transition to develop superior varieties/cultivars with prolonged vegetative phase in Stevia. Current comparative transcriptional analysis of low dose (5 kR) gamma-irradiated mutant genotype (SMG) with prolonged vegetative phase vis-à-vis background genotype (SBG) identified DGEs of major floral transition pathways, and expressed according to their physiological fate irrespective to SMG & SBG. Contrarily, reduced expression of floral integrator genes (FT and LEAFY) in mutant genotype suggests their involvement in prolonged vegetative phase phenotype. Likewise, GO and KEGG enrichment of photosynthesis and carbon assimilation efficiency might be associated with prolonged vegetative phase and higher accumulation of Stevioside content in mutant genotype. Furthermore, deviation of flowering related transcription factors (higher expressions except MIKS-type MADS-box SMG_PV compared to SBG_F) may possibly be correlated with low expression of floral integrator genes. Findings of current studies will facilitate the genetic manipulations and crop improvement efforts in Stevia through conventional breeding and genome editing approaches for increased SGs biosynthesis.

Photoperiod and elicitors increase steviol glycosides, phenolics, and flavonoid contents in root cultures of Stevia rebaudiana

The establishment of green root cultures of Stevia rebaudiana Bertoni, and the effect of elicitors such as hydrogen peroxide (H2O2) and methyl jasmonate (MeJA), is shown in the present study. Stevioside, rebaudioside A, and the isomers steviol/isosteviol were identified through DFI-ESI-IT-MSn and UPLC-TOFMS spectrometric systems, in combination with solid-phase extraction. The accumulation of steviol glycosides increased by 2.4 times (compared to the control value of 22.35 μgSG per gDW), with the addition of 250 μM H2O2. The non-enzymatic antioxidant response, which resulted from production of phenolic and flavonoid compounds, was modified based on the elicitor and the dose used. The maximum accumulation of flavonoids was induced on the third day with the addition of H2O2 (250 or 500 μM), and with MeJA (250 or 500 μM); the increase was observed on the fifth day. The enzymatic antioxidant response of the catalase and peroxidase from the roots under elicitation confirmed the stress conditions.