Accumulation Of Steviol Glycosides Research Articles

Physiological and morphological responses to abiotic stresses in two cultivars of Stevia rebaudiana (Bert.) Bertoni

Stevia rebaudiana (Bert.) Bertoni is cultivated for its sweet leaves as they contain steviol glycoside sweeteners such as stevioside and rebaudioside A. Two cultivars of S. rebaudiana, Shoutian-2 and Fengtian, were evaluated in a greenhouse for their tolerance to abiotic stresses. The seedlings were exposed to salinity, drought or waterlogging stresses for 4 weeks. The cultivars differed significantly in their biomass accumulation, tissue ion concentrations and steviol glycoside accumulation. The salt tolerant cv. Fengtian contained higher concentrations of K+ which led to higher K+/Na+ ratio in the leaves, as compared to the less salt tolerant cv. Shoutian-2. Reduced biomass accumulation in cv. Shoutian-2 coincided with increased Na+ and Cl˗ concentrations in the leaves. The waterlogging stress reduced growth of both cultivars, but the salinity stress increased rebaudioside A concentration in the tolerant cv. Fengtian, and the drought stress increased stevioside concentration in the cv. Shoutian-2. This study concludes that the two cultivars of S. rebaudiana differ in their tolerances to salinity and drought stresses.

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.

Investigation of different concentrations of MS media effects on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is one of two species that contains steviol glycosides. Among steviol glycosides that extracted from leaves, stevioside and rebaudioside A are the two major and the sweetest glycosides that are about 200-300 times sweeter than sucrose with zero calories. The best method for stevia propagation is tissue culture. So, for investigation of nutrients in medium, we studied the effect of different concentrations of MS media (MS, 0.5 MS, 0.25 MS, 0 MS) on morphological traits, UGT74G1 and UGT76G1 genes expression and accumulation of steviol glycosides in stevia leaves. The best growth rate (0.472 mm/d) has occurred in plants grown in MS media. Also, the highest gene expression of UGT74G1 gene (1.000 Total lab unit) was seen under MS treatment. However, the highest expression level of UGT76G1 gene (1.701 Total lab unit) was observed at plants grown in 0 MS. The highest amount of both Stevioside and Rebaudioside A (14.23 and 8.12, respectively) were accumulated in plants under MS treatment. Obviously, dilution of MS media associated with decreasing in both expression of the intended genes and accumulation of steviol glycosides.

Effects of various glutamine concentrations on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is one of the most important biologically sourced and low-calorie sweeteners that contains a lots of Steviol glycosides. Tissue culture is the best method for propagation of stevia and micro nutrients can affect both morphological traits and steviol glycosides production. In the present study, we investigated the effect of different concentrations of glutamine (10, 20, 30 and 40 g/l) on expression of UGT74G1 and UGT76G1 genes and stevioside and rebaudioside A accumulation in the leaves of stevia under in vitro conditions. The highest level of expression for UGT74G1 (1.000 Total lab unit) was seen at plants grown in MS media without glutamine and the highest gene expression level for UGT76G1 (1.321 Total lab unit) was observed at plants grown in 2% glutamine. Based on HPLC results, the highest amount of stevioside (22.74) was accumulated in plants which were under 3% glutamine treatment and the lowest production level of stevioside (16.19) was resulted under MS (0 glutamine) medium. The highest rebaudioside A (12.19) accumulation was observed under 2% glutamine treatment and the lowest accumulation of rebaudioside A (8.41) was seen at plants grown in MS medium.

Effects of life cycle and leaves location on gene expression and glycoside biosynthesis pathway in Stevia rebaudiana Bertoni

Stevia rebaudiana Bertoni is One of the most important biologically sourced and low-calorie sweeteners that known as "Sweet Weed". It contains steviol glycosides that they are about 200-300 times sweeter than sucrose. Tissue culture is the best method with high efficiency that can overcome to problems of traditional methods, and it is the most useful tools for studying stress tolerance mechanisms under in vitro conditions to obtain drought tolerance. In the present research, we investigated the impact of life cycle, leaves location and the harvesting time on expression of UGT74G1 and UGT76G1 as well as steviol glycosides accumulation. The highest gene expression of both UGT74G1 and UGT76G1 (207.677 and 208.396 Total Lab unit, respectively) was observed in young leaves in the second vegetative year. Also, the highest amount of stevioside accumulation (13.04) was due to the old leaves in vegetative stage which had significant differences with other effects whereas the lowest accumulation (7.47) was seen at young leaves at vegetative stage. Interestingly, the highest level of rebaudioside a production (15.74) was occurred at the young leaves at vegetative stage. There was significant differences between life cycle and leaves location on steviol glycoside production in stevia.

Hydrogen Peroxide-Induced Steviol Glycosides Accumulation and Enhancement of Antioxidant Activities in Leaf Tissues of Stevia rebaudiana Bertoni

Hydrogen peroxide (H2O2) functions as an abiotic stress elicitor for plants. In this study, the effect of oxidative stress caused by H2O2 on steviol glycosides (SGs) production and antioxidant activities of an in vitro grown antidiabetic plant, Stevia rebaudiana, has been investigated. Direct shoot organogenesis was performed, and after 4 weeks of culture, the shoots of S. rebaudiana were transferred to MS medium containing H2O2 (10 or 20 mM) and incubated in growth room for 6 h. It resulted in increased SGs production, such as rebaudioside A (Reb-A) and stevioside (ST), and enhancement of total phenolic content, total flavonoid content, total antioxidant capacity, total reducing power and DPPH free radical scavenging activity when compared with control. The results clearly showed highest amount of SGs produced in MS medium containing 10 mM H2O2 [producing 2.97% Reb-A and 2.32% ST (w/w)], followed by the medium employing 20 mM H2O2 and control group. It is also evident that H2O2 pre-treatment caused significant increase in non-enzymatic antioxidants that play defensive role against an oxidative stress induced by H2O2. The described protocol has a great potential to be utilized for Reb-A production on commercial scale.

Impact of blue, red, and far-red light treatments on gene expression and steviol glycoside accumulation in Stevia rebaudiana

Stevia rebaudiana (Bertoni) Bertoni is a plant that biosynthesizes a group of natural sweeteners that are up to approximately 400 times sweeter than sucrose. The sweetening components of S. rebaudiana are steviol glycosides (SGs) that partially share their biosynthesis pathway with gibberellins (GAs). However, the molecular mechanisms through which SGs levels can be improved have not been studied. Therefore, transcription levels of several SG biosynthesis-related genes were analyzed under several light treatments involved in GA biosynthesis. We detected higher transcription of UGT85C2, which is one of the UDP-glycosyltransferases (UGTs) involved in catalyzing the sugar-transfer reaction, under red/far-red (R/FR) 1.22 light-emitting diodes (LEDs) and blue LEDs treatment. In this study, it was demonstrated that transcription levels of SG-related genes and the SGs content are affected by light treatments known to affect the GA contents. It is expected that this approach could serve as a practical way to increase SG contents using specific light treatments.

Gene transcription and steviol glycoside accumulation in Stevia rebaudiana under polyethylene glycol-induced drought stress in greenhouse cultivation.

Stevia rebaudiana is a sweet herb of the Astraceae family, which is cultivated for the natural sweeteners it contains. The aim of this study was to assess the effect of drought, simulated by the application of polyethylene glycol (5%, 10%, and 15% w/v), on the content of steviol glycosides (SVglys) and transcription levels of six genes involved in the biosynthesis of these natural sweeteners. The transcription levels of ent‐kaurene synthase, ent‐kaurene oxidase, ent‐kaurenoic acid hydroxylase, and three UDP‐dependent glycosyltransferases, UGT85C2,UGT74G1 and UGT76G1 were downregulated under polyethylene glycol treatment. Polyethylene glycol treatment significantly decreased the amount of stevioside, rebaudioside A, B, C and F, steviolbioside, dulcoside A, rubusoside, and total SVglys. These results strongly suggest a close relationship of SVglys content with the transcription of genes involved in the SVglys biosynthesis pathway. Comparing the observations of the present study with other reports provided the knowledge that the Stevia response to drought stress can be influenced by different environmental and experimental factors, in addition to intensity of drought stress. In conclusion, these results strongly suggest that polyethylene glycol‐induced drought stress has a negative effect on the content of SVglys and transcription of SVglys biosynthetic genes and that this should be investigated further. We recommend that sufficient irrigation of Stevia is required to obtain a high content of SVglys.

Methanol elicits the accumulation of bioactive steviol glycosides and phenolics in Stevia rebaudiana shoot cultures

Stevia rebaudiana Bertoni is a plant that generates interest mainly due to the presence in their leaves of steviol glycosides that can be used as non-caloric sweetener agents. But S. rebaudiana is also interesting for its medicinal properties related to the accumulation in leaves of steviol glycosides and other bioactive compounds, such as phenolics. In order to find ways to increase the production of secondary metabolites with commercial interest, S. rebaudiana in vitro shoot cultures were elicited with methanol at different concentrations. Analysis of the extracts obtained from plant materials revealed that the addition of 1.5% (v/v) methanol to culture media resulted in the highest antioxidant capacity determined. Similar trends were observed when levels of total soluble phenols, flavonoids, and hydroxycinnamic acids were assessed pointing to a close association between these compounds and antioxidant capacity in S. rebaudiana shoots. Regarding steviol glycosides, incubation of shoots in the presence of 0.1% (v/v) methanol led to the maximum levels with an increase of approximately 2-fold over control values. Taken together, these results seem to indicate a differential sensitivity to methanol of metabolic pathways leading to the accumulation of steviol glycosides and phenolics in S. rebaudiana leaves. Moreover, from this study methanol emerges as a potential, concentration-dependent, selective elicitor that opens up new opportunities for the production of valuable natural bioactive compounds.

Effects of growth stage and fulvic acid on the diversity and dynamics of endophytic bacterial community in Stevia rebaudiana Bertoni leaves.

The aim of this study was to learn the interactions among the endophytic bacteria, the plant growth, the foliar spray of fulvic acid, and the accumulation of steviol glycosides in the leaves of Stevia rebaudiana. Metagenomic DNA was extracted from the Stevia leaves at different growth stages with or without the fulvic acid treatment; and the diversity of endophytic bacteria in Stevia leaves was estimated by pyrosequencing of 16S rRNA genes. As results, Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were found to be the dominant phyla despite the growth stages and fulvic acid application. Stevia growth stages strongly regulated composition of endophytic community. The genera Agrobacterium (12.3%) and Erwinia (7.2%) dominated in seedling stage were apparently declined in the vegetable and initial flowering stages, while Sphingomonas and Methylobacterium increased in mature leaves at harvest time, which showed that the mature leaves of Stevia preferred to accumulate some certain endophytic bacteria. Sphingomonas and Methylobacterium constituted an important part of the core endophytic community and were positively correlated with the stevioside content and UGT74G1 gene expression, respectively; while Erwinia, Agrobacterium, and Bacillus were negatively correlated with the stevioside accumulation. Fulvic acid treatment accelerated the variation of endophytes along the growth stages and increased the steviol glycosides content. This is the first study to reveal the community composition of endophytic bacteria in the Stevia leaves, to evidence the strong effects of growth stage and fulvic acid application on the endophytes of Stevia, and to demonstrate the correlation between the endophytic bacteria and the steviol glycosides accumulation.

RNA-Seq for gene identification and transcript profiling of three Stevia rebaudiana genotypes

BackgroundStevia (Stevia rebaudiana) is an important medicinal plant that yields diterpenoid steviol glycosides (SGs). SGs are currently used in the preparation of medicines, food products and neutraceuticals because of its sweetening property (zero calories and about 300 times sweeter than sugar). Recently, some progress has been made in understanding the biosynthesis of SGs in Stevia, but little is known about the molecular mechanisms underlying this process. Additionally, the genomics of Stevia, a non-model species, remains uncharacterized. The recent advent of RNA-Seq, a next generation sequencing technology, provides an opportunity to expand the identification of Stevia genes through in-depth transcript profiling.ResultsWe present a comprehensive landscape of the transcriptome profiles of three genotypes of Stevia with divergent SG compositions characterized using RNA-seq. 191,590,282 high-quality reads were generated and then assembled into 171,837 transcripts with an average sequence length of 969 base pairs. A total of 80,160 unigenes were annotated, and 14,211 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Gene sequences of all enzymes known to be involved in SG synthesis were examined. A total of 143 UDP-glucosyltransferase (UGT) unigenes were identified, some of which might be involved in SG biosynthesis. The expression patterns of eight of these genes were further confirmed by RT-QPCR.ConclusionRNA-seq analysis identified candidate genes encoding enzymes responsible for the biosynthesis of SGs in Stevia, a non-model plant without a reference genome. The transcriptome data from this study yielded new insights into the process of SG accumulation in Stevia. Our results demonstrate that RNA-Seq can be successfully used for gene identification and transcript profiling in a non-model species.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-571) contains supplementary material, which is available to authorized users.

Yield components, light interception and marker compound accumulation of stevia (Stevia rebaudiana Bertoni) affected by planting material and plant density under western Himalayan conditions

A field experiment was conducted to study the effect of planting material and plant density on stevia (Stevia rebaudiana Bertoni) under western Himalayan conditions during 2011 and 2012. The experiment conducted in a split plot design consisted of two types of planting material (rooted slips and fresh seedlings) in the main plot and five inter- and intra-row spacing in subplots with three replications. Yield attributes and dry leaf biomass yield of stevia were not affected by the type of planting material; however, plant density significantly influenced the yield attributes and leaf and stem dry biomass. Although the wider spacing (60 × 45 cm) gave more leaves, higher leaf area index, higher leaf dry mass per plant as compared to closer spacing, it resulted in lower values of these attributes per unit area. Plants spaced in 30 × 30 cm accumulated 41.2% and 42.8% more total biomass than 60 × 45 cm. Steviol glycoside content did not change due to different planting materials and plant densities; however, closer plant spacing (30 × 30 cm) recorded 114.8% and 70.0% higher steviol glycoside accumulation compared to wider row spacing (60 × 45 cm) in 2011 and 2012, respectively.

Agrobacterium mediated transient gene silencing (AMTS) in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway.

BackgroundSteviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins.Methodology/Principal FindingsRNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes.Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route.

Glucose, sucrose, and steviol glycoside accumulation in Stevia rebaudiana grown under different photoperiods

The amount of free glucose and sucrose within the leaves of Stevia rebaudiana has been evaluated under different photoperiods and related to steviol glycoside (SVgly) accumulation. The diurnal dynamics of glucose and sucrose in leaves, internodes, apices, and roots revealed considerable fluctuations. Within leaves, glucose and sucrose content decreased up to three-fold during the night whereas SVgly amounts did not show any significant diurnal fluctuations. Ontogenetic variations in glucose content was markedly different under long-day (LD) and short-day (SD) regimes. Under LD, glucose content increased two-fold after the onset of flower bud formation, whereas under SD, a stagnation or small decrease was observed. This increase under LD was already noticed in the upper sink leaves at the end of the vegetative stage, possibly acting as a metabolic trigger for the phase transition to reproductive development. Under SD, glucose content appeared more controlled resulting in a better linear relationship with dry matter and SVgly content. The increased sink demands of the transitional apex led to a rapid decline in leaf sucrose which showed little or no correlation with SVgly amounts. From our results, it becomes clear that the correlation between glucose or sucrose as substrates and SVglys as products is very dynamic and is significantly influenced by day-length and ontogeny.

Stimulation of steviol glycoside accumulation in Stevia rebaudiana by red LED light

The aim of this study was to determine whether steviol glycoside accumulation is under phytochrome control. The results indicate that Stevia rebaudiana Bertoni plants grown under short-day conditions showed precocious flowering and stagnation of steviol glycoside accumulation. Long night interruption by red LED light stimulated and sustained the vegetative growth as well as the accumulation of steviol glycosides in the leaves. After 7weeks of treatment, steviol glycoside content was about two-fold higher in LED-treated plants than in the short-day control group. The effects of red LED light were measured both in a greenhouse and in a phytotron, irrespective of cultivar-specific differences. Therefore, it can be concluded that a mid-night interruption by red LED light during short photoperiods provides an easy and inexpensive method to increase vegetative leaf biomass production with an increased steviol glycoside yield.

UDP-dependent glycosyltransferases involved in the biosynthesis of steviol glycosides

A short-term experiment was designed to measure the transcript levels of downstream genes contributing to the biosynthesis of steviol glycosides. Stevia rebaudiana plants were subjected to long- and short-day conditions for different time intervals. Samples from both lower and upper leaves were collected. Using quantitative real-time polymerase chain reaction, the transcript levels of three UDP-dependent glycosyltransferases, UGT85C2, UGT74G1 and UGT76G1, were studied. The results were compared with the steviol glycoside contents measured in the leaves, which were quantified by reversed phase HPLC. In the same daylength condition, steviol glycoside concentration and the transcript levels of the three UGT genes were higher in upper leaves than in lower leaves. Steviol glycosides accumulated more in plants under short-day conditions. Under these conditions, a highly significant correlation was found between UGT85C2 transcription and total steviol glycoside accumulation in the upper leaves. This suggests that the glycosylation of steviol to form steviolmonoside is the rate-limiting step in the glycosylation pathway of steviol glycosides. In these upper leaves, a relatively high accumulation of rebaudioside A compared to stevioside was also observed, however, without correlation with the transcription of UGT76G1.

Morphology and ultrastructure of trichomes of intact and in vitro plants of stevia rebaudiana Bertoni with reference to biosynthesis and accumulation of steviol glycosides

The ultrastructure of trichomes of intact and in vitro plants of stevia were investigated under scanning and transmission electronic microscopy. Three types of trichomes were found on both the upper and lower surfaces of the leaf: viz. rounded glands and large and small trichome. The density of trichomes on the surface of upper leaves is higher than on lower leaves, while in vitro plants are characterized with lower density of trichomes than intact ones. The positive correlation between number of trichomes on leaves and content of steviol glycosides (SGs) in them was found. Three basic stages of ontogeny of gland cells were revealed which reflect the sequence of secrete accumulation.

Chloroplast ultrastructure, photosynthetic apparatus activities and production of steviol glycosides in Stevia rebaudiana in vivo and in vitro

The accumulation of steviol glycosides (SGs) in cells of Stevia rebaudiana Bertoni both in vivo and in vitro was related to the extent of the development of the membrane system of chloroplasts and the content of photosynthetic pigments. Chloroplasts of the in vitro plants, unlike those of the intact plants, had poorly developed membrane system. The callus cells grown in the light contained proplastids of almost round shape and their thylakoid system was represented by short thylakoids sometimes forming a little number of grana consisting of 2-3 thylakoids. In cells of the etiolated in vitro regenerants and the callus culture grown in the dark, only proplastids practically lacking the membrane system were observed. All the chloroplasts having developed thylakoids and forming at least a little number of grana were equipped with photochemically active reaction centers of photosystems 1 and 2. Leaves of in vivo plants accumulated greater amount of the pigments than leaves of the in vitro plants. In both the callus culture grown in the light and the etiolated in vitro regenerants, the content of the pigments was one order of magnitude lower than that in leaves of the intact plants. The callus tissue grown in the dark contained merely trace amounts of the pigments. Leaves of the intact and the in vitro plants did not exhibit any significant differences in photosynthetic O2 evolution rate. However, photosynthetic O2 evolution rate in the callus cells was much lower than that in the differentiated plant cells. The in vitro cell cultures containing merely proplastids did not practically produce SGs. However, after transferring these cultures in the light, both the formation of chloroplasts and the production of SGs in them were detected.

Effects of nutrient medium composition on development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycosides

Effects of sugars, mineral salts and plant growth regulators on the development of Stevia shoots cultivated in the roller bioreactor and their production of steviol glycosides (SGs) were investigated. In the medium with fructose or glucose, extension of the shoots and development of their root system were much better than in the medium supplemented with sucrose. Under these conditions, however, accumulation of leaves dry mass decreased, and the content of the SGs in leaves declined. At elevated sucrose concentrations (from 1 to 5%), enhanced development of the root system and an increase in plant dry mass and number of leaf pairs was observed. At the same time, 3% sucrose gave optimal SG accumulation. Twofold elevating the concentration of mineral salts considerably stimulated growth of the shoots, whereas the content of the SGs in their leaves decreased by about order of magnitude. Addition of 0.1 mg/l BA or 6-benzylaminopurine (BA) together with α-naphthaleneacetic acid resulted in an 1.5-fold increase in the number of shoots. However, the shoots grown on the BA-supplied medium displayed a strong inhibition of the development of their root system. When the medium was supplied with gibberellic acid, lengthening of shoots and roots of Stevia were observed. All the plant growth regulators used strongly inhibited production of SGs. The changes in nutrition medium composition had practically no effect on the ratio of individual glycosides in Stevia leaves.