Steviol Glycosides Biosynthesis Research Articles

Development of Photoaffinity Probe for the Discovery of Steviol Glycosides Biosynthesis Pathway in Stevia rebuadiana and Rapid Substrate Screening.

Functional discovery and characterization of the target enzymes responsible for the biosynthesis pathway coded for the genes is ongoing, and the unknown functional diversity of this class of enzymes has been revealed by genome sequencing. Commonly, it is feasible in annotating of biosynthetic genes of prokaryotes due to the existence of gene clusters of secondary metabolites. However, in eukaryotes, the biosynthetic genes are not compactly clustered in the way of prokaryotes. Hence, it remains challenging to identify the biosynthetic pathways of newly discovered natural products in plants. Steviol glycosides are one class of natural sweeteners found in high abundance in the herb Stevia rebaudiana. Here, we applied the chemoproteomic strategy for the proteomic profiling of the biosynthetic enzymes of steviol glycosides in Stevia rebaudiana. We not only identified a steviol-catalyzing UDP-glycosyltransferase (UGT) UGT73E1 involved in steviol glycoside biosynthesis but also built up a probe-based platform for the screening of potential substrates of functional uncharacterized UGT rapidly. This approach would be a complementary tool in mining novel synthetic parts for assembling of synthetic biological systems for the biosynthesis of other complex natural products.

Elicitor-Induced Transcriptional Changes of Genes of the Steviol Glycoside Biosynthesis Pathway in Stevia rebaudiana Bertoni

Stevia rebaudiana is a medicinal plant widely used as a sweetener, and the sweetening power comes from the diterpenoid glycosides known as steviol glycosides (SGs). The studies of gene expression patterns in response to elicitors treatment are of extreme importance for biotechnological purposes. Methyl jasmonate (MeJa), spermidine (SPD), salicylic acid (SA), and paclobutrazol (PBZ) (100 µM) were applied to stevia plants growing in a hydroponic system to examine the effect of these elicitors along the time (24, 48, 72, and 96 h). SGs’ contents and transcription levels of fifteen genes involved in the three stages comprising their biosynthesis pathway were evaluated. The results provide direct evidence that HDR, GGDPS, CDPS, KS, KO, and KAH are elicitor-responsive genes and they can be effectively regulated at least at the transcriptional level. MeJa and SPD treatments resulted in positive effects on the transcription of SGs biosynthetic genes, with SPD exhibiting the highest number of regulated genes at 48 h of exposure. PBZ treatment down-regulated genes encoding kaurenoid enzymes. SA treatment did not affect transcription of UGT85C2, UGT74G1, and UGT76G1 and decreased stevioside levels. Overall, this study offers new insights into the transcriptional response mechanisms in S. rebaudiana plants under the effect of the elicitors MeJa, SPD, SA, and PBZ.

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.

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.

Quantitative analysis of uridine diphosphate glycosyltransferase UGT85C2, UGT74G1 and UGT76G1 genes expression in Stevia rebaudiana under different irrigations

Stevia rebaudiana Bertoni is known to the scientific world for its sweetness and steviol glycosides. Steviol glycosides are the secondary metabolites responsible for the sweetness of stevia and have many medical applications. They are synthesized by a biosynthesis pathway operating in the leaves. Stevioside (Stev) and rebaudioside A (Reb A) are two main glycosides compounds of Stevia. Translation products of three Uridine diphosphate glycosyltransferase candidate genes, UGT85C2, UGT74G1 and UGT76G1, were found to have an activity towards the synthesis of Stev and Reb A. In this study, a experiment was designed to measure the expression level of three candidate genes contributing to the biosynthesis of steviol glycosides under three different irrigation intervals using quantitative real-time PCR analysis technique. Irrigation was done at 3, 6 and 9 days irrigation intervals, based on soil moisture content 90, 75 and 60% of field capacity (FC) respectively. The results showed that soil water depletion up to 75% FC lead to significant increase in expression of UGT85C2 and UGT74G1 genes, whereas a significant growth in expression of UGT76G1 gene occurred at 60% FC. Measurement of Reb A contents in treated leaves showed that a significant correlation between UGT76G1 transcription and Reb A accumulation under 60% FC. Also, the comparison of expression patterns of the three target UGT transcripts at 75 and 60% FC showed a highly significant difference in UGT74G1 transcripts in comparison of UGT85C2 and UGT76G1 transcripts. These results indicated that the highest value of Reb A/ Stev ratio as sweetness quality index likely belongs to 60% FC and suggested 9 days irrigation intervals to improve the quantity and quality of glycoside profile by up-regulation and down-regulation of mentioned genes.

From Caá-ehé to a commercial sweetener - the diterpenoid glycosides of Stevia rebaudiana.

The isolation, structure elucidation, chemistry, biosynthesis and biological activity of the sweet steviol glycosides from Stevia rebaudiana, are reviewed.

Transcript Quantification of Genes Involved in Steviol Glycoside Biosynthesis in Stevia rebaudiana Bertoni by Real-Time Polymerase Chain Reaction (RT-PCR).

Stevia (Stevia rebaudiana Bertoni) is a medicinal plant having sweet, diterpenoid glycosides known as steviol glycosides which are 200-300 times sweeter than sucrose (0.4 % solution). They are synthesized mainly in the leaves via plastid localized 2-C-methyl-D-erythrose-4-phosphate pathway (MEP pathway). Fifteen genes are involved in the formation of these glycosides. In the present protocol, a method for the quantification of transcripts of these genes is shown. The work involves RNA extraction and cDNA preparation, and therefore, procedures for the confirmation of DNA-free cDNA preparation have also been illustrated. Moreover, details of plant treatments are not mentioned as this protocol may apply to relative gene expression profile in any medicinal plant with any treatment. The treatments are numbered as T0 (Control), T1, T2, T3, and T4.

Daily Light Integral Influences Steviol Glycoside Biosynthesis and Relative Abundance of Specific Glycosides in Stevia

The biosynthesis of steviol glycosides is affected by both genetic and environmental factors. To evaluate the influence of total daily solar radiation or daily light integral (DLI) under long-day conditions on steviol glycoside concentration, we grew Stevia rebaudiana under ambient irradiance or varying levels of shading at different times of the year in both greenhouse and field environments, resulting in DLIs ranging from 3.55 to 20.31 mol·m−2·d−1 in the greenhouse and 10.32 to 39.7 mol·m−2·d−1 in the field. Total steviol glycoside concentration of selected leaves from greenhouse-grown plants increased as DLI increased up to ca. 10 mol·m−2·d−1, remaining constant with further increases in DLI, and was similar across the range of DLIs evaluated in the field. DLI influenced both the concentration and the relative proportions of specific steviol glycosides. Rebaudioside A concentration increased as DLI increased from 3.55 to 8.53 mol·m−2·d−1, remaining similar with further increases in DLI. Rebaudioside D and stevioside concentration of selected leaves from field-grown plants decreased by 22% and 13%, respectively, as DLI increased from 10.32 to 39.7 mol·m−2·d−1, while rebaudioside A and M concentrations remained similar across this DLI range. Collectively, these results indicate that the greatest influence of DLI on steviol glycoside concentration occurs under relatively low DLIs (<10 mol·m−2·d−1). However, higher DLIs can significantly affect the synthesis of minor glycosides of increasing commercial importance including rebaudioside D.

In silico analysis, expression profiling and correlation of miRNAs targeting genes of steviol glycosides biosynthesis with steviol glycosides contents in different tissues of Stevia rebaudiana

In silico analysis, expression profiling and correlation of miRNAs targeting genes of steviol glycosides biosynthesis with steviol glycosides contents in different tissues of Stevia rebaudiana

Enhanced production of steviol glycosides in mycorrhizal plants: A concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes

Stevia rebaudiana (Bertoni) produces steviol glycosides (SGs) – stevioside (stev) and rebaudioside-A (reb-A) that are valued as low calorie sweeteners. Inoculation with arbuscular mycorrhizal fungi (AMF) augments SGs production, though the effect of this interaction on SGs biosynthesis has not been studied at molecular level. In this study transcription profiles of eleven key genes grouped under three stages of the SGs biosynthesis pathway were compared. The transcript analysis showed upregulation of genes encoding 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway enzymes viz.,1-deoxy-d-xylulose 5-phospate synthase (DXS), 1-deoxy-d-xylulose 5-phospate reductoisomerase (DXR) and 2-C-methyl-D-erytrithol 2,4-cyclodiphosphate synthase (MDS) in mycorrhizal (M) plants. Zn and Mn are imperative for the expression of MDS and their enhanced uptake in M plants could be responsible for the increased transcription of MDS. Furthermore, in the second stage of SGs biosynthesis pathway, mycorrhization enhanced the transcription of copalyl diphosphate synthase (CPPS) and kaurenoic acid hydroxylase (KAH). Their expression is decisive for SGs biosynthesis as CPPS regulates flow of metabolites towards synthesis of kaurenoid precursors and KAH directs these towards steviol synthesis instead of gibberellins. In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. While higher transcription of all the three characterized UGTs in M plants explains augmented production of SGs; higher transcript levels of UGT76G1, specifically improved reb-A to stev ratio implying increased sweetness. The work signifies that AM symbiosis upregulates the transcription of all eleven SGs biosynthesis genes as a result of improved nutrition and enhanced sugar concentration due to increased photosynthesis in M plants.

Impact of nitrogen supply on growth, steviol glycosides and photosynthesis in Stevia rebaudiana Bertoni

This work investigated the agronomic, physiological and biochemical response of Stevia rebaudiana Bertoni grown under different nitrogen (N) rates. A pot trial in open air conditions was set up in 2012 with the aim to evaluate the effect of four N rates on the biometric and productive characteristics, steviol glycoside (SG) content as well as on leaf gas exchanges, chlorophyll fluorescence, photosynthetic pigments, Rubisco activity and N use efficiency. N deficiency caused a decrease in leaf N content, chlorophylls and photosynthetic CO2 assimilation, resulting in a lower dry matter accumulation as well as in reduced SG production. The application of 150 kg N ha− 1 seems to be the most effective treatment to improve rebaudioside A (Reb A) content, Reb A/stevioside ratio, photosynthetic CO2 assimilation, stomatal conductance, N use efficiency, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) and PSII efficiency. The results demonstrate that by using an appropriate N rate it is possible to modulate the SG biosynthesis, with a significant increase in the Reb A content and in the ratio between Reb A and stevioside. This finding is of great relevance in order to obtain a raw material designed to meet consumer needs and bio-industry requirements for high-quality, Reb A content, and safe and environmentally friendly products.

Environmental cues induce changes of steviol glycosides contents and transcription of corresponding biosynthetic genes in Stevia rebaudiana

Plant growth and secondary metabolism are commonly regulated by external cues such as light, temperature and water availability. In this study, the influences of low and high temperatures, dehydration, photoperiods, and different growing stages on the changes of steviol glycosides (SGs) contents and transcription levels of fifteen genes involved in SGs biosynthesis of Stevia rebaudiana Bertoni were examined using HPLC and RT-PCR. The observations showed that the transcript levels of all the fifteen genes were maximum under 25 °C treatment, and the transcription of SrDXS, SrDXR, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI, SrGGDPS, SrCPPS1, SrUGT85C2 and SrUGT76G1 were restrained both in low temperature (15 °C) and high temperature (35 °C). Most genes in SGs biosynthesis pathway exhibited down-regulation in dehydration. To elucidate the effect of photoperiods, the plants were treated by different simulated photoperiods (8L/16D, 10L/14D, 14L/10D and 16L/8D), but no significant transcription changes were observed. In the study of growing stages, there were evident changes of SGs contents, and the transcript levels of all the fifteen genes were minimal in fast growing period, and exhibited evident increase both in flower-bud appearing stage and flowering stage. The obtained results strongly suggest that the effect of environmental cues on steviol glycosides contents and transcription of corresponding biosynthetic genes in S. rebaudiana is significant. It is worth to study deeply.

Study on the bioactivity of steviol and isosteviol in stevia (Stevia rebaudiana Bertoni)

Steviol and isosteviol are diterpene metabolites that are produced from a shared biosynthesis pathway with gibberellins in stevia leaves. These compounds share a chemically similar structure with gibberellins. An experiment was conducted to find the bioactivity properties of steviol and isosteviol in stevia. During vegetative growth, external solutions of steviol and isosteviol (1.6 × 10−5 and 1 × 10−5 M, respectively) were sprayed on stevia shoots and then the stevia growth and metabolites were assessed. The results showed that the stevia leaf growth was reduced by external application of steviol. The leaf growth was not affected by external isosteviol while the stevia height and stem dry weight significantly increased. The antioxidant capacity and steviol glycosides content were increased by external steviol. Our results implicated that the isosteviol bioactivity is similar to that of gibberellin hormone. On the other hand, steviol induces the antioxidant system and stimulates the steviol glycosides biosynthesis in stevia leaves.

Transcriptional profiling of genes involved in steviol glycoside biosynthesis in Stevia rebaudiana bertoni during plant hardening.

Stevioside is a diterpene glycoside found in Stevia rebaudiana Bertoni (Asteraceae) and is 200-300 times sweeter than sucrose. It is synthesized through a plastid localized 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. Fifteen genes are involved in the formation of steviol glycosides (stevioside and rebaudioside A). In the present investigation, micropropagated plants were allowed to harden for one month during which transcriptional profiling of candidate genes was carried out. Sampling from all the plants was carried out during hardening at different time intervals (day 10, 20, and 30) along with control plants (day 0). Stevioside content was also measured. Of 15 genes, 9 were up-regulated two-fold or greater. Nine genes were expressed at higher levels after 30 days than in the untreated controls. Moreover, these transcriptional differences were correlated with a significant enhancement in stevioside content from 0- (11.48%) to 30- (13.57%) day-old plants. MEP pathway genes in stevia are expressed at higher levels during hardening, a change to vegetative growth from reproductive growth. Although there were higher transcript levels of candidate genes at the initial phase of hardening, the highest stevioside content was found after 30 days of hardening, suggesting translational/posttranslational regulatory mechanisms.

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.

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.

Gene transcription of steviol glycoside biosynthesis in Stevia rebaudiana Bertoni under polyethylene glycol, paclobutrazol and gibberellic acid treatments in vitro

This experiment was carried out to measure the transcript levels of some genes involved in biosynthesis of steviol glycosides. Stevia rebaudiana plants were treated with polyethylene glycol (PEG), paclobutrazol (PBZ) and gibberellic acid (GA). Using quantitative real-time poly- merase chain reaction, the transcript levels of kaurene synthase (KS), kaurene oxidase (KO), kaurenoic acid hydroxylase (KAH) and three UDP-dependent glycosyl- transferases, UGT85C2, UGT74G1 and UGT76G1, were studied. The transcription of ent-KS1-1, ent-KAH and UGT74G1 were fairly stable under different treatments. The transcription of ent-KO, UGT85C2 and UGT76G1 significantly decreased by PBZ and PEG treatments. The results indicate that both PBZ and PEG treatments resulted in the same negative effect on genes transcription, which could not be reversed by GA treatment.

Insights into Steviol Glycoside Biosynthesis Pathway Enzymes Through Structural Homology Modeling

Insights into Steviol Glycoside Biosynthesis Pathway Enzymes Through Structural Homology Modeling

Steviol Glycosides from Stevia: Biosynthesis Pathway Review and their Application in Foods and Medicine

Stevia rebaudiana, a perennial herb from the Asteraceae family, is known to the scientific world for its sweetness and steviol glycosides (SGs). SGs are the secondary metabolites responsible for the sweetness of Stevia. They are synthesized by SG biosynthesis pathway operating in the leaves. Most of the genes encoding the enzymes of this pathway have been cloned and characterized from Stevia. Out of various SGs, stevioside and rebaudioside A are the major metabolites. SGs including stevioside have also been synthesized by enzymes and microbial agents. These are non-mutagenic, non-toxic, antimicrobial, and do not show any remarkable side-effects upon consumption. Stevioside has many medical applications and its role against diabetes is most important. SGs have made Stevia an important part of the medicinal world as well as the food and beverage industry. This article presents an overview on Stevia and the importance of SGs.

Effect of Sucrose on Steviol Glycoside Biosynthesis Pathway in Stevia rebaudiana

Effect of Sucrose on Steviol Glycoside Biosynthesis Pathway in Stevia rebaudiana