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 7 weeks 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.     

Iron nano modulated growth and biosynthesis of steviol glycosides in Stevia rebaudiana

Plant Cell, Tissue and Organ Culture (PCTOC) - Two East Asian grape species Vitis amurensis and Vitis coignetiae have been investigated for their increased cold and drought tolerance and high level...     

A beta-glucosidase gene from Stevia rebaudiana may be involved in the steviol glycosides catabolic pathway

Molecular Biology Reports - We herein report the preparation of a full-length raucaffricine-O-beta-d-glucosidase gene of stevia rebaudiana Bertoni (named SrRG1, GenBank accession number MK920450)....     

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.     

The positive role of steviol glycosides in stevia (Stevia rebaudiana Bertoni) under drought stress condition

Steviol glycosides (SVglys) are a group of diterpenoids mainly present in the leaves of stevia (Stevia rebaudiana Bertoni). An experiment was conducted to find the functional role of SVglys compounds in stevia affected by drought stress. In this study, a liquid blend of SVglys (200 ppm) was sprayed on stevia plants grown in well-watered (90% field capacity) and drought-stress conditions (45% field capacity) and then the morphological traits and metabolites were evaluated. It was observed that leaf losses caused by drought stress were stopped through external application of SVglys and consequently the harvest index of stevia was increased. Metabolite analysis of stevia leaves showed that the total SVglys content was significantly decreased due to drought stress, but was compensated by external application of SVglys. Among the SVglys, Rebaudioside A responded more to external SVglys. A slight promotion in total antioxidant activity of stevia leaves was observed when external SVglys was applied. The glucose availability in stevia leaves was increased by external application of SVglys but only in well-watered plants. According to our findings, it can be concluded that in stevia, SVglys may have a positive function in drought stress tolerance by exerting a protective role under such conditions.     

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.     

Effect of two plant growth retardants on steviol glycosides content and antioxidant capacity in Stevia (Stevia rebaudiana Bertoni)

Two greenhouse experiments were conducted to study the effect of two plant growth retardants, Chlorocholine chloride (CCC) and Paclobutrazol (PBZ), on growth, Steviol glycosides (SVglys) content and antioxidant capacity in Stevia (Stevia rebaudiana Bertoni). Five concentrations of CCC (0, 250, 500, 750 and 1,000 ppm) and PBZ (0, 6, 12, 18 and 24 ppm) with three replications were applied to Stevia plants as treatments based on completely randomized design. CCC was sprayed on Stevia shoots, but PBZ was applied as a drench. The obtained results showed that CCC reduced plant height but improved leaf and stem dry weight, especially with 750 ppm concentration. Total SVgly content and consequently SVglys yield were significantly reduced by CCC application, and 1,000 ppm of CCC concentration was more effective than other treatments. PBZ had no effect on Stevia height while it significantly enhanced stem and dry weight at 12 ppm. Moreover, PBZ remarkably increased total SVglys contents, SVglys yield, and Rebaudioside A/Stevioside ratio. Total antioxidant capacity significantly varied with CCC and PBZ and the highest activity was obtained with 1,000 and 12 ppm of CCC and PBZ, respectively. The results of these experiments indicated that, although CCC and PBZ are plant growth retardants and act as anti-gibberellins, only CCC reduced plant height and SVglys production in Stevia. On the contrary, PBZ at 12 ppm concentration, improved Stevia growth, SVglys production, and antioxidant capacity.     

A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni)

Stevia [Stevia rebuaidana (Bertoni); family: Asteraceae] is known to yield diterpenoid steviol glycosides (SGs), which are about 300 times sweeter than sugar. The present work analyzed the expression of various genes of the SGs biosynthesis pathway in different organs of the plant in relation to the SGs content. Of the various genes of the pathway, SrDXS, SrDXR, SrCPPS, SrKS, SrKO and three glucosyltransferases namely SrUGT85C2, SrUGT74G1 and SrUGT76G1 were reported from stevia. Here, we report cloning of seven additional full-length cDNA sequences namely, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI and SrGGDPS followed by expression analysis of all the fifteen genes vis-à-vis SGs content analysis. SGs content was highest in the leaf at 3rd node position (node position with reference to the apical leaf as the first leaf) as compared to the leaves at other node positions. Except for SrDXR and SrKO, gene expression was maximum in leaf at 1st node and minimum in leaf at 5th node. The expression of SrKO was highest in leaf at 3rd node while in case of SrDXR expression showed an increase up to 3rd leaf and decrease thereafter. SGs accumulated maximum in leaf tissue followed by stem and root, and similar was the pattern of expression of all the fifteen genes. The genes responded to the modulators of the terpenopids biosynthesis. Gibberellin (GA₃) treatment up-regulated the expression of SrMCT, SrCMK, SrMDS and SrUGT74G1, whereas methyl jasmonate and kinetin treatment down-regulated the expression of all the fifteen genes of the pathway.     

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.     

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

Molecular Biology Reports - Stevia rebaudiana produces sweet steviol glycosides that are 300 times sweeter than sugar and have the beneficial effects on human health including anti-hyperglycaemic....     

Utilisation of steviol glycosides from Stevia rebaudiana (Bertoni) by lactobacilli and bifidobacteria in in vitro conditions

In the current study, eight strains of bifidobacteria and seven strains of lactobacilli were tested for their ability to grow in the presence of rebaudioside A and steviol glycosides from the sweetener Natusweet M001 originating from herb Stevia rebaudiana (Bertoni). Stevia is gaining popularity as a natural, non-caloric sugar substitute, and recently, it was allowed as a food additive by European Union too. Utilisation of steviol glycosides by intestinal microbiota suggests that they might have potential prebiotic effect. Based on the evaluation of bacterial density and pH values in our in vitro study, it was found that lactobacilli and bifidobacteria tested were able to utilise steviol glycosides as a carbon source only to a very limited extent. All strains tested showed significantly lower change in the absorbance A₅₄₀ (P?<?0.05) and pH decrease of the growth media as compared with the positive controls (medium containing glucose as a carbon source and de Man Rogosa Sharpe broth). We concluded that a suggested prebiotic effect was not confirmed either in the case of rebaudioside A or in the case of the sweetener Natusweet M001 containing a mixture of steviol glycosides.     

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.

     

Diterpene synthesis in Stevia rebaudiana: recruitment and up-regulation of key enzymes from the gibberellin biosynthetic pathway

Stevia rebaudiana Bertoni leaves accumulate a mixture of at least eight different glycosides derived from the tetracyclic diterpene steviol. These natural products taste intensely sweet and have similar biosynthetic origins to those of gibberellic acid (GA). The initial steps leading to the formation of GA result from the two-step cyclization of geranylgeranyl diphosphate (GGDP) to (-)-kaurene via the action of two terpene cyclases (-)-copalyl diphosphate synthase (CPS) and (-)-kaurene synthase (KS). Steviol biosynthesis probably uses the same mechanism although the genes and enzymes from S. rebaudiana that are involved in the cyclization of GGDP have not been characterized. We have isolated both the CPS and KS genes from S. rebaudiana and found that recombinant CPS and KS were catalytically active, suggesting that the CPS and KS genes participate in steviol biosynthesis. The genes coding for CPS and KS are usually present in single copies in most plant species and their expression is normally low and limited to rapidly growing tissues. The KS gene has been duplicated in the S. rebaudiana genome and both the KS and CPS genes are highly expressed in mature leaves, a pattern opposite to that found with GA biosynthesis. This pattern may, at least in part, lead to temporal and spatial separation of GA and steviol biosynthesis and probably helps to prevent over-expression from interfering with normal GA metabolism. Our results show that CPS and KS are part of the steviol glycoside biosynthetic pathway and that Stevia rebaudiana has recruited two genes to secondary metabolism from a highly regulated pathway involved in hormone biosynthesis.     

Synthesis of GA20 glucosyl derivatives and the biological activity of some gibberellin conjugates

GA₂₀-13-0-glucoside (7a) and GA₂₀ glucosyl ester (6a), potential endogenous conjugates in maize, were synthesized chemically. The biological activities of these compounds and of nine more GA glucosyl derivatives were determined using theZea mays dwarf- 5 seedling andOryza sativa cv. “Tan-ginbozu” assays. The relative bioactivities of the conjugates were also calculated.     

The Fertilization and Early Development of Embryo and Endosperm in Stevia rebaudiana Bertani

The mature embryo sac is surrounded by endothelium tapetum. It is composed or an egg apparatus, one central cell with secondary nucleus, and 1–6 antipodal cells. About the 6th hour after pollination, female and male nuclei fuse with each other. The syngamy occurred almost simultaneously with the fusion of an other sperm nucleus and the secondary nucleus, but the velocity of the latter is faster than that of the syngamy. The fertilization of Stevia rebaudiana Bertani belongs to the premitotic type. About the 8th hour after pollination, primary endosperm nucleus is in mitosis, its dividing orientation may parallel or at right angle to the long axis of the embryo sac, and gives rise to two initial endosperm cells. The first five divisions of the endosperm cells are of synchronism. At the stage of heart-shaped embryo, the endosperm cells show the signs of digestion and absorbed. The endosperm development is of the cellular type. About the 10th hour after pollination, zygote divides for the first time. The division of the zygote is always transverse. The embryo development conforms to the Asterad type.     

Synthesis of GA20 glucosyl derivatives and the biological activity of some gibberellin conjugates

GA₂₀-13-0-glucoside (7a) and GA₂₀ glucosyl ester (6a), potential endogenous conjugates in maize, were synthesized chemically. The biological activities of these compounds and of nine more GA glucosyl derivatives were determined using theZea mays dwarf- 5 seedling andOryza sativa cv. Tan-ginbozu assays. The relative bioactivities of the conjugates were also calculated.