Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

Guleria, Praveen; Yadav, Sudesh Kumar

doi:10.1371/journal.pone.0074731

Cited by 38 publications

(19 citation statements)

References 32 publications

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“…Plants exhibit phenotypic changes such as dwarfism and reduced internode length under reduced GA content (Thomas and Sun, ). It has been reported that transient knockdown of the SrUGT76G1 in Stevia led to an increase in GA levels so the overexpression of SrUGT76G1 may have an opposite effect (Guleria and Yadav, ). Hence, we monitored the growth and development of the transgenic Stevia plants.…”

Section: Resultsmentioning

confidence: 99%

Overexpression of SrUGT76G1 in Stevia alters major steviol glycosides composition towards improved quality

Kim

Zheng

Liao

et al. 2018

Plant Biotechnology Journal

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Summary Steviol glycosides ( SG s) are extracted from Stevia leaves for use as a natural sweetener. Among SG s, stevioside is most abundant in leaf extracts followed by rebaudioside A (Reb A). However, Reb A is of particular interest because of its sweeter and more pleasant taste compared to stevioside. Therefore, the development of new Stevia varieties with a higher Reb A to stevioside ratio would be desirable for the production of higher quality natural sweeteners. Here, we generated transgenic Stevia plants overexpressing Stevia UDP ‐glycosyltransferase 76G1 ( Sr UGT 76G1 ) that is known to convert stevioside to Reb A through 1,3‐β‐ d ‐glucosylation in vitro . Interestingly, by overexpressing Sr UGT 76G1 , the Reb A to stevioside ratio was drastically increased from 0.30 in wild‐type ( WT ) plants up to 1.55 in transgenic lines without any significant changes in total SG s content. This was contributed by a concurrent increase in Reb A content and a decrease in stevioside content. Additionally, we were able to find an increase in the Reb C to dulcoside A ratio in transgenic lines. Using the glutathione S‐transferase‐tagged Sr UGT 76G1 recombinant protein for an in vitro glucosyltransferase assay, we further demonstrated that Reb C can be produced from the glucosylation of dulcoside A by Sr UGT 76G1. Transgenic Stevia plants having higher Reb A to stevioside ratio were visually indistinguishable from WT plants. Taken together, our results demonstrate that the overexpression of Sr UGT 76G1 in Stevia is an effective way to generate new Stevia varieties with higher proportion of the more preferred Reb A without compromising on plant development.

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Section: Resultsmentioning

confidence: 99%

Overexpression of SrUGT76G1 in Stevia alters major steviol glycosides composition towards improved quality

Kim

Zheng

Liao

et al. 2018

Plant Biotechnology Journal

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“…In the present study, we characterize the function of these miRNAs. For this, we over-expressed miR319g and miRStv_11 in leaves of S. rebaudiana plant by syringe agroinfiltration method [5, 31] and the samples were collected after 2, 4, 6, 8 and 10 days post infiltration. Infiltration of over-expression construct corresponding to miRStv_11 was found to significantly up-regulate the expression of its target gene, KAH up to 8.16 fold than the control.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2019

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Background miRNAs are major regulators of gene expression and have proven their role in understanding the genetic regulation of biosynthetic pathways. Stevioside and rebaudioside-A, the two most abundant and sweetest compounds found in leaf extract of Stevia rebaudiana , have been used for many years in treatment of diabetes. It has been found that the crude extract is more potent than the purified extract. Stevioside, being accumulated in higher concentration, imparts licorice like aftertaste. Thus, in order to make the sweetener more potent and palatable, there is a need to increase the intrinsic concentration of steviol glycosides and to alter the ratio of rebaudioside-A to stevioside. Doing so would significantly increase the quality of the sweeteners, and the potential to be used on a wider scale. To do so, in previous report, miRNAs associated with genes of steviol glycosides biosynthetic pathway were identified in S. rebaudiana. In continuation to that in this study, the two miRNAs (miR319g and miRStv_11) targeting key genes of steviol glycosides biosynthetic pathway were modulated and their impact was evaluated on steviol glycosides contents. Results The over-expression results showed that miRStv_11 induced, while miR319g had repressive action on its target genes. The knock-down constructs for miR319g and miRStv_11 were then prepared and it was demonstrated that the expression of anti-miR319g produced inhibitory effect on its target miRNA, resulting in enhanced expression of its target genes. On the other hand, anti-miRStv_11 resulted in down-regulation of miRStv_11 and its target gene. Further miRStv_11 and anti-miR319gwere co-expressed which resulted in significant increase in stevioside (24.5%) and rebaudioside-A (51%) contents. Conclusion In conclusion, the role of miR319g and miRStv_11 was successfully validated in steviol gycosides biosynthetic pathway gene regulation and their effect on steviol gycosides contents. In this study, we found the positively correlated miRNA-mRNA interaction network in plants, where miRStv_11 enhanced the expression of KAH gene. miRNAs knock-down was also successfully achieved using antisense precursors. Overall, this study thus reveals more complex nature and fundamental importance of miRNAs in biosynthetic pathway related gene networks and hence, these miRNAs can be successfully employed to enhance the ratio of rebaudioside-A to stevioside, thus enhancing the sweetening indices of this plant and making it more palatable. Electronic supplementary material The online version of this article (10.1186/s12870-019-1871-2) contains supplementary material, which is available to authorized users.

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“…2) in determining SVgly total content (Kumar et al 2012;Guleria and Yadav 2013). UGT85C2, which catalyze the formation of steviol from kaurenoic acid and of steviolmonoside from steviol, might also be a rate limiting step for the SVgly biosynthesis route (Guleria and Yadav 2013;Mohamed et al 2011). On the other hand, the relative stability of the SVgly composition suggests that the UGTs are less regulated by the environment.…”

Section: Discussionmentioning

confidence: 99%

Towards a semi-perennial culture of Stevia rebaudiana (Bertoni) Bertoni under temperate climate: effects of genotype, environment and plant age on steviol glycoside content and composition

Barbet-Massin

Giuliano

Alletto

et al. 2015

Genet Resour Crop Evol

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Stevia rebaudiana (Bertoni) Bertoni is a perennial shrub native to Paraguay whose leaves accumulate sweetening compounds of high value: steviol glycosides (SVglys). These diterpenoids differ in the number (1-4) and the nature of sugar units bound to a steviol skeleton: mainly glucose but also rhamnose and xylose. The two main SVglys are stevioside (ST; 3 glucose units) and rebaudioside A (RA, 4 glucose units). Along with an evaluation of genotypic variability for SVglys content and composition, this study investigates the winter survival and the effect of perennial cultivation on S. rebaudiana genotypes through a 2-year experiment conducted on two locations. The first year, a population of 96 genotypes was examined in a nursery field (site A) and a subset of five genotypes was grown in a field trial (site B). The winter surviving genotypes (16 on site A and 5 on site B) were studied the following year, alongside new plantings of the same lines (8 on site A and 5 on site B). The genotypes showed a high variability in SVgly content (from 45.7 to 186.2 mg g -1 DM) and composition along four distinct clusters. There was no correlation between total SVglys content and composition. Genotypes without RA had no other SVglys with more than three conjugated sugars. SVgly content varied among environments and generally increased between 1-and 2-year-old plants, while SVgly composition remained stable. This study showed that breeding for winter survival in temperate conditions would allow an increase in SVgly productivity in a semi-perennial stevia production.

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Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

Cited by 38 publications

References 32 publications

Overexpression of SrUGT76G1 in Stevia alters major steviol glycosides composition towards improved quality

Overexpression of SrUGT76G1 in Stevia alters major steviol glycosides composition towards improved quality

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

Towards a semi-perennial culture of Stevia rebaudiana (Bertoni) Bertoni under temperate climate: effects of genotype, environment and plant age on steviol glycoside content and composition

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