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.
Stevia rebaudiana (stevia) contains commercially important steviol glycosides, stevioside and rebaudioside A, these compounds have insulinotropic and anti-hyperglycemic effect. Steviol, stevioside and rebaudioside-A have taste modulation and insulin potentiation activity. Stevia leaves are composed of steviol (2–5%), stevioside (4–13%) and rebaudioside-A (1–6%). Stevioside has after-taste bitterness, rebaudioside-A is sweetest in taste among all the glycosides present. Therefore, lower ratio of rebaudioside-A to stevioside has bitter after-taste, which makes stevia plants unpalatable. By over-expressing the genes, SrUGT76G1 and SrKO, we propose to increase the ratio of RebA to stevioside in stevia. Various lines were generated and amongst them, seven lines had both the transgenes present. Co-overxpresion of SrUGT76G1 and SrKO led to the increased concentration of RebA in all the seven transgenic lines (KU1-KU7) than control plant and RebA to stevioside ratio also increased significantly. Steviol, stevioside and RebA showed a differential concentration in all the seven lines, but the pattern was the same in all of them and the ratio of RebA to stevioside increased dramatically. In transgenic line 2 (KU2), RebA showed a steep increase in concentration 52% the rebaudioside-A to stevioside ratio increased from 0.74 (control) to 2.83. In overall all the lines, RebA showed a positive correlation with steviol and stevioside. Overexpression of SrKO led to an increase in steviol which increased the stevioside, overexpression of SrUGT76G1 ultimately increased RebA concentration. In conclusion, concentration of RebA increased significantly with co- overexpression of SrUGT6G1 and SrKO genes. Lines with increased RebA are more palatable and commercially viable.
MicroRNAs (miRNAs)
play an important role in the regulation of
gene expression. They play a regulatory role in various nutrient assimilatory
pathways of plants; however, their role in the regulation of sulfur
uptake and assimilatory pathways in mustard cultivars under high/low
sulfur conditions is not elucidated. Sulfur is essential for plant
growth and development, and its deficiency can cause a decline in
oil seed content and thus lower the economic yield in
Brassica
juncea
. In this study, different miRNAs involved in the regulation
of sulfur uptake and assimilation pathways in
B. juncea
were identified using a psRNA target analyzer and miRanda database
tools. The predicted miRNAs that belong to 10 highly conserved families
were validated using stem-loop RT-PCR. The
B. juncea
cultivars Pusa Jaikisan, Pusa Bold, and Varuna were kept in sulfur-excessive
(high) and -deficient (insufficient) conditions, and expression studies
of miRNAs and their target mRNAs were carried out using qRT-PCR. The
correlation between the expression pattern of miRNAs and their target
genes showed their potential role in sulfur uptake and assimilation.
Analysis with 5′ RACE revealed the authentic target of miRNAs.
The influence of S treatments on metabolites and sulfur content was
also studied using GC-MS and a CHNS analyzer. Our study showed the
potential role of miRNAs in the regulation of sulfur uptake and assimilation
and put forward the implications of these molecules to enhance the
sulfur content of
B. juncea.
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