Unraveling microRNA mediated gene regulation in Centella asiatica (L.) Urb. by high-throughput sequencing based small-RNA profiling

Centella asiatica is a widely spread herb mostly found in the tropics having extensive medicinal values. Here, we report for the first time, transcriptome-wide characterization of miRNA profile from the leaves of C. asiatica using high-throughput Illumina sequencing. We identified 227 conserved and 109 putative novel miRNAs. Computational screening revealed potential mRNA targets for both the conserved and novel miRNAs encoding diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signaling pathways. Gene ontology annotation and KEGG analysis revealed the miRNA targets to be involved in a wide range of metabolomic and regulatory pathways. The differential expression of the miRNA encoding genes in diverse tissues was determined by real-time PCR analysis. We also found that gene expression levels of miR156, 159 and 1171 was reduced in salicylic acid treated axenic shoot cultures of C. asiatica compared to its control. Furthermore, RLM-RACE experiments mapped miRNA-mediated cleavage at two of the mRNA targets. The present study represents the large-scale identification of microRNAs from C. asiatica and contributes to the base for the up-coming studies on miRNA-mediated gene regulation of plant secondary metabolite pathways in particular Gouri Priya Ranjith, Jisha Satheesan, K K Sabu. 2023. Unraveling microRNA mediated gene regulation in Centella asiatica (L.) Urb. by high-throughput sequencing based small-RNA profiling. Tropical Plant Biology, Springer. https://10.1007/s12042-023-09342-0

Impact of mannitol and poly ethylene glycol 6000 induced water deficit on plant biomass and major secondary metabolites in Centella asiatica (L.) Urb. in vitro

Jisha S, Aiswarya S, Gouri PR, Manikantan K & Sabu KK. 2022. Impact of mannitol and poly ethylene glycol 6000 induced water deficit on plant biomass and major secondary metabolites in Centella asiatica (L.) Urb. in vitro. Indian Journal of Experimental Biology Vol. 60, March 2022, pp. 184-191.

Low water potential related stresses are regulated by modifying water uptake and loss to avoid low water potential, accumulating solutes which in turn enhance active principles and its gene expressions. Present study examined effect of in vitro induced absorption of mannitol and PEG (poly ethelene glycol) 6000 in Indian pennywort, Centella asiatica (L.) Urb., neutraceutical plant, evidenced by phenotypic, molecular and phytochemical analyses. Both mannitol and PEG 6000 induce water deficit conditions in plants and retarded normal plant biomass in terms of fresh and dry weights. These effects were significantly less severe in plants subjected to mannitol, compared to PEG. PEG and mannitol imposed water deficit, resulted in decline in major active compound, asiaticoside evidenced by HPTLC of asiaticoside content. Differential expression of some selected key genes in the asiaticoside pathway including squalene synthase and β amyrin synthase by qPCR, confirmed decrease in transcript level expression of asiaticoside, whereas upregulated transcript level expression was observed in cycloartenol synthase for synthesis of phytosterols. Estimation of total flavonoids and phenolics under different water deficit conditions were found declined. In conclusion, water deficit by mannitol and PEG 6000 can significantly affects processes associated with biomass growth and ability to synthesize secondary metabolites in C. asiatica.

Download/Read article at http://nopr.niscair.res.in/handle/123456789/59289

Genetic diversity and structure revealed by genomic microsatellite markers in Centella asiatica (L.) Urb., a plant with medicinal potential

 Abstract

Background: Around the world, medicinal plants are utilised for various purposes. Centella asiatica is one of the important medicinal plants widely used in many medicinal systems. Nevertheless, analysis of the genetic diversity would pave the way for its most suitable utilisation.

Methods and results: The present study analyses the genetic diversity and structure of eighty C. asiatica accessions collected from the southern states of India, using ten genomic microsatellite markers. The mean Nei’s gene diversity (0.46) indicates considerable genetic diversity. Analysis of molecular variance (82.48%) exhibited significant genetic variance between samples within the population. The cluster analysis brought out the structure of the analysed populations as three subpopulations based on the genetic differentiation.

Conclusions: The study showed significant intra-population variation, predominant inbreeding and population differentiation in C. asiatica. The findings will help better understanding of the genetic structure and gene pool of the plant.

Full article link: https://rdcu.be/cAvFI

Impact of potential stimulants on asiaticoside and madecassoside levels and expression of triterpenoid-related genes in axenic shoot cultures of Centella asiatica (L.) Urb

Gouri Priya Ranjith, S. Jisha, Achutan Sudarsanan Hemanthakumar, Chinthu V. Saji, Rajesh A. Shenoi, Kallevettankuzhy Krishnannair Sabu

Phytochemistry, Volume 186, 2021, 112735

The triterpenoid saponins, asiaticoside and madecassoside from Centella asiatica (L.) Urb. are known to have a wide range of applications in pharmaceutical and cosmetic industries. The effect of addition of Potential Metabolite Stimulants (PMSs) - casein acid hydrolysate, meat peptone, salicylic acid, copper sulphate, and silver nitrate, on the concentrations of these saponins and transcript levels of associated genes encoding important biosynthetic enzymes, was assessed in axenic shoot cultures of C. asiatica. Among the stimulants, silver nitrate induced asiaticoside content approximately 6-fold increase in madecassoside levels, after three weeks post-treatment with a decrease in biomass compared to its control. Gene expression analysis of essential genes involved in triterpenoid synthesis such as β-amyrin synthase showed an upregulation of approximately 50-fold at the third week of silver nitrate treatment compared to control. These findings suggest that silver nitrate can act as a metabolite stimulant, to enhance the formation of triterpenoids in axenic shoot culture of C. asiatica, which could be utilized in studying the regulation of terpenoid biosynthesis and biotechnological application for the increased production of these bioactive molecules.

https://doi.org/10.1016/j.phytochem.2021.112735

Dataset on Discovery of MicroRNAs in Centella asiatica (L.) Urb.

Gouri, P. R., Jisha, S., and K. K. Sabu. 2020. Dataset on Discovery of MicroRNAs in Centella asiatica (L.) Urb. Data in Brief 106451. Available online 22 October 2020. https://doi.org/10.1016/j.dib.2020.106451

MicroRNAs (miRNAs) are small (21–24 nucleotides), non-coding, riboregulators that regulate gene expression in eukaryotes. Pentacyclic triterpenoid saponins and sapogenins known as centellosides of the plant Centella asiatica (L.) Urb. are known for their broad spectrum medicinal properties. Two C. asiatica accessions viz.,CA301 and CA308 were selected for the miRNAome profiling. Total RNA isolated from fresh young leaves of both accessions along with their replicas was used for library construction. Illumina® sequencing of the four small RNA libraries generated a total of 59,234,923; 58,487,817; 59,520,376; 64,093,228 raw reads. The raw reads were quality filtered and used for the prediction of conserved and novel miRNAs. A total of 227 conserved and 109 novel microRNAs were identified from the libraries. Target gene prediction done using psRNAtarget and PANTHER™GO helped in localization of predicted targets. KEGG (Kyoto Encyclopedia for Genes and Genomes) was used for pathway prediction of the targets of predicted miRNAs. The present study provides first elaborated glimpse of miRNA pool of C. asiatica. The outcome of this research could help understand miRNA dependent regulation of centelloside biosynthesis and to design further metabolic engineering experiments to enhance their content in this important medicinal plant.

Expression profiling of selected microRNAs coupled with molecular and biochemical analyses of asiaticosides in Centella asiatica (L.) Urb in vitro

Jisha, S., Hemanthkumar A. S., Gouri P. R. and K. K. Sabu. 2019. Expression profiling of selected microRNAs coupled with molecular and biochemical analyses of asiaticosides in Centella asiatica (L.) Urb in vitro. International Journal of Pharmacy and Pharmaceutical Sciences Vol. 11, no. 5, Mar. 2019, https://innovareacademics.in/journals/index.php/ijpps/article/view/32097

ABSTRACT
Objective: Centella asiatica (L.) Urb from Umbelliferae is a potential source of secondary metabolites having immense medicinal value. Asiaticoside is the major therapeutic compound. In the present study, Identification of a possible relationship between concentration/transcript level expression of asiaticoside and concentrations of growth hormones at different growth stages was observed. The current study includes molecular and biochemical evaluation of stress generated in C. asiatica at different time intervals in vitro.

Methods: The enhancement in auxin, cytokinin and final asiaticoside content were determined using immunoassay kits for auxin, cytokinin and HPLC analysis respectively. Transcript level expression at different growth phases was carried out using real-time RT-PCR. For isolation of stress-related miRNAs, reverse transcription of total RNA using miScript II RT Kit PCR System was carried out as per instructions. The differential expression of five selected miRNAs was done by Real-Time RT-PCR. The analysis of stress in vitro was done by quantification of Hydrogen Peroxide (H2O2), total phenolics and total antioxidants by H2O2 assay kit, total antioxidant assay kit and Folin Ciocalteau reagent respectively. The final asiaticoside content was determined by HPLC.

Results: Differential expression of key genes involved in asiaticoside pathway showed significantly higher transcript expression, which is in correlation with the final asiaticoside content. The enhanced expression of miRNAs and the analysis of H2O2, total antioxidant capacity and total phenolics are suggestive of generation of oxidative stress under controlled conditions.

Conclusion: The present study shows a direct correlation between oxidative stress and transcript/phytochemical estimation of asiaticoside content under in vitro conditions.

KEYWORDS:  Asiaticoside, Centella asiatica, In vitro, microRNA, Real-time RT-PCR

The protective role of Piriformospora indica colonization in Centella asiatica (L.) in vitro under phosphate stress

Jisha, S., K. N. Anith and K. K. Sabu. 2019. The protective role of Piriformospora indica colonization in Centella asiatica (L.) in vitro under phosphate stress. Biocatalysis and Agricultural Biotechnology Vol. 19. Article 101088. https://doi.org/10.1016/j.bcab.2019.101088

Abstract
The core of present study was to determine potential role of Piriformospora indica, mutualistic Basidiomycete endosymbiont on co-cultivation with Centella asiatica (L.) Urb., a neutraceutical plant. The analysis of major secondary metabolite asiaticoside and differential expression of key genes involved in pathway under varying phosphate (P) concentrations maintained in vitro were carried out. Maximum asiaticoside content was documented in plants maintained in MS medium containing lowest level (62.5 μM) tried (p < 0.001). At phosphate concentration of 125 μ M (10% of normal P), significant stimulation of plant growth and asiaticoside production were observed in colonized plants. P levels at 125 μM however favored maximum fresh weight, root and shoot length, root and leaf number of plants. The transcript accumulation of β Amyrin Synthase, the key gene in asiaticoside pathway was recorded at the P level of 62.5 μM, which is significantly higher (p < 0.001) than those at other P concentrations tried which was evidenced by overproduction of auxin, Indole-3-Acetic Acid (IAA). A significantly higher level of IAA was recorded in P. indica colonized plants maintained in low (125 μM) P medium. The presence of P. indica under low phosphate concentrations has protective role in mitigating effects of stress, as evidenced by non-significant hydrogen peroxide production, acid/alkaline phosphatase activity, total phenolics and increased super oxide dismutase (SOD) activity in colonized plants. The study clarifies positive correlation of the asiaticoside production in P. indica colonized plants under low P conditions with significant up regulation of asiaticoside pathway gene transcripts with less stress.

Sakthipriya M., S. S. Vishnu, S. Sujith, P. Rajeesh Kumar, and K. K. Sabu. 2018. Analysis of genetic diversity of Centella asiatica using SSR markers. International Journal of Applied Sciences and Biotechnology Vol 6(2): 103-109. http://dx.doi.org/10.3126/ijasbt.v6i2.19583

Abstract
Genetic diversity represents the heritable variation within and between populations of organisms. A better understanding of genetic diversity and its distribution is essential for its conservation and use. In this study the genetic diversity analysis was performed in 30 accessions of Centella asiatica (L.) Urb. using 10 SSR markers. C. asiatica is a tropical medicinal plant from Apiaceae family native to Southeast Asian countries. It is among the top herbs in the category of anti-aging and CNS drugs used worldwide. The whole plant and aerial parts are used widely in traditional and alternative medicines due to its ample pharmacological activities. The molecular screening through microsatellite markers showed low polymorphism (0.019) between the samples analyzed. Further studies, including the effect of environmental factors, genetic composition or possibility of inbreeding, are required to analyze the probable reason for the low variability exists in the species.

Int. J. Appl. Sci. Biotechnol. Vol 6(2): 103-109

Keywords: Centella asiatica; genetic diversity; SSR; microsatellites; medicinal plant

Physiological and Molecular Plant Pathology

Volume 103, August 2018, Pages 8-15

Stress analysis and cytotoxicity in response to the biotic elicitor, Piriformospora indica and its' cell wall extract in Centella asiatica L. Urban

Author links open overlay panelS.Jishaa   P.R.Gouria   K.N.Anithb  K.K.Sabua

https://doi.org/10.1016/j.pmpp.2018.04.001

Highlights

•

Oxidative stress analysis in Centella asiatica(L.) Urban on co-cultivation with Piriformospora indica and its extract is proposed.

•

The presence of Piriformospora indica had protective role in alleviating stress.

•

No variation in the cytotoxicity levels in control and treated groups, as evidenced by MTT assay.

•

The cell integrity appeared intact in P. indicacolonized C. asiatica.

Abstract

Study reports prominent findings on the analysis of oxidative stress generated in Centella asiatica, a multifunctional medicinal plant on co-cultivation with P. indica and its extract, P. indica Cell Wall Extract. The presence of P. indica had protective role in alleviating stress, evidenced by lack of significant change in H2O2, increased total antioxidant capacity and phenolics in P. indica colonized and PiCWE treated plants. No variation was observed between IC50 values and the cell integrity in P. indica colonized C. asiaticaappeared intact by TEM analysis. The study preludes the possible role of P. indica in conferring drought tolerance to C. asiatica.

Keywords

Endosymbiosis, 

Centella asiatica, 

Oxidative stress, 

Piriformospora indica, 

Total antioxidant capacity

Plant Physiol Biochem. 2018 Apr;125:106-115. doi: 10.1016/j.plaphy.2018.01.021. Epub 2018 Feb 7.

Piriformospora indica cell wall extract as the best elicitor for asiaticoside production in Centella asiatica (L.) Urban, evidenced by morphological, physiological and molecular analyses.

Jisha S1, Gouri PR1, Anith KN2, Sabu KK3.

Author information

Abstract

Vascular plants synthesise a multitude of organic molecules or phytochemicals, referred to as "secondary metabolites". These molecules are involved in a variety of roles in the life span of plants, ranging from structural ones to protection. Centella asiatica (L.) Urban has probably been used since prehistoric times and has been reported to have been used for various medicinal and cosmetic purposes. The plant contains several active constituents, of which the most important is asiaticoside, a triterpenoid. Asiaticoside content in C. asiatica can be enhanced by the use of biotic elicitors like Piriformospora indica. P. indica has been used as a model to study the mechanisms and evolution of mutualistic symbiosis. P. indica is similar to Arbuscular Mycorrhizal (AM) fungi in terms of plant growth promotional effects. The autoclaved fraction from P. indica (PiCWE) was found to be the most active fraction in promoting the plant biomass and asiaticoside content. To date, there are no reports on the potential role of PiCWE in enhancement of asiaticoside over the control and P. indica colonized plants, which was evidenced by the differential expression of key genes involved and final asiaticoside content along with the determination of phytohormones. Moreover, differential expression of selected miRNAs in PiCWE - C. asiatica root interactions over the control and P. indica treated C. asiatica leaf samples was also scrutinized. The important consequence of induction with PiCWE was the significant enhancement of asiaticoside in the PiCWE induced plants in comparison with the asiaticoside content in control and P. indica-C. asiatica interaction. In addition, the role of miRNAs in C. asiatica - PiCWE would enable more in-depth studies for deciphering the molecular and physiological mechanisms of the association and regulation of PiCWE - C. asiatica interactions.

KEYWORDS:

Centella asiatica; HPLC; Piriformospora indica cell wall extract; β amyrin synthase

PMID:

 

29438895

 

DOI:

 

10.1016/j.plaphy.2018.01.021