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

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.

Role of miRNAs in biotic and abiotic stress management in crop plants

Anjali N & Sabu K K (2020). Role of miRNAs in biotic and abiotic stress management in crop plants. In: Sustainable Agriculture in the Era of Climate Change. Edited by R. Roychowdhury (Springer New York, USA). Pp. 513-532. https://doi.org/10.1007/978-3-030-45669-6_22

MicroRNAs (miRNAs) are endogenous, 18–25 nucleotide in length, and noncoding RNAs found in animals, plants, and in some viruses. They have been recognized as an important class of gene expression regulators and are involved in a multitude of biological processes in plants, including organ differentiation and development, metabolism, and response to biotic and abiotic stress factors, to mention a few. The method of action of miRNA is by binding to a ribonucleoprotein complex which is incorporated with a protein of Argonaute family. This RNA-induced silencing complex (RISC) targets the mRNA and inhibits its expression by degrading mRNA or by translational repression. It has been reported that posttranscriptional regulation mediated by miRNAs has important roles in adverse stress conditions. Some miRNAs respond to a number of abiotic and biotic stresses, and some others are stress specific. Not only characterization of miRNAs but also identification of their targets is crucial in this field of research. The participation of miRNAs in a species-specific manner to biotic and abiotic stress responses reveals the functional diversification of miRNAs among plant species and highlights the importance of conducting miRNA studies in diverse plant species. MicroRNA-based genetic modification technology is a very powerful technique to develop high-yielding crops which can resist biotic and abiotic stress factors.

Small RNA manipulation in plants: Techniques and recent developments

The term “RNA silencing” refers to the processes that depend on small RNAs (approx. 18–24 nt) to regulate the mRNA expression in eukaryotes. In plants, small RNA regulation plays crucial roles in development, in responses to biotic or abiotic stress, in regulating metabolic pathways, in maintaining genome integrity, and in defense response mechanisms. In the last few years, small RNA manipulation has been used as a research tool to identify and validate gene expression, and studies now extensively focus on applying this technology in commercial applications, especially in medical therapeutics and flower color in ornamental plants. This chapter covers current progress on the application of RNA silencing to produce plants resistant to plant viruses, insects, and fungal pathogens, and also in applying these technologies toward the improvement of agricultural crops and medicinal plants. This chapter will also discuss the challenges, limitations, and biologic safety concerns related to small RNA manipulation in crops.

https://www.sciencedirect.com/science/article/pii/B9780128171127000183

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

Identification and characterization of drought responsive microRNAs and their target genes in cardamom

Anjali, N, Nadiya, F, Thomas, J, and K. K. Sabu. 2018. Identification and characterization of drought responsive microRNAs and their target genes in cardamom (Elettaria cardamomum Maton). Plant Growth Regulation. doi: 10.1007/s10725-018-0462-9 Online First: 2018-12-06

Abstract
Plant miRNAs are found to be present throughout the genome and they regulate gene expression either by cleaving mRNA or inhibiting the translational process at the post transcriptional level. Drought is one of the major limiting factors that negatively affect productivity of plants. Cardamom cultivation is having good production potential but the plants are vulnerable to biotic and abiotic stress factors. To date, nothing is known about the regulatory roles of miRNAs in response to drought stress in cardamom. Ion Torrent sequencing of two small RNA libraries prepared from control (C) and treated (T) plants raised under well irrigated and drought stressed treatments respectively created 3,938,342 and 4,083,181 primary reads. A total of 150 conserved and 20 novel microRNAs were identified from both the control and treated libraries. Discovery of 17 differentially expressed miRNAs under drought stress suggests that these miRNAs might have involved in various biological processes to improve plant tolerance to water stress. Several target genes for drought stress regulating miRNAs were identified including miR156l and miR169c which cleave the target mRNA involved in response to water deprivation. miR530b and miR156a target mRNAs which respond to water deprivation and inhibit the translational process. The expression patterns of some of the miRNAs and their targets were validated by qRT-PCR. This study is the first report of drought responsive miRNAs and their targets in cardamom. The outcome of this research could provide insights into the miRNA based regulatory mechanisms in response to drought stress in monocot plants.

See at Journal website - https://link.springer.com/article/10.1007%2Fs10725-018-0462-9

Nadiya, F., N. Anjali, Jinu Thomas, A. Gangaprasad, K. K. Sabu. 2018. Deep sequencing identified potential miRNAs involved in defense response, stress and plant growth characteristics of wild genotypes of cardamom. Plant Biology. Published online: 11 August 2018 https://doi.org/10.1111/plb.12888

Abstract

• Cardamom has long been used as a food flavouring agent and in ayurvedic medicines for mouth ulcers, digestive problems and even depression. Extensive occurrence of pests and diseases adversely affect its cultivation and result in substantial reductions in total production and productivity. Numerous studies revealed the significant role of miRNAs in plant biotic stress responses.
• In the current study, miRNA profiling of cultivar and wild cardamom genotypes was performed using an Ion Proton sequencer.
• We identified 161 potential miRNAs representing 42 families, including monocot/tissue‐specific and 14 novel miRNAs in both genotypes. Significant differences in miRNA family abundance between the libraries were observed in read frequencies. A total of 19 miRNAs (from known miRNAs) displayed a twofold difference in expression between wild and cultivar genotypes. We found 1168 unique potential targets for 40 known miRNA families in wild and 1025 potential targets for 42 known miRNA families in cultivar genotypes. The differential expression analysis revealed that most miRNAs identified were highly expressed in cultivars and, furthermore, lower expression of miR169 and higher expression of miR529 in wild cardamom proved evidence that wild genotypes have stronger drought stress tolerance and floral development than cultivars.
• Potential targets predicted for the newly identified miRNAs from the miRNA libraries of wild and cultivar cardamom genotypes involved in metabolic and developmental processes and in response to various stimuli. qRT‐PCR confirmed miRNAs were differentially expressed between wild and cultivar genotypes. Furthermore, four target genes were validated experimentally to confirm miRNA–mRNA target pairing using RNA ligase‐mediated 5′ Rapid Amplification of cDNA Ends (5′RLM‐RACE) PCR.

Keywords: Cardamom, microRNA, next generation sequencing, RNA sequencing

Discovery of MicroRNAs in Cardamom (Elettaria cardamomum Maton) under Drought Stress

Dataset Papers in Science
Volume 2017 (2017), Article ID 9507485, 4 pages
https://doi.org/10.1155/2017/9507485

Dataset Paper

Discovery of MicroRNAs in Cardamom (Elettaria cardamomum Maton) under Drought Stress

N. Anjali, F. Nadiya, Jinu Thomas, and K. K. Sabu

Jawaharlal Nehru Tropical Botanic Garden and Research Institute (JNTBGRI), Palode, Thiruvananthapuram 695562, India

Correspondence should be addressed to K. K. Sabu

Received 31 December 2016; Revised 21 March 2017; Accepted 19 April 2017; Published 31 July 2017

Academic Editor: Michael Hackenberg

Copyright © 2017 N. Anjali et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

MicroRNAs (miRNAs) are endogenous, 19–24-nucleotide-long, noncoding RNAs found in animals, plants, and some viruses. Drought is one of the major limiting factors that negatively affect productivity of plants. To date, nothing is known about the regulatory roles of miRNAs in response to drought stress in cardamom. Cardamom collected from the natural forest area in Therakkudi in the Idamalayar forest range was selected for the study. Total RNA was isolated from leaves and stems of control and drought treated cardamom plant, which was used for library construction. Ion torrent sequencing of the two small RNA libraries prepared from plants raised under well irrigated and drought stressed treatments, respectively, created 3,938,342 and 4,083,181 primary reads. A total of 150 conserved and 20 novel microRNAs were identified from both the control and the treated libraries. Discovery of 17 differentially expressed miRNAs under drought stress suggests that these might have been involved in various biological processes to improve plant tolerance to water stress. This study is the first report of drought responsive miRNAs and their targets in cardamom. The outcome of this research could provide insights into the miRNA based regulatory mechanisms in response to drought stress in monocot plants.

Development of a New Pipeline for Identification and Characterization of MicroRNAs from Plants

Open source microRNA analysis pipelines for next generation sequencing data (NGS) often make necessary use and working knowledge of command line interface, massive data processing resources and expertise which is a daunting task for biologists. Further, the microRNA data generated from NGS platforms will not be in a form from which one could understand or make use of it. Hence a comprehensive pipeline has been developed by integrating several open source NGS tools along with a graphical user interface called sRNAbench. It is useful for expression profiling of small RNAs and prediction of microRNAs from NGS data. The pipeline features functionalities such as read processing, sequence identification, target prediction and enrichment analysis. It provides even prediction of novel microRNAs and its sequences. The pipeline will be very useful for plant genomics community and it does not require knowledge in computational biology in order to discover miRNAs and utilize the same in genomics studies.

Citation
Thomas, J. and K. K. Sabu. 2016. Development of a New Pipeline for Identification and Characterization of MicroRNAs from Plants. International Journal for Computational Biology 5(1): 21-27.

MiRNA Isolation from Plants Rich in Polysaccharides and Polyphenols

MicroRNAs (miRNAs) are a group of 18–22-nucleotide-long noncoding RNAs, which show wide array of roles in various biological and metabolic processes in both animals and plants. They are formed endogenously and have evolutionary conserved sequences. RNA quality is extremely important for sequencing miRNAs using next-generation sequencing platforms. However, isolation and quantification of miRNAs from plant samples are often technically difficult and recovery of miRNAs from total RNA might be problematic. Degradation of RNA weakens the true miRNAs present in the sample, hence it is crucial to use a protocol that effectively retains good integrity miRNAs. In this chapter, we outline few protocols that can be used to maximize the retrieval of good-quality miRNA and total RNA to be used in miRNA analysis for plants rich in polysaccharides and polyphenols.

Citation
Sabu, K.K., Nadiya. F. and N. Anjali. 2017. MiRNA Isolation from Plants Rich in Polysaccharides and Polyphenols. In: MicroRNA Profiling. Vol. 1509. Pp 25-36. Sweta Rani (Ed). Springer. ISBN:978-1-4939-6522-9. PMID: 27826915 DOI: 10.1007/978-1-4939-6524-3_4

High quality RNA extraction from small cardamom tissues rich in polysaccharides and polyphenols

Abstract

Due to the presence of a diverse array of metabolites, no standard method of RNA isolation is available for plants. We noted that polysaccharide and polyphenol contents of cardamom tissues critically hinder the RNA extraction procedure. Hence, we attempted several methods for obtaining intact mRNA and small RNA from various cardamom tissues. It was found that protocols involving a combination of commercial kits and conventional CTAB (cetyl trimethylammonium bromide) methods yielded RNA with good purity, higher yield, and good integrity. The total RNA isolated through this approach was found to be amenable for transcriptome and small RNA analysis through next-generation sequencing platforms.

Keywords

• RNA isolation; Next-generation sequencing (NGS); mRNA; miRNA; RIN value
• • http://dx.doi.org/10.1016/j.ab.2015.05.017 
  • Full paper: http://authors.elsevier.com/a/1RJrWlbACuNf
• 
  
• Analytical Biochemistry

• Fasiludeen Nadiya^a, 
• Narayanannair Anjali^a, 
• Appukuttannair Gangaprasad^b,
• Kalluvettankuzhy Krishnannair Sabu^a, , 

• ^a Biotechnology and Bioinformatics Division, Jawaharlal Nehru Tropical Botanic Garden & Research Institute (JNTBGRI), Thiruvananthapuram 695562, Kerala, India
• ^b Department of Botany, University of Kerala, Thiruvananthapuram 695037, Kerala, India

Received 22 May 2015, Accepted 28 May 2015, Available online 3 June 2015

Corresponding author

Kalluvettankuzhy Krishnannair Sabu - sabu@jntbgri.res.inBiotechnology and Bioinformatics Division, Jawaharlal Nehru Tropical Botanic Garden & Research Institute (JNTBGRI), Thiruvananthapuram 695562, Kerala, India.