miRNAs are small, non-coding, single stranded regulatory elements, subjected to deep research since last two decades. Recently, identified plant miRNAs were found to help in plant adaptation during stress conditions. These findings concluded that altered expressions of miRNAs regulate plant growth and development in several plant species subjected to abiotic stress conditions such as drought, salinity, extreme temperatures, nutrient deprivation, and heavy metals. So, miRNAs can be used further for genetic manipulations to make crop plants more stress tolerant. In the past few years, scientific endeavors have been directed towards understanding the post-transcriptional regulation of secondary metabolites involving miRNAs. Effective role of miRNAs to directly alter the plant biochemicals are already reported, understanding of this mechanism will help to further improve secondary metabolite concentration in plants. First such report was published in 2011, which disclosed the role of miR156 in regulating the amount of anthocyanin by targeting the SPL genes . After this, a number of studies reported the role of miRNAs in regulation and biosynthesis of many secondary metabolties such as flavonoids, terpenoid and alkaloids . Zhang et al. 2012 have shown in their landmark study that miRNA from food products can travel safely to mammalian gut and enter the bloodstream. This suggests the plant miRNAs can be used to regulate expression of target genes even for the treatment of many human diseases. Hence, miRNAs are reported to be used as bioengineering tool to alter gene expression in plants and animals . Till now, many plants have been explored e.g. in rice, miR168 was found to bound human/mouse low-density lipoprotein receptor adapter protein 1 (LDLRAP1) mRNA and reduce its protein level in liver, and consequently decrease LDL removal from mouse plasma . In Curcuma longa, ath-miR167d homolog was found to hybridize with EIF2AK2 (Tyrosine-protein kinase) and ZFYVE16 (Zinc Finger FYVE-Type) genes responsible for blocking the pathway of protein processing in endoplasmic reticulum and regulation of membrane trafficking in the endosomal pathway. This can be helpful in treating diseases like arteriosclerosis and hyperglycemia . In Gmelina arborea
, 6 putative miRNAs were found to play a significant role in preventing diseases like cancer, blood borne disease, and other urinary infections . Apart from this, 36 medicinal plants belonging to families like Fabaceae, Asteraceae, Brassicaceae, Theaceae, Caricaceae, Apocyanaceae, Rutaceae, Rubiaceae, Zingiberaceae, Scrophulariaceae, Myrtaceae, Verbenaceae, Linaceae, Euphorbiaceae, Solanaceae, Araliaceae, Coniferophyta, Salicaceae, Lamiaceae, Malvaceae, Ericaceae, Vitaceae, and Gramineae have been studied to explore their miRNA pool . This is the first attempt to study miRNA of a medicinal monocot herb from Liliaceae family. Extensive therapeutic and medicinal properties of C. borivilianum made it a choice for miRNA study. C. borivilianum has been focused for the study of secondary metabolites biosynthetic pathways since a decade. Till now, miRNAs and their function in C. borivilianum are not known. Knowledge of miRNA can help in understanding the loop hole in other molecular and metabolic studies in this plant. In this study, we applied deep sequencing combined with bioinformatic analysis to identify and characterize miRNAs and their targets in C. borivilianum. There is no information present worldwide about sRNA of any liliaceae family plant. So this study may serve as a foundation for further exploration and application of complex metabolic mechanism of bioactive substances found in C. borivilianum. Larger amount of data produced by deep sequencing helped to identify even less abundant and small sized miRNAs, because analyzing larger amount of reads increases the odds of recovering rare transcripts .
A total of 442 known miRNAs belonging to 47 families and 5 novel miRNAs were identified from C. borivilianum leaf sRNA library. It signifies that, diverse and highly complex small RNA population exists in C. borivilianum. An almost similar trend has been observed in other monocot plants e.g. in Aegilops tauschii, Brachypodium distachyon, O. sativa, Sorghum bicolor, Triticum aestivum, and Zea mays, number of mature miRNAs reported are 173, 525, 713, 241, 119, and 321, respectively . Although the miRNA gene sequences may vary, the seed regions of the miRNAs belonging to a same family can be identical . Another difficulty regarding miRNA annotation is the repetitive presence of paralogous MIRNA loci in genome producing identical or nearly identical mature miRNAs. Based on this rationale, these miRNAs can be categorized into same families . Highly conserved miRNAs like miR156, miR157, miR159, miR160, miR162, miR164, miR165, miR166, miR167, miR168, miR169, miR171, miR172, miR319, miR390, miR393, miR394, miR395, miR396, miR397, miR398, miR399, miR408, and miR444 in other species  were also observed in C. borivilianum in high abundance. Novel miRNAs or species specific miRNAs considered to play specific functions in plants whereas conserved miRNAs are thought to be involved in generalized functions like signal transduction, development of leaf and flower etc. . Conserved miRNAs have unique property of being represented by multiple loci in sequenced genomes; most of them were generated through genome duplication events, giving some indication of their antiquity .
Conservation pattern of miRNAs vary from species to species. Most often conservation of miRNAs is associated with its sequence features such as base content and cleavage sites. Plant miRNAs show a negative/positive correlation between conservation and AU/GC content. At the 5′ end, conserved miRNAs usually starts with base U, while less-conserved miRNAs have a non-U base at start position in mammals. But this is not true in case of insects and plants . On the basis of data analysis, it can be predicted that miR159 and miR166 have maximum expression in leaf during the period of its active growth. Highest abundance of same miRNAs is also reported in other plants like Panax ginseng, Stevia rebaudiana, and A. thaliana [53,54,55]. miR159 represents one of the most ancient miRNAs in the plant kingdom . miR159 family members were reported to regulate ABA stress response and seed germination in plants by regulating the level of MYB transcription factor in A. thaliana [56, 57]. In tomato, miR159 regulates leaf and flower development by targeting the SGN-U567133 . As the sample was collected during the active plant growth period, high abundance of miR159 suggests its active regulation of leaf and root development.
Monocot specific miRNAs such as miR437, miR444, miR396 were reported in monocot plant species like rice, maize, sorghum, and sugarcane . In our study, we found the presence of only miR396 and miR444 but not miR437.
Complete reference genome should be the primary limiting factor for sRNA-seq. Unfortunately, the genome of C. borivilianum has not been published, but transcriptomic study of root and leaf tissue of C. borivilianum has already been conducted. Transcriptomic data from previous studies was used as reference for novel miRNA prediction. But many more miRNAs from C. borivilianum can be annotated in future when complete genome information will be available. Further, our study revealed 5 putative novel miRNAs in C. borivilianum using the available sequence data of this plant as reference. MFE (Minimum Fold Energy), sequence length and base composition are important features in predicting plant miRNAs. So, predicted novel miRNAs were confirmed by forming secondary structure of their precursor. All novel miRNA precursors have stem loop hairpin structure, and this fold-back hairpin structure has a low free energy as predicted by Mfold software. Novel miRNAs in C. borivilianum found to originate from precursor with AU percentage ranged from 40 to 53%. Seed region of miRNA can bind to the 3’-UTR, 5’-UTR, and ORF region of target mRNA but out of all these 3’-UTR targeting is much more frequent than other two .
In the present study, along with transcriptomic data of leaf and root of C. borivilianum, O. sativa and A. thaliana transcriptome have also been used to find maximum miRNAs targets. Target inhibition by cleavage was found to be more frequent. This result supports the fact that cleavage by Argonaute2 mediation maybe the main mode of gene suppression for many known plant miRNAs . Moreover, we found that in A. thaliana ARF6 and ARF8 were targeted by miR167 and ARF10, ARF16, and ARF17 by miR160 and in O. sativa ARF16 by miR160; ARF6, ARF8 were targeted by miR167. In C. borivilianum ARF18 and ARF17 were found to be targeted by miR160; and ARF12 by miR167. Down-regulation of ARF6 and ARF8 by miR167 in tomato, leads to affects the floral development and female sterility . This suggests the functional similarity of miRNAs in C. borivilianum, O. sativa, and A. thaliana.
Pathway specific target gene prediction in this study shown, miRNA can target transcripts coding for all-trans-nonaprenyl-diphosphate synthase, farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, hydroxymethylglutaryl-CoA synthase, isopentenyl-diphosphate delta-isomerase, 1-deoxy-D-xylulose-5-phosphate synthase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, mevalonate kinase, heptaprenyl diphosphate synthase, and (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase.
Targets of known miRNAs from leaf transcriptome of C. borivilianum were found to code for protein toll, ARF, transcription factor GAMYB, growth-regulating factor, D-tyrosyl-tRNA(Tyr) deacylase, SPL protein, transport protein Sec, NAC domain-containing protein, HD-Zip protein, UDP-glucuronate decarboxylase protein, putative ABC1 protein, ethylene-insensitive 2 isoform, UDP-N-acetylglucosamine diphosphorylase, leucine-rich repeat receptor-like serine/threonine-protein kinase, tryptophan synthase, DEA(D/H)-box RNA helicase family protein, and phosphoenolpyruvate phosphatase-like. During target analysis, it was observed that the some miRNAs that were previously reported to regulate primary metabolic pathway are regulating both primary and secondary metabolic pathways in C. borivilianum. For example, miR172d-3p.2 target ABC1 protein and Chloroplast 1-deoxy-d-xylulose-5-phosphate synthase; miR172c target APETALA2-like protein and Farnesyl pyrophosphate synthase; miR172c-5p Ethylene-Insensitive 2 isoform X2 and 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase; miR156g.2, miR156e.3 and miR156m.3 target SPL12 and Farnesyl pyrophosphate synthase. Detailed list of such miRNAs are mentioned in Additional file 9.
Target prediction results were further analyzed using Blast2GO. Biological processes like signaling, response to stimulus, developmental process, reproductive process, were found to be regulated by miRNAs. This suggested the wide regulation by miRNA in C. borivilianum. The two most important pathways identified were terpenoid backbone biosynthesis and sesquiterpenoid and triterpenoid biosynthesis. This suggested the specific role of miRNAs in secondary metabolism. Predicted target genes and the functions of their products may provide valuable clues for research into essential biological processes and metabolism in C. borivilianum.
RT-qPCR was carried out to calculate the expression levels of miRNAs and their target mRNAs. The expression was measured in leaves at two growth stages of plant. All 11 miRNAs were found with increased expression during dormancy. Maximum variation was observed in the expression of miR172c-5p and miR398a-3p.5 which are targeting 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase. This concluded that multiple miRNAs can regulate same gene collectively. Respective up and down regulation of miRNAs and their targets during dormancy indicate vital role played by miRNAs in regulation of secondary metabolite accumulation. It ensures that miRNAs are directly involved in negative regulation. This present study follows the same scenario as discussed previously by some other investigators .