Biosynthesis of Secondary Metabolites Based on the Regulation of MicroRNAs

MicroRNA (miRNA), a noncoding ribonucleic acid, is considered to be important for the progression of gene expression in plants and animals by rupture or translational repression of targeted mRNAs. Many types of miRNA regulate plant metabolism, growth, and response to biotic and abiotic factors. miRNA characterization helps to expose its function in regulating the process of post-transcriptional genetic regulation. There are a lot of factors associated with miRNA function, but the function of miRNA in the organic synthesis of by-products by natural products is not yet fully elucidated. The current review is aimed at observing and characterizing miRNAs and identifying those involved in the functioning of the biosynthesis of secondary metabolites in plants, with their use in controlled manipulation.


Introduction
miRNAs are eighteen to twenty-eight nucleotides containing single-stranded RNA molecules which are not transcribed into proteins during transcription. It takes part in posttranscriptional regulation by binding to the messenger RNA and inhibiting the expression of specific genes [1]. These types of molecules are usually expressed in eukaryotes such as animals and plants and some viruses [2,3].
The first miRNA was reported in Caenorhabditis elegans is Lin-4; then, further studies identify more than 18,226 other types of miRNAs in the same organisms such as 22 (nt) lin-4 and 21 (nt) let-7 [4]. While miRNA-targeted gene interconnections are extensively prolonged, this technique is restrained among domains [5]. miRNAs are disbursed in genetic material as clumps exhibit as a polycistronic segment with features [6]. Most miRNAs in flora are encrypted with their prime transcript and some precedents of miRNA clumps described as miR395. The basic identified hotspots for their beginning are introns [7]. Most of the processes in which they affect are the expansion of time and hostpathogen connection in addition to cell differentiation, proliferation, apoptosis, and tumorigenesis.
In eukaryotes and metazoans, miRNAs are a form of noncoding (22-nucleotide) ribo regulators that control gene expression which has an exciting role in the biosynthesis of plant secondary metabolites like flavonoids, terpenoids, alkaloids, and some other compounds [8,9]. RNA polymerase II is a precursor RNA also described as the pri-miRNA that helps to stimulate the production of miRNA, processed by DICER-LIKE 1 (DCL1) to fully grown or developed miRNA [8].
In this review, we have updated the knowledge about the present understanding of miRNA-based regulation of biosynthesis and accumulation of secondary metabolites in plants. The data written in English were collected, using scientific search engines such as PubMed/Medline, Science-Direct, Web of Science, Scopus, and Google Scholar. The search terms used were microRNA, biosynthesis, secondary metabolites, miRNA, flavonoids, alkaloids, and terpenoids.
The scientific names of the plants have been validated according to the Plant List [10,11].

miRNA as a Secondary Metabolite Regulator
The polyadenylated caps and RNAs are the precursor molecules for the synthesis of miRNA both in plants and animals, and RNA polymerase II (RNAPII) transcribed many coding RNAs, although in plants, nucleus RNAase dicer-like 1 (DCL1) and their essential proteins SERRATE (SE) and hyponastic leaves (HYL1) synthesize primary miRNA but the Drosha gene is not found in plants [5,12]. In Arabidopsis thaliana, the precursor miRNA, dsRNA, is synthesized in the nucleus by the splitting of pri-miRNA by HYL1 and DCL1. The second splitting is done by the activity of HYL1 and DCL1 which results in releasing of miRNA; methylation of two nucleotides 3 ′ overhangs is performed by methyltransferase HUA enhancer1 (HEN1). Adult single-stranded cytoplasmic miRNA activated by AGO1 is found in the RISC (RNA-induced silencing complex) ribonucleoprotein and by breaking mRNA suppresses its function [13]. The miRNA binds with mRNA at the 3 ′ untranslated region (3 ′ UTR) which inhibits the activity of miRNA [14]. The transcriptional factors regulate the expression of miRNA. The transcription of miRNA genes and effective use of DCL1 in the biosynthesis of miRNA is facilitated by At-Negative on TATA-less 2 (NOT2) [15]. Cell division cycle 5 (CDC5) is associated with miRNA which is a positive transcription factor. The drosophila exportin-5 ortholog HASTY (HST) factor exports miRNA duplex to the cytoplasm.
The miRNA-targeted genes may be solitary members of a gene group or synchronize various family groups. However, more than one type of miRNA gene can regulate a single individual with tissues and a single-miRNA gene can control many family individuals. The territorial and worldly articulation of plenitude-developed miRNAs is firmly managed; they differ enormously within various miRNAs, and the bounty likewise fluctuates relying upon the type of tissues or growth and embryonic stages [16].
The miRNAs inhibit the activity of mRNA to control the gene expression at the level of post-transcription. In contrast to animals, plants have an ideal relationship between mRNA and miRNA [17]. For the silencing, a ribonucleoprotein RNA-induced silencing complex (RISC) is formed [18]. Different types of slicers have been reported: AGO10, AGO7, AGO4, AGO2, and AGO1. However, AGO1 is mostly related to miRNA [19]. The RNA splitting is activated by AGO1 by inhibiting specific genes [20].
In plants, the genes coding for transcription, stress response proteins, and other factors which are associated with different biological mechanisms are controlled by miRNA-like conservation of genetic materials, maintenance of metabolism, and enlargement of plants, signal transduction, energy pathway, homeostasis, natural immunity, and adaptive feedback to biotic and abiotic stress [21,22]. The phytochemicals are peripheral metabolites that play an important role in different processes which are related to the association between plants and its surrounding [23][24][25]. The mechanism of action of miRNAs in plants is shown in Figure 1.
These compounds involve isoprenoids, tannins, alkaloids, phenols, glycosides, and triterpene glycosides, which protect the plants from different living and nonliving stress-causing factors [26,27]. Such kinds of molecules are produced by herbs for self-protection; these compounds are also used in various industries like nutraceuticals, flavouring F compounds, dyes, and insecticides as they are providing a great response in terms of human health [28,29]. Materialistic significance has brought about an extraordinary enthusiasm for considering the prospects of improving its creation [30]. It concluded that miRNAs regulate several biological cycles at the stage of post-transcription. Several recent investigations concluded that the miRNA plays an important part in the transduction of the peripheral metabolic series [31]. So, the products of peripheral metabolites can be regulated by miRNAs. The production of any metabolite can be regulated by a positive or negative feedback 2 BioMed Research International mechanism, so we can stop the production of undesired products and enhance the production of desired metabolites [32]. Computational evaluation executed in two transcriptomes of Swertia ensured to identify miRNAs connected with secondary metabolites, miR-11320, miR-168, miR-156a, miR-11071, miR-166a, and miR-166b focused on metabolic enzymes consisting of premnaspirodiene oxygenase, aspartate aminotransferase, phosphoglycerate mutase, ribulosephosphate 3-epimerase, acetyl-CoA, and acetyltransferase. Additionally, it consists of a gene encrypting a homeoboxleucine zipper protein (HD-ZIP) along with secondary metabolite organic production in Swertia chirayita (Roxb.) Buch.-Ham. ex C.B.Clarke [33].
The production of self-protecting metabolites is reduced in diseased plants due to alteration in gene expression so these may be maintained by miRNAs. When Solanum tuberosum L. is exposed to sunlight, brilliance-reactive miR-NAs are produced which plays an important role in nicotine metabolism, UMP salvage, fat production, and cellulose catabolism [34].
The stress of cadmium in oil seed rape (Brassica napus L.) described miRNA as a crucial element in the synchronization of transcription factors, living thing stress protection, ion balancing, and peripheral metabolism production [35].
Nicotiana tabacum tainted with tobacco mosaic virus (TMV), at the beginning phase of the disease (5 dpi), shows a bunch of miRNAs with low aggregation, while a large portion of the miRNAs was upregulated at 15 and 22 dpi including both miRNAs and miRNA targets [36,37].

Flavonoid Biosynthesis and miRNA
Flavonoids have a hydroxylated phenolic structure and contain benzo-γ-pyron, which is obtained from phenylpropanoid [38,39]. Flavonoids have different compounds including chalcones, catechins, flavonols, flavonols, anthocyanins, flavanones, and flavanonols. These compounds are synthesized with the help of others in plants and microorganisms, and their concentration varies as per the environmental conditions [40,41].
The biological functions of flavonoids vary greatly with the structure and result in various bioactivities such as antimutagenic, antioxidative, anticarcinogenic, antiinflammatory, and antiplatelet aggregation [42]. These biological activities of flavonoids impart different applications such as medicinal, nutraceutical, cosmetics, and pharmaceutical [43][44][45].
miRNA-initiated reactions take part in the assemblage of peripheral metabolites (Table 1).

BioMed Research International
Anthocyanins are one of the important members of the flavonoid family [67,68]. In the tuberous root of sweet potato, the expression of ib-miR156 is its target ibSPL, which aids in anthocyanin production [49]. Kiwifruit, miR858, reported accelerating anthocyanin accumulation [69]. High microR156 movement initiates aggregation of anthocyanin along with movement initiated by flavonols. The current study also illustrated that squamosa promoter-bindinglike protein 9 SPL9 adversely initiated anthocyanin aggre-gation with the disruption of an MYB-bHLH-WD40 transcriptional-initiated system. Diospyros kaki L.f. fruit results acquired at tested periods (15 and 20 WAF) confirmed the contrasting appearance of the messenger RNAs, suggesting that those miRNAs throughout the growth and a number of them are miRNAs 858 and 56 which adjust the synthesis of PA. PA production genes are positively initiated, miR858 but negative effects are observed by miR156. Another miR395 and miR858b play their role in biosynthesis proanthocyanidin [51]. Some miRNAs (U4351355, U3938865, U805963, U977315, and U436803) initiate lipid and flavonol organic production in Lonicera japonica Thunb. [2,3]. Yang et al. [52] imply that salty situations modify miR-NAs in which few salt pressure-associated organic channels consist of flavonoid biosynthesis, calcium signaling pathway, and plant hormone signal transduction [52].
Gou and colleagues reported that in the stem of Arabidopsis thaliana anthocyanins accumulate in an acropetal manner, this model is controlled by SPL (squamosa binding protein-like) genes and stimulated by miR156 [53]. The depiction of A. thaliana indicates that miR858a targets myeloblastosis (MYB) transcript elements which are engaged in flavonoid organic production, development, and growth. MYB transcription factors down synchronize with overexpression of miR858a and the redirection of metabolic flux initiated with the excessive appearance of MYBs concerning the composition of flavonoids [54]. The biosynthesis of  BioMed Research International flavonoids (anthocyanins) is regulated by miR156, miR858a by stimulating the transcription factors R2R3-MYB and SPL9 [53,54]. Rauvolfia serpentine (Indian snakeroot) has two essential types of miRNA such as miR396b and miR828a, both of which targets kaempferol 3-O-β-D-galactosyltransferase and anthocyanin regulatory C1 protein for synthesizing flavonol glycoside and anthocyanin biosynthesis, respectively [55].
Podophyllum hexandrum Royle (May apple) is an endangered medicinal plant [56]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway study demonstrated that miR1438 and miR1873 regulate various metabolic pathways, especially the biosynthesis of secondary metabolites like lignin and flavonoid via caffeoyl-CoA Omethyl transferase and dihydroflavonol 4-reductase C gene, respectively [56]. Biswas et al. [56] further revealed that miR5532 upregulates the 2-hydroxyisoflavanone dehydratase gene for synthesizing isoflavonoid in the May apple tree. Flavonoid biosynthesis also occurred in May apple through inducing chalcone synthase and 4-coumarate-CoA ligase. Both of them are targeted by miR829.1 and miR172i, respectively [56]. 4-Coumarate-CoA ligase catalyzes the activation of the corresponding thiol esters by 4-coumarate and other 4-hydroxycinnamates. These activated thiol esters are subsequently employed as flavonoid and monolignol precursors [70]. miR5015 has been shown to influence the production of gingerol by suppressing phenyl ammonia-lyase (PAL) which is a basic enzyme in the synthesis [57]. Glycine max (L.) Merr. (soybean) CHS-siRNA targets chalcone synthase enzyme to the biosynthesis of flavonoids [58,59].
Squamosa promoter binding and ATHB13 were observed to be promoted by miRNAs (1533, 5021, and 5658) [76]. Sesquiterpene biosynthesis is mediated by miRNA156connected SPL gene [77]. miR414 is involved in the triterpenoid and sesquiterpenoid organic syntheses while miR5021 also plays a role in terpenoid formation (Table 1) [66]. Investigators revealed that SPL9 specifically connects to and triggers the TPS21 promoter. Due to the great conservation of miR156-SPLs in plants, the findings establish a molecular connection between the developmental time and sesquiterpene synthesis and provide a novel method for engineering plants to grow faster while producing more terpenoids [77]. In all, 130 unique candidate transcripts for eight miRNA families were anticipated. All anticipated miRNA targets govern development, reproduction, stimulus response, signaling, and various metabolic processes. Additionally, a network of miRNA-mediated gene regulation was built using the hybridized lowest free energy of discovered miRNAs and their targets. The study discovered that the mint family's miR414, miR156, and miR5021 might regulate the essential oil biosynthesis gene control mechanism. Additionally, three miRNA candidates were identified as important in trichome formation (miR5021, miR156, and miR5015b) [66]. However, apart from providing first-hand knowledge 7 BioMed Research International    10 BioMed Research International on Rauvolfia serpentina (L.) Benth. ex Kurz miRNAs and their objectives, the outcomes in this work help to a profound comprehension of miRNA-mediated gene regulation mechanisms in plants [55]. Another study demonstrated that among 452 recognized miRNAs, which equate to 589 precursor miRNAs, 62 miR-NAs are expressed exclusively in the base, 95 miRNAs are expressed exclusively in the stem, 19 miRNAs are expressed exclusively in the leaf, and 71 miRNAs are expressed exclusively in flower. The degradome study found 69 targets that may be cleaved by 25 miRNAs. Among these, miR5072 cleaved acetyl-CoA C-acetyltransferase, which is implicated in the production of tanshinones. This investigation expanded significantly to our comprehension of the tissuespecific expression patterns of miRNAs in Salvia miltiorrhiza Bunge and laid the groundwork for future exploration on the production of tanshinone mediated by miRNAs [78].
Moreover, recent findings indicated that most anticipated miRNAs were engaged in rhizome formation control. miR854, miR5021, and miR838 have been revealed as miR-NAs that control ginger rhizome growth and essential oil production [57]. It is demonstrated that miRNA-4995 performs a key role in regulating terpenoid biosynthesis, subsequently influencing the development of picroside-I. miR-5532 and miR-5368 expression rates were significantly lower in field-grown specimens than in vitro-cultured samples, implying a role in controlling Picrorhiza kurroa Royle ex Benth. development under culture environments [73]. Additionally, an investigation concluded 60 mature miR-NAs and 6 precursor miRNAs in Podophyllum hexandrum Royle using 454 pyrosequencing. The finding contributes fundamental knowledge regarding the control of secondary metabolite production in Podophyllum hexandrum Royle by miRNAs [56].
A study demonstrated to pinpoint the trichome-specific miRNAs and mRNA targets. The findings from the experiment give a basis for additional research in Xanthium strumarium L. glandular cells to elucidate the governing strategy of miRNAs underpinning the production of secondary, in particular, terpenoids [75]. Researchers discovered that a large number of the miRNAs pinpointed had tissuespecific expression. By scanning the ginseng EST database for anticipated objectives of the estimated 69 conserved miRNAs, 346 promising targets were recognized. The anticipated targets were primarily engaged in secondary metabolic processes responding to transcription regulator activities and biotic and abiotic stress, among other metabolic pathways [79]. Conversely, miR390 was expected to engage a gene associated with trichome formation, the location of artemisinin production, and therefore may be possible for genetic transformation to increase the artemisinin content [72]. Also, a new study demonstrated insight into the role of miRNAs in the control of secondary metabolite production in the herbal plant Swertia chirayita (Roxb.) Buch.-Ham. ex C.B.Clarke. Additionally, the putatively discovered miRNAs can increase secondary metabolite production in S. chirayita by genetic modification [33].
Another study revealed that three miRNAs (miR156b, miR1168.2, and miR1858) were involved in curcumin pro-duction. Other miRNAs were discovered to be engaged in turmeric's development and growth. Additionally, a phylogenetic analysis of selected miRNAs was done in this study [48]. Moreover, another investigation demonstrated the identification of miRNA associated with control gene channels regulating terpenoid biosynthesis in Camellia sinensis (L.) Kuntze at various growth stages [65]. Investigating the regulation of terpenoid biosynthesis by miRNA outcomes indicates that six miRNAs examined control terpenoid production in P. minor post-transcriptionally. This governmental function of miRNAs suggests that they may be used as a genetic tool to control terpenoid production in Persicaria minor (Huds.) Opiz [80]. Another study also evaluated miRNAs and their presumed target genes involved in transthyretin-like (TTL) biosynthesis, and the results showed the existence of a complex miRNA-mRNA regulatory network involved in Ginkgo biloba L. TTL metabolism [81]. Figure 3 shows few examples in which miRNA assists in the improved production of terpenoid compounds.
The investigation uncovered multiple previously unknown miRNA families in Artemisia annua L., including miR399, miR396, miR319 miR858, miR6111, and miR5083. The expression patterns and correlations between miRNAs and their associated targets in A. annua plant are different, depending on its developmental phases [82]. Saifi and coworkers found nine miRNAs (miR319c, miR319a, miR319f, miR319b, miR319h, miR319d, miRstv-7, miR319e, and miRstv-9) and authors reported that these miRNAs are correspondingly connected to the expression thresholds of their target mRNAs and steviol glycosides were detected to be directly connected to the expression stages of their target mRNAs. This work established a foundation for a better knowledge of the steviol glycoside biosynthetic process, and these miRNAs may be used to control the production of these metabolites in Stevia rebaudiana (Bertoni) Bertoni to increase their concentration and productivity [83].
In plants, a myriad of bioactive compounds is synthesized naturally by several pathways. Among them, the involvement of miRNA is the most common way used for the biosynthesis of naturally occurring alkaloids. Pyrrolidine, purine, imidazole, indole, quinolizidine, pyrrolizidine, isoquinoline, and tropane are among the various types of alkaloids listed [90]. Disparate to other bioactive 11 BioMed Research International compounds, this group is extremely diverse and heterogeneous with an estimated 12,000 alkaloids in nature [89]. Alkaloids according to their toxic nature react as resistance compounds against many kinds of herbivores and pathogens. The understanding of alkaloid biosynthesis balancing is critical for its production.
In the opium poppy, the botanical name of Papaver somniferum L., a member of the Papaveraceae family, it has been identified that transcription of pso-miR408, pso-miR13 and pso-miR2161 (Table 1) has an important role in the biosynthesis of organic benzylisoquinoline alkaloids (BIA) [92]. pso-miR13, pso-miR2161, and pso-miR408 would possibly break 7-O-methyltransferase, S-adenosyl-lmethionine:3 ′ -hydroxy-N-methylcoclaurine, 4 ′ -O-methyltransferase 2/4 ′ -O-methyltransferase 2 (4-OMT)/FADbinding, and BBE domain-containing protein, respectively, transcript concerned with inside the transformation of Sreticuline to morphinan alkaloids (BIA). 4-OMT is the target of pso-miR216 and moderates the assembly of S-reticuline; this is additionally an intermediary particle in BIA organic production. On the opposite side, pso-microR408 feasibly reacts with messenger RNA from reticuline oxidase-like protein in the rate of transformation of S-reticuline to (S)-scou-lerine within the BIA channel [92]. Figure 4 showed the role of miRNA in the synthesis of alkaloid and other Ncontaining metabolites.
In Madagascar periwinkle, there are some other miRNAs such as cro-miR160, cro-miR164a, cro-miR164b, and cro-miR393d, which have a regulatory role in targeting CrARF16, ORCA3, ORCA4, and BIS1 genes in the TIA biosynthetic pathway and synthesis indole alkaloids [95]. In addition, Robert-Seilaniantz et al. [96] demonstrated that miR393 with its related auxin receptor genes is involved in alkaloid (camalexin) biosynthesis in Thale cress (Arabidopsis thaliana (L.) Heynh.) and in turmeric also named as Curcuma longa; miR5021 is illustrated to have a role to synthesize isoquinoline alkaloid interacting with two enzyme genes like aspartate transaminase and aromaticamino-acid transaminase [48]. Furthermore, in sunflower plants, U3938865 and U4351355 involved glucosinolates and camalexin (indole alkaloid) biosynthesis through regulating the MYB34 gene [2] (Table 3).

Fatty Acids and Other Compound
Biosynthesis and miRNA miRNA plays an important role in fatty acid secondary metabolite biosynthesis [97]. Lonicera japonica targets regulatory genes which help in the process of synthesis such as acyl-CoA synthetase, acyl carrier protein, and fatty acid hydroxylase. miRNAs involved in the fatty acid synthesis are U436803, U977315, and U805963 [2]. Besides this, in Paeonia × suffruticosa Andrews (tree peony) plant, fatty acids are synthesized by miR156b and miR7826 via regulating 1-acyl-sn-glycerol-3-phosphate acyltransferase and ACCase gene activities [98].
In Corynespora cassiicola plant, some novel miRNA was found that positively or negatively regulates secondary metabolite production in plants. These novel types of miR-NAs are novel-miR1 to novel-miR7 which targets genes in phenylpropanoid synthesis [15]. Furthermore, phenylpropanoid biosynthesis is also regulated by miRNA (miR2673a and miR396b) in Podophyllum hexandrum Royle [99].

Conclusion
miRNAs are microscopic particles related to growing functions that direct gene expression. The method assumed post-transcriptional and transductional procedures. miRNA secondary metabolism dominance is the latest kind of field and better information about the process of peripheral metabolism in plants will play a critical role in achieving new kinds of outcomes in controlled systems. These outcome by-products have good economic values due to their utilization in cosmetics product, food, agronomy, and other industries. Further investigations for the production of secondary metabolites by maneuvering the role of miRNA in various other crops such as spices, flowering plants, and medicinal plants may result in improving the profitability of the food and pharmaceutical industries.