MicroRNAs Associated with Secondary Metabolites Production

Abstract

MicroRNAs (miRNAs) are noncoding RNAs that play an important role in the regulation of the genetic expression in animals and plants by targeting mRNAs for cleavage or translational repression. Several miRNAs regulate the plant development, the metabolism, and the responses to biotic and abiotic stresses. Characterization of an miRNA has helped to show its role in fine tuning the mechanisms of posttranscriptional gene regulation. Although there is a lot of information related to miRNA regulation of some processes, the role of miRNA involved in the regulation of biosynthesis of secondary plant product is still poorly understood. In this chapter, we summarize the identification and characterization of miRNAs that participate in the regulation of the biosynthesis of secondary metabolites in plants and their use in the strategies to manipulate a controlled manipulation.

1. Introduction

1.1. Definition of miRNA

mRNAs are noncoding single-stranded RNA molecules that range from a length of 18 to 28 nucleotides. These molecules play an important role in posttranscriptional regulation by the inhibition of the expression of target genes by binding to mRNA [1]. Eukaryotic organisms, such as plants and animals, and some viruses express this type of molecules [2, 3]. Lin-4 is the first miRNA that was identified in Caenorhabditis elegans, although later in the same organism were found 22-nt lin-4 and 21-nt let-7; currently, more than 18,226 miRNAs are reported [4]. Although miRNA:target-gene interactions are widely conserved, this process is limited between kingdoms [5]. miRNAs are distributed in genome as single or clusters expressed as polycistronic units and share function relationships [6]. In plants, most miRNAs are encoded by their own primary transcript; few examples of miRNA cluster are reported (i.e., miR395). Introns are the main hotspots for their origination [7]. Some of the mechanisms where they act are the development of time and host-pathogen interactions, as well as cell differentiation, proliferation, apoptosis, and tumorigenesis.

1.2. Mechanism of action of miRNAs

The biogenesis of miRNAs in plants and animals presents some differences. In both plants and animals, the precursors of miRNAs are polyadenylated caps and RNAs, and transcribed as for any coding RNA by RNA polymerase II (RNAPII) [5, 8]. However, in the plants, the primary transcript (pri-miRNA) that gives rise to the miRNA is produced by the nuclear RNAase dicer-like 1 (DCL1) and its accessory proteins SERRATE (SE) and hyponastic leaves (HYL1) [8]. Also, Drosha gene is absent in plants [5].

In Arabidopsis thaliana, DCL1 and hyponastic leaves 1 (HYL1) cleave the pri-miRNA in the nucleus of the cell, which gives rise to the precursor-miRNA (pre-miRNA) dsRNA. Subsequently, there is another cleavage by the action of DCL1 and HYL1 to release the miRNA; the two nucleotide 3′ overhangs are methylated by the action of the sRNA-specific methyltransferase HUA enhancer1 (HEN1). When the mature single-stranded miRNA is found in the cytoplasm, it is loaded onto AGO1 that is present in RNA-induced silencing complexes (RISC), repressing the expression by mRNA cleavage [9]. 3′ untranslated region (3′ UTR) is the union region of the miRNAs to its target mRNAs which allows it to be repressed [10].

The expression of miRNAs is regulated by transcription factors. Negative on TATA less 2 (NOT2) promotes the transcription of protein miRNA genes and facilitates efficient DCL1 recruitment in miRNA biogenesis [11]. Cell division cycle 5 (CDC5) acts as a positive transcription factor associating with miRNA genes [12]. Pleiotropic regulatory locus 1 (PRL1) has the ability to bind to DCL1 and pri-miRNAs. The miRNA duplex is transported to the cytoplasm by nuclear export factor Drosophila Exportin-5 ortholog HASTY (HST).

The miRNA target genes can be a single member of a gene family or regulate a multiple family members. Thus, multiple miRNA genes could be targeting a single member, with tissues and stage specificity, and/or a single miRNA gene could be regulating multiple family members. The spatial and temporal expression and abundance of mature miRNAs are tightly regulated; they vary greatly among different miRNAs; and the abundance also varies depending on the tissue types or developmental stages [13].

1.3. Regulatory processes involving miRNAs

In the cytoplasm of cells, the miRNAs regulate the expression of genes at the posttranscriptional level via mRNA degradation and/or translational repression [14]. Unlike animals, in plants, there is a perfect complementarity between miRNA and target mRNA [14]. To carry out the silencing, a ribonucleoprotein RNA-induced silencing complex (RISC) is formed [15]. AGO1, AGO2, AGO4, AGO7, and AGO10 slicer activity has been reported, even though AGO1 is associated with most miRNAs [16]. AGO1-catalyzed RNA cleavage (slicing) represses miRNA targets [17].

2. miRNAs and secondary metabolism in plants

In plants, miRNAs control the expression of genes that encode transcription factors, stress response proteins, and others, which have an impact on biological processes. The miRNAs regulate the biological processes in the plans such as maintenance of genome integrity, primary and secondary metabolism, development, signal transduction, signaling pathways, homeostasis, innate immunity, and adaptive responses to biotic and abiotic stress [18]. Secondary metabolites are a group of phytochemicals that regulate various processes related to the interaction of the plant with its environment [19]. These compounds include terpenoids, alkaloids, phenolics, glycosides, tannins, and saponins, and defend plants from several biotic an abiotic stressors [20]. Even though these types of compounds are synthesized by plants to help in self-defense, they have diverse industrial uses such as insecticides, dyes, flavoring compounds, and nutraceuticals having a positive effect on human health. Commercial importance has resulted in a great interest in studying possibilities of enhancing its production [21]. It is known that miRNAs control several biological processes at the posttranscriptional level. Currently, some studies reveal the role that miRNAs have in the regulation of secondary metabolic pathways [20]. Therefore, the production of compounds derived from secondary metabolism can be managed through the miRNAs. Since they are positively or negatively regulated, the production of desired metabolites can be induced, the production of toxic metabolites can be limited, and new metabolites can be produced [22].

Computational analysis carried out in two transcriptomes of Swertia resulted in the identification of miRNAs associated to secondary metabolites biosynthesis; miR-156a, miR-166a, miR-166b, miR-168, miR-11071, and miR-11320 targeting metabolic enzymes, such as aspartate aminotransferase, ribulose-phosphate 3-epimerase, acetyl-CoA acetyltransferase, phosphoglycerate mutase, and premnaspirodiene oxygenase, also include a gene encoding a homeobox-leucine zipper protein (HD-ZIP) with a possible association in secondary metabolites biosynthesis in Swertia chirayita [23]. Elicited or infected plants induce change in gene expression and production of defensive metabolites and these might be regulated by miRNAs. Solanum tuberosum L. under light stimulus found light-responsive miRNAs that are important regulators in alkaloid metabolism, UMP salvage, lipid biosynthesis, and cellulose catabolism [24]. Cadmium stress in oilseed rape (Brassica napus L.) reported miRNAs involved in the regulations of TFs, biotic stress defense, ion homeostasis, and secondary metabolism synthesis [25]. Nicotiana tabacum plants infected with tobacco mosaic virus (TMV), at the early stage of infection (5 dpi), show a cluster of miRNAs with down-accumulation, while most of the miRNAs were upregulated at 15 and 22 dpi, including both miRNAs and miRNA targets [26].

2.1. Flavonoids

Flavonoids are secondary metabolites that possess a polyphenolic structure. Those compounds consist of hydroxylated phenolic substances having a benzo-𝛾-pyrone structure and derived of phenylpropanoid pathway [27]. Within the subgroups of the flavonoids are flavones, flavonols, flavanones, flavanonols, flavanols or catechins, anthocyanins, and chalcones [28]. For plants, this type of compounds is synthesized as a result of the interaction with the environment, other plants, and microorganisms. They have diverse biological functions as anti-oxidative, anti-inflammatory, anti-mutagenic, and anti-carcinogenic properties, which are structure dependent [28]. The above makes flavonoids a compound with nutraceutical, pharmaceutical, medicinal, and cosmetic applications [28]. The production of secondary metabolites is found in cases regulated by the miRNAs (Table 1). Little is known about the miRNAs involved in flavonoid biosynthesis. In Helianthus, 323,318 ESTs were computationally screened for the miRNAs identification of them, and a miR911 family was found related to the biosynthesis of tocopherols. Gou et al. [51] demonstrate that accumulation of anthocyanins in the stems of A. thaliana is under the regulation of miR156-targeted squamosa promoter binding protein-like (SPL) genes. High miR156 activity promotes accumulation of anthocyanins and activity-induced of flavonols. This study also demonstrates that SPL9 negatively regulates anthocyanin accumulation through destabilization of a MYB-bHLH-WD40 transcriptional activation complex. Diospyros kaki fruits collected at two examined stages (15 and 20 WAF) showed differential expression of the mRNAs, indicating that these miRNAs might regulate PA synthesis during development, and some of them are miR858 and miR156, which regulate PA synthesis. miR858 positively regulates the genes responsible for the production of PA, while miR156 does so in a negative way. miR395 is another miRNA that has an influence on PA biosynthesis [30]. Some miRNAs (U436803, U977315, U805963, U3938865, and U4351355) regulate fatty acid and flavonoid biosynthesis in Lonicera japonica [2, 3]. The characterization in A. thaliana shows that miR858a targets MYB transcription factors that are involved in flavonoid biosynthesis, growth, and development. Over-expression of miR858a downregulates several MYB transcription factors, and the higher expression of MYBs in MIM858 lines leads to the redirection of the metabolic flux toward the synthesis of flavonoids [31]. Yang et al. [32] indicate that salt stress conditions regulate miRNAs; some salt stress-related biological pathways includes calcium signaling pathway, MAPK signaling pathway, plant hormone signal transduction, and flavonoid biosynthesis [32]. Himalayan mayapple (Podophyllum hexandrum), miR1438 target caffeoyl-CoA O-methyltransferase and is related to phenylalanine metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, stilbenoid, diarylheptanoid, and gingerol biosynthesis. miR1873 targets dihydroflavonol 4-reductase C related to flavonoid biosynthesis. miR5532 2-hydroxyisoflavanone dehydratase is related to isoflavonoid biosynthesis [33].

Plant species	miRNA	Target	Function	References
Flavonoids
Sunflower	miR2911	Gamma-tocopherol methyl transferase	Tocopherols biosynthesis	[29]
A. thaliana	miR156	SPL transcription factor	Accumulation of anthocyanins, whereas reduced miR156 activity results in high levels of flavonols	[51]
Diospyros kaki	miR395p-3p and miR858b	bHLH and MYB, respectively	Proanthocyanidin biosynthesis	[30]
Lonicera japonica	miRNAs (U436803, U977315, U805963, U3938865 and U4351355)	R2R3-MYB transcription factors	Flavonoid biosynthesis	[2, 3]
A. thaliana	MicroRNA858a	R2R3-MYB transcription factors	Flavonoid biosynthesis	[31]
Halostachys caspica	miR6194 and miR5655	Flavanone 3-hydroxylase	Flavonols, anthocyanidins proanthocyanidins synthesis	[32]
Podophyllum hexandrum	miR1873/miR5532	Dihydroflavonol 4-reductase C/-hydroxyisoflavanone dehydratase	Flavonoid/isoflavonoid biosynthesis	[33]
Alkaloids
Opium poppy (Papaver somniferum)	pso-miR13, pso-miR2161, and pso-miR408	7-O-methyltransferase, S-adenosyl-l-methionine:3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase 2/ 4′-O-methyltransferase2 (4-OMT)/FAD-binding and BBE domain-containing protein, also known as reticuline oxidase-like protein	Benzylisoquinoline alkaloids	[34]
Tobacco	miRX17, miRX27, miRX20, and miRX19	QPT1, QPT2, CYP82E4, and PMT2	Nicotine biosynthesis and catabolism	[35]
T. baccata	miR164 and miR171	Taxane 13α hydroxylase and taxane 2α-O-benzoyltransferase	Paclitaxel biosynthetic genes	[36]
R. serpentina	miR396b	Targets kaempferol 3-O-beta-D-galactosyltransferase	Flavonol glycosides	[37]
Mentha spp.	miR156	Basic helix-loop-helix (bHLH)	Flavone/flavonol biosynthesis	[38]
Terpenoids
P. kurroa	iRNA-4995	3-Deoxy-7-phosphoheptulonate synthase (DAHP synthase)	Terpenoid biosynthesis ultimately affecting the production of picroside-I	[39]
Korean ginseng (Panax ginseng Meyer)	miR854b and miR854c	3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), farnesyl diphosphate synthase (FPS), geranyl-diphosphate synthase, squalene synthase, and squalene epoxidase (SE)		[40]
C. roseus	mir-5021	MYB transcription factor, geranyl diphosphate synthase, GCPE protein, UDP-glucose iridoid glucosyltransferase	Primary and secondary metabolism, Isoprenoid/terpenoid biosynthesis iridoid production in higher plants	[41]
Xanthium strumarium L.	miR7539, miR5021, and miR1134	Nontranscriptional factor proteins, such as DXS, HMGR, IDS, and IDI, essential to produce IPP and DMAPP	Terpenoid biosynthesis	[42]
Xanthium strumarium L.	miR7540, miR5183, miR6449, miR5255, miR5491, and miR6435	R-linalool synthase, gibberellin 3-oxidase, ent-kaurene synthase, squalene epoxidase, beta-amyrin synthase, and germacrene A oxidase	Mono-, sesqui-, di-, and tri-terpenoids biosynthesis	[42]
Ferula gummosa	miR2919, miR5251, miR838, miR5021, and miR5658	SPL7, SPL11, and ATHB13 TFs	Terpene biosynthesis	[43]
Pogostemon cablin	miRNA156	Squamosa promoter binding protein-like (SPL)	Sesquiterpene biosynthesis	[44]
A. thaliana	miR156	SPL transcription factor	Modulate sesquiterpene synthase gene TPS21 responsible for the biosynthesis of (E)-β-caryophyllene	[44]
Mentha spp.	miR156, miR414, and miR5021	Basic helix-loop-helix (bHLH) geranyl di-phosphate synthase subunit alpha-like protein (NACA), respectively	Terpenoid backbone biosynthesis, sesquiterpenoid and triterpenoid biosynthesis	[38]
Others
S. chirayita	miR-168, miR-11320, miR-166a, miR-11071, miR-156a and miR-166b	Acetyl-CoA acetyltransferase (AACT), aspartate aminotransferase (PHAT), premnaspirodiene oxygenase (PSO), ribulose-phosphate 3-epimerase (RPE), phosphoglycerate mutase (PGM), and a gene encoding homeobox-leucine zipper protein (HD-ZIP)	Secondary metabolites biosynthesis	[23]
A. thaliana	miR163	Family of small molecules of methyltransferases	Secondary metabolism	[45]
A. thaliana	miR393	Auxin receptors (TIR1, AFB2 and AFB3)	Increase of glucosinolate and decrease of camalexin	[46]
Potato	mirn79	AP2/ERF transcription factor	JA-responsive secondary metabolites	[24]
S. rebaudiana	miRstv_7	UDP-glycosyltransferase 76G1 (ugt76g1), kaurenoic acid hydroxylase (KAH), and kaurene oxidase (KO)	Steviol glycoside biosynthesis	[47]
Arabidopsis thaliana	miR826 and miR5090	AOP2	Glucosinolate biosynthesis	[48]
A. thaliana	miR826	Alkenyl hydroxyalkyl producing 2	Glucosinolate synthesis	[49]
Salvia miltiorrhiza	miR5072	Acetyl-CoA C-acetyl transferase	Tanshinones biosynthesis	[50]

Table 1.

miRNA related to secondary metabolite production.

2.2. Alkaloids

Alkaloids are naturally compounds that have one or more of their nitrogen atoms. Alkaloids are classified into different groups: indole, piperidine, tropane, purine, pyrrolizidine, imidazole, quinolizidine, isoquinoline, and pyrrolidine alkaloids [52]. Because of their toxicity, alkaloids act as defense compounds against diverse pathogens or herbivores. Understanding the regulation of alkaloid biosynthesis is crucial for its production. Target transcript identification analyses in Opium poppy (Papaver somniferum) revealed that pso-miR13, pso-miR2161, and pso-miR408 (Table 1) might be involved in BIA biosynthesis. pso-miR13 might cleave 7-OMT transcript, involved in the conversion of S-reticuline to morphinan alkaloids. 4-OMT is the target of pso-miR216 and mediates the production of S-reticuline that is also an intermediate molecule in BIA biosynthesis. On the other hand, pso-miR408 possibly targets mRNA from reticuline oxidase-like protein in charge of the conversion of S-reticuline to (S)-scoulerine in the BIA pathway [34]. Studies in tobacco (Nicotiana tabacum), identified four unique tobacco-specific miRNAs miRX17, miRX27, miRX20, and miRX19 that were predicted to target key genes of the nicotine biosynthesis and catabolism pathways, QPT1, QPT2, CYP82E4, and PMT2 genes, respectively [35]. In Taxus baccata, two paclitaxel biosynthetic genes, taxane 13α hydroxylase and taxane 2α-O-benzoyltransferase, are the cleavage targets of miR164 and miR171, respectively [36]. In silico analysis reveals that miR396b in Rauwolfia serpentina targets kaempferol 3-O-beta-D-galactosyltransferase whose activity as transferase activity, transferring hexosyl groups is essential for formation of flavonol glycosides [37]. A computational approach in Mentha spp., revealed that miR156, miR414, and miR5021 are essential for regulation of essential oil biosynthesis. miR156 participates in flavone, flavonol biosynthesis, and terpenoid backbone biosynthesis [38].

2.3. Terpenoids

Plant terpenoids secondary metabolites are synthesized from C5 precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). They are classified according to the number of carbon atoms as monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), carotenoids (C40), and polyprenols (>45) [53]. Like the alkaloids and the flavonoids, the biological characteristics and the applications of interest in the industry. Computational identification of miRNAs was done in six transcriptomes of Picrorhiza kurroa revealed that miRNA-4995 has a regulatory role in terpenoid biosynthesis (Table 1), ultimately affecting the production of picroside-I [39]. In silico profiling of microRNAs (miRNAs) in Korean ginseng (Panax ginseng Meyer) indicate that 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), farnesyl diphosphate synthase (FPS), geranyl-diphosphate synthase, squalene synthase, and squalene epoxidase (SE) were predicted been regulated by miR854b and miR854c, especially SE [40]. miR-5021 was identified in Catharanthus roseus which targets two enzymes involved in biosynthesis of terpenoid indole alkaloids (TIAs), GCPE protein, and Terpenoid cyclase [41]. miR7539, miR5021, and miR1134 might be involved in regulating terpenoid biosynthesis by targeting upstream terpenoid pathway genes; nontranscriptional factor proteins, such as DXS, HMGR, IDS, and IDI, essential to produce IPP and DMAPP, the common precursors for all the downstream end terpenoids [42]. miRNAs miR7540, miR5183, miR6449, miR5255, miR5491, and miR6435 target downstream enzymes in the biosynthesis of mono-, sesqui-, di-, and tri-terpenoids; they were R-linalool synthase, gibberellin 3-oxidase, ent-kaurene synthase, squalene epoxidase, beta-amyrin synthase, and germacrene A oxidase [42]. miR2919, miR5251, miR838, miR5021, and miR5658 were found to be related to the pathway of terpene biosynthesis in Ferula gummosa. SPL7, SPL11, and ATHB13 TFs are putatively regulated by miR1533, miR5021, and miR5658, respectively [43]. miRNA156-targeted squamosa promoter binding protein-like (SPL) intervenes in the temporal space regulation of sesquiterpene biosynthesis [44]. miR5021 is also involved in terpenoid backbone biosynthesis and miR414 is related to sesquiterpenoid and triterpenoid biosynthesis [38].

2.4. Other secondary metabolites

miRNAs were identified from in vitro culture of roots and leaves tissues of the transcriptome of Withania somnifera; miR159, miR172, miR5140, and miR5303 in root tissue and miR477, miR530, miR1426, and miR5079 of leaf tissue. These miRNA were associated in the regulation of secondary metabolites. Endogenous miRNAs (miR159 and miR5140 from roots, miR477 and miR530 from leaves) may be help to increase the metabolites (withanoides) yield. Also, miR159, miR172 from roots, and miR530 from leaves were involved in the regulation of secondary metabolite associated with mRNAs [54]. Chlorophytum borivilianum, Oryza sativa, and Arabidopsis thaliana target gene prediction indicate that miR9662, miR894, miR172, and miR166 might be involved in regulating saponin biosynthetic pathway [55]. miR8154 and miR5298b increase taxol, phenylpropanoid, and flavonoid biosynthesis in subcultured Taxus cells [56]. In silico analysis indicate that miRstv_7* target ugt76g1, KAH, KO, for steviol glycoside biosynthesis [47]. In Arabidopsis thaliana, miR826 and miR5090 share the target AOP2, which encodes a 2-oxoglutarate-dependent dioxygenase that is involved in glucosinolate biosynthesis [48]. Salvia miltiorrhiza miR5072 targets acetyl-CoA C-acetyl transferase that is involved in the biosynthesis of tanshinones [50]. miR826 targets alkenyl hydroxyalkyl producing 2 oxoglutarate dioxygenase, which is involved in glucosinolate synthesis [49].

3. Conclusion

miRNAs are small molecules associated with developmental processes controlling gene expression. The mechanisms involved posttranscriptional and transductional processes. The miRNA secondary metabolism control is a relative new field of study; the knowledge of the regulation of secondary metabolism in plants will help to understand the production of these products in controlled systems. Some of these products have an important economical value because of their use in agricultural, food, and cosmetic industries making these areas (miRNA regulation) very attractive.

Acknowledgments

The authors thank Universidad De la Salle Bajío campus Campestre for financial support. MVH thank to CONACyT for postdoctoral fellowship (288773).