MINI REVIEW
published: 29 March 2017
doi: 10.3389/fpls.2017.00374
Frontiers in Plant Science | www.frontiersin.org 1 March 2017 | Volume 8 | Article 374
Edited by:
Xiaoya Chen,
Shanghai Institute of Plant Physiology
and Ecology, China
Reviewed by:
Jin-Ying Gou,
Fudan University, China
Jonathan Gershenzon,
Max Planck Institute for Chemical
Ecology, Germany
*Correspondence:
Om P. Gupta
opguptaiari@gmail.com
†
Present Address:
Om P. Gupta,
Division of Biochemistry, ICAR-Indian
Agricultural Research Institute,
New Delhi, India
Specialty section:
This article was submitted to
Plant Metabolism and Chemodiversity,
a section of the journal
Frontiers in Plant Science
Received: 17 January 2017
Accepted: 03 March 2017
Published: 29 March 2017
Citation:
Gupta OP, Karkute SG, Banerjee S,
Meena NL and Dahuja A (2017)
Contemporary Understanding of
miRNA-Based Regulation of
Secondary Metabolites Biosynthesis
in Plants. Front. Plant Sci. 8:374.
doi: 10.3389/fpls.2017.00374
Contemporary Understanding of
miRNA-Based Regulation of
Secondary Metabolites Biosynthesis
in Plants
Om P. Gupta
1
*
†
, Suhas G. Karkute
2
, Sagar Banerjee
3
, Nand L. Meena
4
and Anil Dahuja
3
1
Division of Quality and Basic Sciences, ICAR-Indian Institute of Wheat and Barley Research, Karnal, India,
2
Division of
Vegetable Improvement, ICAR-Indian Ins titute of Vegetable Research, Varanasi, India,
3
Division of Biochemistry, ICAR-Indian
Agricultural Research Institute, New Delhi, India,
4
Division of Basic Sciences, ICAR-Indian Institute of Millets Research,
Hyderabad, India
Plant’s secondary metabolites such as flavonoids, terpenoids, and alkaloids etc. are
known for their role in the defense against various insects-pests of plants and for
medicinal benefits in human. Due to the immense biological importance of these
phytochemicals, understanding the regulation of their biosynthetic pathway is crucial. In
the recent past, advancement in the molecular technologies has enabled us to better
understand the proteins, enzymes, genes, etc. involved in the biosynthetic pathway
of the secondary me tabolite s. miRNAs are magical, tiny, non-coding ribonucleotides
that function as critical regulators of ge ne expression in eukaryotes. Despite the
accumulated knowledge of the miRNA-mediated regulation of several processes, the
involvement of miRNAs in regulating secondary plant product biosynthesis is still poorly
understood. Here, we summarize the recent progress made in the area of identification
and characteriz ati ons of miRNAs involved in regulating the biosynthesis of secondary
metabolites in plants and discuss the future perspect ive s for designing the viable
strategies for their targeted manipulation.
Keywords: miRNAs, terpenoids, alkaloids, flavonoids, phenolics, glycosides
INTRODUCTION
Since the age of human civ iliza tion, plants are used as a source of nutrition and medicine,
which is evidenced by the numerous texts from China and India (
Kirtikar and Basu, 1918; Tang
and Eisenbrand, 1992). The nutritional and medicinal properties of the plants are due to the
presence of numerous metabolites. These metabolites are of two types: primary and secondary.
Unlike primary metabolites, secondary metabolites are a huge group of phytochemicals, which
are not directly involved in plant’s vital processes such as growth, development, and reproduction
(
Fraenkel, 1959) but they are major components in defense mechanism of plants in order to protect
them from any possible harm in the ecological environment (Stamp, 2003) and other interspecies
protection (Samuni-Blank et al., 2012). Humans have exploited secondary metabolites in the form
of flavoring agents, fragrances, insecticides, dyes, drugs, etc., More than 100,000 phytochemicals
have been isolated from different plant sources so far (Mahajan et al., 2011). These second ary
metabolites are broadly categorized as terpenoids, alkaloids, phenolics, glycosides, tannins, and
saponins (Verpoorte, 1998). These phytochemicals are synthesized in the plants for a specialized
need in a specific set of ecologic a l conditions as their biosynthesis are highly energy consuming.
This kind of biosynthesis and accumulation behavior of secondary metabolites in plants is the
Gupta et al. miRNA-Based Regulation of Plant’s Secondary Metabolites
result of tight regulation of their biosynthetic machinery.
Metabolic engineering may further pave a way for enhancing
biosynthesis of economically important phytochemicals or for
producing desired combinations of such chemicals. One of the
ways to tinker with biosynthetic path ways is through modulating
miRNA levels as miRNAs are the ultimate regulators in plants.
miRNAs are small (21–24 nucleotides), non-coding,
riboregulators that regulate gene expression in eukaryotes
(Jones-Rhoades et al., 2006). miRNA is transcribed by RNA
polymerase II as a precursor RNA known as the primary miRNA
(pri-miRNA), which is subsequently processed by DICER-LIKE
1 (DCL1) to release the mature miRNAs. These mature miRNAs
are then loaded into the RISC complex to bind mRNAs for
cleavage (Jones-Rhoades et al., 2006). miRNAs are well-known
molecules for their role in regulating various plants processes
under biotic and abiotic stresses (
Gupta et al., 2014a,b; Shriram
et al., 2016
). Recently, various reports suggested their roles
in regulating the biosynth esis and accumulation of secondary
metabolites in plants (see review
Bulgakov and Avramenko,
2015). In the present review, we have updated the knowledge
about present understanding on miRNAs based regulation of
biosynthesis and accumulation of secondary metabolites in
plants.
ROLE OF miRNAs IN FLAVONOID
BIOSYNTHESIS
Flavonoids such as flavonols, flavones, isoflavones, anthocyanins,
proanthocyanidins, and phlobaphene pigments are low
molecular weight phenylpropanoid compounds which are
widely distributed throughout the plant kingdom (
Taylor and
Grotewold, 2005; Lepiniec et al., 2006; Buer et al., 2010). These
polyphenolic met abolites play a variety of significant biological
roles such as protection against UV radiation, as signaling
molecules, as phytoalexins in plant-microbe interaction, and
as re gulators of phytohormones such as auxin transport in
plants (Santelia et al., 2008; Buer et al., 2010). The flavonoid
backbone is synthesized by the central phenylpropanoid
pathway a nd different flavonoid metabolites share common
enzymes and substrates. Phenylpropanoid pathway is one of
the most extensively studied pathways of secondary metabolites
for transcriptional regulation in plants (Quattrocchio et al.,
2006; Stracke et al., 2007; Li, 2014). In the past few years,
scientific endeavors are directed toward understanding the post-
transcriptional regulation of this pathway involving miRNAs.
The schematic representation of the general phenylpropanoid
pathway leading to major branches of flavonoid biosynthesis
and their possible interaction with miRNAs has been depicted in
Figure 1A.
About 17 SQUAMOSA PROMOTER BINDING PROTEIN-
LIKE (SPL) proteins are encoded by the Arabidopsis genome
(Riese et al., 2007) . These SPL transcription factors are reported
to affect numerous processes of plant growth and development,
such as vegetative phase transition by enhancing the expression
of miRNA172, flowering induction by LEAFY and MADS box
genes, embryonic development, cell size, trichome formation,
and fertility (
Wu et al., 2009; Yamaguchi et al., 2009; Xing
et al., 2010; Yu et al., 2010). In addition, miR156 targeted SPL9
protein has been shown to regulate the metabolic flux during
flavonoid biosynthetic pathway. Anthocyanins accumulate in an
acropetal manner in Arabidopsis stems, wit h the highest level
at the junction between the stem and the rosette leaves. This
array of anthocyanin accumulation is regulated by the miR156
targeted SPL9 gene in Arabidopsis (
Gou et al., 2011). The tissues
having high anthocyanin concentration accumulate higher levels
of miRNA156 leading to reduced SPL activity which in turn
enhance the expression of F3
′
H, DFR, and other anthocyanin
biosynthetic genes. As a result, dihydroflavonols are direc ted
into the anthocyanin branch. On the other hand, expression of
SPLs gradually increases along the growing stem because miR156
levels decline as the plant progresses during development
(Gou et al., 2011). Therefore, increased accumulation of SPL
leads to decreased expression of anthocyanin biosynthetic
genes resulting in the increased production of flavonols
by FLS. It has been demonstrated that MYB-bHLH-WD40
transcriptional activation complex is destabilized by SPL9, a
target of miRNA156, by competing with bHLHs for t h eir binding
to PAP1 which in turn inhibits expression of anth ocyanin
biosynthetic genes (anthocyanidin synthase, flavanone 3-
hydroxylase, dihydroflavonol reductase, and UDP-glucosyl
transferase 75C1 etc.) influencing anthocyanin accumulation
in Arabidopsis (
Gou et al., 2011). Similarly, miRNA156-
SPL9 pair influences anthocyanin production by targeting
dihydroflavonol 4-reductase (Cui et al., 2 014). Therefore,
an antagonistic relationship exists between anthocyanin and
flavonol biosynthesis in Arabidopsis. Recently,
Biswas et al.
(2016) have computationally identified several miRNAs such as
miR172i, miR829.1, miR1438, miR1873, and miR5532 targeting
mRNAs coding for enzymes of phenylpropanoid pathway,
such as 4-coumarate–CoA ligase, Chalcone synthase, Caffeoyl-
CoA O-methyl transferase, Dihydroflavonol 4-reductase C,
2-hydroxyisoflavanone dehydratase respectively in Podophyllum
hexandrum (Table 1). Overexpression of miR8154 and miR5298b
in sub-cultured Taxus cell lines revealed their crucial role in the
regulation of taxol, phenylpropanoid, and flavonoid biosynthesis
pathways (Zhang et al., 2015). Similarly, several other miRNAs
of phenylpropanoid pathway, such as miR395p-3p/ targeting
bHLH mRNA in D. kaki (
Luo et al., 2015), miR396b and
miR828a targeting mRNAs coding for Kaempferol 3-O-beta-
D-galactosyltransferase and anthocyanin regulatory C1 protein
respectively in R. serpentina (Prakash et al., 2016), miR858 a
targeting R2R3-MYB mRNA in A. thaliana (Sharma et al., 2016),
miR6194 targeting Flavanone 3b-hydroxylase mRNA (F3H) in
H. caspica (Yang et al., 2015), miR1061-3p and miR1318 in pear
fruit (Wu et al., 2014) etc., (Table 1) have been reported.
Further, the use of advanced computational tools
complementing the experimental methods has accelerated
the accumulation of reports on new as well as existing miRNAs
implying their regulatory role during flavonoid pathway in
plants. Therefore, further work on functional characterization
of these tiny miRNAs-target networks using reverse genetic
approach would certainly pave a way for understanding post-
transcriptional regulatory mechanism of the flavonoid pathway.
Frontiers in Plant Science | www.frontiersin.org 2 March 2017 | Volume 8 | Article 374
Gupta et al. miRNA-Based Regulation of Plant’s Secondary Metabolites
FIGURE 1 | (A) Schematic representation of the general phenylpropanoid pathway leading to major branches of flavonoid biosynthesis and their possible interaction
with miRNAs. Phe ammonia-lyase (PAL); cinnamate-4-hydroxylase (C4H); 4-coumaroyl:CoA-ligase (4CL); chalcone reductase (CHR), chalcone synthase (CHS);
(Continued)
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Gupta et al. miRNA-Based Regulation of Plant’s Secondary Metabolites
FIGURE 1 | Continued
stilbene synthase (STS); chalcone isomerase (CHI); flavanone 3-hydroxylase (F3H); isoflavone synthase (IFS); dihydroflavonol 4-reductase (DFR); isoflavone
O-methyltransferase (IOMT); isoflavone 2
′
-hydroxylase (I2
′
H); isoflavone reductase (IFR); vestitone reductase (VR); 2
′
-dihydroxy, 49-methoxyisoflavanol dehydratase
(DMID); leucoanthocyanidin dioxygenase (LDOX); O-methyltransferase (OMT); UDPG-flavonoid glucosyl transferase (UFGT); rhamnosyl transferase (RT); flavonol
synthase (FLS); leucoanthocyanidin reductase (LAR); anthocyanidin reductase (ANR); anthocyanidin synthase (ANS). (B) Schematic representation of biosynthetic
pathway of volatile terpenoid and their possible interaction with miRNAs. acetoacetyl-CoA thiolase (AACT); HMG-CoA synthase (HMGS); HMG-CoA reductase
(HMGR); mevalonate kinase (MVK); phosphomevalonate kinase (PMK); mevalonate diphosphate decarboxylase (MVD); isopentenyl diphosphate isomerase (IDI);
geranyl diphosphate synthase (GDS); farnesyl diphosphate synthase (FDS); terpene synthase (TPS); DOXP synthase (DXS); DOXP reductoisomerase (DXR);
2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT); CDP-ME kinase (CMK); 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS);
(E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS); (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR); geranyl geranyl diphosphate
synthase (GGDS).
This information could further be used for metabolic engineering
of t h e entire pathway for human benefits.
ROLE OF miRNAs IN TERPENOID
BIOSYNTHESIS
Owing to their numerous biological roles, isoprene (C5),
monoterpenes (C10), and sesquiterpenes (C15) establish the
biggest class of plant volatile compounds. In plants, these
volatile compounds act as defense molecules against biotic
stresses, attracts pollinators and se ed disseminators, and help
improve thermo-tolerance (
Dudareva et al., 2006). In addition,
they are used as aroma compounds and natural flavor
enhancers which have the beneficial impact on human health
(Wagner and Elmadfa, 2003). Considering the importance
of these compounds, understanding the regulatory schema
of their biosynthetic pathway and accumulation stands on
priority. These volatile compounds are synthesized from
isopentenyl diphosphate (IPP) and dimethylallyl diphosphate
(DMAPP), which are derived from two alternate biosynthetic
pathways localized in different subcellular compartments.
During the past several years, there has been a significant
progress in identification and characterization of genes and
enzymes involved in the biosynthesis of volatile terpenoids
(Figure 1B), determination of their spati otemporal expression
and compartmentalization, and metabolic engineering. However,
the regulatory role of miRNAs in their biosynthesis and
accumulation is poorly understood, which opens a new window
for furt her investigations.
Terpene synthases (TPSs) Catalyses the conversion of farnesyl
diphosphate (FPP) into sesquiterpenes (C15). Transcription
factor SPL9, the target of miRNA156, directly binds to and
activates promoter of terpene synthases 21 (TPS21) gene and
positively regulates its transcription thereby regulating the
synthesis of sesquiterpenoid (Yu et al., 2015). Similarly, miR-
4995 was predicted to target mRNA of an enzyme 3-deoxy-7-
phosphoheptulonate synthase, which is involved in t he picroside
biosynthetic pathway in a medicinal herb P. kurroa (Vashisht
et al., 2015). In addition, Saifi et al. (2015), have mined and
validated 11 miRNAs which are involved in steviol glycoside
biosynthetic pathway (Table 1) in Stevia and established the
relationship pattern with the expression levels of their target
mRNAs as well as steviol glycoside contents. Using NGS
technology, several miRNAs involved in the sesquiterpene
biosynthesis pathway have been mapped and validated in X.
strumarium. For example, mRNAs of the upstream enzymes
in the pathways of terpenoid biosynthesis, including 1-
deoxy-D-xylulose 5-phosphate synthase (DXS), 3-hydroxy-3-
methylglutaryl coenzyme A reductase (HMGR), isopentenyl
diphosphate (IPP)/dimethylallyl diphosphate (DMAPP) synthase
(IDS), and isopenteyl diphosphate isomerase (IDI) were
predicted to be targeted by miR7539, miR5021, and miR1134
(
Fan et al., 2015). The complete list of miRNAs and their
target genes have been provided in Table 1. Most recently,
bioinformatics approaches have been utilized to mine miRNAs
involved in terpenoid metabolism in Mentha spp. (Singh et al.,
2016a), Ginger (Singh et al., 2016b), C. roseus (Pani and
Mahapatra, 2013), and P. hexandrum (Biswas et al., 2016;
Table 1).
THE ROLE OF miRNAs IN THE
REGULATING BIOSYNTHESIS OF
ALKALOID AND OTHER N-CONTAINING
METABOLITES
Alkaloids are nitrogen containing low molecular-weight
compounds which are mostly derived from amino acids. They
are known to play signific ant roles in defense against herbivores
and pathogens and are being widely used as pharmaceuticals,
stimulants, narcotics, and poisons. Unlike other secondary
metabolites, this class is highly diverse and heterogenous in
nature and around ∼12,000 alkaloids have been characterized
till date (
Ziegler and Facchini, 2008). These compounds are
synthesized through diverse metabolic pathways. Recent genome
based technological advancement have led us to add to on
our current understanding of their biosynthetic pathways and
regulation. However, knowledge on the role of miRNAs during
alkaloid biosynthesis and accumulation in plant kingdom has
just started to proliferate.
Boke and his coworkers in 2014 have extensively worked
on regulation of the alkaloid biosynthesis by miRNA in
opium poppy. They identified pso-miR13, pso-miR2161, and
pso-miR408 as potential miRNAs involved in the alkaloid
biosynthetic pathway. Pso-miRNA2161 targets the mRNA
of gene encoding S-adenosyl-L-methionine: 30-hydroxy-
N-methylcoclaurine 40-O-methyltransferase 2 (4O MT)
enzyme which converts S-norcoclaurine into S-reticuline, an
intermediate mole cule in benzylisoquinoline alkaloids (BIA)
biosynthesis. Similarly, pso-miR13 targets mRNA of 7-O-
methyltransferase (7O MT) gene, which converts S-reticuline to
morphinan alkaloids. pso-miR408 targets mRNA of reticuline
Frontiers in Plant Science | www.frontiersin.org 4 March 2017 | Volume 8 | Article 374
Gupta et al. miRNA-Based Regulation of Plant’s Secondary Metabolites
TABLE 1 | List of miRNAs involved in regulating biosynthesis and accumulation of common secondary metabolites in plants.
Sr.
no.
miRNA Plant species Target Target function Phytochemical biosynthesis Validation/
detection
References
FLAVONOIDS
1. miR156* A. thaliana SPL9 Destabilizes MYB-bHLH-WD40 transcriptional
activation complex
Anthocyanin biosynthesis Transgenic
approach
Gou et al., 2011
2. miR172i P. hexandrum 4-coumarate–CoA ligase Catalyses the activation of 4-coumarate and
other 4-hydroxycinnamates to the respective
thiol esters
Flavonoid biosynthesis Computational
Biswas et al., 20 16
3. miR395p-3p/* D. kaki bHLH Regulates genes of proanthocyanidin
biosynthetic pathway
Proanthocyanidin biosynthesis Illumina Luo et al., 2015
4. miR396b R. serpentina Kaempferol 3-O-beta-D-
galactosyltransferase
Transferase activity, transferring hexosyl groups Flavonol glycoside Computational Prakash et al.,
2016
5. miR828a R. serpentina Anthocyanin regulatory C1
protein
DNA/chromatin binding Anthocyanin biosynthesis Computational Prakash et al.,
2016
6. miR829.1 P. hexandrum Chalcone synthase Catalyses the conversion of 4-coumaroyl-CoA
and malonyl-CoA to naringenin chalcone
Flavonoid biosynthesis Computational Biswas et al., 2016
7. miR858a* A. thaliana R2R3-MYB transcription
factors
Regulate genes of flavonoid biosynthetic
pathway
Flavonoid biosynthesis Transgenic
approach
Sharma et al.,
2016
8. miR858b* D. kaki MYB protein Regulates genes of proanthocyanidin
biosynthetic pathway
Proanthocyanidin biosynthesis pathway Illumina Luo et al., 2015
9. miR1438 P. hexandrum Caffeoyl-CoA O-methyl
transferase
Cat- alyzes methylation of caffeoyl-CoA to
produce feruloyl-CoA.
Lignin biosynthesis Computational Biswas et al., 2016
10. miR1873 P. hexandrum Dihydroflavonol 4-reductase C Flavanone 4-reductase activity Flavanoid biosynthesis Computational Biswas et al., 2016
Z. officinale Phenylalanine ammonia lyase
(PAL)
Conversion of L-phenylalanine to ammonia and
trans-cinnamic acid
Gingerol (phenolic) biosynthesis,
Flavanoid biosynthesis
Computational Singh et al., 2016b
11. miR5532 P. hexandrum 2-hydroxyisoflavanone
dehydratase
Catalyses conversion of
2,7,4’-trihydroxyisoflavanone into diadzein
Isoflavonoid biosynthesis Computational Biswas et al., 2016
12. miR6194 H. caspica Flavanone 3b-hydroxylase
(F3H)
Catalyses the conversion of flavanone into
dihydroflavonol
Biosynthesis of flavonols, anthocyanidins
and proanthocyanidins
HiSeq deep
sequencing
Yang et al., 2015
13. CHS-siRNA G. max Chalcone synthase Catalyses the conversion of 4-coumaroyl-CoA
and malonyl-CoA to naringenin chalcone
Flavonoid biosynthesis Transgenic
approach
Cho et al., 2013;
Tuteja et al., 2009
14. miR1061-3p Pyrus spp Naringenin 3-dioxygenase Catalyses the 3-beta-hydroxylation of
2S-flavanones to 2R,3R-dihydroflavonols
Flavonoid biosynthesis Computational Wu et al., 2014
TERPENOIDS
15. miR156* P. cablin SPL9 Activate TPS21 gene Sesquiterpenoid and triterpenoid
biosynthesis
Transgenic
approach
Yu et al., 2015
M. spp. 1-deoxy-D-xylulose
5-phosphate synthase (DXS)
Catalyses conversion of 1-deoxy-D-xylulose
5-phosphate into pyruvate and
D-glyceraldehyde 3-phosphate
Terpenoid biosynthesis Computational
Singh et al., 2016a
16. miR396b R. serpentina Secologanin synthase Oxidoreductase activity Secologanin Computational Prakash et al.,
2016
17. miR414 M. spp. Terpene synthase 21 (TPS21) Catalyses reaction for terpene s ynthesis Sesquiterpenoid and triterpenoid
biosynthesis
Computational Singh et al., 2016a
(Continued)
Frontiers in Plant Science | www.frontiersin.org 5 March 2017 | Volume 8 | Article 374