MicroRNA and Transcription Factor: Key Players in Plant Regulatory Network

1The recent discovery of complex regulatory network in higher organisms like; plants and animals have been recognized in plants. These complex networks which consist of chromatin modification (at epigenetic level), mRNA splicing, cell signaling, polyadenylation, and mechanisms of protein activation and degradation demanded substantial attention in order to achieve complete understanding on how plant system are being regulated. This review has been intended to focus on gene regulation at transcriptional and post-transcriptional levels which involving microRNAs (miRNAs) and transcription factors (TFs) as key regulatory players.

2At the transcriptional level, the interaction of TFs interact with enhancers to coordinate gene expression has been well established in the past decade. This can be obviously seen in the discovery of many types of TFs families which play a diverse role in plant system. At the post-transcriptional level, another attractive mechanism of gene regulation has been discovered a couple of decades ago, involving a large class of small non-coding RNAs, known as miRNAs. These miRNAs act as gene regulators in plants and animals by negatively regulate mRNAs. With the increasing number of regulators involved in gene networks, it is interesting to observe and understand a dynamic relationship between miRNAs, TFs, and mRNAs.

3To date, about 28,645 miRNAs from plants, animals, and viruses have been registered in public miRNA database. These miRNAs are able to modulate and fine-tune majority of biological processes by regulating a large number of target genes. Thus, screening of the potential target genes can provide an efficient and critical approach to explore the miRNA-mediated regulatory functions in depth at post-transcriptional level. Early exploration of some empirical parameters and algorithms deduced for known miRNA-target interactions using computational prediction in Arabidopsis had been applied to determine miRNA targets in other plants. The prediction approaches have further been validated through wet lab techniques and methods like PAGE, Northern Blot, Rapid Amplification of cDNA Ends at 5′ (5′-RACE), and Degradome Sequencing analysis.

4Currently there are 58 families of TFs which consist of 320,370 members from 165 plant species. miRNAs only downregulate their targets while TFs activate or repress the transcription of their targets, eventually determining the fate of particular gene, either to be switched “on” or switched “off”. Interestingly, majority of miRNA targets are TFs. Since both regulators demonstrate great impact toward plant genetic system, the circuiting of miRNAs-TFs will allow orchestration of numerous biological processes with high reliability.

5Recent trends in miRNA research were focused on plant responses to abiotic rather than biotic stresses. The prevailing environmental stresses like; drought, salinity, and cold, which significantly affect plant growth and development, are the prominent factors of plant research shift toward abiotic stress. The present review will provide a better understanding of miRNAs regulation and their interaction with the TFs, which can assist the researchers to explore more about plant survival mechanism under unfavorable environmental conditions. The newly developed relationship among the above mentioned gene regulators will assist the plant scientists to gain insight into the relationship among these regulators in different plant species.

6In plants, miRNA genes are transcribed by RNA polymerase II to produce primary miRNA (Pri-miRNA) and their length is highly variable between themselves. Unlike animal, plant miRNA processing is accomplished inside the nucleus because they lack protein processor like Drosha and DGCR8. In plant, DICER-LIKE 1 (DCL1) process most of pri-miRNAs by cleavage. Pri-miRNAs are stabilized with a type of RNA binding protein, DAWDLE (DDL), which interacts with DCL1 in nuclear foci, named dicing bodies (D-bodies). The combined action of couple of proteins like; SERRATE (SE) and HYPONASTIC LEAVES 1 (HYL1), followed by DCL1 and the nuclear cap-binding complex, led to form a short duplex miRNA which consist of mature miRNA guide cleavage and passenger miRNA strand (miRNA∗). Further processing of this duplex leds to the 2′-O-methylation at 3′ by the methyltransferase HEN1. A family of enzymes, called SMALL RNA DEGRADING NUCLEASE (SDN) genes, is responsible for the accumulation of miRNAs. SDN1 have shown 3′-5′ exoribonuclease activity against short and single-stranded RNA substrates. In plants, HASTY (HST), which is homolog to EXPORTIN5 (EXP5), plays a crucial role in exporting pre-miRNAs or mature miRNAs to cytoplasm. Another export pathway of miRNAs seems to be involved but the exact mechanism is still not clear.

7In the cytoplasm, ARGONAUTE (AGO) proteins form an assembly with miRNA, known as RNA-induced silencing complex (RISC). AGO1 in the RISC is the major player for the miRNA pathway. AGO protein consist of PAZ and PIWI domain. Particularly, PIWI domain form RNaseH-like fold which catalyze endonuclease activity. This endonuclease activity is capable of chopping RNA targets that are complementary to the miRNA strand loaded inside the AGO. AGO proteins in Arabidopsis thaliana such as AGO1, AGO2, and AGO10 has been reported to have the endonuclease activity, which leds to splicing the mRNA targets. Identification of the sliced mRNA targets by miRNA can be discovered through sequencing of mRNA degradome. Previous study reveals that plant miRNAs bind to their targets with high complementary which results in the cleavage of target mRNA. Beside of cleavage, there are several cases where the miRNA target is regulated at protein level without significant changes in mRNA level. These findings suggest that plant miRNAs are capable in interfering the translation process of mRNA.

8Transcription factors are essential for the regulation of gene expression, and usually belong to members of multigene families. Generally TFs exist as modular proteins containing of DNA-binding domain that interacts with cis-elements of their target genes. Besides, it also consists of protein–protein interaction domain that assists oligomerization between TFs or with other regulators. Many TFs have been recognized by X-ray crystallography and Nuclear Magnetic Resonance spectroscopy. TFs families can be evolved in many ways such as exon capture, duplication, translocation and mutation. In plants, the regulation of TFs genes occurs at transcriptional and post-transcriptional levels. They participate in genetic system via many ways such as developmental control, elicitation of defense, and stress responses by expressing the gene at right time and right place.

9Hence, understanding the activity of TFs expression is crucial for building regulatory networks. Mode of action of TFs is considered to occur mainly through the binding of TFs to cis-regulatory element within the promoter regions of their targets genes. However, with various post-transcriptional regulatory mechanisms that recently have been discovered, including; miRNA regulation, nonsense-mediated mRNA decay, and nuclear export control, it evidences that mRNAs are regulated at many layers of gene regulation. Undoubtedly, there is a potential for altering expression patterns mediated by cis-elements through post-transcriptional regulation. Beside, a single TF has the ability to regulate multiple genes in certain metabolic pathways. Further, it is also quite clear through recent investigations that changes in gene transcription are closely related to changes in the expression of TFs. Therefore, alteration in the expression of TF genes normally results in remarkable changes during plant growth and development. As a future consequence, engineering of transcription factor genes may provide a valuable means for manipulation of desired traits in plants. Here we have reviewed briefly TF families that involve in plant growth and development under normal and stress environmental condition.

10NF-Y or also known as Nuclear factor Y, are TFs that consist of three subunits, NF-YA (CBF-B or HAP2), NF-YB (CBF-A or HAP3), and NF-YC (CBF-C or HAP5). All of these subunits are essential for DNA binding. NF-Y, in the promoter region, recognize CCAAT box with high specificity and affinity due to the presence of its highly conserved trimeric activator. These transcription factors have different functions according to their subunits. For instances, NF-YA and NF-YB involve in drought tolerance and NF-YC, appears to be important regulator in flowering and photomorphogenesis. Nf-y mutant plant exhibited dark grown phenotype, although in the present of light, thus indicate NF-Y TF were positive regulators of photomorphogenesis. NY-FA participated in flowering process when in complex with NF-YB/NF-YC by activating FLOWERING LOCUS T gene. Transgenic Arabidopsis plants overexpressing NFYA5 resulted to high drought tolerance.

11MYB (myeloblastosis) is a large family of proteins, playing diverse role in gene network in eukaryotes. Most MYB proteins act as TFs with different numbers of MYB domain repeats; MYB-related, R2R3-MYB, R1R2R3-MYB, and atypical MYB family which exhibited their ability to bind DNA. They are widely distributed in plants and also interact with other TFs. MYB have been involved in growth and development of different plant species, e.g., in Glycine max, they are involved in flower color development, and in signal transduction pathways in A. thaliana, Oryza sativa, and cassava. In A. thaliana and Medicago truncatula they regulate the biosynthesis of secondary metabolites.

12APETALA2 (AP2) family of TFs plays a pivotal role in regulating the complex developmental process of floral development. AP2 family, also called class “A” of TFs, interacts with other two classes (B and C) of TFs and determines the final development of the floral organs, and this interaction was summarized as ABC model. Complexity of floral formation shown by co-regulation of three classes of TFs, class A, B, and C genes, which determine the four floral organ types. Family of class A TFs (AP2) itself alone regulate the identity of sepal in whorl 1. It’s co-action with B class genes, PISTILLATA (PI), determines petal identity in whorl 2. Further the interaction of class B TFs with class C, AGAMOUS (AG), determines stamen identity in whorl 3. Carpel identity in whorl 4 specified by AG itself. AP2 which belong to Class A gene, interacts with class C gene and AG by suppressing each other’s roles in order to determine the identities and properties of the reproductive organs and perianth. Loss-of-function of AP2 turns sepals and petals into carpels since there are in excess of AG activity into the outer two whorls of the flower.

13Most of the miRNA targets are TFs which regulate plant growth and developments. One of the important plant developmental processes is flowering stage, which is regulated by complex gene networks that integrate multiple environmental and endogenous cues to ensure flowering at the appropriate time. This mechanism is eventually regulated by the induction and activity of three main TFs; LEAFY (LFY), FRUITFULL (FUL), and APETALA1 (AP1). Among these three genes, LFY has been considered to play a role as major regulator, since, the loss of LFY function causes the most remarkable delay in flowering process. However, these three genes are controlled by TF SQUAMOSA PROMOTER BINDING PROTEINLIKE 3 (SPL3). In general, SQUAMOSA PROMOTER BINDING PROTEINLIKE (SPL) genes are featured by their SQUAMOSA PROMOTER-BINDING (SBP) domain, which consists of a novel zinc finger with two zinc ion binding sites.

14TCP TF contains a TCP domain, which codes a motif that is predicted to form basic helix-loop-helix structure known for distinct DNA-binding domains. TCP TF is named after the first four characterized members, namely TEOSINTE BRANCHED1 (TB1) from maize, CYCLOIDEA (CYC) from snapdragon, and PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR1 (PCF1) from rice. Previous finding unravel that TCP have been involved in different ways to promote leaf development by cell division, growth, and differentiation. TCP TFs also involve in flower development, leaf senescence, auxin and jasmonic acid signaling, development of male and female gametophyte, mitochondrial biogenesis, and interaction with the circadian clock. In Arabidopsis, there are 24 predicted TCP proteins. These predicted TCP were classified into two groups; class I and class II proteins. Class I made up of 13 proteins, whereas, remaining 11 proteins were placed in class II proteins. Both of these classes act as activator and repressor.

15WRKY TFs belong to huge and diverse family of TFs. Till now, 74 members had been identified in A. thaliana and 109 in O. sativa. Members of this family have at least one conserved DNA-binding region, WRKY domain, comprising of a conserved WRKYGQK peptide sequence and a zinc finger motif. In general, this domain binds to the W box, a DNA element, even though alternative binding sites also have been identified. WRKY TFs involve in various networks in genetic system to govern multiple responses at once; whether it is biotic and abiotic stresses, or physiological. Besides, WRKY TFs are also responsible to regulate production of some secondary metabolites such as phenolic compounds along with lignin, flavanols, and tannins.

16NAC [no apical meristem (NAM), Arabidopsis transcription activation factor [ATAF1/2], and cup-shaped cotyledon (CUC2)] are among of major families of transcriptional regulators in plants, and present in a wide range of land plants. In Arabidopsis, 9 of the 10, NAC domains are known to bind with conserved DNA target sequence having a CGT[GA] core with different affinity levels. Interestingly, NAC TFs play diverse roles in plant system which includes; regulation of plant development and responses to biotic and abiotic stresses.

17Plant-specific TFs, growth-regulating factor (GRF) were initially identified for their role in developing stem and leaf. But later studies revealed that in addition to stem and leaves, other important for other developmental processes including root growth, flower and seed development, and plant responses under extreme environmental conditions. GRF forms complexes by combining with GRF-interacting factors (GIFs), a type of transcriptional co-activators.

18Homeo domain-leucine zipper (HD-Zip) proteins are among the TFs that belong to plants kingdom. In A. thaliana, these TFs are encoded by more than 25 genes. Two important domains HD-Zip proteins are characterized by the presence of a homeo domain (HD) and a leucine zipper domain (Zip) which are responsible for DNA binding and involved in protein–protein interaction, respectively. Based on previous sequence similarities findings, these proteins have been divided into four groups. Among these groups, HD-Zip I proteins are involved in plant responses related to abiotic stress, blue light, de-etiolation, abscisic acid (ABA), and embryogenesis. Second group, HD-Zip II proteins take part in auxin signaling, light response, and shade avoidance. Similarly, HD-Zip III governs embryogenesis, lateral organ initiation, leaf polarity, and meristem function. Whereas, HD-Zip IV proteins play important role during trichome formation, root development, differentiation of epidermal cells, and anthocyanin accumulation.

19It is essential to illustrate an integrated picture for the regulatory relationships between miRNAs, TFs, and target genes. However, it is quite difficult to develop a clear cut regulatory relationship between miRNAs and TFs, because, in addition to the interaction of these regulators with their target genes, they sometimes interact with each other; leading to some different results. Here, we have summarized the similarities (Table 1) and differences (Table 2) between miRNAs and TFs mediated regulatory system. We also propose a model to relate these two regulators with their target genes and the consequences of this model to the plant regulatory network under normal (Figure 1) and stress condition (Figure 2). The existence of both miRNAs and TFs in gene regulatory networks will reveal the regulatory role involving both direct and indirect regulatory relationships. In this review, we have tried to bring together previous findings, related to the interaction between miRNAs and TFs, mostly in model plants and some non-model plant (Table 3). Rigorous and time consuming web lab/experimental work was a big hurdle in developing interactions between miRNAs and TFs in plants. But now, with the advancement in the public data bases and bioinformatics tools, to develop an interaction between above mentioned regulators it is relatively easy.

20Plant growth and development are reflection of genes expression. Appropriate timing and pattern of gene expression and production of proteins are required to ensure proper growth and development in plant. miRNAs and TFs are among the key regulators which determine how gene expression being regulated, thus affect the physiology and phenotype of the plant.

21In Arabidopsis, four isoforms of miR169 were identified to target NF-YA transcription factor. Those miR169 isoforms (miR169defg) and the NF-YA2 TF were recognized to control the root architecture since loss-of-function miR169defg led to improper root initiation. On the other hand, interaction of miR169 and NF-YA transcription factor also seems to be affect nodule formation in Arabidopsis. The overexpression of miR169 against NFYA transcription factor family member, HAP2-1, resulted in late nodulation and detained meristem development, thus led to improper development of nodules.

22Loss-of-function of miR858 plants led to the robust growth and early flowering. Further expression of artificial miRNA (amiRNA) target mimic (MIM858) cause the reduction of plant growth and delayed flowering. MYB transcription factor was able to activate miR399, which responsed to phosphate (Pi) starvation in Arabidopsis. Overexpressing AtMYB2 showed high miR399f expression and tissue Pi contents which may resulted via elevated expression of a subset of Pi starvation-induced genes.

23In maize inbred line, ELS-1, zma-miR159d which targeted MYB transcription factor was significantly downregulated in the leaves during senescence, while in another inbred line, Yu87-1, zma-miR159d was upregulated. In cotton, ghr-miR447a and ghr-miR5255a/b/c/e/f/g/h targeted CPC, a type of MYB transcription factor showing that ghr-miR447a and ghr-miR5255a/b/c/e/f/g/h might play a significant role in root and fiber development under drought and salinity stresses by regulating CPC in cotton. Similar study was carried out in cotton where two miRNAs; miR828 and miR858, targeted MYB2, which is responsible for fiber development.

24Couple of decades ago, there had been numerous studies which illustrated that different miRNA families play role in regulating floral timing and development, by targeting transcription factors involved in these processes. At least 11 different miRNA families have been involved in regulating the induction of floral development at each stage. In plant miR156, miR172, and miR390 were involved during transition from juvenile to adult stage. Similarly, in transition from vegetative to reproductive stage, miR159, miR169, miR172, and miR399 were involved. A large group of miRNAs, including; miR159, miR160, miR164, miR166/5, miR167, miR169, miR172, and miR319 regulate flower development stage.

25In Arabidopsis, miRNA172 targeted mRNA of a floral homeotic gene AP2 that involve in floral development. Unlike most plant miRNAs which mostly turn off their own targets by cleavage, miR172 regulate its target via translational inhibition. Prominent miRNA172 accumulation causes defect in floral organ identity, which looks similar to those loss-of-function ap2 mutants. High levels of mutant ap2 mRNA with disrupted base for miR172 base pairing resulted floral pattern defects.

26In common beans and soybean miR172 and its target; AP2, involved in nodule formation. Nodulation occurs when plant roots establish a symbiotic relationship with nitrogen-fixing bacteria (rhizobia) to obtain nitrogen. Rhizobial infection on bean caused the expression level of miR172c to increase until during nodule development stage, while uninfected nodules show low level of miR172c and high level of AP2. In addition, overexpression of miR172c resulted in enhanced root growth, improved rhizobial infection, improved expression of early nodulation and autoregulation of nodulation genes, and improved nodulation and nitrogen uptake.

27In Arabidopsis, SPL gene family is a well evident target of miR156. Out of 17 SPL genes, 11 have been reported as downregulated by miR156 through mRNA cleavage and translational inhibition. A reduced level of miR156 over time with increasing plant age, resulted in increased expression level of SPL transcription factors which induced flowering through the activation of FT, LFY, and MADS-box gene. In contrast, overexpressing miR156 in transgenic plant resulted delayed flowering and extended juvenile phase. Interestingly, miR156 was downregulated when temperature was increased by elevated carbon dioxide concentration. miR156 have conserved role in regulating flowering in rice, tomato, and maize.

28In Arabidopsis, role of TCPs and their regulation by miR319 was first identified using microarray in jaw-D mutants. Overexpressing miR319 in Arabidopsis mutants delayed the flowering phenotype in long day conditions. Late-flowering phenotype was observed due to loss-of-function; where miR319 had targeted TCP4. Another functional analysis revealed that loss-of-function of miR319, which was regulating TCP genes, led to slightly increase in the leaves size. Additionally, misexpression of tissue-specific promoters at later stages of leaf development had significantly reduced the leaf size in Arabidopsis. Beside, miRNA319 targeted TCPs which involved in regulation of KNOTTED1-Like HOMEOBOX (KNOX) genes, BREVIPEDICELLUS (BP) and KNAT2 through ASYMMETRIC LEAVES 2 (AS2) which can affect the leaf morphogenesis. Recent study reported that TCP transcription factor was regulated by RABBIT EARS (RBE) during leaf development. In tomato, overexpression of miR319 resulted into formation of a giant tomato leaf.

29In model plant, Arabidopsis, miR159 and miR319 targeted the MYB and TCP transcription factors, respectively. Interestingly, both miRNA-target nodes had abilities to regulate miR167 and the target, ARF6/ARF8 node. The direct interaction of MYB and TCP transcription factor may contribute to the functional redundancy. Regardless of high sequence similarity of these miRNAs, the regulation of target TCP and MYB transcripts remain conserved for each miRNA families. Although, miR319 was able to bind MYB transcripts, but it exhibited an incomplete temporal and spatial expression pattern corresponding to miR159. On the other hand, miR159 was unable to bind TCP transcripts. This unique interaction suggested that miR159 and miR319 were able to perform different regulatory roles in plant development.

30Previous evidences suggested that NAC TFs could enhance lateral root development. In Arabidopsis, endogenous and transgenic NAC1 transcript were cleavaged by miR164, producing a 39 nucleotide long specific fragments. However, the action of miR164 was blocked by NAC1 mutations that interrupted the base pairing with miR164. Arabidopsis mir164a and mir164b mutant plants had reduced miR164 expression, thus upregulated NAC1 transcript level, resulting to more lateral roots as compared to wild type plants. Overexpression of ZmNAC1 in transgenic Arabidopsis had enhanced lateral roots formation in comparison to the wild type plant.

31In Arabidopsis, miR396a and miR396b were involved in regulating the leaf morphology by targeting GRF TFs family. Liu and his colleagues, using northern blot hybridizations approach, found that miR396 was predominantly expressed in leaf and seedling. Overexpression of miR396a or miR396b in Arabidopsis resulted into a phenotype with narrow leaves, which probably was due to decreased in cell numbers. The overexpression of miR396 had also led to suppression of six GRF genes and GIF1 which acted as key players in cell division in leaves. Additionally, the overexpression of miR396 resulted in reduced stomata density, a feature that favors drought tolerance in plants. Moreover, additional target for miR396 had been identified which were basic Helix-Loop-Helix (bHLH74) TF, required for Arabidopsis normal growth and development. Further, in Populus trichocarpa, precursor of the miR396c, which possess mature sequence identical to miR396b in Arabidopsis, was expressed in tobacco plant using CaMV35S promoter. The transgenic plant exhibited altered organ development, where the third and fourth whorls were turned into stigmatoid anthers and fasciated carpels and delayed the flower development process.

32Recent findings has discovered that miR396 and GRF regulatory network may regulates the transition of stem cells which are located at specific cellular context or stem cell niche (SCN) to transit-amplifying cells (TACs) in the Arabidopsis root meristem. In SCN, miR396 is expressed, but in TACs GRFs are expressed. The GRFs are essential for the function of the TACs. Low expression level of GRFs in TACs resulted in a low rate of the cell cycle. Additionally, it would affect TACs by generating periclinal cell divisions typical of stem cells. In opposite, the regulation of miR396 is required to repress the GRFs from the SCN.

33In maize, in an attempt was carried out to explore the profile changes profiles of miR396 and GRF TF and also to analyze their potential regulatory roles during maize effective grain filling period. RNA sequencing was carried out in developing maize. It was observed that miR396 was highly expressed at initial stages, and gradually declined during later grain filling stages. By contrast, its target GRF TF was initially negatively regulated, decreased at the beginning, but increased continuously at later stages. Further analysis of expression pattern provide the information that other miRNAs like miR319, miR166, and RNA dependent RNA polymerase may involve in the interaction between miR396 and GRF TF during grain development in maize. Similar study was carried out in rice, where LOC_Os02g47280, which is responsible for grain shape, was downregulated by miR396. The evidences obtained from A. thaliana, Zea mays, and O. sativa clearly support that networking between miR396 and GRF TF plays an important role in plant leaf growth and grain development.

34Among four classes of HD-Zip TFs, the interaction of HD-Zip III with miRNA families, 165 and 166, has been well studied. In Arabidopsis, an overexpression of miR166 had downregulated three HD-ZIP III genes; ATHB-9/PHV, ATHB-14/PHB, and ATHB-15, resulting in recapitulate phenotypes. The same phenotype was also observed in loss-of-function mutations of these genes. Similarly, overexpression of miR165 had turned off all five HD-ZIP III genes, which led to recapitulated phenotypes caused by loss-of-function of mutations of hd-zip III genes, such as loss of shoot apical meristem (SAM), changed in organ polarity and defects in vascular tissues development and inter fascicular fibers. Beside, miR165 and miR166 were also reported to regulate SAM and floral development through WUSCHEL (WUS)-CLAVATA (CLV) pathway. Although miR165 and miR166 target the same genes, individual miR165 and miR166 display different expression domains in different part of plant tissues. For instances, overexpression of miR165 and miR166 plant resulted in the alterations in SAM activities and floral formation.

35Additionally, in both Arabidopsis and maize, miR165 and 166 were observed with abundance on the abaxial side of leaf, and in developing phloem of the leaf primordium, since both miRNAs play critical role in leaf asymmetry patterning. Adaxialization and overexpression of the rolled leaf1 gene occur when miRNA target site of a REV homolog was mutated in maize rolled leaf1 (rld1) mutant. Hence, in both eudicots and monocots, HD-ZIP III which is responsible for vascular patterning of leaves and stems has been suppressed by miRNA from abaxial domains.

36MicroRNAs and TFs are the gene regulators which play an important role under biotic and abiotic stresses in plant. In this section, we have summarized latest information on the interaction between miRNAs and TFs under biotic and abiotic stresses and their effect on phenotypic and physiological changes in plants.

37In Arabidopsis, interaction between miR169 and NF-YA transcription factor regulates the drought tolerance. Coexpression of miR169 members and NFYA5 revealed that miR169a was more effective than miR169c in suppressing the NFYA5 at mRNA level. Arabidopsis nfya5 mutants and transgenic plants overexpressing miR169a showed more susceptibility toward drought as compared to wild type plants. In contrast, overexpression of NFYA5, resulted plants with enhanced drought tolerance. However, during salinity stress, miR169 was significantly induced which halted the expression of nfya5 in Arabidopsis. In addition, ABA treatment to the Arabidopsis had significantly induced NFYA5 TF and caused the downregulated of miR169 level.

38A study had been carried out to functionally characterized miR858a in Arabidopsis. The miR858a, which putatively targets R2R3-MYB transcription factors involved in flavonoid biosynthesis. Overexpression of miR858a in Arabidopsis resulted in the downregulation of several MYB TFs involved in flavonoid biosynthesis pathway, hence decrease the flavonoid production. In contrast, knockdown of miR858a by target mimic led to plant growth reduction and delayed flowering.

39In cotton, MYB TF was found to be upregulated in response to high temperature. Like the previous study, MYB TF was targeted by miR828a and miR858. From this finding, we suggest MYB TF and miR828 and miR858 may have dual role in cotton, during fiber development and adaptation against high temperature.

40In sunflower, when plant exposed to high temperatures, a WRKY TF (HaWRKY6) exhibited inverse correlation with miR396. High level of miR396 was observed in older leaves in contrast to the distal portion where the expression was low. In rice treated with arsenic, miR396 was downregulated which resulted in the upregulation of its target, WRKY TF. Currently, as per our knowledge, no functional study involving the overexpression or loss-of-function of miRNA and WRKY TF has been carried out.

41Last decade, a combination of genome-wide, biochemical and genetic studies discovered TCP were responsible for the jasmonic acid biosynthesis. Leaf extracts analysis from plants with high activity of miR319 regulate the expression of the biosynthetic genes, which subsequently led to change in jasmonic acid levels. Moreover, recent finding demonstrated that root-knot nematode (RKN) resistance in tomato was established by using reverse genetic approaches in the interaction of miR319 and TCP4. These interactions affected both jasmonic acid synthetic genes and the endogenous jasmonic acid level in leaves. These finding suggest that the negative interaction between miR319 and TCP4 acted as a systemic signal responder and regulator that modulated the systemic defensive response, mediated via jasmonic acid responsive to RKN.

42The interaction between miR164 and NAC TF is well known in developmental process in Arabidopsis. In addition to that, miR164 and NAC TFs play an important role in regulating drought resistance in rice; where overexpression of miR164 against NAC TFs led to susceptibility toward drought.

43In wheat, interaction between miR164 and novel TF NAC21/22 was confirmed experimentally via co-transformation of both genes in tobacco leaves. Transcript accumulation of NAC21/22 and miR164 exhibited contrasting expression patterns in wheat response to Puccinia striiformis f. sp. tritici (Pst). Silencing of the NAC21/22 showed reduced stripe rust resistance in wheat. These results indicate that the target of miR164 is a novel NAC TF from wheat and plays an essential role in developing stripe rust resistance in host plants.

44Gathering all information above, interaction between miRNAs and TFs will help in understanding the regulatory networks influenced directly by these regulators and cross talking between various biological processes in plants. As miRNAs has been found to be related in secondary metabolite regulation which need to be further explored. In Arabidopsis, the interaction between miR156 and SPL resulted in the negative regulation of anthocyanin biosynthesis. Moreover, in Arabidopsis, loss-of-function of miR163 also led to the accumulation of methyl farnesoate. miR163 also found to regulate another mRNA encoding S-adenosylmethionine dependent methyltransferases that is responsible for methylation of secondary metabolites and different signaling molecules.

45In opium, miR13, miR2161, and miR408 were involved in indole alkaloid biosynthesis. In medicinal herb, Picrorhiza kurroa, miR4995 was involved in the regulation of terpenoid biosynthesis. These interactions can be utilized as a tool to enhance secondary metabolite production either by overexpressing miRNA or transcription factor. Other approach can be the knockdown of miRNA/TF which interfere in the secondary metabolite production. In Persicaria minor plant, an interaction among different miRNAs and TFs investigated. For instance, the targets of miR156 and miR172 (SPL and AP2) were downregulated, whereas targets of miR858 and miR894 (MYB and WRKY) were upregulated under elicitation by Fusarium oxysporum. These finding supported the previous studies; especially, in model plant where the TFs that played role in defense mechanism were upregulated while those TFs which mostly involved in plant development were downregulated by miRNA under stress condition.

46One of the most important global issues is food security to ensure everyone living in this globe can access sufficient food. Since world population is increasing on an alarming rate every effort must be taken in account to obtain higher food production. Genetic modification technology based on miRNA and TFs approaches, can be one of the solutions that contribute to crop yields directly by developing superior plants which can survive under environmental stresses, with high yield and nutrients. This technology will also promote a healthy environment due to less pesticide usage, and this reduced pesticide cost will be used to elevate the living standard of the poor agricultural community across the globe.

47After the breakthrough of miRNA discovery, extensive studies had been done which led to development of new version of miRNA called amiRNA. This approach utilize the unique stem-loop structure of endogenous pri-miRNAs, in which the miRNA/miRNA∗ duplex sequences are being replaced with amiRNA/amiRNA∗ sequences that direct the silencing of target gene with high efficiency. AmiRNA technique exhibited some advantages when compared with conventional RNA interference (RNAi), where amiRNA can be useful for targeting groups of closely related genes, including tandem arrayed and the prediction of gene targeted by amiRNA could be more precise. This approach was effectively used for the downregulation of Chalcone synthase genes in Arabidopsis.

48Beside, amiRNA can be a new approach for developing pathogen tolerant plants, especially virus. T2 transgenic tomato plants expressing amiR-AV1-1 were highly tolerant to Tomato leaf curl New Delhi virus (ToLCNDV), while those plants expressing amiR-AV1-3 showed moderate tolerance. Moreover, recent study in cassava showed transgenic plants which carry four amiRNA challenged with Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) isolates, showed resistance levels that ranged between ∼20 and 60%.

49In the present article, we have reviewed the regulatory relationships between miRNAs and various families of TFs like; NF-YA, MYB, AP2, TCP, WRKY, NAC, GRF, and SPL, from different plant species. The studied interactions between various miRNAs and above mentioned TFs have important roles during drought tolerance, signal transduction and biosynthesis of secondary metabolites, floral development and nodule formation, leaf development, multiple stresses tolerances, lateral root growth, and plant transition from juvenile to adult, respectively. Being the major gene regulators, miRNAs and TFs determine the phenotype, physiology and response to various environmental stresses. Our current review, with lots of newly developed relations between different miRNAs and TFs, will help plant scientists to develop plants with desired phenotypes and stress tolerance ability against particular stress. The plants with stresses tolerance will help to secure the food production for the ever increasing world population. Moreover, some studied interactions have important role in regulation of secondary metabolites biosynthesis and can be used as tool for the production of plant based medicinal biomolecules.

50The first version of this manuscript was written by AS, MS, NN, and IF. AM, ZZ, and II revised the manuscript and advised the writing style. All authors made substantial contribution and approved the final version of the manuscript.

51The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.