Appl Biochem Biotechnol DOI 10.1007/s12010-014-0914-2
Role of MicroRNAs in Biotic and Abiotic Stress Responses in Crop Plants Rajesh Kumar
Received: 30 October 2013 / Accepted: 9 April 2014 # Springer Science+Business Media New York 2014
Abstract MicroRNAs (miRNAs) are small non-coding endogenous RNAs (18–24 nucleotides) which regulate gene expression at posttranscriptional level either by degrading the target mRNA (plants) or by blocking the protein translation through binding with 3′ UTR of the target mRNA (animals). Though miRNAs are known to play key roles in animal development, miRNAs that are involved in plant developmental timing, cell proliferation, and several other physiological functions need to be investigated. In addition, plant miRNAs have been shown to be involved in various biotic (bacterial and viral pathogenesis) and abiotic stress responses such as oxidative, mineral nutrient deficiency, drought, salinity, temperature, cold (chilling), and other abiotic stress. miRNA expression profiling reveals that miRNAs which are involved in the progression of plant growth and development are differentially expressed during abiotic stress responses. The high-throughout techniques can provide genome-wide identification of stress-associated miRNAs under various abiotic stresses in plants. Various web-based and nonweb-based computational tools facilitate in the identification and characterization of biotic/ abiotic stress associated miRNAs and their target genes. In the future, miRNA-mediated RNA interference (RNAi) approach might help in developing transgenic crop plants for better crop improvement by conferring resistance against biotic (pathogens) as well as abiotic stress responses. Keywords MicroRNAs . Post-transcriptional-regulation . Untranslated region (UTR) . miRNA expression profiling . Differential expression . High throughout technique . Biotic and abiotic stress Abbreviations
miRNA siRNA SBP ARF DCL1
microRNA Small interfering RNA Squamosa promoter Binding Protein Auxin response factors Dicer-Like 1
R. Kumar (*) Department of Internal Medicine I and Clinical Chemistry, Im Neuenheimer Feld 410, University of Heidelberg, Heidelberg 69120, Germany e-mail: [email protected]
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HD-ZIP AGO-1 SCL TCP TIR1 APS AST GRF CSD CCS PHO2 UBC2 PPR ABA ATP MYB PARE PCD PCR RACE RISC RNA stRNA UTR RNAi HSR PR ABF ABRE PRR PAMP RSV VSR TYMV TuMV CMV bHLH DREB CBF DRE SDC APS1 SULTR ETR ROS AFB RAS PCD PLC
Class III homeodomain-leucine zipper Argonaute-1 Scarecrow-like Teosinte branched1-cycloidea-PCF Transport inhibitor response1 ATP sulfurylase Sulfate transporter Growth regulating factor Cu/Zn superoxide dismutase Copper chaperone for superoxide dismutase Phosphate transporter Ubiquitin-conjugating enzyme Pentatrico peptide repeat protein Abscisic acid Adenosine triphosphate Myoblastosis Parallel analysis of RNA ends Programmed cell death Polymerase chain reaction Rapid amplification of cDNA ends RNA-induced silencing complex Ribonucleic acid Short temporal RNA Untranslated regions RNA interference Hypersensitive reaction Pathogenesis-related ABA response element binding factor ABA response element Pathogen recognition receptor Pathogen-associated molecular pattern Rice stripe virus Viral suppressor of RNA silencing Turnip yellow mosaic virus Turnip mosaic virus Cucumber mosaic virus Basic helix-loop-helix Dehydration-responsive element binding C-repeat binding factor Dehydration response element Sulfur-containing defense compound ATP sulfurylase Sulfate transporter Ethylene receptors Reactive oxygen species Auxin signaling F-Box Root system architecture Programmed cell death Phospholipase C
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ABF-ABRE-binding factor CRT DRE LTRE COR FRY1
AREB-ABA-responsive element binding protein C-repeat element Dehydration-responsive element Low-temperature-response element Cold responsive Inositol polyphosphate 1-phosphatase
Introduction Several biotic and abiotic (environmental) stress factors such as salt, drought, cold (chilling), and extreme temperatures significantly reduce crop productivity and grain quality throughout the world . In the past decades, tolerance towards these stress responses was conferred using traditional as well as modern breeding approaches that led crop improvement to some extent. Several mechanisms that control signal recognition, transduction and downstream regulatory elements, are currently being investigated which can provide valuable information to understand the molecular mechanism involved in abiotic stress response pathways [73, 35]. Several plants have developed a protective mechanism called as hypersensitive reaction (HSR) to limit the spreading of insects or potentially pathogenic microorganisms by changing composition and physical properties of their cell walls and synthesizing secondary metabolites (salicylic acid and aspirin) . Upon infection with viruses, bacteria, fungi, and nematodes, these HSRs get activated in plants by pathogenesis-related (PR) proteins which comprise protease inhibitors for deactivating proteolytic enzymes (secreted by the pathogens) and lytic enzymes (β-1-3,-glucanase) and chitinase for degrading microbial cell walls. Recently, it was found that jasmonic acid and methyl jasmonate mediate insect and plant disease resistance . Plants respond to acute water stress by closing their stomata that facilitate transpirational water loss from leaf surface. Abscisic acid (ABA) hormone acts as antitranspirant on account of its capacity to induce stomatal closure and thus reduces water loss through transpiration . In addition to hormonal regulation, transcription regulation also plays a key role in controlling the water stress in plants. Transcription factors known as ABA response element binding factors (ABFs) bind to the promoter region [ABA response element (ABRE)] of ABA-induced genes which make enzymes required for the synthesis of osmolytes or compatible solutes [43, 13]. During water-deficit conditions, several compatible solutes such as proline, sorbitol, mannitol, pinitol, glycine betaine, and choline-O-sulfate get accumulated in plants which substantially do not interfere with the normal metabolic activities however provide protection and viability of plants under water stress conditions . A better understanding of adaptive responses against these stress factors will facilitate in developing novel strategies for improving plant stress tolerance. Nowadays, transcriptional activities of stressassociated genes are significantly modulated during abiotic stress response. In spite of stressinducible regulatory proteins and transcriptional factors, recently discovered endogenous small RNAs, such as miRNAs, nat-siRNAs are involved to regulate plant developmental pathways as well as biotic and abiotic plant stress responses [11, 110, 42].
Discovery, Biogenesis, and Mechanism of miRNAs in Plants MicroRNAs (miRNAs) were first discovered in a nematode Caenorhabditis elegans . Initially miRNAs were called as short temporal RNAs (stRNAs) since they were found to be
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expressed temporally in a mutant C. elegans . In C. elegans, the lin-4 gene codes for the 80 nucleotides (nt) untranslated RNA that controls the expression of lin-14 gene . Plant miRNAs are a class of endogenous non-coding small RNAs that are essential for plant growth, development, and survivability. Various plant miRNAs were initially reported in early 2002 [58, 71, 77]. Similar to other protein-coding genes, miRNA genes are transcribed by RNA polymerase II  and form primary transcripts (pri-miRNAs) of about 1,000 bp long as stem-loop structure . Formation of mature miRNAs from pri-miRNAs requires two processing steps (Fig. 1). In the nucleus, the first step is processed by formation of 60- to 70-nt-long precursor miRNAs (pre-miRNAs) [49, 16] from pri-miRNAs with the action of microprocessor complex. The microprocessor complex is comprised of a 169 kDa, RNAse III protein (Drosha) , and dsRNA binding protein Pasha/DGCR8. In plants, Dicer-Like 1 (DCL-1) and HYPONASTIC LEAVES 1 (HYL-1) are orthologs of Drosha and Pasha, respectively, which are involved in the first processing step of miRNA biogenesis pathway [77, 83]. In animals, Drosha generates 2 nt overhangs at 3′ ends on pre-miRNAs . Exportin-5 protein interacts with 3′ overhangs of pre-miRNAs and mediates their transportation from the nucleus to the cytoplasm with the aid of Ran-GTP hydrolysis [10, 117, 114, 60]. In plants, nuclear transport of pre-miRNAs is mediated by HASTY transport protein (ortholog of exportin-5) . In the cytoplasm, the second processing step involves with the formation of 21–24-nt-long mature miRNA-miRNA* duplex from a hairpin dsRNA (pre-miRNA) having 2 nt 3′ overhangs. This step is governed by ATP-dependent RNAse III protein (Dicer) which recognizes 2 nt 3′ overhangs of pre-miRNA  and removes approximately ~21 nt sequence from its ends . The miRNA strand which has G:U base pairs, mismatches, unpaired base pairs at its 5′ end called as antisense miRNA (miRNA) strand while other strand is called as sense strand (miRNA*) . In 2001, Hammond and his colleagues found that generally the antisense strand is incorporated in the RNA-induced silencing complex (RISC) whereas the sense strand is degraded . The single miRNA strand makes a complex with Argonaute-1 (AGO-1) protein which is known to be the part of RISC complex and guides miRNA to target its complementary mRNA sequence  (Fig. 1). The fate of target mRNA depends upon the degree of its complementarity with cognate miRNA sequence. Near-perfect complimentarily leads to complete degradation of the target mRNA while partial complementarity leads the repression of protein translation [78, 45, 31]. In addition, the miRNA biogenesis pathway itself is under the control of feedback regulation by two key players, miR162 and miR168 which lead to cleavage of DCL1 mRNA  and AGO-1 mRNA [78, 57], respectively.
Functional Role of miRNAs in Plant Stress Responses MiRNAs are known to play key roles in animal development, cell proliferation, and programmed cell death (apoptosis); however, their functional roles in the regulation of plant development and abiotic stress responses need to be deciphered. In a sequence analysis of small RNA libraries derived from A. thaliana seedlings, subjected to diverse abiotic stresses as well as computational approaches, identified several miRNAs that play major roles in plant responses to environmental stresses [92, 38]. For example, miR-395 was not detected in plants under normal circumstances; however, its expression was upregulated with low levels of sulfates. MiR-395 targets ATP sulfurylase, an enzyme that catalyzes the first step of inorganic sulfate assimilation and induces upon sulfate starvation, indicating its role in abiotic stress response  (Table 1).
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Fig. 1 miRNA biogenesis pathway in eukaryotes. Primary miRNAs (pri-miRNAs) are transcribed by RNA polymerase II or III which form a hairpin secondary structure of 50–120 nt in length. These pri-miRNAs are cleaved by a multiprotein Microprocessor Complex that includes Drosha and DiGeorge syndrome critical region gene 8 (DCRC8), also known as Pasha and forms approximate 70-nt-long hairpin nuclear pre-miRNA structure. Nuclear pre-miRNAs are exported from the nucleus to the cytoplasm by Exportin-5 with the help of Ran-GTP. In the cytoplasm, dicer along with protein kinase R-activating protein (PACT), TAR RNA-binding protein (TRBP) cleave the hairpin loop of pre-miRNA and makes mature ds miRNA-miRNA* duplex. The mature miRNA duplex is unwound to yield a 22 nt single-stranded mature miRNA which is incorporated into the RNA-induced silencing complex (RISC) along with Argonaute proteins-1 (AGO-1) and other regulatory proteins such as GW182 [also known as trinucleotide repeat containing 6A (TNRC6A)] and poly (A)-binding protein (PABP). Finally, mature miRNA-RISC complex binds to specific mRNA transcripts through complementary base pairing interactions with regions in the 3′ UTR of the target mRNA transcript and leads to either translation repression or target mRNA degradation
Similarly, miR-399 was not detectable in non-stressed plants; however, phosphate starvation induces the accumulation of miR399 [12, 22]. The upregulation of miR-399 targets ubiquitin-conjugating E2 enzyme as a consequence plants competency to cope with phosphate starvation (Table 1). MiR-398 responds to environmental stimulus by targeting two closely related Cu/ Zn superoxide dismutase enzymes (CSD1 and CSD2), which can protect plant cells against superoxide radicals. In this case, oxidative stress reduces the expression level of miR-398 allowing the accumulation of CSD1 and CSD2 mRNA transcripts and mediating oxidative stress tolerance . In addition, the CSD1 and CSD2 also regulate Cu+2 homeostasis and mobilize Cu+2 to plastocyanin protein in thylakoid lumen of chloroplast . MiR-398 was overexpressed in response to Cu+2-limited conditions . At high cold response, miR-319c was upregulated which targets MYB
Appl Biochem Biotechnol Table 1 Stress (biotic/abiotic) associated miRNA family and their predicted targets in plants miRNA family
[38, 92, 56]
[38, 56] [38, 2]
[38, 92, 56]
CSD, CCS, CytC oxidase
[92, 56, 1]
and TCP transcription factors . MiR-389 expression was downregulated in response to cold, dehydration, salt, and ABA stress . It has been shown that the expression of miR-393 is induced by a bacterial flagellin-derived peptide of Pseudomonas syringae . The miR-393 is able to downregulate mRNA of F-box auxin receptors. The repression of auxin signaling results in the restriction of P. syringae growth indicating that miR-393 might play a direct role in host defense mechanism (biotic stress). Biotic Stress (Viral and Bacterial Pathogenesis) Nearly all plants and animals have pathogen recognition receptors (PRRs) which perceive signal from pathogen-associated molecular patterns (PAMPs) such as bacterial flagellin and activate host defenses mechanisms (innate immunity) in response to bacterial infections. Recently, studies showed that Arabidopsis leaves infected with bacterial pathogen P. syringae pv. tomato (Pst) DC3000 showed upregulation of miR393, miR319, miR159, miR160, miR165/166, and miR167 whereas miR390, miR398, and miR408 were downregulated . In Arabidopsis, perception of flagellin increases resistance to the bacterium P. syringae by unknown mechanism. In Arabidopsis, leaves challenged with flagellinderived peptide induce plant microRNA (miR393) that negatively regulates mRNAs for the F-box auxin receptors transport inhibitor response 1 (TIR1), AFB2, and AFB3 . Transgenic plants overexpressing miR393 decrease the transcript level of TIR1 and show enhanced resistance to bacterial infection  thus miR393-mediated suppression of auxin signaling confers antibacterial resistance in plants. In summary, miRNAs which target auxin signalingassociated regulatory proteins and transcription factors are upregulated during bacterial
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pathogenesis and suppress auxin signaling therefore inducing host plant defense mechanisms against these infections. In rice, miRNAs (miR160, miR166, miR167, miR171, and miR396 family of miRNAs) were found to accumulated in response to Rice stripe virus (RSV, a negative sense and ambisense RNA virus) infection  which implies that miRNAs also involved in developing viral resistance against viral infection in plants possibly by targeting viral suppressors of RNA silencing (VSRs). In order to circumvent plant host defense mechanism (RNA silencing), plant viruses activate their counter-defense mechanism by expressing VSRs . In the past few years, some of the miRNAs have been identified that provide a mechanism of viral resistance by targeting transcripts of the viral suppressors. Artificial miRNAs (amiRNAs), modified from Arabidopsis thaliana miR159 precursor were found to target viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV) . Transgenic A. thaliana plants expressing amiR-P69159 and amiR-HC-Pro159 constructs were resistant to TYMV and TuMV, respectively. However, transgenic plants that express both amiR-P69159 and amiR-HC-Pro159 from a dimeric pre-amiR-P69159/amiR-HC-Pro159 transgene in a single Pol II transcription unit showed double viral resistance simultaneously against these two viruses . Similarly, transgenic tobacco plants overexpressing an artificial miRNA (miR2b) conferred effective resistance to cucumber mosaic virus (CMV) infection by inhibiting the expression of silencing suppressor 2b of CMV . Consequently, expression of virus-specific artificial miRNAs could be an effective and predictable novel approach for engineering resistance to CMV and possibly to other plant viruses as well. miRNAs and Environmental (Abiotic) Stress Though abiotic stress responses in plants are regulated by certain stress-inducible regulatory proteins and transcriptional factors, compatible solutes (osmoprotectants) nevertheless miRNAs also play key role in the regulators of abiotic stress responses at posttranscriptional level . In animals, approximately 60 % of protein-coding genes  are regulated by miRNAs while in plants, miRNAs regulate less than 1 % of protein-coding genes [3, 24, 52]. Most of the plant miRNA targets are transcriptional factors  which participate in the developmental processes from seed germination to seed maturation. These miRNAs not only regulate plant developmental processes but also regulate plant stress responses. For instance, miR398 targets two Cu/Zn superoxide dismutase (CSD1 and CSD2) , miR395 and miR399 target sulfate transporter (AST68)  and the phosphate transporter (PHO1) , respectively. MiRNA expression profiling reveals that conserved miRNAs that target various abiotic stress-associated transcription factors and regulatory proteins are significantly altered (upregulated or downregulated) during stresses. MiR393 upregulated during nitrogen availability which targets transcripts that code for a basic helix-loop-helix (bHLH) transcription factor and auxin receptors TIR1, Auxin signaling F-Box proteins 1, 2, 3 (AFB1, AFB2, and AFB3); however, only AFB3 transcript is controlled by miR393 in order to maintain root system architecture (RSA) in response to nitrate supply . Under normal conditions, low levels of miR393, miR160, and miR167 are sufficient for fine tuning of the auxin response factors (ARF) levels that are needed for transcription of the auxin-responsive genes. During stress (biotic/abiotic) condition, miR193 and miR160/67 are upregulated that keep low transcript levels of TIR and ARF, respectively. Therefore, miR393, miR160 and miR167 suppress ARF-mediated expression of auxin-responsive genes, causing the attenuation of plant growth and development under stress, and probably induce plant stress tolerance as well (Fig. 2).
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Fig. 2 miRNA-mediated gene regulation upon auxin perception and signaling under normal growth and stress condition. Upon binding of auxin to its nuclear-located auxin receptor, transport inhibitor response1 (TIR1) it forms an auxin-TIR1 complex. This complex mediates the expression of auxin-responsive genes that are necessary for plant growth and development via dissociation of the auxin response factors (ARFs) from Aux/ IAA (auxin/indole-3-acetic acid)-mediated heterodimerization. At low auxin concentration, ARFs are heterodimerized with Aux/IAA repressors which prevent transcription of auxin-responsive genes. Auxin increases the affinity of TIR1 for Aux/IAA and facilitates the dissociation of Aux/IAA from the ARF, relieving its inhibitory effect on ARFs. TIR1 also recruits E3 ubiquitin-ligating enzyme (SCF) which polyubiquitinates Aux/ IAA repressors and that are subsequently degraded by 26S proteasomal pathway. MiR393 downregulates TIR1 transcripts while miR160 and miR167 downregulate five different ARF transcripts by guiding the degradation of their corresponding target mRNAs
Drought and Salinity Drought and salinity are the major external factors that constrain crop productivity and seed quality worldwide. To cope with water-deficit crop, plants adapt several strategies at the physiological, biochemical, and molecular levels. Researchers have identified different genes, biomolecules, and compounds that genetically could be altered to make crop plants as drought resistant without reducing their yield. In addition, overexpressing stress-responsive genes (transcription factors) that are induced under drought and salt stress conditions or manipulating other regulatory genes associated with stress signaling pathways would improve plant stress tolerance. During osmotic stress, plants accumulate organic compounds referred as osmoprotectants (compatible solutes) which assist in efficient water uptake thereby preventing water loss. These compounds have no inhibitory effect on the enzyme activity even in high cellular concentration. Two types of osmoprotectants have been found to be accumulated inside plant cells such
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as polyols which comprise glycerol, mannitol, sorbitol, pinitol, and sucrose and quaternary ammonium compounds that include proline, glycine betaine, proline betaine, b-alanine betaine, and choline-O-sulfate . Thus osmoprotectants are considered as protective compounds which protect plants with severe water stress, making them drought tolerant and preventing their cell death. Glycine betaine accumulates in plants naturally during water stress and in other organism such as arthrobacter. Some plants such as rice and potato do not accumulate glycine betaine. Thus introduction of genes, involved in the synthesis of glycine betaine can accumulate glycine betaine in rice plants and thereby can make them drought tolerant and resistant. The plant stress hormone ABA has been shown to trigger stomatal closure in plants thus a reduction in water loss and tolerating them with the conditions of drought stress. During drought stress conditions, various transcription factors are either upregulated or downregulated depending on their cellular function in triggering tolerance to drought stress. For example, dehydration-responsive element binding (DREB) transcription factors have been shown to control the expression of genes involved in plant drought tolerance (Fig. 3). Thus, overexpressing the DREB transcription factor, responsible for the activation of these genes, will result in developing drought tolerant and resistant plants. Other transcription factors such as AtMYB60 have been shown to downregulate in plants as it encourages opening of the stomata pores. In addition, drought tolerance is triggered through signaling mechanism
Fig. 3 Transcriptional regulatory network of cis-acting elements and ABA-dependent/independent transcription factors, involve in drought, cold, and salinity stress-inducible gene expression. ABA‐independent abiotic stress signaling mediated by phospholipase C (PLC) activates DREB2 transcription factors that bind to DRE and induce ABA‐independent transcription of stress-responsive genes. Inositol polyphosphate 1-phosphatase (FRY1) functions as a negative regulator of drought, salinity, and ABA responses which involves in the catabolism of inositol 1, 4, 5-trisphosphate (IP3) (Xiong et al., 2001). ABA‐dependent pathway regulates stress‐responsive gene expression through CBF4 (DREB1D), AREB (ABF), and MYC/MYB transcription factors, which bind to the CRT/DRE, ABA Responsive Elements (ABRE), and MYC/MYB Recognition Sequences (MYCR/MYBR) promoter elements, respectively. During cold stress, ICE is activated by ABA-independent pathway which activates downstream transcription factor CBF/DREB1 that binds to C-repeat elements (CRT)/low-temperature-response elements (LTRE) and induces the expression of cold-responsive (COR) genes
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through second messenger molecules such as nitric oxide (NO) and cyclic guanosine monophosphate (cGMP). NO regulates detoxification of superoxides and enhances the activity of the enzyme that degrades the hydrogen peroxide which is produced when superoxides are degraded. Thus, NO prevents plant cells from loss of function and cell death, making the plants to survive under osmotic stress conditions. In plants, during drought and/or salt stress, miR393, miR160, and miR167 were found to be upregulated . Additional miRNA, miR169 which plays major role in drought and salt stress response, was downregulated through an ABA-dependent pathway in Arabidopsis  therefore leading overexpression of its target Nuclear Factor YA5 (NFYA5) transcription factor which increases the expression of a number of drought stress-responsive genes. In rice, two miRNAs, miR169g and miR393, were observed to be upregulated during drought stress response . The upregulated miR393 targets TIR1 therefore causing attenuation in plant growth and development during drought condition . Medicago truncatula, a model legume nitrogen-fixing plant, showed upregulation of miR398a/b and miR408 levels in both roots and shoots while downregulation of miR169 only in the roots in response to water deficiency state . In water-stressed M. truncatula plants, it was observed that there was no direct inverse correlation between miR169 and its target MtHAP2-1 transcript which implies that miR169 may not involve in the regulation of drought response . The upregulated miR398a/b and miR408 showed a strong inverse correlation with downregulation of their respective target transcripts, copper superoxide dismutase (CSD1/2), mitochondrial cytochrome c oxidase, and plastocyanin which establish a direct link in adaptation of M. truncatula to drought and copper homeostasis  (Fig. 4). The miRNA expression profiling between two maize lines  and different tissues such as roots and shoots  showed that some miRNAs respond differently among different plants and tissues (tissue specific expression) during stress conditions.
Fig. 4 The regulatory network of drought and salinity stress-associated miRNAs in Arabidopsis (non-nitrogenfixing model plant) and M. truncatula (nitrogen-fixing model plant). The proposed network describes molecular mechanisms in Arabidopsis and M. truncatula that leads to phenotypic changes in response to drought and salt stress. Upon stress, miRNAs are differentially expressed which subsequently silence their respective target transcripts. Most of the miRNA targets are transcription factors which regulate downstream stress-responsive genes. The miRNA-mediated stress regulation in Arabidopsis and M. truncatula is shown by solid lines. Green line: In Arabidopsis; Light yellow line: In M. truncatula; Pink line: In both Arabidopsis and M. truncatula
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Heat, Chilling, and Freezing Plants maintain the homeostasis by reprogramming their gene expression profiles in response to environmental temperature fluctuation. In Arabidopsis, C-repeat binding F factors (CBFs) transcription factors were identified which bind to the dehydration-responsive element (DRE) and increase the expression of cold-responsive proteins during low temperature and dehydration stress; however, miRNAs are also involved in the regulatory network of these proteins. For example, signaling of the ABA hormone which involves in the cold acclimation of herbaceous tissues is controlled by various miRNAs. During cold acclimation, the ABA level was observed significantly higher in several cold-tolerant varieties compared to cold-sensitive varieties. Further research is required to elucidate the precise role and mechanism of ABA in cold acclimation and induction of genes associated with cold tolerance. In plants such as Arabidopsis, Brachypodium, and popular, miR397 and miR169 were found to be upregulated in response to cold stress . Similarly miR168 was upregulated in popular and Arabidopsis ; however, it was downregulated in rice  during cold stress. Metal Ion Starvation Phosphate Starvation Phosphorus, one of the macronutrients of plants, is untaken by roots primarily via the high-affinity inorganic phosphate (Pi) transporters. Phosphate (Pi) availability in soil regulates plant growth, development, and crop productivity. In this process, not only transcription factors or signaling molecules such as WRKY75  play a key role in the regulation of Pi starvation responses but also miRNA-mediated posttranscriptional regulation. Upon Pi deprivation, the expression of miR156, miR399, miR778, miR827, and miR2111 was induced, whereas the expression of miR169, miR395, and miR398 was repressed . During Pi starvation, the upregulated miR399 regulates Pi homeostasis by suppressing the expression of PHO2 encoding ubiquitin-conjugating E2 enzyme, UBC24 [22, 6] which acts as a negative regulator and suppresses Pi uptake and translocation when Pi supply is inadequate. For example, the accumulation of E2 transcripts was suppressed in transgenic Arabidopsis plants overexpressing miR399 . Upon Pi starvation, several transcription factors and Pi transporter proteins that regulate phosphate uptake from the soil are degraded via ubiquitin proteasome pathway (UPP). Therefore, overexpressing the miRNAs that suppress E2 and E3 (ubiquitin-conjugating and ubiquitin-ligating enzyme, respectively) would release the downstream targets from proteasomal degradation pathway and maintaining Pi homeostasis during Pi deprivation. Sulfate Starvation Plants absorb sulfur in the form of sulfate from the soil and assimilate into the amino acid cysteine which subsequently converted into sulfur-containing defense compounds (SDCs) such as glutathione, phytoalexins, and glucosinolates. These SDCs are essential for plant growth and survival under optimal conditions as well as conditions of biotic and abiotic stresses . During sulfate starvation condition, synthesis of SDCs was induced via jasmonic acid and/or other signaling molecules . In addition, miRNAs also have been shown to involve in the regulatory pathway of sulfate metabolism. The expression of miR395 was significantly upregulated upon sulfate starvation that targets two families of genes ATP sulfurylase (APS1, APS3, APS4)  and sulfate transporter 2;1(SULTR2;1/AST68) , both of them are involved in the sulfate metabolism pathway. A negative correlation between APS1 transcript (not with AST68 transcript) and miR395 was observed in the shoot of Arabidopsis during sulfate starvation condition . The miR395 target transcripts are strongly suppressed in transgenic Arabidopsis overexpressing miR395, which over-accumulates sulfate in the
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shoot but not in the root . APS1 knockdown mutants were also shown to accumulate two fold much sulfate as compared to wild-type . Copper Starvation Copper is an essential cofactor of several proteins such as plastocyanin (PC), Cu/Zn super oxidase (CSD), ethylene receptors (ETRs), laccase, and cytochrome c oxidase that are associated with photosynthesis, scavenging reactive oxygen species (ROS), ethylene signaling, metabolism of phenolic compounds (anthocyanin and lignin) and electron transport, respectively . Some of the copper containing proteins for instance CSDs, plastocyanin, laccases were posttranscriptionally silenced during cupper limitation by upregulated copper responsive miRNAs (miR397, miR398, miR408, miR857) [112, 1] therefore these miRNAs facilitate in retaining and accumulating more copper in other essential copper containing proteins like cytochrome c oxidase as a cofactor. MiR398 is most significantly altered (upregulated) miRNA in copper limited condition that targets two different proteins, Cu/Zn superoxide dismutase (CSD1 and CSD2) and copper chaperone for superoxide dismutase (CCS1) [90, 14, 7]. In addition, miR398 is also modulated (downregulated) in response to oxidative stress . These observations indicate that miR398 is involved in the regulation of both copper deprivations as well as oxidative stress but in opposite manner. The expression of CSDs is controlled by miR398 at both posttranscriptional as well the posttranslational level through CCS transcript regulation. During oxidative stress, reactive oxygen species (O2−) are detoxified by overexpressed CSD1 and CSD2 proteins which are facilitated through low expression of miR398. In the reverse way during copper starvation, upregulated miR398 silences CSD1/CSD2/CCS1 transcripts as a consequence copper becomes available for other essential proteins for their proper functionality in the adverse conditions (Fig. 5).
Fig. 5 Stress-dependent regulation of miR398 in Arabidopsis. Apo-CSDs are produced by translation of CSD1/ CSD2 mRNAs which become biologically functional upon incorporation of copper ion as a cofactor by CCS1. Both CSDs and CCS1 transcripts are targeted by miR398. In normal condition, miR398 level leads to fine tuning of the expression of CSD transcripts. Under oxidative stress, miR398 is downregulated that causes restoration of the CSDs as well as CCS1 transcripts and therefore leading to high expression of CSD proteins. During copper starvation, miR398 is upregulated, leading to posttranscriptional silencing of CSDs and CCS1 expression, consequently copper will be available for other cupper-containing essential proteins as a cofactor. The level of miR398 transcript under different stress conditions is shown by thickness of yellow line in the box
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Nitrogen Starvation Nitrogen is an essential component of three plant macronutrient N, P, and K. Together with regulatory proteins, miRNAs also participate in adaptation of plants during low nitrogen availability. In Arabidopsis, the miR393/AFB3 module controls RSA in response to external and internal nitrogen availability . miR393 silences transcripts of the bHLH  transcription factor and auxin receptors TIR1, AFB1, AFB2, and AFB3 [38, 92, 67]. It was demonstrated that in nitrogen treated Arabidopsis seedling, upregulated miR393 downregulate auxin signaling F-Box 3 (AFB3), which in turns controls primarily and lateral root growth development . Another nitrogen responsive module was identified as miR167/ARF8 module that regulates lateral root initiation and emergence in Arabidopsis . Nitrogen availability (glutamate/glutamine) suppresses the miR167 expression, therefore ARF8 would be free from antagonistic regulation (silencing) by miR167; as a consequence, overexpressed ARF8 initiates lateral root formation in Arabidopsis . In summary, nitrogen availability modulates miRNA expression that regulates auxin signaling pathway by targeting TIR1, AFB3, or ARF8, which further regulates the root system architecture (RAS). In maize, total nine miRNAs were identified to be differentially expressed in response to chronic low nitrogen availability in which three of the miRNAs (miR164, miR172, and miR827) were upregulated while six miRNAs (miR167, miR169, miR395, miR399, miR408, and miR528) were downregulated .
Oxidative Stress In plants, aerobic metabolic pathways such as respiration and photosynthesis produce hydrogen peroxide (H2O2) and other ROS as by-products . Though H2O2 is highly reactive and toxic, however, it also involves in regulating basic biological activities, such as growth, development, hormone signaling, biotic/abiotic stress responses, and programmed cell death (PCD) in plants [64, 65]. In general, during stress (biotic and abiotic) conditions, H2O2 production is increased which acts as a secondary messenger in stress-response signal transduction pathway. In H2O2 stress condition, global gene expression profiling studies reveal that proteins associated with redox homeostasis are overexpressed while normal growth and development associated proteins are downregulated . The high throughput sequencing combined with computational analysis and subsequently followed by northern blot analysis from the normal and H2O2 treated rice seedlings showed that seven miRNA families differentially expressed under H2O2 stress (oxidative stress) . The predicted H2O2 responsive miRNA targets are involved in several plant metabolic activities such as nutrient transport, auxin homeostasis, PCD.
A Plausible Strategy to Develop Abiotic Resistant Crop Plants Environmental stress is a major force that adversely affects plant growth, development, crop productivity, and quality in the tropics. Some of the food, oil, and fruit crop plants like rice, maize, soybean, mustard, apple, etc. lose their productivity and fruit/grain quality because of various environmental stress responses (drought, cold, temperature, salinity, nutrient depletion). Among all abiotic stress factors, drought has caused significant crop losses in most of the cereal crops.
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In order to cope up with such environmental stresses, plants have developed several tolerance mechanisms for instance accumulation of compatible solutes (osmolytes), induction of stress-associated proteins (transcription factors), and induction of ROS scavenging system. Till today, the whole genome of some of the crop plants have been sequenced that can be used for the functional annotation of abiotic stress-associated miRNAs (Table 2). In the future, miRNAs-based approach could be a sustainable approach for the improvement and management of crops in the stressful environmental conditions.
Computational Identification of Plant-Specific miRNAs and Their Targets Deep sequencing technologies have become very powerful tools in the identification and quantification of small RNAs involved in gene regulation in different types of responses in plants and animals. In 1993, Lee and his colleagues first discovered miRNAs by genetic approaches; however, due to its time-consuming property, low efficiency, and high cost, these approaches were found to be limited . Since these miRNAs are naturally occurring, found in plants, animals, and invertebrates, consequently they show significant variations in their level of expression among different tissues and/or under different environmental conditions . For miRNA identification, several alternative computational approaches were developed to complement the limitations of genetic approaches [26, 120, 104]. The computational (bioinformatics) tools that are commonly used to predict differentially expressed miRNA and their targets genes in different organism are shown in the Tables 3 and 4. These computational tools are based on three major characteristic features of miRNAs: hairpin-shaped stem-loop secondary structures , high evolutionary conserved “seed” region , and high minimal folding free energy index . About 6–8 nucleotide long seed region  located at 5′ end of the miRNA shows Watson-Crick base pairing with the 3′ UTR of the targeted transcript. These computational tools provide efficient ways to predict miRNAs and their target genes in animals, human, fungi, invertebrates, and plants. In contrast, to identify and characterize miRNAs by Table 2 The predicted genome size and % coverage of crop plants Crop plant
Protein coding genes
Rice (Oryza sativa)
Maize (Zea mays)
Populus (Populus trichocarpa)
Grapes (Vitis vinifera) Sorghum (Sorghum bicolor)
4.87×108 bp 7.30×108 bp
Soybean (Glycine max)
Mustard (Brassica rapa)
Jatropa (Jatropa curcas)
Peaches (Prunus persica)
Apple (Malus domestica)
cucumber (Cucumis sativus)
Barrel Clover (Medicago truncatula)
Appl Biochem Biotechnol Table 3 Useful tools/approaches for miRNA prediction Program
Plants, animals Plants, animals
experimental approach requires cloning and sequencing of small RNAs which seems slightly complicated than computational approach. Nevertheless, major challenges with the computational approach is with finding the miRNAs which are species specific and unrelated to other organisms since these algorithms function by searching evolutionary conserved “seed” sequence of miRNAs.
Experimental Validation of miRNAs and Their Targets (by Gain/Loss of Function) The predicted miRNAs and their targets through above bioinformatics tools can be confirmed by direct cloning technique, northern blotting, quantitative real-time PCR, and/or 5′ rapid amplification of cDNA ends (5′ RACE) . Although the whole genome sequence of some
Table 4 Major computational tools used for miRNA targets prediction in plants Program
Plants, animals Plants
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of food and oil crop plants like rice, maize, soybean, mustard, Jatropha, and Barrel Cloverare are available, miRNA sequences associated with biotic and abiotic stresses, however, have not been annotated completely. Consequently, genes of small RNAs which are present in these sequences can be used for analysis of stress tolerance in the conditions of abiotic stresses. All small RNAs can be cloned and sequenced by parallel analysis of RNA ends (PARE)  method which involves a modified 5′-rapid amplification of cDNA ends and deep sequencing of 3′ cleavage products of mRNAs. From sequencing, miRNAs can be selected from a pool of small RNAs (smRNAs) such as small interfering RNA (siRNAs), trans-acting siRNA (ta-siRNAs), natural-antisense transcriptderived siRNA (nat-siRNAs), and repeat-associated siRNA (ra-siRNAs) and subjected to miRNA chip preparation . For miRNA expression profiling, nowadays several webbased servers such as Capital Biochip Corporation (http://www.capitalbio.com/) and LC SCIENCES Corporation (http://www.lcsciences.com/) are available which provide advanced tools for direct detection of miRNAs in plants and animals with high sensitivity and specificity. In spite of microarray, these tools provide direct services for sequencing of small RNAs and qRT-PCR where multiple data sets can be analyzed simultaneously by normalization, ANOVA, t test and clustering analysis as well. The miRNA microarray, a high throughput technique can monitor the expression pattern of thousands of genes simultaneously to determine microRNA profiling in stress-induced plant tissues/cells. The differentially expressed miRNAs further could be validated by quantitative real-time PCR (qRT-PCR) or by northern blot analysis. The conventional techniques such as cloning, northern hybridization, and miRNA microarray can lead in drastically variation in miRNA expression levels during profiling. Though the microarray technique provides an efficient genome-wide screening of miRNA expression, qRT-PCR, however, is the most advantageous approach for validating the microarray expression data as it can detect and quantify mature miRNAs in a fast, accurate, specific, and reliable manner. In addition, the specificity of qRT-PCR expression data can facilitate to understand the true biological significance of the novel abiotic stress-associated miRNAs among various crop plant species.
Abiotic Stress Induction and Functional Annotation From above approaches, a particular crop plant might be selected and subjected to various abiotic stresses. The total RNAs with a pool of small RNAs can be isolated from stressinduced as well as normal plant tissues, followed by the miRNA profiling. Functional annotation of novel stress-associated miRNAs and their targets could be performed by antisense chemistry. Loss of function studies can be carried out by overexpressing antisense oligonucleotides (antimiRs)  and siRNAs for the stress-associated miRNA and their targets, respectively.
Conclusion and Future Prospective MicroRNAs are 18–24 nucleotides long endogenously produced non-coding small RNAs which regulate gene expression at posttranscriptional level and play key role in developmental timing, growth, stem cell maintenance, cancer, apoptosis (programmed cell death), and biotic/ abiotic stress responses in animals and plants. These miRNAs have been identified as frontiers in plant functional genomics where they assist in crop improvement by reducing pathogenicity through sequence-specific silencing of plant-specific pathogen transcripts. In order to
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counteract adverse conditions, these miRNAs have been shown to regulate various stressresponsive genes, proteins, and transcription factors which are involved in the maintenance of stress tolerance during adverse conditions. Although the whole genome sequences of some of crop plants like rice, maize, soybean, mustard, Jatropha, Barrel Cloverare, etc. have been completed, the sequences of miRNAs which are associated with biotic (viral, bacterial, and fungal infection) and abiotic (drought, cold/chilling, salinity, temperature, nutrient deficiency, heavy metals, hypoxia, etc.) stresses have not been annotated completely. Therefore, genes of small RNAs (miRNAs) which are present in these sequences can be used for analysis of stress tolerance in biotic as well as abiotic stress conditions. Differentially expressed miRNAs and their target(s) which provide defense mechanisms against various biotic and abiotic stresses can be identified by computational methods and experimentally verified by high throughput technique like miRNA microarray, real-time PCR, or northern blot. The miRNAs which are modulated during abiotic stress responses in different crop plants can be selected by deep sequencing methods such as PARE method  which further could be used for miRNA profiling with respect to normal plant tissue. With the utilization of high throughput methods and bioinformatics tools, novel miRNAs and their targets can be identified that assist or curtail in survival of a plant during different type of stresses. Transgenic crop plant harboring either miRNA or their target gene will be resistant to abiotic stress (drought, salinity, temperature, etc.). Manipulation of miRNA-guided gene regulation represents a novel and feasible approach to improve various type of plant stress tolerance. The expression of virus-specific amiRNAs in transgenic plants confer viral resistance by targeting viral suppressor transcripts and therefore amiRNA-mediated approach may provide broad applications for engineering multiple virus resistance in economically important crop plants. In summary, the miRNA-mediated gene silencing approach may help in developing transgenic abiotic stress-resistant crop plants for better crop improvement under unfavorable environmental conditions (abiotic stresses). In the future, these approaches will lead to reduction in water used for irrigation in agriculture and also an increase in crop productivity in dry areas of the world. Acknowledgments The author gratefully acknowledges the Indian Institute of Technology, Guwahati (India) for providing M.Tech. fellowship in the course of his manuscript preparation. The author also expresses gratitude to Prof. Dr. Vikash Kumar Dubey for critical comments on the manuscript.
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