19

Impact of Climate Change on MicroRNA Expression in Plants

Vallabhi Ghorecha, N.S.R. Krishnayya, and Ramanjulu Sunkar

Abstract

Climate change is an alarming issue of the twenty-first century, and the effect ofclimate change on plant resources, biodiversity, and global food security is a majorconcern. For assured food production in the projected climates of 2030 and beyond,it is essential that we understand at the molecular level how plants respond andadapt to higher temperatures, drought, high CO2, ozone, and UV-B radiation,which are some of the major stresses that will be associated with the changingclimates. In plants, stress-responsive gene expression largely constitutes nuclearprocesses such as transcriptional regulation involving interaction between DNA(cis-elements) and proteins (transcription factors). However, recently discoveredmicroRNA (miRNA)-mediated post-transcriptional gene regulation is also criticalfor adaptation to stress conditions. In this chapter, we discuss the responses androles of plant miRNAs in changing climates.

19.1

Introduction

Climate change is an alarming issue of the twenty-first century. Anthropogenicactivities such as increased burning of fossil fuel coupled with industrial gaseousemissions contribute to the rise of greenhouse gases (GHGs) such as carbondioxide (CO2), carbon monoxide (CO), methane (CH4), and nitrous oxide (NOx) inthe troposphere. Current estimated atmospheric CO2 levels are 385 parts permillion by volume (ppmv) and these levels are speculated to rise to 450�600 ppmvby the end of this century [1]. GHGs affect the natural energy flow by trapping theoutgoing radiation from the Earth to space, leading to global warming [2,3]. Thecurrent projections suggest that the world’s temperature will rise 2�4 �C by 2100[4�6]. Global warming mediated by GHGs and the rise in atmospheric aerosolscauses a rise in the amount of water vapor, altered precipitation rates andfrequency, drying of the land surface, and tropical cyclones, affecting severely boththe biosphere and hydrological cycle [2,7,8]. The increase in water vapor accounts

507

Climate Change and Plant Abiotic Stress Tolerance, First Edition. Edited by Narendra Tuteja and Sarvajeet S. Gill.� 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

for more intense but erratic precipitation rates with total precipitation remainingalmost constant [2,9]. Erratic intense precipitation causes waterlogging, affectingcrop yield by creating anoxic conditions in the soil. In contrast, land surface dryingcauses loss in surface and underground water, resulting in drought. Reduction inrainfall by 13�16% may be observed against a predictive rise in CO2 by 600 ppmvin regions of southern Europe, western Australia, northern Africa, southwesternNorth America, eastern South America, and southern Africa [1]. Warming coupledwith drought increases the vulnerability of forests to higher background treemortality rates and die-off, affecting the world’s forest ecosystem, which isnecessary to maintain planetary energetics, the hydrological cycle, and atmosphericcomposition [10,11]. Warmer temperatures have affected the phenology oforganisms, the range and distribution of species, and the composition anddynamics of communities, highlighting climate change-induced affects on biota[12]. Significant impacts of a rise in temperature are already visible in terms ofaltered growing seasons (like early spring in Europe and North America) as well asa decrease in crop yields [6,13,14]. Adaptation of crop plants to the climateprojected for 2030 is needed for future food assurance [15]. Exploring theunderlying physiological, metabolic, and gene regulatory mechanisms underprojected climatic conditions is of significant importance to implement breedingor biotechnological strategies.Plant domestication/cultivation is being carried out for maximum growth and

yield. Cultivated plants invest more in the structures desired by humans. This leadsto a trade-off between yield and stress resistance [16]. Wild plant species, however,have higher tolerance to extreme abiotic stresses than their relative model plantspecies [17]. The evolution of stress tolerance in many wild plant species is anongoing process and such plants represent an important source of genetic variationthat can be exploited for a better understanding of stress tolerance at the molecularlevel. Moreover, some of the wild plant species have a unique ability to adapt andsurvive under a wide range of conditions in the field. For instance, Ipomoeacampanulata in the field can tolerate both drought and waterlogging, whereas itsrelative Jacquemontia pentantha is susceptible to these stresses. I. campanulatagrowing in water-deficit and waterlogged conditions exhibits differential geneexpression including microRNA (miRNA)-dependent post-transcription generegulation compared to J. pentantha (our unpublished work). In-depth molecularinformation from such closely related plant species exhibiting differentialsensitivities is likely to be useful for improving tolerance of crop plants underchanging climates.

19.2

Small Non-Coding RNAs in Plants

Plants encode, process, and accumulate small RNAs 20�40 nucleotides in length,which are classified into two major categories: miRNAs and other endogenoussmall interfering RNAs (siRNAs). The major distinction between miRNAs and

508 19 Impact of Climate Change on MicroRNA Expression in Plants

siRNAs is that miRNAs are derived from single-stranded RNA that adopts ahairpin-like structure, whereas siRNAs are derived from double-stranded RNAsthat are generated as result of RNA-dependent RNA polymerase activity or pairingof mRNAs derived from natural antisense pairs of genes. On the basis of length,origin, and function, endogenous siRNAs can be further divided into severalsubcategories, such as trans-acting siRNAs (ta-siRNAs), natural antisense siRNAs(nat-siRNAs), heterochromatic siRNAs, and long siRNAs (lsiRNAs) [18,19].ta-siRNAs encoded by a TAS loci have a miRNA-dependent biogenesis pathwayand have a mode of regulation of gene expression similar to that of miRNAs[19,20]. nat-siRNAs are derived from pairing of mRNAs derived from naturalantisense pairs of genes [21,22]. Heterochromatic siRNAs are specifically asso-ciated with repetitive sequences (centromeric repeat sequences, retroelements,transposons, and ribosomal DNA), and appear to play a role in DNA and histonemethylation [23�26]. lsiRNAs, which are 30�40 nucleotides in length compared toother classes of siRNAs that are 21�24 nucleotides, have been recently reported tofunction in response to bacterial infection in Arabidopsis. Despite the fact thatplants accumulate numerous endogenous siRNAs that are not very abundant, butvery diverse, little is known about their functions with respect to development orstress responses. Only nat-siRNAs and lsiRNAs are implicated in stress responsesin plants [27�30].

19.3

Biogenesis and Function of miRNAs in Plants

miRNA genes are transcribed by RNA polymerase II, generating long primarymiRNA transcripts. These transcripts can adopt hairpin-like structures due toimperfect self-complementarity and such structures serve as substrates for theenzyme Dicer-like 1 (DCL1), releasing miRNA duplexes around 21 nucleotides inlength. Although DCL1 is the major protein implicated in miRNA biogenesis,several other double-stranded RNA-binding protein(s) and DCL1-interactingproteins such as HYL1 (hyponastic leaves 1), SE (serrate), DDL (dwadle-like),CBP80 (cap-binding protein 80), CBP20 (cap-binding protein 20), STA1 (stabilized 1),and CPL1/FRY2 (C-terminal domain phosphatase-like 1/FIERY 2) are also essential inreleasing the miRNA and miRNA� (miRNA� is the complementary strand of themature miRNA) duplex from the hairpin-like structure [20,31�36]. The processedmature miRNAs are loaded into RNA-induced silencing complex (RISC), whereasmiRNA� species are usually degraded rapidly. Guided by base pairing betweenmiRNAs and complementary target mRNAs, the RISC causes largely transcriptcleavage or translational inhibition, thus contributing to post-transcriptional generegulation in plants (Figure 19.1) [20,31,32,37].Upon exposure to stress, plants use multiple gene regulatory mechanisms to

restore cellular homeostasis as well as to decrease the detrimental effects of stress.Transcriptional regulation during stress is the predominant mode of generegulation and therefore has been extensively investigated [38]. The importance of

19.3 Biogenesis and Function of miRNAs in Plants 509

post-transcriptional gene regulation during stress was realized when correlationsbetween mRNA abundance and respective protein levels were not evident [39]. Post-transcriptional gene regulation is dependent on various factors such as small RNAs(miRNAs and other endogenous small RNAs) and proteins (RNA-binding proteins)

Figure 19.1 Primary miRNA transcripts are

transcribed from miRNA genes by RNA

polymerase II and these transcripts can adapt

hairpin-like structures that are recognized

and processed by the RNase III enzyme DCL1

with the assistance of several RNA binding

proteins such as CBP20/80, HYL1, SE, DDL,

TGH (tough) and CPL-1. DDL interacts with

DCL1 and stabilizes the primary miRNA

transcript. TGH is a component of the

DCL1�HYL1�SE complex and facilitates

primary miRNA recruitment to HYL1.

CPL1dephosphorylates HYL1 for optimal

activity. The miRNA/miRNA� duplex is

stabilized by HEN1 (HUA enhancer 1, a

methyltransferase), which adds a methyl

group to the 20-OH group at the 30-end. Themature miRNA is transported to cytoplasm

by HST (hasty, exportin), where only the

miRNA strand gets loaded into the RISC

complex containing AGO1 protein. The RISC

complex is guided by the miRNA to target

mRNA causing target mRNA cleavage or

translational repression.

510 19 Impact of Climate Change on MicroRNA Expression in Plants

or processes such as alternate splicing. Of these, miRNA-regulated target mRNAabundance has emerged as one of the ubiquitous pathways of post-transcriptionalgene regulation. One major advantage with miRNA-controlled gene regulation isthat the miRNAs can respond spontaneously to stress and thereby regulate theexisting pool of mRNA targets without any de novo synthesis [40]. Altered miRNAseventually impinge upon their targets and modulate their expression.Several miRNAs responsive to climate change-associated abiotic stresses, such as

heat, drought, UV-B radiation, and ozone (O3), have been reported in plants(Table 19.1) [41�48]. The altered expression of miRNAs and the impact on theirmRNA targets in plant species exposed to climate change-associated stresses is nowdiscussed.

19.4

Heat Stress

Plants that are exposed to 3�5 �C more than their optimal temperatures willexperience heat stress. High temperature is known to affect primarily photosynth-esis, growth, and development as well as diverse physio-biochemical processes.Heat stress has been reported to alter miRNA expression in diverse plant species,such as Triticum aestivum, Brassica rapa, Populus tomentosa, and Panax ginseng[41,42,49,50]. miRNAs responsive to heat stress are largely either upregulated, asobserved in T. aestivum and B. rapa, or downregulated, as reported in P. tomentosa.In T. aestivum, miR156, miR159, miR160, miR166, miR169, miR827, miR2005, andmiR168 were upregulated, whereas miR172 was downregulated under heat stress(40 �C for 2 h) [41]. In B. rapa seedlings exposed to high temperature (46 �C for 1 h),miR156, miR5714, miR5718, and miR5726 levels were upregulated, while miR399b,miR827, miR5716, and miR1885b.3 levels were decreased [42]. In contrast to theobserved upregulation of miRNAs in T. aestivum and B. rapa, the woody plant P.tomentosa showed mostly downregulation of miRNAs under heat stress (37 �C for8 h) [49]. Similarly miRNAs are largely downregulated in Populus trichocarpa growingat 37 �C for 24 h [51]. On the other hand, miR160, miR168, and miR169 aredownregulated under heat stress in P. tomentosa, while these miRNA levels wereupregulated in T. aestivum.A heat-susceptible cultivar of T. aestivum (Chinese Spring) exposed to heat stress

(40 �C) showed initial (30min after stress imposition) upregulation of miR159,which later decreased (2 h after stress) [41]. Surprisingly, such a downregulation ofmiR159 was not seen in the tolerant cultivar (TAM107). Upregulated miR159appears to delay heading time by negatively regulating its target TaGAMYB inT. aestivum [52]. Similar differences in the magnitude of regulation of othermiRNAs was observed in heat-tolerant cultivar (TAM107) compared to susceptiblecultivar (Chinese Spring) of T. aestivum [41].miR398 levels were downregulated by more than 3-fold in B. rapa exposed to

heat stress [42]. miR398 mediates the upregulation of Cu/Zn-superoxidedismutase (SOD) expression in A. thaliana, which is important for decreasing

19.4 Heat Stress 511

oxidative stress levels [53]. A similar response of miR398 in B. rapa as observedin Arabidopsis is likely to upregulate the Cu/Zn-SOD levels and thereby provideprotection against oxidative stress caused by heat stress. However, miR398 levelswere found to be upregulated during heat stress in Arabidopsis [54] and inP. tomentosa [49].

Table 19.1 Differentially regulated miRNAs in response to climate change-associated stresses.

miRNA Target Heat Drought UV-B Ozone References

miR156 Squamosapromoter-binding protein(SBP)-like tran-scription factors

Tae",Bra"

Mtr¼ ,Tdo",Hvu"

Ath",Ptr"

Ath" [41,42,45,47,58,59]

miR159 MYB transcrip-tion factors

Tae" � Ath",Ptr #

Ath" [41,46]

miR160 Auxin responsefactors (ARF10,ARF16, ARF17)

Tae",Pto#

� Ath",Ptr"

� [41,46,47,49]

miR166 HD-ZIP tran-scription factors

Tae" Tdo#,Mtr,Hvu"leaf,Hvu# root

Ath",Ptr"

� [41,46,47,58,59]

miR167 Auxin responsefactors (ARF6,ARF8)

� Ath" Ath",Ptr"

� [44,46,47]

miR168 Argonaute-1(AGO1)

Tae",Pto#

Ath",Pvu"

� � [41,44,49]

miR169 NF-YA tran-scription factors

Tae",Pto#

Osa",Mtr,Hvu

Ath" � [41,48,57�59]

miR171 GRAS domainor SCL proteintranscriptionfactors

� Osa",Ath",Tdo#,Mtr¼ ,Hvu"

Ath" � [44�46,58,59]

miR172 AP2-like tran-scription factors

Tae# Osa# Ath" � [41,45,46]

miR319 TCP transcrip-tion factors

� � Ath" Ath" [46,48]

miR393 F-box protein(TIR1 andAFBs)

� Ath",Mtr¼

Ath",Ptr#

� [43,46,47,59]

miR398 Cu/Zn-SODs(CSD1, CSD2),copper chaper-one for SODs(CCS)

Ath" Ath#,Tdo",Mtr"

Ath",Ptr"

Ath"(initial),#(later)

[43,46�48,54,58,59]

Triticum dicoccoides, Tdo; Oryza sativa, Osa; Arabidopsis thaliana, Ath; Medicago truncatula, Mtr;Phaseolous vulgaris, Pvu; Populus trichocarpa, Ptc; Triticum aestivum, Tae; Brassica rapa, Bra;Populus tomentosa, Pto; Populus tremula, Ptr; Hordeum vulgare, Hvu; ", upregulated; #, down-regulated; ¼, no response.

512 19 Impact of Climate Change on MicroRNA Expression in Plants

miR156 levels were upregulated in response to heat and drought in wheat [41,49].miR156 is a highly conserved miRNA regulating the expression of SPL transcrip-tion factors. Overexpression of miR156 delays flowering initiation as well asaccumulates anthocyanin levels by negatively regulating its target genes [55,56].The accumulation of anthocyanin may provide tolerance against heat stress whiledelaying the reproductive phase and can help the plant to escape the unfavorablecondition, at least temporarily. Taken together, these studies revealed that miRNAscould play important regulatory roles in response to heat stress and some of theresponses can mediate adaptation to stress.

19.5

Drought

One of the major consequences of changing climate is the erratic and reducedprecipitation rates resulting in more frequent and intense drought spells. Droughtis known to adversely affect plant growth and development, including grain yield. Itaffects plant physiology, and induces molecular reprogramming at the transcrip-tional and post-transcriptional levels by regulating gene expression, therebyaltering biochemical and physiological processes.Drought-responsive miRNAs have been identified in several plants species, such

as A. thaliana, Oryza sativa, Triticum dicoccoides, Medicago truncatula, Phaseolousvulgaris, and P. trichocarpa [43�45,51,57�60]. Drought upregulated the expressionof miR393, miR397, miR402, miR167, miR168, miR171, and miR396, whereas itdownregulated the expression of miR398 in Arabidopsis seedlings [43,44]. Micro-array-based analysis of miRNAs in rice seedlings exposed to Polyethylene glycol(PEG)-mediated water deficit has identified upregulation of miR169f and miR169g,in roots, but not in shoots [57]. However, O. sativa plants exposed to drought hasidentified 11 downregulated miRNAs (miR170, miR172, miR397, miR408, miR529,miR896, miR1030, miR1035, miR1050, miR1088, and miR1126) and eightupregulated miRNAs (miR395, miR474, miR845, miR851, miR854, miR901,miR903, and miR1125) [45]. In T. dicoccoides, the ancestor of domesticated Triticumdurum, 13 drought-responsive miRNAs (miR1867, miR896, miR398, miR528,miR474, miR1450, miR396, miR1881, miR894, miR156, miR1432, miR166, andmiR171) from leaf and root tissues have been identified using a microarrayplatform [58]. Drought-tolerant and -sensitive genotypes of soybean showeddifferences in miRNA expression; miR166-5p, miR169f-3p, miR1513c, miR397a,b,andmiR-Seq13 levels were upregulated in sensitive genotypes, while these miRNAswere downregulated in tolerant genotypes [61].Upregulation of miR398a,b and miR408 as well as a transient downregulation of

miR169 and miR166 was observed in M. truncatula, a model leguminous plantexposed to drought stress [59]. In response to drought stress, P. vulgaris showedsignificant upregulation of miR2118, miR159.2, miRS1, miR1514a, and miR2119,and a moderate upregulation of miR168, miR395, miR397, miR399, miR403, andmiR408 [60]. The woody plant P. trichocarpa showed differential expression

19.5 Drought 513

miR171l�n, miR1445, miR1446a�e, and miR1447 during drought [51]. miR896was downregulated in T. dicoccoides leaf tissue after 4 h, but induced by 8 h of stress[58]. Similarly,O. sativa showed both up- and downregulation ofmiR896 dependingon the developmental stage and organ considered (abundantly downregulated ininflorescence) [45]. miR474 levels were upregulated in T. dicoccoides [58], and in O.sativa and P. trichocarpa under drought [45,51]. miR474 is thought to play a role inRNA processing and regulation of organelle gene expression [45,62].Drought caused upregulation of miR396 in A. thaliana, while it was down-

regulated in T. dicoccoides and O. sativa [44,45,58]. miR396 is known to target GRLtranscription factors. Upregulated miR396 downregulates GRL expression, and thisappears to decrease stomatal density and narrow the leaf blade, which couldcontribute towards drought tolerance [63]. However, the role of downregulatedmiR396 as observed in T. dicoccoides and O. sativa under drought needs furtherinvestigation. miR171 is induced by drought stress in A. thaliana, Solanumtuberosum, and P. trichocarpa, while it is downregulated in T. dicoccoides roots, butunaltered in M. truncatula. miR171 is known to target SCL (scarecrow-like)/GRAStranscription factors that are known to participate in various developmentalprocesses in plants [64].miR169 is downregulated in A. thaliana andM. truncatula, while it is upregulated

in O. sativa in response to drought. miR169-mediated induction of its targetNF-YA5 transcription factor is important for expression of number of droughtstress-responsive genes [65]. Transgenic plants overexpressing NF-YA5 showedreduced leaf water loss and were more resistant to drought [65].Drought triggers downregulation of miR398 in A. thaliana, while upregulation in

M. truncatula and T. dicoccoides [43,58,59]. In Arabidopsis, miR398 targets Cu/Zn-SOD (CSD1 and CSD2) and copper chaperone for SODs (CCS). Stress-inducedCSD1 and CSD2 enzymes are thought to provide protection against oxidative stressthat commonly occurs during a variety of biotic and abiotic stresses [38,53].

19.6

UV-B Radiation

A rise in chlorine and bromine compounds (such as chlorofluorocarbons) leadsto stratospheric ozone depletion, which increases penetration of UV-B radiation[66]. UV-B stress induces rapid alterations in gene expression (within 1 h ofexposure) [67,68]. miRNAs regulating gene expression in response to UV-Bstress have been reported in A. thaliana and Populus tremula [46,47]. Inresponse to UV-B radiation, P. tremula showed downregulation of 11 miRNAsand upregulation of 13 miRNAs as studied by using miRNA array [47].Although none of the computational predictions were confirmed experimentally,miR156/157, miR159/319, miR160, miR165/166, miR167, miR169, miR170/171,miR172, miR393, and miR398 were predicted as responsive to UV-B radiation inArabidopsis [46]. miR398 was found to be upregulated in P. tremula within 1 h ofexposure to UV-B radiation. UV-B exposure usually leads to oxidative stress;

514 19 Impact of Climate Change on MicroRNA Expression in Plants

upregulation of miR398 is in contrast to the downregulation observed underother oxidative stress conditions such as high Cu2þ, Fe3þ, methyl viologen, highlight, ozone fumigation, and salt stress [53,69].

19.7

Ozone

Some of the GHGs, such as CO and NOx, react with volatile organics, whichundergo photochemical oxidation to produce tropospheric ozone [3]. Althoughneeded in the stratosphere, ozone of the troposphere is a GHG and a primaryconstituent of smog. Ozone levels in the preindustrial era were 10�15 ppb inthe Northern hemisphere, which has currently risen to 35 ppb [70,71]. Ozoneenters the leaves through stomata, causes oxidative stress, decreases photo-synthesis, damages DNA, protein, and cell membranes, and thereby negativelyaffects plant productivity [72�76]. A recent study investigated the response ofmiRNAs in Arabidopsis under ozone stress, which recorded altered response of22 miRNAs [48]. Most of these miRNAs showed similar expression under UV-Bstress, indicating the activation of similar regulating pathways under both typesof stressors [48]. Such similarities in miRNA responses during ozone and UV-Bstress could be expected, as both stresses lead to the production of reactiveoxygen species (ROS) causing oxidative stress. Under oxidative stress, miR398mediates the upregulation of CSD gene expression, which is known to decreaseROS accumulation [53]. Therefore, downregulation of miR398 under ozone andUV-B stress [47,48,69] may induce CSD gene expression and thereby protect theplant against oxidative damage. The importance of miR398-mediated upregula-tion of CSDs can be explained by the fact that miR398 levels accumulate tonormal levels immediately after being relieved from ozone treatment [74]. OthermiRNAs like miR390, miR319, miR159, and miR156 showed upregulationwithin 1 h of ozone stress, and their cognate mRNA targets were downregulatedrapidly [54].

19.8

Conclusions and Future Directions

miRNAs regulate the expression of approximately 60% of the protein-coding genesin animals [77]. In plants, this number is extremely small (only about a couple ofhundred) compared to their animal counterparts [78�80]. However, the overallimpact of miRNA-regulated gene expression in plants cannot be underestimatedbecause the majority of the target genes are transcription factors such as SPLs(squamosa promoter-binding protein-like transcription factors), MYBs (MYB-domain containing transcription factors), ARFs (auxin response factors), TCPs(teosinte branched 1, cycloidea, and PCF (TCP)-domain protein family), NACs(NAM, ATAF1/2, and CUC2 domain-containing transcription factors), HD-ZIPs

19.8 Conclusions and Future Directions 515

(homeodomain leucine zipper family transcription factors), NF-YA subunits/CBFs(CAAT box-binding factors), SCLs, AP2 (apetala 2-like transcription factors), andGRFs (growth-regulating transcription factors) that are proven or predicted tofunction as critical regulators of plant growth and development. Additionally, theF-box proteins and E3 ligases such as TIR1 (transport-inhibitor response 1) andUBC24 that are implicated in targeted proteolysis; phosphate and sulfatetransporters such as PHO1 and AST68 and enzymes involved in sulfateassimilation (ATP sulfurylases such as APS1, APS3 and APS4); CSDs and CCS1that are required for detoxification of ROS as well as laccases and plantacyaninwhose roles are unclear, are also targeted by miRNAs in plants. Of all these targets,only CSDs have established roles in plant stress responses [43,53,81]. However, asdiscussed in this chapter, most miRNAs (both conserved as well as novel species-specific miRNAs) that appear to be critical for plant growth and development arealso altered during stress, implying an important role not only for CSDs (targetedby miR398), but also for diverse families of transcription factors (targeted byconserved miRNAs such as miR156, miR160, miR164, miR166, miR167, miR169,miR171, miR172, and miR396) in adaptation to stress conditions.On the basis of the above discussions, several inconsistencies were evident with

respect to miRNA regulation under a given stress in different plant species. It isreasonable to argue that some of the contrasting observations could be genuine,whereas some could be attributed to the age of the plant, tissue, duration, andseverity of stress treatment and how stress treatment is being imposed (e.g., fasterversus slow drying) are some of the possible causes [82,83]. Thus, more uniformand systematic studies are required to compare the expression profiles of miRNAsduring stress between different plant species.Undoubtedly, miRNAs play critical roles in response to heat, drought, UV-B, and

ozone stresses (i.e., the stresses that are likely to be associated with predictedclimate change). However, under natural environmental conditions all of the abovefactors causing climate change act in combination: a rise in ozone and CO2

together can cause warming, which in combination with drought coupled withoxidative stresses (UV-B radiation and ozone) will affect plants more severely thanthe effect of the individual stressor that have been investigated. Therefore, it isnecessary to carry out miRNA analysis in combinations of these stresses to mimicfuture climate changes. This analysis will certainly reveal the small RNA regulatorymechanisms that better assist the adaptability of plants under field conditions.Such knowledge has the potential to incorporate novel molecular mechanisms incrop plants that will sustain crop productivity in changing climates.

Acknowledgments

Research in R.S.’s laboratory is supported by NSF-EPSCoR award EPS0814361,Oklahoma Center for the Advancement of Science and Technology (OCAST), andthe Oklahoma Agricultural Experiment Station. VG and NSRK are thankful toUGC-DRS programme for financial assistance.

516 19 Impact of Climate Change on MicroRNA Expression in Plants

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