Small RNAs as big players in plant abiotic stress responses and nutrient deprivation

Small RNAs as big players in plant abiotic stress responses and nutrient deprivation

Ramanjulu Sunkar 1,Viswanathan Chinnusamy 2,Jianhua Zhu 3and Jian-Kang Zhu 3

1

Department of Biochemistry and Molecular Biology,246Noble Research Center,Oklahoma State University,Stillwater,OK 74078,USA 2

Water Technology Centre,Indian Agricultural Research Institute,New Delhi,India 3

Department of Botany and Plant Sciences and Center for Plant Cell Biology,2150Batchelor Hall,University of California,Riverside,CA 92521,USA

Abiotic stress is one of the primary causes of crop losses worldwide.Much progress has been made in unraveling the complex stress response mechanisms,particularly in the identi?cation of stress responsive protein-coding genes.In addition to protein coding genes,recently discovered microRNAs (miRNAs)and endogenous small interfering RNAs (siRNAs)have emerged as important players in plant stress responses.Initial clues suggesting that small RNAs are involved in plant stress responses stem from studies showing stress regulation of miRNAs and endogenous siRNAs,as well as from target predic-tions for some miRNAs.Subsequent studies have demonstrated an important functional role for these small RNAs in abiotic stress responses.This review focuses on recent advances,with emphasis on integ-ration of small RNAs in stress regulatory networks.Small RNAs are ubiquitous regulators of gene expression

Plant development,metabolism and stress responses,as well as a myriad of other functions,depend on the correct regulation of gene expression.This is achieved by multiple mechanisms,with perhaps the most important control being exerted at the level of transcription.However,post-transcriptional events also play a crucial role in regulating gene expression.The stability of mRNAs is regulated by a variety of signals acting on speci?c sequences within the RNAs.This regulation is often mediated by speci?c RNA-binding proteins (RBPs)that bind to elements in the untranslated regions (UTRs)of mRNAs and regulate the stability,translation or localization of the mRNA [1–3].The recent discovery of microRNAs (miRNAs,see Glossary)and small interfering RNAs (siRNAs)revealed another ubiquitous mode of post-transcriptional regulation.These small RNAs are known to silence genes post-transcription-ally by guiding target mRNAs for degradation or by repres-sing translation [4–8].The role of miRNAs in controlling developmental processes has been at the forefront of plant miRNA research.This is based in part on the fact that many

proteins (dcl1,hen1,hyl ,se and hst )required for miRNA generation and miRNA target genes (phb and ago1)were ?rst identi?ed through genetic screens for developmental defects [9–15].A comprehensive examination of miRNAs in plant development is provided by several excellent recent reviews [5–8,16–18].In this review,the emerging roles of miRNAs and endogenous siRNAs in plant stress responses are discussed.

Overview of the role of miRNAs in plant stress responses

Plants are sessile organisms that must endure stressful environments.A large proportion of plant genes are regulated by stresses such as drought,soil salinity and extreme temperatures [19–23].Of the many gene regulatory mechanisms such as transcriptional,post-transcriptional and post-translational regulation,transcriptional regula-tion is the most widely studied mechanism.The action of speci?c transcription factors that bind to conserved cis -acting promoter elements is well documented as a cause of changes in gene expression,particularly those induced by abiotic stress [20].Furthermore,post-transcriptional gene regulation under stress conditions has been documented before,although the underlying mechanism was not known [24–26].Considering the important roles of small RNAs in guiding post-transcriptional gene silencing,their involve-ment in stress-regulated gene expression seemed likely [27,28].The discovery that stress can regulate miRNA levels,coupled with the identi?cation of stress-associated genes as miRNA targets provided clues about the role of miRNAs in stress responses.Functional analyses have demonstrated that several plant miRNAs play vital roles in plant resistance to abiotic as well as biotic stresses [29–34].Understanding small RNA-guided stress regulatory networks should provide new tools for the genetic improve-ment of plant stress tolerance (Figure 1).Indeed,it has been shown recently that manipulation of miRNA-guided gene regulation can help to engineer plants that will be more stress-resistant [34].

miRNA and oxidative stress

Under normal conditions,plants maintain a delicate balance between reactive oxygen species (ROS)

production

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Corresponding authors:Sunkar,R.(rsunkar@https://www.sodocs.net/doc/1612779069.html, );Zhu,J.-K.(jian-kang.zhu@https://www.sodocs.net/doc/1612779069.html, ).

Available online 18June 2007.

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1360-1385/$–see front matter ?2007Elsevier Ltd.All rights reserved.doi:10.1016/j.tplants.2007.05.001

and scavenging.Plants have developed a highly sophis-ticated and ef?cient antioxidant system [20,22,35,36].How-ever,exposure to stress conditions such as drought,cold,salinity,high light and heavy metals results in the accumu-lation of excess ROS in plant cells.Superoxide radicals (O 2à)are the primary products of photo-reduction of dioxy-gen in Photosystem I (PSI)of chloroplasts.These reactive O 2àradicals need to be scavenged at the site of their synthesis to limit the generation of more toxic hydroxyl radicals.Cu-Zn superoxide dismutase 2(CSD2)is attached to the thylakoid of chloroplasts,which is also the site of superoxide generation and,thus,plays an important role in localized and immediate scavenging of superoxide radicals.SOD genes are induced under oxidative stress to meet the requirement for superoxide detoxi?cation [37,38].Nuclear run-on assays indicate that CSD2as well as CSD1transcripts are not induced at the transcriptional level during oxidative stress [34].Interestingly,the up-regulation of the two CSD genes was found to be dependent on changes in the miR398levels.miR398targets both the cytosolic CSD1and plastidic CSD2.Under normal growth conditions,the two closely related Cu-Zn SOD genes are transcribed but their mRNAs do not accumulate because

of

Figure 1.Two possible modes of small RNA-guided target gene regulations under abiotic stress and their impact on plant stress tolerance.Small RNAs that are positively regulated by stress might target negative regulators of stress tolerance for enhanced suppression.By contrast,small RNAs that are suppressed during stress likely target positive regulators of stress tolerance resulting in the accumulation of gene products.

Glossary

Argonaute proteins:Argonaute proteins are de?ned by the presence of PAZ and PIWI domains with slicer activity.The PAZ domain interacts with the 30end of single-stranded miRNA or siRNA and orienting and recognition of cleavage on the mRNA substrate.The PIWI (name originates from its initial ?nding in Drosophila,P element induced wimpy testis)domain is similar to an RNase H domain possessing slicer activity.Recent studies indicated that PAZ domains of Argonaute proteins interact directly with the 30ends of the small RNAs and PIWI,and the middle domains of Argonaute proteins with the 50end of small RNAs.

ASRP (Arabidopsis thaliana small RNA project,https://www.sodocs.net/doc/1612779069.html,/):a public resource developed exclusively for the Arabidopsis small RNAs by James Carrington’s laboratory.Arabi-dopsis small RNA sequencing data is deposited in the database.Dicer-like proteins:Dicer-like proteins are a class of RNase III endo-ribonucleases with two RNase III domains and a PAZ (Piwi Argonaut and Zwille)domain.The PAZ domain is an RNA-binding module found in Dicer-like proteins as well as Argonaute (Ago)proteins.Dicer excise the 20–24-nt small RNA duplexes with 2-nt 30overhangs,each strand bearing 50phosphate and 30hydroxyl termini either from single-stranded RNA adopting imperfect hairpin structure (primary miRNA transcript)or from long perfect dsRNAs formed as a result of RNA-dependent RNA polymerase activity or read-through transcrip-tion of inverted repeats or NAT pairs.

MicroRNA:the term short-temporal RNA (stRNA)had been used when this class of small RNA in Caenorhabditis elegans was described in 1993[90,91],but the term microRNA (miRNA)was only coined in 2001.MicroRNAs are genome-encoded $20–24-nucleotide (nt)duplex-structured small RNAs with 50-phosphate and 30-hydroxyl groups with 2-nt overhangs.These are excised by the cropping activity of Dicer-like 1(RNase III-like endoribonucleases)on the primary miRNA transcript (pri-miRNA)that can adopt an imperfect hairpin-like structure.One of the duplexes serves as a guide strand,which is referred to as miRNA (and the antisense strand to miRNA is known as miRNA*),and is selectively loaded onto RISC and serves as a sequence-speci?city determinant in recognizing the target tran-script.Based on complete or partial complementarity between miRNA and its target transcript,RISC can cause either target mRNA degradation or inhibit protein synthesis.Some of these miRNAs are evolutionarily deeply conserved whereas some others are lineage-speci?c and even species-speci?c.

MPSS (massively parallel signature sequencing,https://www.sodocs.net/doc/1612779069.html,/at/):a sequencing-based technology that identi?es short sequence signatures produced from a de?ned position within an mRNA.A modi?ed version of MPSS has been adapted to sequence plant small RNAs.

NATsiRNA:a class of endogenous siRNAs derived from dsRNAs formed by annealing of sense and antisense transcripts encoded by natural cis -antisense gene pairs.NATsiRNAs are capable of reg-ulating target mRNA expression at the post-transcriptional levels by guiding mRNA cleavage.

Natural-antisense transcripts (NATs):NATS formed from partial or complete overlapping genes on opposite strands of DNA from the same locus (cis -NATs)or from transcripts of distinct loci separate from their sense partners (trans -NATs).

RISC (RNA-induced silencing complex):RISC is a cytosolic complex with the Argonaute protein as a slicer and a 21-nt miRNA or siRNA as a guide molecule that can guide post-transcriptional gene silencing of the target transcript either by target mRNA degradation or by inhibiting the translation.

RITS (RNA-induced transcriptional silencing complex):an active RITS complex containing Argonaute protein and a 24-nt siRNA ser-ving as a guide molecule.RITS complex functions in the nucleus and has the ability to cause DNA and histone modi?cation leading to transcriptional gene silencing.

siRNA:‘small interfering RNA’or ‘short interfering RNA’that can be derived from the genome (as shown in plants)or exogenously supplied in the form of a long duplex of nucleic acids.siRNAs are a class of $20–24-nt small regulatory RNAs generated by the activity of a Dicer-like family of ribonucleases (DCL2,DCL3and DCL4in Arabidopsis )acting on a long double-stranded RNA with perfect

duplex structure.Upon processing,only the guide strand is loaded onto the RISC or RITS complex.RISC or RITS loaded with the siRNA is capable of recognizing homologous RNA or DNA sequences and function in silencing the gene expression either at the post-transcrip-tional level (PTGS)or at the transcriptional level (TGS).Both PTGS and TGS pathways are thought to be the genome surveillance mechanisms providing protection against viral infections,transposon proliferations or introgression of aberrant transgenes.Endogenous siRNAs have been classi?ed into at least three subclasses:repeat-associated siRNAs (rasiRNAs),trans -acting siRNAs (ta-siRNAs),and siRNAs derived from natural antisense transcripts (nat-siRNAs).

trans -acting short interfering RNAs (tasiRNA):tasiRNAs form a class of 21-nt regulatory small RNAs found only in plants.tasiRNAs are generated from non-coding RNA precursors that are initially targeted for cleavage by a miRNA.RNA-dependent RNA polymerase acts on the cleavage products and converts them into dsRNAs that are cleaved into 21-nt tasiRNAs.Thus,the accumulation of tasiRNAs is dependant on the components of both miRNA (Dicer-Like1,Argo-naute1,HYL1and HEN1)and siRNA pathways (RNA-dependent RNA polymerase 6and DCL4).tasiRNAs can guide cleavage of target mRNAs and regulate gene expression at the post-transcriptional level like plant miRNAs.

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miR398-guided cleavage.In response to oxidative stress, miR398is transcriptionally down-regulated to release its suppression of CSD1and CSD2genes[34](Table1).Thus, the down-regulation of miR398expression permits the accumulation of CSD1and CSD2mRNAs,which are important for plant stress resistance.Further insight into the role of miR398in regulating the CSD2gene has been obtained from transgenic plants carrying miR398-resist-ant mutations in the CSD2mRNA.These plants showed much improved tolerance to diverse abiotic stress con-ditions compared with transgenic plants carrying the normal,miR398-susceptible CSD2gene[34].

Given the important role of Cu-Zn superoxide dismutases (CSDs),particularly under stress conditions,it is intriguing to ask why plants have evolved a conserved negative regulatory mechanism on the expression of two of the three CSDs involving miR398.First,there are metabolic costs associated with CSDs mRNA synthesis.Second,there is also a cost associated with miR398-guided post-transcriptional silencing.This investment suggests that miR398-guided CSD1and CSD2regulation in plant cells is unlikely to be a futile process,and this regulation could be important for multiple reasons.MicroRNA398plays an important dual but opposite role during normal growth conditions and abiotic stress(Figure2).Both plant development and stress resistance pathways constitute a complex network of multiple pathways.Only genes encoding proteins in the right places at the right times at optimal

levels

Figure2.Model depicting a role for miR398under(a)normal growth conditions and(b)during abiotic stress in regulating the expression of CSD1and CSD2genes at the post-transcriptional level.miR398plays dual but opposite roles during normal growth conditions and abiotic stress.miR398is expressed in a spatial-and temporal-specific manner under normal growth conditions and regulates the expression of CSD1and CSD2transcripts.This miR398-guided regulation might be crucial for the expression of optimal CSD1and CSD2that in turn regulate the levels of superoxide or other ROS required for signaling.This is still relatively unknown,and is indicated by a question mark(?).During abiotic stress,miR398expression is suppressed and this relieves the negative regulation on two SOD genes resulting in the accumulation of CSD1and CSD2transcripts.The enhanced CSD1and CSD2transcripts contribute to enhanced detoxification of ROS,which can accumulate at high levels during stress.

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can determine normal growth and development or stress tolerance.Therefore,protein abundance needs to be tightly coordinated in both space and time,in both processes.The abundant expression of miR398in certain tissues under normal growth conditions suggests a role for miR398in superoxide and H2O2signaling[35]via regulation of the CSD1and CSD2genes.miR398-guided,tissue-speci?c post-transcriptional regulation plays a crucial role in specifying the temporal-and spatial-expression pattern of CSD1and CSD2(Figure2a).Although the precise physiological implication for the differential accumulation of CSD1and CSD2mRNAs in different tissues is not known,it is likely that some tissues require a high level of CSD1and CSD2 expression even under normal growth conditions.Knowl-edge about the physiological concentrations of ROS(super-oxide or H2O2)required for signaling should shed light on the signi?cance of miR398-guided post-transcriptional regulation under normal growth conditions.The role of miR398during stress conditions is to promote the up-regu-lation of two CSD genes(Figure2b).One could speculate that a change in the expression of a miRNA might be an ef?cient strategy for cells to coordinate changes in the expression of multiple target genes participating in the same pathway.Decreased miR398expression during stress enables both target(CSD1and CSD2)mRNA levels to increase so that they can participate in oxidative stress management.Another gene,cytochrome c oxidase subunit V is also targeted by miR398[27,28].Further studies are required to determine whether this gene is also part of the oxidative stress network in plants.

miRNAs and nutrient homeostasis

Inorganic phosphate(P i)is frequently a limiting factor for plant growth[39].Plants adapt to low P i soils in several ways:root growth and architecture are altered to access a larger soil volume;organic acids,phosphatases and nucl-eases are exuded to solubilize P i or release P i from organic sources;the capacity for P i uptake is increased;and internal P i is recycled[40–42].Although some components of P i starvation signaling in plants have been identi?ed,the overall pathway is still poorly understood[32].Recent?nd-ings implicate the involvement of miR399in P i starvation responses.The Arabidopsis genome encodes six MIR399 genes[27,28].miR399was predicted to target two genes belonging to different families:a phosphate transporter[28] and a putative ubiquitin conjugating enzyme(UBC24) possibly involved in protein degradation[27].Both target genes function in P i homeostasis.

miR399has multiple target sites on the50UTR of the UBC transcript[27].miR399levels increase dramatically in P i-deprived plants[29,30,32]but fall rapidly after the addition of P i[32].P i starvation strongly induces the expression of all six MIR399genes and this is rapidly reversed by P i addition[32].An inverse correlation between miR399and the UBC mRNA levels has been observed under low P i conditions and under P i-suf?cient conditions[29–32].Down-regulation of UBC under low P i conditions is important for the attenuation of primary root elongation,induction of high-af?nity P i transporters such as AtPT1,and acquisition of P i to maintain P i homeostasis [29,30].miR399overexpressing transgenic plants accumu-lated?ve to six times the normal P i level in shoots and showed P i toxicity symptoms that were phenocopied by a loss-of-function in the miR399target gene(i.e.the ubc (pho2)mutant)[30–32,43].pho2mutant plants are known to over-accumulate P i in the shoot.Recently,PHO2was cloned and found to be the UBC24gene targeted by miR399 [31,32].

Under P i-suf?cient conditions,miR399expression is suppressed and UBC24or PHO2is expressed and presum-ably participates in a ubiquitin or proteosome pathway that negatively regulates the expression of P i transporters and controls hormonal signaling for root growth regulation to prevent the overloading of P i.However,under limiting P i conditions,miR399is induced and represses UBC24or PHO2expression and,thus,alleviates the repression of P i transporter genes and alters root growth and architecture to maximize P i uptake.Thus,miR399plays a key role in maintaining P i homeostasis.The protein targets of the UBC24or PHO2-mediated ubiquitin or proteosomal path-way are not known,but might include transcriptional acti-vators of P i starvation responsive genes and components of the auxin and ethylene pathways that control root growth and development under P i stress.Interestingly,At4,a non-coding transcript with signi?cant sequence complementar-ity to miR399is also induced under low P i conditions[44]. The miR399complementary sequence in At4homologs is highly conserved in several plant species[44].A hairpin structure could not be predicted for the At4precursor sequences.It is not known whether there might be an interaction between miR399and this non-coding RNA. Recent analysis of the At4loss-of function mutant indicated that the At4non-coding RNA does indeed play a role in redistributing P i from shoots to roots under P i deprivation [44].It is possible that the At4non-coding transcript can modulate the activity of miR399by binding to the miRNA to prevent it from targeting the UBC24or phosphate trans-porter,or from being degraded.

Another example of possible involvement of miRNAs in nutrient homeostasis is miR395in sulfur starvation.For land plants,sulfur is an indispensable inorganic nutrient ranking in need next to N,P i and K.Sulfur is taken up by roots mainly as inorganic sulfate[45].Sulfate starvation leads to a range of physiological changes so that sulfate acquisition can be sustained to some extent and sulfate assimilation is suspended.This involves alterations in the expression levels of sulfate assimilation enzymes. MicroRNA395is represented by six loci arranged in two clusters in Arabidopsis[46].miR395targets ATP sulfur-ylases(APS1,APS3and APS4),enzymes that catalyze the ?rst step of the sulfur assimilation pathway[47].miR395 also targets AST68,a low-af?nity sulfate transporter (At5g10180,AtSULTR2;1)[48](Table1).AST68is impli-cated in the internal translocation of sulfate from roots to shoots[49,50].Thus,miR395potentially plays an import-ant role in coordinating changes in sulfate translocation and assimilation.miR395was induced under conditions of low sulfate,whereas APS1transcript levels decreased[28]. By contrast,APS1transcripts were abundantly expressed when miR395expression could not be detected under sulfur-suf?cient conditions[28].The functional signi?-cance of sulfate regulation of miR395and its targets

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AST68,APS1,APS3and APS4remain to be investigated. Nevertheless,the available evidence implies an important role for miR395in regulating sulfate homeostasis.

Other abiotic stress-regulated small RNAs and their target genes

High-through-put gene expression analysis of plants under abiotic stress indicated that several hundred genes have modulated expression[19–23].Some of these genes are up-regulated or induced,and others are down-regulated under stress conditions.The up-or down-regulation of genes appears to be dependent on their roles.MicroRNAs that are up-regulated by stress might down-regulate their tar-get genes,which might be negative regulators of stress tolerance(e.g.repressors of stress-responsive genes and genes involved in plant processes that are inhibited by stresses e.g.cell division and expansion);downregulation of miRNAs under stress might result in accumulation of their target gene mRNAs,which might positively regulate stress tolerance(Figure1).Sequence analysis of a stress-treated Arabidopsis small RNA library indicated that miR393was the most abundantly expressed miRNA based on the number of times it appeared[27].miR393was also found to be induced in response to?agellin[33]or Pseu-domonas syringae treatment[51](Table1).Stress-speci?c regulation of miRNAs was also observed,for example, miR319c is up-regulated by cold but not by dehydration, salt or ABA[27].Microarray data from Genevestigator (https://www.genevestigator.ethz.ch)[52]suggest that sev-eral miRNA target genes are altered under diverse stress conditions,although this information needs further scru-tiny because of differences in the growth conditions, duration of the treatment and age of the plants used.

The predicted target genes of miR393include four putative ubiquitin E3ligase SCF complex F-box proteins and a basic-helix–loop–helix family protein[27,28].One of the F-box family proteins(At4g03190)is identical to Glucose Repression Resistance-like protein1(GRR1).Ubiquitin E3 ligases confer substrate speci?city to the ubiquitin or26S proteosome pathway,which mediate regulated protein degradation and,thus,stress response and developmental processes.Protein degradation serves at least two physio-logical functions in plant stress responses(i.e.gene regula-tion and senescence).Most of the abiotic stresses induce senescence.Senescence of older leaves helps not only to supply the nutrients(mainly N)to young leaves and repro-ductive parts,but also minimizes water loss under drought stress.Furthermore,ubiquitination also regulates gene expression under stress.During cold acclimation,Inducer of CBF expression1(ICE1)protein is activated,inducing the expression of C-repeat(CRT)-binding factors(CBFs)and other transcription factors[53,54].The HOS1(high expres-sion of osmotically responsive genes1)protein,a ubiquitin E3ligase,negatively regulates cold responses through the ubiquitination of ICE1[55].The miR393target gene, At3G26810,a putative ubiquitin E3ligase,is downregu-lated by0.92,0.66,0.55and0.66-fold under drought,salt, cold and ABA,respectively,compared with non-stress con-ditions(Genevestigator response viewer data).Further-more,other miR393target genes coding for E3ubiquitin ligases At1g12820and At3g62980(an auxin receptor,trans-port inhibitor response1)showed downregulation under cold stress(Genevestigator response viewer https://www. genevestigator.ethz.ch).The Response Viewer tool provides gene expression data for control and stress conditions,that is,biotic and abiotic stresses,hormones and chemicals from several representative experiments[52].Studies of miR393 regulation of these E3ligases and their role in abiotic stress responses should provide insights into the mechanism of regulated proteolysis-mediated abiotic stress tolerance.

Energy and carbon requirements for growth and development are provided by sugars,the primary product of photosynthesis.Hence,many metabolic processes are regulated by sugar concentration,which is in turn in?uenced by abiotic stresses.The plant stress hormone, abscisic acid(ABA)plays a crucial role in cellular sugar budget-mediated regulation of plant growth and develop-ment[56].One of the miR393target genes,At4g03190, encodes an F-box protein that shows similarity to GRR1,a yeast protein involved in glucose repression.Genevestiga-tor response viewer data[52]suggest that At4g03190is down-regulated in response to cold and ABA.Investi-gations into the role of miR393and its target At4g03190 might shed light on sugar sensing in plants under stress.

The levels of several poplar miRNAs(miR156,miR162, mi164,mi475,mi480and mi481)declined under mechanical stress conditions[57].Interestingly,some of these miRNAs are poplar-speci?c and are induced under stress conditions whereas some other miRNAs such as miR156,miR162and miR164are associated with development.The functional roles of these putative mechanical stress-responsive miR-NAs remain to be determined experimentally.

miRNAs and growth and development under abiotic stresses

Crop yield under abiotic stresses depends not only on the mere survival of plants under stress conditions but also on the phenological and developmental plasticity of plants. Under abiotic stress conditions,plants adjust the durations of phenological phases,and the rate of developmental pro-cesses,which modify biomass and harvest index.Changes in the duration of various phenological phases(e.g.vegetative phase,days to?owering and grain development duration) help plants to avoid critical growth phases under stress conditions.Tolerant genotypes often enhance their growth rate to compensate for the reductions in phenological durations.Reproductive development appears to be the phase of crop development that is the most susceptible to abiotic stresses given that any damage at this stage is irrecoverable.Drought stress reduces days to?owering in wheat[58]but delays?owering in rice[59].In maize, drought stress increases anthesis to silking interval[60]. Reduction in reproductive organ number and size helps plants to use the available resources ef?ciently so that some viable healthy seeds are produced.However,to date the molecular basis of phenological and developmental plasticity under abiotic stress is poorly understood.Inter-estingly,our analysis of microarray data from Genevesti-gator[52]suggests that many of the miRNA target genes involved in growth and development are stress-regulated as well.Extensive molecular,physiological and even anatom-ical changes take place in plants in response to a stressful

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environment such that plants under a speci?c stress condition might be viewed as entering a particular developmental phase.MicroRNA160,miR164and their target genes are involved in the regulation of root growth. Roots play a pivotal role in the acquisition and transport of water and nutrients,and root-based hormonal signal(ABA) is an important determinant of stomatal responses.

Under conditions of drought,roots can adapt to continue growth to acquire water and nutrients from deep soil layers.Overexpression of miR160,which targets auxin response factors(ARFs)resulted in agravitrophic roots and increased the number of lateral roots,whereas over-expression of miR160-resistant ARF16resulted in reduced lateral roots and reduced fertility[61].By contrast,trans-genic Arabidopsis overexpressing miR164,which targets NAC transcription factors,exhibited reduced lateral roots, whereas overexpression of miR164-resistant NAC1 resulted in increased number of lateral roots[62].

Leaf development is also regulated by miRNAs.Leaf development determines the source size(photosynthetic area)and area for transpiration and,hence,regulation of leaf development is crucial for abiotic stress tolerance. Plants overexpressing miR159-resistant MYB33exhib-ited reductions in size,petiole length,apical dominance and fertility,and had round leaves[63].Transgenic Ara-bidopsis overexpressing miR160-resistant ARF17had extra cotyledons,leaf defects,extra petals and reduced fertility[64].Similarly,overexpression of miR164-resist-ant CUC1,miR164-resistant CUC2,miR165or miR166-resistant PHB and miR165or166-resistant REV in transgenic Arabidopsis resulted in leaf polarity defects [65–67].

The reproductive phase is also sensitive to abiotic stresses.Plants modulate?owering time and?ower num-ber(sink size)in response to abiotic stresses.Overexpres-sion of some miRNAs results in alterations of?owering time.Overexpression of miR156,which targets the SPL family of transcription factors,showed enhanced leaf initiation,decreased apical dominance and delayed?ower-ing time[68].Similarly transgenic Arabidopsis overexpres-sing gibberellin-regulated miR159and miR319showed a delay in?owering time[69,70].By contrast,overexpression of miR172,which targets AP2-type transcription factors, resulted in early?owering[71,72].In addition to the time of?owering,fertility is highly affected by abiotic stresses. Transgenic overexpression of miR159and miR166resulted in enhanced male sterility and female sterility,respect-ively,under non-stress conditions[70,73].

siRNAs and stress responses

Endogenous siRNAs are synthesized from long double-stranded RNAs(dsRNAs).The endogenous sources of dsRNAs are:(i)miRNA-directed cleavage products of non-coding transcripts,which are then converted into dsRNAs by RNA-dependent RNA polymerases(RDRs);(ii)dsRNAs formed from the mRNAs encoded by natural cis-antisense gene pairs[74];and(iii)dsRNAs generated from hetero-chromatin and DNA repeats[8].The siRNAs produced by miRNA-directed cleavage of mRNAs are referred to as trans-acting siRNAs(ta-siRNAs);the siRNAs derived from dsRNAs formed from the mRNAs encoded by natural cis-antisense gene pairs are called natural anti-sense transcript-derived siRNAs(nat-siRNAs).RDRs and DCL-like proteins process the dsRNAs formed from different sources.The biogenesis of different classes of siRNAs is carried out by speci?c RDR–DCL combinations [7,8].

ta-siRNAs biogenesis begins with miRNA-directed cleavage of target mRNAs and these cleaved single-stranded RNAs are recognized by SUPPRESSOR OF GENE SILENCING3(SGS3,At5g23570),a coiled-coil protein with a zinc?nger domain,followed by synthesis of the complementary RNA strand by RDR6(At3g49500= SUPPRESSOR OF GENE SILENCING2,SILENCING DEFECTIVE1).These dsRNAs are then cleaved by DCL4to produce21nt ta-siRNAs[75–77].

Genome analyses have revealed thousands of genes in convergent overlapping pairs that can generate comp-lementary transcripts[78–80].In addition,various expres-sion pro?ling approaches showed widespread antisense transcription throughout genomes in plants[81,82].From overlapping genes on opposite strands of DNA,cis-natural antisense transcripts(NATs;endogenous coding or non-protein-coding RNAs with sequence complementarity to other transcripts)are generated[74].The biogenesis of nat-siRNAs begins with the formation of dsRNAs by annealing sense and antisense transcripts.These dsRNAs are processed by DCL2,RDR6,SGS3and a plant-speci?c RNA polymerase,NRPD1A,to generate a24-nt nat-siRNA,which then directs the biogenesis of21-nt nat-siRNAs by DCL1[74].The third type of siRNAs(24nt) is generated by DCL3,RDR2and NRPD1A by processing RNAs from transposons,5S rRNA genes and other repeats [8,83].

Work on the founding member of nat-siRNAs,which is derived from a cis-NAT gene pair of SRO5and P5CDH genes,demonstrated an important role of nat-siRNAs in osmoprotection and oxidative stress management under salt stress in Arabidopsis[74](Table1).Salt stress-induced SRO5mRNA complements the P5CDH mRNA to produce a dsRNA,which is processed by a siRNA biogenesis pathway requiring DCL2,RDR6,SGS3and NRPD1A to produce a24-nt nat-siRNA.The24-nt nat-siRNA guides the cleavage of the P5CDH transcript to further produce21-nt nat-siRNAs by DCL1.These nat-siRNAs all guide cleavage of P5CDH mRNAs,suppressing proline degradation and,thus,allowing proline accumu-lation.Downregulation of P5CDH also causes P5C-mediated ROS accumulation.The SRO5protein is tar-geted to mitochondria,the site of proline catabolism[74]. SRO5is similar to RADICAL INDUCED CELL DEATH1 (RCD1),which prevents ROS-induced cell death,given that rcd1plants are hypersensitive to ROS-induced cell death[84].High salt stress causes accumulation of H2O2, and both salt and H2O2induce the expression of SRO5. sro5mutant plants also showed hypersensitivity to ROS (H2O2).These?ndings suggest that ROS detoxi?cation under salt stress is mediated by the SRO5protein[74]. Thus,the SRO5-P5CDH nat-siRNAs together with the P5CDH and SRO5proteins form an important regulatory loop controlling proline and ROS production and stress tolerance[74].

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Conclusions and outlook

Extensive efforts over the past two decades have identi?ed thousands of stress-regulated genes.With the recent identi?cation of miRNAs and siRNAs as components of stress response,another level of gene regulation has been revealed.The evidence thus far suggests important roles for these small RNAs in stress response.The extent of small RNA involvement in abiotic stress response should become clear in the next several years if suf?cient effort can be directed to these studies in Arabidopsis as well as in crop plants.

Most miRNAs show dynamic expression patterns.Some miRNAs are expressed only during certain developmental stages or tissues,whereas others are responsive to hor-mones,nutrient deprivation or other abiotic stresses [11,27,28,34,85].These observations imply that miRNA expression is controlled at the level of transcription.How-ever,it is likely that miRNA expression can also be con-trolled at the level of processing and stability,like other RNAs.An important question is how is miRNA expression controlled at the transcriptional level in response to environmental or developmental changes?Connecting miRNAs to both upstream and downstream events will place them within the regulatory networks that govern diverse physiological processes.A recent study indicated that PHOSPHATE STARVATION RESPONSE1(PHR1), a MYB-like transcription factor,is one of the factors responsible for miR399induction in response to P i depri-vation because the induction of miR399is decreased but not completely blocked in the phr1mutant under low phosphate conditions[32].Future studies aimed at dis-secting the promoter elements of the miRNA genes should shed light on transcriptional regulation of miRNA genes.

NAT-siRNAs have recently emerged as important players in plant stress responses.A pair of natural anti-sense transcript(NAT)genes can give rise to both24nt and 21nt siRNAs in response to salt stress and are important for salt tolerance in Arabidopsis[74].A nat-siRNA that is speci?cally induced by the bacterial pathogen Pseudomo-nas syringae carrying the avirulence gene avrRpt2plays an important role in RPS2-mediated race-speci?c disease resistance[86](Table1).Recent genome-wide analyses have revealed the widespread existence of NATs in eukar-yotic genomes[78–80].NATs might have the potential to generate nat-siRNAs for gene regulation under various stress conditions.This hypothesis is well supported by the presence of several hundred potential nat-siRNAs in small RNA databases,including the MPSS[87]and ASRP databases[88].The current ASRP and MPSS datasets were generated from untreated plants only[89].We believe that many more nat-siRNAs will be discovered in the future by using stress-treated samples.Further studies of stress-regulated miRNAs and siRNAs and their target genes in plants should identify new components in plant stress resistance pathways and help to elucidate the com-plex regulatory network underlying plant stress responses.

Many genes have been shown to confer marginal improvements in stress tolerance when overexpressed in transgenic plants[22].A combination of previously reported as well as novel approaches will be needed to increase plant abiotic and biotic stress resistance to levels high enough for ?eld application in crops.Manipulation of small RNA-guided gene regulation represents a novel and feasible approach to improve plant stress tolerance[34].

In summary,an understanding of post-transcriptional gene regulation by small RNAs under abiotic stress is crucial for understanding and improving stress tolerance in crop plants.In silico identi?cation of miR395,miR398 and miR399homologs in about two dozen diverse plant species suggests that these miRNAs are conserved across species boundaries.Conservation of these miRNAs implies that they have conserved biological functions.Appropriate manipulation of miRNA target genes should help overcome post-transcriptional gene silencing and,thus,might lead to better expression of engineered traits in transgenic plants. Acknowledgements

The work in R.S.’s laboratory is supported by the Oklahoma Agricultural Experiment Station and OCAST,and the work in J-K.Z.’s laboratory is supported by the National Institutes of Health grants R01GM0707501 and R01GM59138.

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