Climate Change and Plant Abiotic Stress Tolerance || Abiotic Stress-Responsive Small RNA-Mediated Plant Improvement Under a Changing Climate

18

Abiotic Stress-Responsive Small RNA-Mediated Plant

Improvement Under a Changing Climate

Basel Khraiwesh and Enas Qudeimat

Abstract

Small, non-coding RNAs are a distinct class of regulatory RNAs in plants andanimals that control a variety of biological processes. In plants, several classes ofsmall RNAs with specific sizes and dedicated functions have evolved through aseries of pathways. The major classes of small RNAs include microRNAs(miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis.miRNAs control the expression of cognate target genes by binding to reversecomplementary sequences, resulting in cleavage or translational inhibition of thetarget RNA. siRNAs have a similar structure, function, and biogenesis as miRNAs,but are derived from long double-stranded RNAs and can often direct DNAmethylation at target sequences. Environmental stress factors such as drought,elevated temperature, salinity, and rising carbon dioxide (CO2) levels affect plantgrowth and pose a growing threat to sustainable agriculture. This has become a hotissue due to concerns about the effects of climate change on plant resources,biodiversity, and global food security. Besides the roles of small RNAs in growth,development, and maintenance of genome integrity, small RNAs are alsoimportant components in plant stress responses. One way in which plants respondto environmental stress is by modifying their gene expression through the use ofsmall RNAs. Thus, understanding how small RNAs regulate gene expression willenable researchers to explore the role of small RNAs in abiotic stress responses foradapting to climate change. Here, we present an overview of small RNA-mediatedplant improvement under a changing climate.

18.1

Introduction

Small non-coding RNAs, which consist of 20–24 nucleotides, have beenincreasingly investigated as important regulators of gene expression; thesesmall RNAs function by causing either transcriptional gene silencing (TGS) or

481

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.

post-transcriptional gene silencing (PTGS) [1]. They were first reported in thenematode Caenorhabditis elegans [2] and are responsible for the phenomenonknown as RNA interference (RNAi), co-suppression, gene silencing, or quelling[3–6]. Shortly after these reports were published, researchers demonstrated thatPTGS in plants is correlated with the activity of small RNAs [7]. These smallRNAs regulate various biological processes, often by interfering with mRNAtranslation. In plants, two main categories of small regulatory RNAs aredistinguished based on their biogenesis and function: microRNAs (miRNAs)and small interfering RNAs (siRNAs). Recently, miRNAs and siRNAs havebeen shown to be highly conserved, important regulators of gene expression inboth plants and animals [8,9]. The modes of action by which small RNAscontrol gene expression at the transcriptional and post-transcriptional levelsare now being developed into tools for molecular biology research. Oneimportant goal of this study is determining how stress affects small RNAs andhow small RNAs, in turn, regulate plant responses to stress for adapting toclimate change.Regarding climate change, the Intergovernmental Panel on Climate Change

(IPCC) asserts that, on average, global temperatures will increase worldwide by0.2 �C per decade. There will be changes in rainfall regimes representing both anincrease and decrease in precipitation, and an increased frequency of droughts andfloods. Climate change will also have an impact on production losses, which couldworsen hunger in developing countries beyond the current 1 billion that are alreadygoing hungry. Many agricultural resources will become more threatened over timeas global climate change will erode genetic diversity and destabilize foodecosystems significantly. Under these circumstances, there is a need to improvecrops by means of genetic modification for food safety and to sustain agriculture. Apowerful technology is to suppress the expression of targeted genes using RNA-mediated gene suppression.Plants have evolved sophisticated mechanisms to cope with a variety of

environmental stresses. Many plant genes are regulated by stresses such asdrought, salt, cold, heat, light, and oxidative stress [10,11]. Recent evidenceindicates that plant miRNAs and siRNAs play a role in biotic and abiotic stressresponses [12]. The first indication for such roles came from bioinformatics/insilico analysis of miRNAs and their target genes, and cloning of miRNAs fromstress-treated Arabidopsis plants, which revealed new miRNAs that had not beenpreviously cloned from plants grown in unstressed conditions [13,14]. Under-standing small RNA-guided stress regulatory networks can provide new insightsfor the genetic improvement of plant stress tolerance. Many studies have alsorevealed complexity and overlap in plant responses to different stresses, andunderstanding this complexity and overlay will likely lead to new ways to enhancecrop tolerance to disease and environmental stress. Researchers recently demon-strated that manipulation of miRNA/siRNA-guided gene regulation can help in theengineering of stress-resistant plants [15,16]. Here, we consider what is currentlyknown and not known about the roles of miRNAs and siRNAs in plant abioticstresses for adapting to climate change.

482 18 Abiotic Stress-Responsive Small RNA-Mediated Plant Improvement Under a Changing Climate

18.2

Classes of Small RNAs

Independent approaches combining traditional cloning, computational predic-tion, and high-throughput sequencing of small RNA libraries have identifiedseveral classes of small RNAs with specific sizes and functions in plants. Theseclasses of small RNAs include miRNAs, repeat-associated siRNAs (ra-siRNAs),natural antisense transcript-derived siRNAs (nat-siRNAs), trans-acting siRNAs(ta-siRNAs), heterochromatic siRNAs (hc-siRNAs), secondary transitive siRNAs,primary siRNAs, and long siRNAs (lsiRNAs) [17–19]. The biogenesis andfunctions of most of these small RNA classes have been well-characterized inthe model plant Arabidopsis thaliana. In general, small RNAs are generatedfrom at least partially double-stranded RNA precursors by the action ofribonuclease III-like Dicer proteins (Dicer-like (DCL)) [20]. The small RNAduplexes generated by DCL activity have a characteristic 2-nucleotide overhangat the 30-end because of an offset cutting of the DCLs. In plants, these30-overhangs are stabilized by 20-O-methylation [21]. Generation of lsiRNAsdepends on the DCL and AGO (argonaute) subfamily protein AGO7. Thisdiffers from the generation of the 25- to 31-nucleotide animal PIWI-interactingRNAs, which are independent of the Dicer and AGO subfamily proteins. Onlyone strand of the processed small RNA duplex subsequently associates with anRNA-induced silencing complex (RISC) that scans for nucleic acids complemen-tary to the loaded small RNA to execute its function [22,23]. In plants, smallRNAs act in gene silencing by mediating RNA slicing [24], translationalrepression [25], and histone modification and DNA methylation [26,27]. Thefirst two mechanisms control gene expression post-transcriptionally, whereasthe later affects gene expression at the transcriptional level.

18.2.1

miRNAs

18.2.1.1 Biogenesis of miRNAs

miRNAs are small RNAs of 20–22 nucleotides that are encoded by endogenousMIR genes. Their primary transcripts form precursor RNAs, which have a partiallydouble-stranded stem–loop structure and which are processed by DCL proteins torelease mature miRNAs [23]. In the miRNA biogenesis pathway, primary miRNAs(pri-miRNAs) are transcribed from nuclear-encoded genes by RNA polymerase II[28], leading to precursor transcripts with a characteristic hairpin structure (Figure18.1a). In plants, the processing of these pri-miRNAs into pre-miRNAs is catalyzedby DCL1 and assisted by HYL1 (hyponastic leaves 1) and SE (serrate) proteins [23].The pre-miRNA hairpin precursor is finally converted into 20- to 22-nucleotidemiRNA/miRNA� duplexes by DCL1, HYL1, and SE. The duplex is then methylatedat the 30-terminus by HEN1 (HUA enhancer 1) and exported into the cytoplasm byHST1 (hasty), an exportin protein [29,30]. In the cytoplasm, one strand of theduplex (the miRNA) is incorporated into an AGO protein, the catalytic component

18.2 Classes of Small RNAs 483

of RISC, and guides RISC to bind to cognate target transcripts by sequencecomplementarity (Figure 18.1a). In addition to the control of targets at the post-transcriptional level, miRNAs regulate gene expression by causing epigeneticchanges such as DNA and histone methylation [26,31].Functional analysis of conserved miRNAs revealed their involvement in multiple

biological and metabolic processes in plants. They regulate various aspects ofdevelopmental programs: including auxin signaling, meristem boundary formationand organ separation, leaf development and polarity, lateral root formation,transition from juvenile to adult vegetative phase and from vegetative to floweringphase, floral organ identity, and reproduction. They also regulate plant responses tostress and the miRNA pathway itself (Table 18.1).

Figure 18.1 Biogenesis and function of

miRNAs and ta-siRNAs. (a) MIR genes are

initially transcribed by RNA polymerase II

into a single-stranded RNA that folds back to

form a hairpin structure (also called pri-

miRNA) thought to be stabilized by the RNA-

binding protein DDL (dawdle). Splicing and

further processing in nuclear dicing bodies

involves the interactive functions of HYL1

and SE and of the cap-binding proteins (CBP)

CBP20 and CBP80. Pri-miRNAs and pre-

miRNAs are generally thought to be

processed from the free-end opposite to the

loop by DCL1 to yield one or several phased

miRNA/miRNA� duplexes. These are then

methylated by HEN1 and transported to the

cytoplasm by HST1. The miRNA guide strand

is selected, incorporated, and stabilized in

dedicated AGO1 protein. miRNA-guided

AGO1-containing RISC directs mRNA

cleavage or translation inhibition of the target

transcript. (b) ta-siRNA pathway. TAS genes

are transcribed by RNA polymerase II into

TAS precursors harboring two miR390/one

miR173 binding sites. After TAS precursor

cleavage at bothmiR390 andmiR173 sites the

middle cleavage product is converted into

double-stranded RNA by RDR6 and

subsequently processed into phased ta-

siRNAs by a DCL4 protein. ta-siRNAs are

loaded into RISC where they act like miRNAs.


Table 18.1 Role of conserved plant miRNAs.

Role miRNA

family

Target genes Reference(s)

Auxin signaling miR160miR164miR167miR390miR393

ARF10NAC1ARF8ARFTIR1/F-box AFB

[108,109][118][108][91][13,110]

Leaf development miR159miR164miR166miR172miR319

MYBNAC1HD-ZIPIIIAP2TCP

[43,113,114][119][120][113][114]

Leaf polarity miR166miR168miR390

HD-ZIPIIIAGO1ARF

[107,120][106][91]

Floral organ identity miR160miR164miR172miR319

ARF10NAC1AP2TCP

[108,109,113,114][119,121][122][113,114]

Flowering time miR156miR159miR172miR319

SBPMYBAP2TCP

[111---113][43,113,114][113,121][113,114]

Responses to stress toadapt to climate change

miR156miR159miR160miR167miR168miR169miR171miR319miR393miR395miR396miR397miR398miR399miR408

SBPMYBARF10ARF8AGO1NF-Y/MtHAP2-1SCLTCPTIR1/F-box AFBAPS/ASTGRFLaccases, b-6-tubulinCSDUBC24/PHO2Plastocyanin

[33,37,38][14,33,37,43,44][33,45][33,37][33][33,37,47,124][33,37][14,33,37][13,14,33,37][13,14,33][14,33][13,14,33][13,16,33,37,48,66][33,69,70][14,33,38]

Regulation of miRNA miR162miR168miR403

DCL1AGO1AGO2

[125][106][91]

Others miR158miR161miR163miR173miR174miR175miR394

At1g64100PPRAt1g66700, At1g66690At3g28460At1g17050At5g18040, At3g43200,

At1g51670F-box


18.2.1.2 Role of miRNAs in Plant Stress Responses for Adapting to Climate Change

Environmental stress causes plants to over- or underexpress certain miRNAs or tosynthesize new miRNAs to cope with stress. Several stress-regulated miRNAs havebeen identified in model plants under various abiotic stress conditions, includingnutrient deficiency [32], drought [33–35], cold [36], high salinity [33], UV-B radiation[37], and mechanical stress [38]. In a recent report, the levels of 117 miRNAs underhigh-salinity, drought, and low-temperature conditions were analyzed usingmiRNA chips representing nearly all known miRNAs identified in Arabidopsis [33].Seventeen stress-inducible miRNAs were detected, and the results were confirmedby detecting their expression patterns and analyzing the cis-regulatory elements intheir promoter sequences [33]. Jones-Rhoades and Bartel [13] identified novelArabidopsis miRNAs that were predicted to target genes such as superoxidedismutases (SODs), laccases, and ATP sulfurylases (APSs). The expression of oneparticular miRNA (miR395) was increased upon sulfate starvation, showing thatmiRNAs can be induced by environmental factors and not only by developmentalprocesses. miR395 targets the genes that encode APS1, APS3, and APS4. Theseenzymes catalyze the first step of inorganic sulfate assimilation [13,14]. Sunkar andZhu [14] constructed a library of small RNAs from Arabidopsis seedlings exposed todifferent abiotic stresses including cold, dehydration, high salt, and abscisic acid(ABA), and identified several new miRNAs that are responsive to abiotic stress [14].For example, miR393 was upregulated by cold, dehydration, high salinity, and ABAtreatments, miR397b and miR402 were slightly upregulated by general stresstreatments, while miR319c was induced by cold but not by the other treatments;miR389a, however, was downregulated by all of the stress treatments. The resultsindicated that stress-induced miRNAs target negative regulators of stress responsesor positive regulators of processes that are inhibited by stresses and that several ofthe newly identified miRNAs exhibit tissue- or developmental stage-specificexpression patterns. More recently, genome-wide profiling and analysis of miRNAswas carried out in drought-challenged rice [39]. Lu et al. [38] also identified 48miRNA sequences from the Populus genome, and found that most of these PopulusmiRNAs target developmental and stress/defense-related genes. The authors alsofound that plant miRNAs can be induced by mechanical stress and may function incritical defense systems for structural and mechanical fitness [38].A number of miRNAs have been linked to biotic stress responses in plants, and

the role of these miRNAs in plants infected by pathogenic bacteria, viruses,nematodes, and fungi has been reported [40]. Moreover, miRNAs are alsoimportant in regulating plant–microbe interactions during nitrogen (N) fixation byRhizobium and tumor formation by Agrobacterium [40]. Additionally, Mishra et al.[41] observed a significant increase in the GC content of stress-regulated miRNAsequences, which further supports the view that miRNAs act as ubiquitousregulators under stress conditions. GC content may also be considered a criticalparameter for predicting stress-regulated miRNAs in plants [41].

miRNAs Involved in ABA-Mediated Stress Responses The phytohormone ABA isinvolved in plant responses to environmental stresses. The first indication that


miRNAs may be involved in ABA-mediated responses came from observations ofABA hypersensitivity in an Arabidopsis mutant containing a “pleiotropic recessiveArabidopsis transposon insertion mutation,” hyl1 [42]. Recently, two researchgroups independently found that either ABA or gibberellin treatment regulatedmiR159 expression [14,43] and controlled floral organ development [43]. Ingerminating Arabidopsis seeds, miR159 was upregulated in ABA-treated seedlings[44]. Sunkar and Zhu [14] reported that the expression of miR393, miR397b, andmiR402 was upregulated by ABA treatment. In contrast, miR389a appears to bedownregulated by ABA [14]. Other studies in Arabidopsis have also reportedupregulation of miR160 [45] and miR417 [46], and downregulation of miR169 [47]and miR398 [48] in response to ABA. In rice, miR319 was upregulated, whereasmiR167 and miR169 were downregulated in ABA-mediated responses [49]. InPhaseolus vulgaris, miR159.2, miR393, and miR2118 were induced under ABAtreatments, whereas miRS1, miR1514, and miR2119 were moderately upregulatedin response to ABA [50]. The dependence of DNA methylation on miRNA levelswas also shown for an ABA-responsive PpbHLH-miR1026 regulon in Physcomitrellapatens. ABA application caused an increase of miR1026 and a decrease of itsPpbHLH target RNA [26].

miRNAs in Response to Drought Stress Studies with Arabidopsis have revealed ageneral role for miRNAs in drought responses. Several stress-related miRNAs wereidentified based on the sequencing of a library of small RNAs isolated fromArabidopsis seedlings exposed to various stresses [14]. Recently, miRNAs such asmiR168, miR171, and miR396 were found to be responsive to high salinity,drought, and cold stress in Arabidopsis, thus supporting the hypothesis of a role formiRNAs in the adaptive response to abiotic stress [33]. miRNA-expression profilingunder drought stress has now been performed in Arabidopsis, rice, and Populustrichocarpa. In Arabidopsis, miR396, miR168, miR167, miR165, miR319, miR159,miR394,miR156,miR393,miR171,miR158, andmiR169 were shown to be droughtresponsive [33]. The upregulation of miR393, miR319, and miR397 in response todehydration in Arabidopsis has been reported [14]. In rice, miR169g was stronglyupregulated while miR393 was transiently induced by drought [34]. Genome-wideprofiling and analysis of miRNAs was carried out in drought-challenged rice acrossa wide range of developmental stages, from tillering to inflorescence formation,using a microarray platform [39]. The results indicated that 16 miRNAs (miR156,miR159, miR168, miR170, miR171, miR172, miR319, miR396, miR397, miR408,miR529, miR896, miR1030, miR1035, miR1050, miR1088, and miR1126) weresignificantly downregulated in response to drought stress. Conversely, 14 miRNAs(miR159, miR169, miR171, miR319, miR395, miR474, miR845, miR851, miR854,miR896, miR901, miR903, miR1026, and miR1125) were significantly upregulatedunder drought stress. Some miRNA gene families, such as miR171, miR319, andmiR896, were identified in both down- and upregulated groups, and nine miRNAs(miR156, miR168, miR170, miR171, miR172, miR319, miR396, miR397, andmiR408) showed opposite expression to that observed in drought-stressedArabidopsis [39]. In Populus, miR171l–n, miR1445, miR1446a–e, and miR1447 were


found to be drought responsive [51]. In P. vulgaris,miRS1,miR1514a, and miR2119showed a moderate but clear increase in accumulation upon drought treatment,while the accumulation was greater by miR159.2, miR393, and miR2118 inresponse to the same treatment [50]. In Medicago truncatula, miR169 wasdownregulated only in the roots, and miR398a,b and miR408 were stronglyupregulated in both shoots and roots under drought stress [52]. More recently,miRNA expression patterns of drought-resistant wild emmer wheat (Triticumturgidum ssp. dicoccoides) in response to drought stress were investigated using aplant miRNA microarray platform [53]. Expression levels were detected for 205miRNAs in control and 438 miRNAs in drought-stressed leaf and root tissues. Thefollowing 13 miRNAs were found to be differentially regulated in response todrought stress: miR1867, miR896, miR398, miR528, miR474, miR1450, miR396,miR1881, miR894, miR156, miR1432, miR166, and miR171 [53]. Similarly, miR474,which targets proline dehydrogenase, was recently shown to be upregulated duringdrought stress in maize [54].

miRNAs in Response to Salt Stress Approximately 6% of the total arable land isaffected by excess salt [55]. Numerous genes and pathways in plants are affected bysalt stress [11]. Several differentially regulated miRNAs have been identified in salt-stressed plants. In Arabidopsis, miR156, miR158, miR159, miR165, miR167,miR168, miR169, miR171, miR319, miR393, miR394, miR396, and miR397 wereupregulated in response to salt stress, while the accumulation of miR398 wasdecreased [33]. In P. vulgaris, Arenas-Huertero et al. [50] observed increasedaccumulation of miRS1 and miR159.2 in response to NaCl addition. In Populustrichocarpa, miR530a, miR1445, miR1446a–e, miR1447, and miR171l–n weredownregulated, whereas miR482.2 and miR1450 were upregulated during saltstress [51]. Later, miR169g and another member of the miR169 family, miR169n,were reported to be induced by high salinity [35]. The authors found a cis-actingABA-responsive element in the upstream region of miR169n, which suggested thatmiR169n may be ABA-regulated. Both miR169g and miR169n targeted nuclearfactor (NF)-Ysubunit A – a factor that was previously shown to be downregulated indrought-affected wheat leaves [56]. Recently, a report used microarray experimentsto explore the miRNA profile in a salt-tolerant and a salt-sensitive line of maize (Zeamays) [57]; the results indicated that members of themiR156,miR164,miR167, andmiR396 families were downregulated, whilemiR162,miR168,miR395, andmiR474families were upregulated in salt-shocked maize roots.

miRNAs in Response to Cold and Heat Stress The expression of miRNAs in coldstress has been examined in Arabidopsis [14,33], Populus [51], and Brachypodium[58]. miR397 and miR169 were upregulated in all three species, and miR172 wasupregulated in Arabidopsis and Brachypodium. In addition to these miRNAs, severalmiRNAs (miR165/166, miR393, miR396, and miR408) were induced under coldstress in Arabidopsis, while other miRNAs (miR156/157, miR159/319, miR164,miR394, and miR398) showed either transient or mild regulation under cold stress[14,33]. In Populus, miR168a,b and miR477a,b were upregulated while miR156g–j,


miR475a,b, and miR476a were downregulated under cold stress [51]. WheatmiRNAs showed differential expression in response to heat stress; by using Solexahigh-throughput sequencing, Xin et al. [59] cloned the small RNAs from wheatleaves treated by heat stress. Among the 32 miRNA families detected in wheat,nine miRNAs were putatively heat responsive. For example, miR172 wassignificantly decreased and eight miRNAs (including miR156, miR159, miR160,miR166, miR168, miR169, miR827, and miR2005) were upregulated under heatstress.

miRNAs in Response to Hypoxia and Oxidative Stress Anaerobic or low-oxygenstress (hypoxia) interferes with mitochondrial respiration [60]. Hypoxia inducesmassive changes in the transcriptome and a switch from aerobic respiration toanaerobic respiration [61]. Recent studies suggested that miRNAs are involved inplant responses to hypoxia [62,63]. Several miRNA were differentially regulatedin seedlings that were submerged; early during submergence, Zm-miR166,Zm-miR167, Zm-miR171, Os-miR396, and Zm-miR399 were induced but Zm-miR159, At-miR395, Pt-miR474, and Os-miR528 were downregulated [63]. Simi-larly, high-throughput sequencing was carried out on small RNA libraries fromhypoxia-treated and control root tissue in Arabidopsis [62]. The abundances of 19miRNAs families significantly changed in response to hypoxia and theirabundances were inversely correlated with those of their specific mRNA targets[62]. Expression levels of miR156g, miR157d, miR158a, miR159a, miR172a,b,miR391, and miR775 increased under hypoxia. The involvement of miRNAs inplant response to hypoxia was further confirmed in experiments with a chemicalthat inhibits mitochondrial respiration and therefore mimics hypoxia [62].Reactive oxygen species (ROS) are inherent to plants because ROS are constantly

produced by aerobic processes in chloroplasts, mitochondria, and peroxisomes [64].Elevated levels of ROS are often associated with plant stress such as high intensitylight, UV radiation, temperature extremes, heavy metals, drought stress, salt stress,and mechanical stress [64]. Superoxide in plants are converted into molecularoxygen and hydrogen peroxide by SODs. Cu/Zn-SODs are encoded by CSD1,CSD2, and CSD3 in Arabidopsis [16]. miR398 was predicted to target CSD1 andCSD2 [13,65], and Sunkar et al. [16] confirmed these targets and discovered thatmiR398 is downregulated under oxidative stress. Downregulation of miR398 isaccompanied by an accumulation of CSD1 and CSD2 transcripts [16]. Thisaccumulation did not result from stress-related transcriptional induction of theCu/Zn-SOD genes, but rather resulted from the relaxation of miR398-directedcleavage [16]. Furthermore, transgenic Arabidopsis plants overexpressing a miR398-resistant form of CSD2 accumulated more CSD2 mRNA than plants overexpres-sing a regular CSD2 and were consequently much more tolerant to high-intensitylight, heavy metals, and other oxidative stressors [16]. Under stress conditions, thenegative regulation of CSD1 and CSD2 by miR398 is relieved, which leads to theincreased expression of CSD1 and CSD2, and therefore reduced accumulationof the highly toxic superoxide free radicals [16]. miR398 was induced underCu2þ-limited conditions to relocate the Cu2þ ion from CSD1 and CSD2 for


synthesis of more essential proteins and processes [66]. A genome-wide study ofH2O2-regulated miRNAs from rice seedlings indicated that expression of somemiRNAs is regulated in response to oxidative stress [67]. The authors identifiedseven H2O2-responsive miRNAs (miR169, miR397, miR528, miR827, miR1425,miR319a.2, and miR408-5p); that is, miRNAs that are expressed differentially inH2O2-treated and control samples; miR169, miR397, miR827, and miR1425 wereupregulated while miR528 was downregulated by H2O2 treatments [67].

miRNAs in Response to UV-B Radiation A computational approach was used toidentify Arabidopsis miRNAs induced by UV-B radiation [37]. Of the 21 miRNAsbelonging to 11 miRNA families identified in that study, the following werepredicted to be upregulated under UV-B stress: miR156/157, miR159/319, miR160,miR165/166, miR167, miR169, miR170/171, miR172, miR393, miR398, andmiR401 [37]. Some of the same miRNA families that were predicted to beupregulated by UV-B radiation in Arabidopsis (miR156, miR160, miR165/166,miR167, miR398, and miR168) were found to be upregulated by UV-B radiation inPopulus tremula [68]. Interestingly, three families (miR159, miR169, and miR393)that were predicted to be upregulated in Arabidopsis were downregulated in P.tremula, suggesting that some responses to UV-B radiation stress may be speciesspecific [68].

miRNAs and Nutrient Homeostasis miRNAs are involved in plant responses tonutrient stress. According to recent studies in Arabidopsis, miR399, miR395, andmiR398 are induced in response to phosphate-, sulfate-, and copper-deprivedconditions, respectively [13,16,66,69,70]. Phosphate is required for the synthesis ofnucleic acids and membrane lipids, and is frequently a limiting nutrient for plantgrowth. Phosphate homeostasis is partially controlled through miR399, whichtargets a gene encoding a putative ubiquitin-conjugating enzyme (UBC24) [13,14].miR399 is upregulated in low phosphate-stressed plants [32,70,71] and is notinduced by other common stresses, whereas its target, UBC24mRNA, was reducedprimarily in roots of plants exposed to low-phosphate stress. It is likely that an MYBtranscription factor, PHR1 (phosphate starvation response 1), is involved inmiR399expression. PHR1 is expressed in response to phosphate starvation and positivelyregulates a group of phosphate-responsive genes by binding to GNATATNC cis-elements [72–74]. This cis-element has been found upstream of all known miR399genes in Arabidopsis [70,72]. Furthermore, phr1 mutants show a significant declinein miR399 induction under phosphate stress [70,72]. miR399 has been isolatedfrom phloem, and its abundance in phloem increases upon phosphate starvation[75,76]. These studies strongly suggest that miR399 acts as a phosphate starvationsignal that is translocated from shoot to root where it promotes phosphate uptakeby downregulating PHO2, which encodes E2-UBC24 [69,70]. miRNAs themselvescan be subject to post-transcriptional regulation, as revealed by the discovery of theIPS1 (induced by phosphate starvation 1) gene that acts as a target mimic to controlmiR399 action [77]. IPS1 has imperfect sequence complementarity to miR399. Thecomplementarity is interrupted by a mismatched loop at the expected miRNA


cleavage site. Owing to this complementarity interruption, the IPS1 RNAsequesters miR399 and prevents it from causing degradation of PHO2mRNA [77].Sulfur (S) is one of the essential macronutrients and is available in the form of

sulfate in the soil. miR395 targets both APSs and the sulfate transporter AST68[13]. Sulfate deprivation induces the expression of miR395, with a concomitantdecrease in transcript levels of APS1 [13]. The abundance of miR395 in the phloemincreases for Brassica plants deprived of S and the increase was much stronger inthe phloem than in the root, stem, or leaf tissue [75,78].The plant micronutrient copper (Cu) is essential for photosynthesis, oxidative

responses, and other physiological processes [79]. miR398 is a key regulator of Cuhomeostasis. When Cu is limiting, the level of miR398 increases, and thisreduces the allocation of Cu to CSDs (CSD1 and CSD2) and therefore makes Cuavailable for other essential processes [66,80]. In higher plants, the Cu/Zn-SODsare replaced by Fe-SODs [81]. In Brassica under Cu deprivation, miR398 isupregulated not only in leaf, stem, and root tissue, but also in phloem sap [78].According to more recent research, Brassica phloem sap contains a specific set ofsmall RNAs that is distinct from those in leaves and roots, and the phloemresponds specifically to stress [75]. Upon S and Cu deficiencies, the phloem sapreacts with an increase of the same miRNAs that were earlier characterized inother tissues, while no clear positive response to iron was observed. However,iron (Fe) led to a reduction of Cu- and phosphate-responsive miRNAs. Graftingexperiments also indicated that miR395 and miR399 are phloem-mobile,suggesting that such translocatable miRNAs might be candidates for informa-tion-transmitting molecules [75].Cadmium (Cd) is one of the most toxic metals in agricultural soils. Under high

concentrations of Cd or other heavy metals, plants encounter cation imbalancesthat result in many physiological and biochemical disorders. Recent studiesidentified a set of conserved and non-conserved miRNAs that are differentiallyregulated in response to heavy metals in rice [82], M. truncatula [83], Brassica napus[84], and Arabidopsis [85]. These results suggest that miRNAs help regulate plantresponses to heavy metal stress in addition to other abiotic stresses. In B. napus,expression of miRNAs shows different responses to sulfate deficiency and Cdexposure [84]. miR160 was transcriptionally downregulated by sulfate deficiencyand by Cd exposure, but miR164b and miR394a–c in roots and stems wereupregulated by sulfate deficiency. Similarly, treatment with Cd induced expressionof miR164b and miR394a–c in all tissues, except that miR164b was downregulatedin leaves [84]. miR156a and miR167b in roots and miR156a and miR167c in leaveswere upregulated under sulfate deficiency. In contrast, miR167a and miR168 inroots and miR167a, miR167b, and miR168 in leaves were downregulated [84].Under Cd stress, most B. napus miRNAs were induced. Notably, miR156a,miR167a, and miR167c in roots and miR167a and miR167c in leaves were stronglyupregulated. miR393 expression in leaves was also upregulated by Cd exposure[84]. Xie et al. [86] reported that Cd exposure increased the expression of miR156,miR171, miR393, and miR396a in B. napus roots, but the accumulation of thetranscripts for these miRNAs was suppressed [86].


Huang et al. [82] constructed a library of small RNAs from rice seedlings thatwere exposed to toxic levels of Cd. In most cases, the rice miRNAs showed differentpatterns of expression in leaves and roots. miR601, miR602, and miR603 in rootswere upregulated, while miR602 and miR606 in leaves and miR604 in roots weredownregulated by Cd exposure. The rest of rice miRNAs, such as miR601 in leavesand miR605 and miR606 in roots, do not appear to be regulated by Cd stress [82].Cd, mercury (Hg), and aluminum (Al) also regulated the expression of miRNAs inM. truncatula [83]. In that study, miR171, miR319, miR393, and miR529 wereupregulated in response to Hg, Cd, and Al, and miR319 showed weak constitutiveexpression in leaves; it was upregulated by Cd and Al, but was not affected by Hg.Similarly, expression ofmiR393 was not affected by Al, but was slightly upregulatedby Hg and Cd. In contrast, miR166 and miR398 were downregulated by Hg, Cd,and Al exposure [83].Rice miR1432 and miR444d, which were identified by high-throughput sequen-

cing, are predicted to target a calmodulin-binding protein and an EF-hand protein,respectively, which suggests a role for these miRNAs in calcium (Ca) signaling [87].miRNAs also affect the growth of specific cells in roots under N-limiting conditions[88]. In the presence of N, miR167a was repressed and its target, ARF8, wasinduced in the pericycle, resulting in the initiation of many lateral roots. When N islimiting, lateral root extension is stimulated and this permits roots to search for Nin the soil further from the plant [88].

18.2.2

siRNAs

18.2.2.1 Biogenesis of siRNAs

siRNAs are generated from perfectly double-stranded RNA that can originate fromdifferent sources such as RNA transcribed from inverted repeats, naturalcis-antisense transcript pairs, the action of RNA-dependent RNA polymerases(RDRs) that convert single-stranded RNA into double-stranded RNA, the replica-tion of RNA viruses, and regions of the genome rich in retroelements. The double-stranded RNA is cleaved into 21- to 24-nucleotide siRNAs by DCLs proteins and thesize of the released siRNAs depends on the specific catalytic activity of therespective DCL protein. Double-stranded RNA is usually cleaved by multiple DCLproteins, thereby generating siRNA classes with different sizes. Like miRNAs,siRNAs are loaded into AGO protein-containing RISC that guides target regulationat the post-transcriptional level or at the transcriptional level through a pathwaytermed RNA-directed DNA methylation.

18.2.2.2 Role of siRNAs in Plant Stress Responses for Adapting to Climate Change

The first evidence that siRNAs are involved in abiotic stress responses in plants wasprovided by Sunkar and Zhu [14]. Work on the founding member of nat-siRNAs,which is derived from a natural cis-antisense transcript pair of SRO5 and P5CDHgenes, demonstrated an important role of nat-siRNAs in osmoprotection andoxidative stress management under salt stress in Arabidopsis [89]. P5CDH is


constitutively expressed, whereas SRO5 is induced by salt stress. Under high saltstress, the 24-nucleotide nat-siRNA corresponding to the SRO5 mRNA isproduced; this nat-siRNA targets the P5CDH mRNA for degradation and leads tothe production of a population of 21-nucleotide nat-siRNAs. Downregulation ofP5CDH leads to proline accumulation, which is an important step contributing tothe plant’s ability to tolerate excess salt [89]. However, reduced P5CDH activity alsoleads to the accumulation of P5C (a toxic metabolic intermediate) and ROS; thataccumulation is probably countered by the SRO5 protein through direct detoxifica-tion activity in the mitochondria [89]. The initial induction of SRO5 mRNA by saltstress may be mediated by oxidative stress that is an inevitable consequence of saltstress. Thus, the SRO5–P5CDH nat-siRNAs together with the P5CDH and SRO5proteins are key components of a regulatory loop controlling ROS production andsalt stress response [89].Abiotic stress responsiveness has also been observed in a pool of Triticum aestivum

small non-coding RNAs [90]. In wheat seedlings, cold, heat, salt, or drought stressessubstantially change the expression of four siRNAs: siRNA002061_0636_3054.1 isstrongly downregulated by heat, salt, and drought; siRNA 005047_0654_1904.1 isgreatly upregulated by cold stress and downregulated by heat, salt, and drought;siRNA080621_1340_0098.1 is slightly upregulated by cold and downregulated byheat but not by salt and drought; and siRNA007927_0100_2975.1 is downregulatedby cold, salt, and drought, but not by heat stress [90].The ta-siRNAs are a specialized class of siRNAs that are generated by

miRNA processing of a TAS gene transcript, resulting in the production of21-nucleotide RNAs that are phased with respect to the miRNA cleavage site(Figure 18.1b). Four families of TAS genes have been identified in Arabidopsis,with TAS1 and TAS2 transcripts recognized by miR173, TAS3 recognized bymiR390, and TAS4 targeted by miR828 [91]. TAS1, TAS2, and TAS3 ta-siRNAsall showed increased expression in hypoxia-treated samples in Arabidopsis [62].These changes in ta-siRNA levels are reflected in the changes to TAS-targetingmiRNAs; both miR173 and miR390 showed increased expression [62]. Mostpentatricopeptide repeats (PPRs) targeted by hypoxia-responsive small RNAsare from the P subfamily, which is predicted to localize to the mitochondria[62]. It is likely that some of the observed changes in ta-siRNAs are the resultof decreased mitochondrial function during hypoxia because TAS1 is inducedby chemical inhibition of the cytochrome and alternative oxidase respirationpathways in the mitochondria. The downregulation of these PPR genes mayeither protect mitochondria during hypoxia or simply reflect a decreasedrequirement for these gene transcripts.Stress responses in plants also involve novel long non-protein coding RNAs

(npcRNAs). In Arabidopsis, salt stress resulted in a dramatic increase in npcRNA60and npcRNA536 and a decrease in npcRNA72 and npcRNA82 accumulation [92]. Inthe same study, phosphate deprivation caused substantial upregulation ofnpcRNA43 and npcRNA536, substantial downregulation of npcRNA33, slightupregulation of npcRNA60, and slight downregulation of npcRNA311 [92]. InCraterostigma plantagineum, an endogenous siRNA has been identified that is


induced during dehydration and that may contribute to desiccation tolerance [93].The authors describe the T-DNA tagging of a Craterostigma gene that inducesdesiccation tolerance by activating ABA-inducible genes. Activation tagging wasused to identify constitutively desiccation-tolerant (CDT) callus lines and this led tothe identification of CDT-1 callus lines. In wild-type C. plantagineum, the transcriptlevels of CDT-1 were upregulated by ABA and dehydration treatments, butrepressed when rehydrated. Interestingly, sense and antisense siRNAs correspond-ing to the CDT-1 locus were detected, suggesting that during stress (ABA ordehydration) the locus generates small RNAs that may confer stress tolerancethrough induction of stress-responsive genes [93].

18.3

Artificial miRNAs

In plants and animals the miRNA sequence within a miRNA precursor can beexchanged without affecting the processing of the miRNA as long as the number ofmatches and mismatches in the foldback structure remains unaltered [94,95]. Thisallows us to modify miRNA sequences and to create artificial miRNAs (amiRNAs)that are able to target any gene of interest and to knockdown its expression at thepost-transcriptional level. This method was successfully applied in different seedplants [96–99] and subsequently adapted for specific gene knockdown inPhyscomitrella [100,101].In Arabidopsis, a similar approach was reported relying on the expression of

artificial ta-siRNAs (ata-siRNAs) by engineering the TAS1c locus to silence theFAD2 gene [102]. Thus, when using gene engineering to improve stress-responsivegenes in crop plants, amiRNAs can be designed to target any gene of interest withgreat specificity. Thus, the amiRNA-mediated approach should have broadapplicability for engineering multiple stress-responsive genes under climatechange. It has also been suggested that amiRNAs pose fewer biosafety orenvironmental problems when applied to agriculture than other strategies.

18.4

Stress---miRNA Networks for Adapting to Climate Change

The spectrum of action by miRNAs seems to be extremely wide and includesvarious aspects of development, adaptive responses to stresses, and the regulationof the miRNA pathway itself. Most miRNAs do not function independently butrather are involved in overlapping regulatory networks (Table 18.1). One obviousfeature of the adaptation to stress is a change in gene expression profiles for genesinvolved in a broad spectrum of biochemical, cellular, and physiological processes[103]. Under optimal conditions, all resources are used for supporting plant growthand development. Under stress, however, growth and development are stalled, andthe resources are mobilized toward adaptive responses to stress. Most conserved


miRNAs target mRNAs encoding diverse families of transcription factors. Forexample, miR156, miR159/319, miR160, miR166, and miR169 target squamosapromoter-binding proteins (SBPs), MYBs/TCPs, auxin response factors (ARFs),class III homeodomain leucine zipper proteins (HD-ZIPs), and the NF-Y subunit,respectively, but miR168, miR393, miR395, and miR398 target mRNAs encodingAGO1, TIR1, ATS/APS, and CSD1/2, respectively [18]. The level of those conservedmiRNAs appears to be regulated during stress and their target genes appear to bestress regulated as well (Figure 18.2), suggesting that plant growth and develop-ment are modulated during stress.The stress-responsive miRNAs could be involved in many pathways that

reprogram complex processes of metabolism and physiology. For example,

Figure 18.2 Regulatory network of stress-

responsive miRNAs for adapting to climate

change in Arabidopsis. A network is proposed

that describes the molecular mechanisms

underlying the response ofArabidopsis plants to

different abiotic stresses. The network is based

on the changes in expression profiles ofmiRNA

and subsequent target transcripts in plants

under stress. Green boxes: upregulated RNAs;

red boxes: downregulated RNAs. (Modified

from [12] with kind permission by Elsevier.)

18.4 Stress---miRNA Networks for Adapting to Climate Change 495

upregulation of miR395 is involved in S metabolism (Figure 18.2); miR395 targetsAPSs (APS1, APS3, and APS4), which catalyze the first step of the S assimilationpathway [104]. miR395 also targets AST68, a low-affinity sulfate transporter [91].AST68 is implicated in the internal translocation of sulfate from roots to shoots[105]. Thus, miR395 potentially coordinates changes in sulfate translocation andassimilation. In addition, downregulation of miR398 during stress enables themRNA levels of both targets (CSD1 and CSD2) to increase and detoxify ROS(Figure 18.2). miR398 also targets the gene cytochrome c oxidase subunit V [13,14];further studies are required to determine whether this gene is also part of theoxidative stress network in plants. Downregulation of miR169a and miR169c allowsexpression of the mRNA of NF-YA5, which is important for drought responses inArabidopsis [47]. Researchers have hypothesized that NF-YA5 expressed in guardcells controls stomatal aperture, whereas NF-YA5 expressed in other cells maycontribute to the expression of stress-associated genes (Figure 18.2). As AGO1 is akey miRNA pathway regulator, the difference in AGO1 mRNA expression undersalt and drought stress may induce further changes of numerous miRNA activities(Figure 18.2). In addition, AGO1 and miR168 seem to be transcriptionallycoregulated, allowing AGO1/miR168 homeostasis to be maintained in every cellwhere the miRNA pathway is functioning [106]. Data also indicate that AGO1 isrequired for stem cell function and organ polarity [106].The accumulation of miR166 and miR160 together with miR393 could

modulate morphological and hormone homeostasis by regulating transcriptsof HD-ZIPIII, ARF10, and TIR1 (Figure 18.2). HD-ZIPIII was found toregulate the pattern of vasculature and the establishment and maintenance ofabaxial–adaxial polarity in lateral organs [107]. ARFs regulate the expression ofauxin-inducible genes, such as GH3 and auxin/indole-3-acetic acid (IAA), bybinding auxin-responsive promoters (ARPs) [108,109]. In related studies,increased or decreased levels of ARF10 altered GH3-like gene expression andled to severe developmental defects, miR160-resistant ARF plants were foundto have dramatic developmental abnormalities, and ARF10 was found toregulate floral organ identity and to be involved in seed germination[45,108,109]. The auxin receptor TIR1 is an important SCF E3 ubiquitin ligasethat functions in degrading auxin/IAA proteins in response to auxin [110] andthen triggers adventitious root formation and lateral root development.miR393-mediated inhibition of TIR1 would downregulate auxin signaling andseedling growth under biotic and abiotic stress conditions. Also, the upregula-tion of miR156 and miR319/159 could help the plant adapt to stress bymodulating plant morphological characteristics via regulation of transcripts ofSBPs and MYBs/TCPs transcription factors (Figure 18.2). SBP-like is involvedin floral transition and regulation of flowering [111,112]. Recent resultsindicate that overexpression of miR156 affects phase transition from vegetativegrowth to reproductive growth by causing a rapid initiation of rosette leaves, asevere decrease in apical dominance, and a moderate delay in flowering [111–113]. TCP transcription factors direct the developmental processes determiningleaf size, leaf form, and flower symmetry [113,114]. Overexpression of miR319,


which specifically downregulates TCP mRNAs, results in uneven leaf shapeand delayed flowering [114]. Overexpression of miR159a specifically reducesMYB mRNA accumulation and results in male sterility, whereas plants thatexpress miR159-resistant MYB33 have upwardly curled leaves, reduced stature,and shortened petioles [114,115]. Reyes and Chua [44] demonstrated how ABAinduces the accumulation of miR159 in association with the seed-specifictranscription factor ABI3. In turn, miR159 mediates cleavage of MYB101 andMYB33 transcripts, which encode positive regulators of ABA responses.

18.5

Application of Small RNA-Mediated Suppression Approaches for Plant Improvement

Under a Changing Climate

Food production, food quality, and food security are expected to be negativelyinfluenced by the overall impact of climate changes. Plants are vulnerable toclimate change; therefore it is important to modify crops to overcome the expectedincrease in temperature, increase in carbon dioxide (CO2), increase of frequency ofdrought, and increase in the salinity level. Crops can be modified by engineeringsmall RNA pathways by which small RNA molecules can influence gene expressionin plants, at both the transcriptional and post-transcriptional levels (Figure 18.3).The very first commercial genetically modified crop was produced using RNA-mediated gene suppression was tomato for the purpose of delaying fruit ripening[116].Examples of RNA-mediated gene suppression methods are: (i) the over-

expression of a transgene, either from antisense (antisense RNA silencing) orfrom sense (co-suppression silencing), (ii) constitutive hairpin expression(hairpin pRNA), (iii) siRNAs, and (iv) amiRNA. In addition, an induciblepromoter can be used in cases where a transgene with an inverted repeat thatsilences the essential gene constitutively is known to be lethal. In this case anindividual that expresses the transgene allows the transgene to survive and cansilence the gene of interest under specific conditions so that the resultingphenotype can be studied (Figure 18.3).A potential benefit of RNA-mediated gene suppression techniques is the ability to

silence multiple target genes at the same time and, in many cases, a single invertedrepeat transgene can be designed to silence multiple, homolog genes. If pointmutations or insertional mutations are being used to silence multiple genes thismight require multiple generations of crosses to generate genetic stocks that aredouble or triple mutants for a specific gene family.Although RNAi is an effective system for silencing many genes in many

organisms, including several agriculturally significant plants, it can still generate agreat number of siRNAs complementary to distinct regions of the desired gene. Itis estimated that 50–70% of the genes in an organism can produce siRNAscomplementary to unwanted targets if they are used in RNAi methods [117].Furthermore, the transgenes can be self-silenced, resulting in loss of silencing

18.5 Application of Small RNA-Mediated Suppression Approaches for Plant Improvement 497

Figure 18.3 Simplified illustration for

developing crops that confer tolerance to

climate changes such as drought and high

temperature. Examples of small RNA (sRNA)-

mediated suppression constructs are shown. I,

antisense expression; II, co-suppression; III,

constitutive hairpin expression; IV, short

interfering RNAs; V, artificial miRNAs. Small

RNA-mediated constructs are driven by plant

genetic transformation and they guide target

regulation at the post-transcriptional level

(degradation of mRNA or inhibition of

translation) or at the transcriptional level

throughDNAmethylation. It is anticipated that

applications of small RNA-mediated

approachesshouldfacilitatethedevelopmentof

improved crops that are better adapted to

abiotic stresses and thereby better able to

tolerate future climate variability.


after several generations. Nevertheless, in plants and animals the miRNA sequencewithin a miRNA precursor can direct site-specific cleavage of target mRNA withmiRNA-complementary motifs without affecting the processing of the miRNA aslong as the number of matches and mismatches in the foldback structure remainsunaltered [94,95]. This allows us to modify miRNA sequences and to createamiRNAs that are able to target any gene of interest, and to knockdown itsexpression at the post-transcriptional level (Figure 18.3).

18.6

Conclusions and Outlook

A complete understanding of the actions of small RNAs depends on theidentification of the target genes. Identification of entire sets of miRNAs andsiRNAs and their targets will lay the foundation needed to unravel the complexmiRNA- and siRNA-mediated regulatory networks controlling development andother physiological processes. Given that miRNAs and siRNAs are crucialcomponents of gene regulatory networks, we believe that a complete under-standing of the functions of miRNAs and siRNAs will greatly increase ourunderstanding of plant tolerance to abiotic stress.Analysis of the DNA methylation profiles and the small RNA profiles will identify

genes or regions that are regulated by miRNA/siRNA-mediated DNA methylation,which may contribute to epigenetic inheritance of stress effects. Also, the amiRNA-mediated approach should have broad applicability for engineering multiple stress-responsive genes in crop plants under climate change.Manipulating new RNAi pathways, which generate small RNA molecules to

amend gene expression in crops, can produce new quality traits, having betterpotentiality of protection against abiotic stresses. Furthermore, conserved miRNAscan be analyzed in crop plants; this will provide the possibility to exploit thepotential of endogenous miRNAs for the improvement of quality traits in cropplants under climate change.Current agricultural technology needs more and more molecular tools to

reduce current crop loss and feed extra mouths, which according to a recentestimate by the UN Food and Agriculture Organization will increase by 2billion over the next 30 years. Small RNA-mediated crop improvement under achanging climate describes one such powerful innovation (Figure 18.3).Hopefully, the technology that has been developed by scientists from developedcountries will be available to any laboratory, including those in developingcountries, where work utilizing RNAi is either in progress or will be launchedshortly. The technology is well-developed and can be applied directly to evolvestress-resistant crops. Since this technology offers a great potential for under-standing gene functions, and utilizing them to improve crop quality andproduction, it is only a matter of time before we see the products of this RNAiresearch in the farmers’ fields around the world.

18.6 Conclusions and Outlook 499

Note

Parts of this chapter have previously been published in [12] and are reprinted herewith kind permission from Elsevier.

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