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Seminars in Cell & Developmental Biology 19 (2008) 586–595

Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology

journa l homepage: www.e lsev ier .com/ locate /semcdb

eview

miRacle in plant development: Role of microRNAs in cell differentiationnd patterning

amien Garcia ∗

nstitut de Biologie Moléculaire des Plantes du CNRS, UPR2357, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France

r t i c l e i n f o

rticle history:

a b s t r a c t

MicroRNAs (miRNAs) are endogenous small regulatory RNAs, which control gene expression in eukary-

vailable online 30 July 2008

eywords:iRNA

a-siRNAevelopment

otes. In plants they repress mRNA targets containing a highly complementary site, either by cleavageor translational repression. Studies of individual miRNA/target interactions highlight the involvement ofthe miRNA-based regulations in a broad range of developmental programs, throughout plant lifecycle.MicroRNAs can have distinct regulatory functions on their targets: some determine their spatial accu-mulation, some have a buffering role that ensures the robustness of their expression pattern, and finally

.

xpression patternobustness

others establish the temporal expression of targeted genes.© 2008 Elsevier Ltd. All rights reserved

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5862. Small RNA pathways involved in plant development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587

2.1. miRNA biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5872.2. Trans-acting siRNA biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587

3. Lessons from activation tagging screens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5873.1. Evidence for transcript cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5873.2. Evidence for translational repression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589

4. Dominant mutations in miRNA binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5895. Regulatory functions of miRNAs in development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589

5.1. Spatial restriction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5905.2. Buffering function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5915.3. Temporal regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592

6. Subfunctionalization of miRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5926.1. miR164 family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5926.2. miR159/319 family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593. . . .. . . . .

MicroRNAs (miRNAs) were discovered in Caenorhabditis ele-ans through the study of lin-4 and let-7 mutants [1,2]. lin-4 andet-7 miRNAs control the temporal expression of heterochronicenes and are required for C. elegans developmental timing.

∗ Tel.: +33 3 88 41 72 49; fax: +33 3 88 61 44 42.E-mail address: Damien.Garcia@ibmp-ulp.u-strasbg.fr.

Sd[aRTiIsm

084-9521/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcdb.2008.07.013

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594

ubsequently to this discovery, small RNA cloning experimentsemonstrated the presence of this class of regulatory RNAs in plants3]. MicroRNAs are produced from larger RNA precursors containing

self-complementary fold-back structure. This double-strandedNA (dsRNA) structure contains the sequence of the future miRNA.he miRNA is precisely excised and loaded into an effector complex,

7. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

nvariably containing an ARGONAUTE protein, called RISC for RNAnduced Silencing Complex. The miRNA loaded RISC induces theequence specific repression of target genes containing a sequenceotif partially complementary to the miRNA. A specificity of plant

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D. Garcia / Seminars in Cell & Dev

iRNAs over animal miRNAs, is that they share very high comple-entarity to their targets with few or no mismatches. In addition,

nlike other types of plant small RNAs, especially small interferingNAs (siRNAs), miRNAs generally act in trans to repress genes unre-

ated to the loci encoding the miRNA itself. Since the first report ofhe developmental potential of miRNAs in plant [4], an increasingumber of studies showed the importance of miRNA-based repres-ion in the control of a wide variety of developmental processes. Themportance of miRNAs for plant development varies depending onhe nature of their targets and the regulatory function of the miRNAtself. Different strategies have been used to study miRNA func-ion: redundancy among miRNA family members can be bypassedn gain-of-function strategies using either miRNA overexpression orngineered miRNA-resistant versions of the targets. In a few cases,he recovery of miRNA knock out mutants allowed to pinpoint thepecialization of individual loci. This review highlights major semi-al discoveries that paved the way to our current understanding ofiRNA function. It also points out more recent studies to illustrate

he different ways by which miRNAs might control crucial eventsndispensable for the function of their targets. The involvement ofnother class of small RNAs, called trans-acting siRNAs (ta-siRNAs),hich depend on miRNAs for their generation, will also be dis-

ussed. To date 184 miRNAs are annotated in miRBase for the modellant Arabidopsis thaliana (http://www.microrna.sanger.ac.uk). Theollowing paragraphs only compare and comment on miRNAs forhich a clear function in development have been established, it

epresents only a small subset among known miRNAs and the widepectrum of plant small RNAs.

. Small RNA pathways involved in plant development

.1. miRNA biogenesis

The importance of miRNAs for plant development was hypoth-sized since the first small RNA cloning experiment, based on theleiotropic phenotypes of mutants affecting small RNA production3]. Indeed, every mutant affecting miRNA biogenesis or functionas originally identified by forward genetics, based on morphologi-

al abnormalities. MicroRNAs are produced from highly structuredNA stem loops. The release of the 21 nt miRNA/miRNA* duplex

rom this structure requires the ‘‘dicer’’ activity of the riboendonu-lease DICER-LIKE1 (DCL1) and its interacting partner, the RNAinding protein HYPONASTIC LEAVES1 (HYL1) [5,6]. The C(2)H(2)inc finger protein SERRATE (SE) is also involved in this processy a mechanism that also involves the CAP BINDING PROTEIN80CBP80) (Fig. 1) [7–9]. The initial identification of dcl1 mutant alle-es illustrates the pleiotropic phenotypes induced by mutations inhe miRNA pathway. They were initially characterized from threendependent phenotypes: an embryo lethal phenotype (suspen-or1), defects in seed integuments growth (short integuments1),nd changes in floral organ identity (carpel factory) [10]. Simi-arly hyl1 and se were first isolated based on their developmentalefects [11,12]. Strong alleles of dcl1 and se are embryo lethal,ointing out the essential role of miRNAs for embryo development7,10].

The miRNA/miRNA* duplex needs to be protected from degra-ation by addition of methyl groups on the 3′ ends. The HUANHANCER1 (HEN1) methyltransferase has the enzymatic activityequired for this step [13]. Mutants in HEN1 were originally iden-ified in a morphological screen for floral organ identity defects,

lthough this mutant also displays a pleiotropic range of vegetativehenotypes [14].

MicroRNAs are produced in the nucleus and nuclear export haseen proposed to rely on the Arabidopsis homologue of humanxportin 5, HASTY (HST) [15]. Mutants in HST induce an accelerated

3

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ental Biology 19 (2008) 586–595 587

egetative phase change and early flowering phenotype, but alsoave a more general impact on development [16].

Finally the two strands of the miRNA duplex are separated andsingle-stranded mature miRNA is loaded into an ARGONAUTE

AGO) protein (generally AGO1). The AGO protein induces theequence specific repression of the miRNA target genes, either byleavage or translational repression. The AGO1 protein is an effec-or enzyme of RISC and is known to exhibit the ‘‘slicer’’ activityequired for target cleavage [17]. The founder member of the AGOrotein family, AGO1, was named from the strong leaf polarityefects observed in the ago1 mutants, which made them look like a

ittle squids (argonauts) [18]. The phenotypes observed in mutantsf the miRNA pathway are probably due to the deregulation of manyiRNA targets. In this context, the evaluation of the importance of

ndividual miRNAs in particular developmental processes requireshe study of specific miRNAs/targets interactions.

.2. Trans-acting siRNA biogenesis

Trans-acting siRNAs represent a second type of small RNAsnvolved in plant development. miRNA and ta-siRNA pathways areightly connected as the latter is dependent on the former forts initiation. But in contrast to the very high number of miRNAsescribed, only four ta-siRNAs loci have been discovered in Ara-idopsis [19–21]. This section will only present data concerning therans-acting siRNA locus 3 (TAS3) biogenesis, as it is the only ta-iRNA known to be necessary for proper plant development. Theain difference between ta-siRNAs and miRNAs resides in their

iogenesis. miRNAs are produced from a precursor forming a sec-ndary structure, whereas ta-siRNAs are produced from a linearrecursor containing miRNAs binding sites (Fig. 2). A particularityf TAS3 resides in its double targeting by miR390 through ARG-NAUTE7 (AGO7) instead of AGO1. miR390 is specifically associated

o AGO7 and excluded from AGO1 by a mechanism that remains toe clarified. This riboprotein complex induces the cleavage of theAS3 precursor only at the most 3′ miR390 site, the other remainsncleaved [22]. The 5′ cleavage product is further stabilized by theUPPRESSOR OF GENE SILENCING 3 (SGS3) protein [23], in ordero allow the recruitment and activity of the RNA DEPENDENT RNAOLYMERASE 6 (RDR6) protein [19]. RDR6 catalyses the produc-ion of double-stranded RNA, which is initiated specifically fromhe cleavage site to the second miR390 binding site occupied byGO7 [22]. The dsRNA molecules are subsequently processed byhe DICER-LIKE4 (DCL4) enzyme [24,25] and its interacting part-er, dsRNA BINDING PROTEIN4 (DRB4) [26]. The production ofa-siRNAs is precisely controlled and the site of miR390 cleavageredetermines the initiation point for DCL4-dependent productionf adjacent, and phased, secondary siRNAs including the actual ta-iRNAs species [20,22]. Ta-siRNAs are very similar to miRNAs inheir mode of action, as they are known to repress in trans mRNA tar-ets containing a complementary binding site (Fig. 2). All mutantsffecting TAS3 biogenesis show an accelerated vegetative phasehange, with precocious production of trichomes on the abaxialventral) side of the leaf [25,27,28]. The developmental importancef this genetic pathway is conserved in maize and rice for whichutants also exhibit defects in leaf polarity and shoot apical meris-

em formation, respectively [29–31].

. Lessons from activation tagging screens

.1. Evidence for transcript cleavage

Activation tagging uses random insertion of a T-DNA constructontaining multiple copies of the 35S enhancer to induce the tran-

588 D. Garcia / Seminars in Cell & Developmental Biology 19 (2008) 586–595

Fig. 1. Plant miRNA biogenesis and miRNA pathway mutants. (A) A model for plant miRNA biogenesis. After transcription the pri-miRNA is processed by the Dicer activity ofthe DCL1/HYL1 complex into 21 nt miRNA/miRNA* duplex. This step also requires the cooperation of the SE and CBP80 genes. The miRNA duplex is further methylated (Me)by the methylase activity of HEN1. Methylated miRNA duplex needs to be exported from the nucleus a step that requires HST. Within the cytoplasm the mature miRNA isl in ind( efectsP nts ard

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oaded into the AGO1 protein, while the miRNA* strand is degraded. The AGO1 proteB) Vegetative phenotypes of mutants in the miRNA pathway. Leaf morphological dictures correspond to dcl1-9, hyl1-1, hen1-1, hst-1, ago1-25. dcl1-9 and ago1-25 mutao not survive after the seedling stage.

criptional activation of genes neighboring the site of insertion [32].xperimental data supporting the role of a miRNA in plant develop-

ent initially came from an activation tagging screen in Arabidopsis,

n which miR-JAW, later renamed miR319a, was overexpressed (jaw-mutant) [4]. Microarray analysis of jaw-D reveals the specific drop

n transcript levels of five TEOSINTE BRANCHED1, CYCLOIDEA, ANDCF FAMILY (TCP) transcription factors containing a 21 nt conserved

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able 1lant miRNAs important for development, their targets and overexpression phenotypes

amily Target family Studied targets

iR156 SBP SPL2, 3, 4, 9, 10, 15

iR156 SBP TGA (Z.m)iR159 GAMYB MYB33, MYB65iR160 ARF ARF10, ARF16, ARF17

iR164 NAC CUC1, CUC2, NAC1iR166 HD-ZIPIII PHB, PHV, REV, ATHB8, ATHB15

iR167 ARF ARF6, ARF8iR172 AP2 AP2, TOE1, 2, 3iR319 TCP TCP2, 3, 4, 10, 24iR319 TCP LA (S.l)iR390 TAS TAS3 (indirect ARF2, 3, 4)iR824 MADS AGL16

lant miRNAs involved in development and their overexpression phenotypes. Species are

uces either cleavage through the ‘‘slicer’’ activity of AGO1 or translation inhibition.can be observed including, smaller rosettes, leaf curling and reduced leaf number.e hypomorph alleles, dcl1 strong alleles are embryo lethal, while ago1 strong alleles

otif complementary to miR319. The efficient complementation ofaw-D by expression of a miRNA-resistant form of TCP2 (mTCP2),

emonstrates the predominant impact of TCP deregulation for theutant phenotype. This study demonstrates the sequence specific

nteraction of TCP genes and miR319. It represents the first exam-le showing the developmental consequences of having the mRNAleavage activity of a miRNA, expressed ectopically. The valida-

miRNA overexpression phenotype

Increased leaf initiation, decreased apical dominance [40,44], delayedflowering time [45]Extended juvenile phase [48]Male sterility, delayed flowering time [38,40]Root cap defects, loss of gravitropism, increased lateral rooting [44],germination sensitivity to ABA reduced [47]Organ fusion [37,39,40], reduced lateral rooting [41]Seedling arrest, fasciated apical meristem, female sterility, altered vascularsystem [42] leaf polarity defects [43]Twisted leaves, short inflorescences, arrested flower development [46]Early flowering, floral organ identity defects [35,36]Distorded leaf development, late flowering, male sterility [4,51]Delayed differentiation of leaf margin [50]Not testedDecreased rate of stomata in high order complexes [49]

indicated if not Arabidopsis; Z.m, Zea mais; S.l, Solanum lycopersicum.

D. Garcia / Seminars in Cell & Developm

Fig. 2. TAS3 biogenesis pathway. The miR390 loaded AGO7 targets TAS3 precursoron two miR390 binding motifs. It cleaves on the 3′ miR390 site while the other site,containing a central mismatch remains uncleaved. The TAS3 5′ cleavage product isstabilized by the action of the SGS3 protein, and is converted by RDR6 in dsRNAbetween the 3′ end and the uncleaved miR390 binding site. The dsRNA produced isprocessed by the action of the DCL4/DRB4 complex into 21nt ta-siRNAs duplexes.These duplexes are methylated (Me) by the enzymatic activity of HEN1. The matureta-siRNAs are loaded into an AGO1 containing complex to induce the cleavage ofTmo

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AS3 targets, ARF2, ARF3 and ARF4. The function of SDE5, an homologue of humanRNA export factor, is necessary for an efficient ta-siRNA production but its mode

f action remains to be investigated.

ion of miRNA targets is commonly achieved by the detection ofhe cleavage product by 5′ race and this method has been exten-ively used as a simple way to confirm the validity of predictediRNA targets [33,34]. In addition, the experimental overexpres-

ion of miRNAs has been widely tested and helped to discover theegulatory potential of numerous miRNAs (Table 1) [35–51].

.2. Evidence for translational repression

The description of another activation tagging line in Arabidop-is describes the effect of miR172 overexpression on the control ofowering time and floral organ identity [35]. In contrast to miR319,iR172 overexpression do not change the stability of APETALA2

AP2) and its APETALA2-like (AP2-like) mRNA targets, TARGET OFAT1 (TOE1) and 2 (TOE2). The availability of an AP2 antibodyllowed to show that plants overexpressing miR172 accumulateimilar amounts of AP2 transcripts but show a dramatic reduc-ion of AP2 protein level [35,36]. This observation demonstrates

he existence of another mode of action for miR172, translationalepression [35,36]. The study of miR172 indicates that ectopic trans-ational repression activity can perturb established developmentalrograms. The second example of translational repression in plant

5

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ental Biology 19 (2008) 586–595 589

evelopment was provided last year with the effect of miR156 onhe SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3) gene,hich also determines flowering time [52]. Only a few examples

f translational inhibition are described in plants [35,36,52,53]. Forhis reason, mRNA cleavage was considered for years as the pre-ominant activity for plant miRNAs, while translational inhibitionas seen as an exception to the rule. A recent report provides new

xperimental clues suggesting a widespread coexistence betweenranslational repression and mRNA cleavage [54]. Translationalnhibition is the predominant mode of action of animal miRNAs55]. The poor study of this process in plants was probably due tohe limited availability of plant specific antibodies against miRNAargets. A reevaluation of the actual activity of known miRNAs, inhe light of this recent discovery will probably clarify the respectiveoles of mRNA cleavage and translational repression activities forlant development. Interestingly two examples of miRNAs guidedranslational activation have been described in animals [56,57], anctivity that remains to be discovered for plant miRNAs.

. Dominant mutations in miRNA binding sites

One of the most extensively studied family of miRNA targets ishe class III HD-ZIP transcription factors, PHABULOSA (PHB), PHAVO-UTA (PHV), REVOLUTA (REV), CORONA (CNA) and HOMEOBOX GENE8ATHB8) [58]. These genes contain a binding site for miRNAs of theiR165/166 family. The importance of miRNA-based repression of

hese genes for plant development is illustrated by the spectacularhenotypes and the large number of dominant mutations identi-ed, disrupting the miRNA binding site of these genes (Table 2)59–64]. Dominant mutants in PHB and PHV have similar pheno-ypes with strong adaxialisation (dorsalisation) of the leaves [59].ain-of-function in REV and CNA affects leaf polarity to a lesserxtent but leads to dramatic patterning defects in the vasculaturend shoot apical meristem [61,63]. These observations point outoth the redundancy and the specialization among class III HD-ZIPranscription factors. These gain-of-function mutations, identifiedy forward genetic screens, also fall into a putative sterol-bindingomain, the START domain. Before the discovery of miR166, theseutations were hypothesized to disrupt the perception of a sterol

ased adaxialisation signal [59]. This possibility was ruled out byhe observation that experimental disruption of the miRNA bindingite without changes in protein sequence recapitulates the domi-ant mutant phenotype [65].

In a recent report, five mutations in the miRNA binding site ofhe TCP transcription factor LANCEOLATE (LA) were identified byorward genetics in tomato. The semi-dominant La-2 mutation fallsn the miR319 binding site of LA. Despite its strong phenotype, this

utation does not induce changes in protein sequence, a clear indi-ation that only miRNA-based regulation and not protein function isisturbed in La-2 [50]. Disruption of the miR319 binding site leads torecocious differentiation of leaf margin and production of simple

eaves instead of the normal composite leaves. The opposite effects observed upon overexpression of miR319, which increases leafomplexity by delaying differentiation. miR319 and LA have over-apping expression pattern in leaf primordia suggesting that miR319cts in the fine tuned regulation of LA accumulation (see paragraph.3) [50]. The experimental introduction of synonymous mutations

n the miRNA binding sites became a widespread method to studyhe importance of miRNA repression of a given target (Table 2) [66].

. Regulatory functions of miRNAs in development

One major challenge to understand miRNA function is to definehe actual regulatory role of miRNAs. This point needs to be investi-

590 D. Garcia / Seminars in Cell & Developmental Biology 19 (2008) 586–595

Table 2Phenotypes of miRNA-resistant targets

miRNA Target Species Line Promoter Phenotype

miR156 SPL3 A.t SPL3m 35S Early vegetative phase change and flowering [46]SPL4 SPL4� 35S Early vegetative phase change and flowering [46]SPL5 SPL5� 35S Early vegetative phase change and flowering [46]SPL9 rSPL9 endo Reduced leaf initiation rate [80]

miR159 MYB33 A.t mMYB33 endo Pleiotropic developemntal defects, hyponastic leaves and sterility [71]miR160 ARF10 A.t mARF10 endo Serrated leaves, curled stems, distorded flowers siliques, germination

ABA hypersensive [47]miR160 ARF16 A.t mARF16 endo Reduced lateral roots initiation, pleiotropic vegetative and

reproductive deffects [44]miR160 ARF17 A.t 5mARF17 endo Serrated leaves, early flowering, altered phylotaxis and root growth

[66]miR164 CUC1 A.t 5mCUC1 endo Altered floral organ number, cotyledons orientation and leaf shape

[39]miR164 CUC2 A.t CUC2m4 2x35S, alcA Wrinkled leaves, boundary enlargement [37]miR164 CUC2 A.t CUC2gm4 endo Increased leaf serration, enlarged boundary domain, carpel fusion

defects [74] altered phylotaxis [75]miR164 NAC1 A.t NACm 35S Increased lateral roots production [41]miR166 PHB A.t phb-1D endo locus Adaxialised leaves [59]miR166 PHV A.t, N.s phv-1D endo locus Adaxialised leaves [60,64]miR166 REV A.t avb-1, rev-10d endo locus Adaxialised leaves, meristem and vascular patterning defects [61,62]miR166 ATHB15 A.t icu4 endo locus Adaxialised leaves, meristem patterning [63]miR166 RLD1 Z.m Rld1-O endo locus Adaxialised leaves [69]miR167 ARF6/ARF8 A.t mARF6, mARF8 endo Ovule integuments and anthers growth defects [50]miR172 AP2 A.t AP2m1, AP2m3 35S Loss of floral organ identity [35]miR172 TOE3 A.t TOE3m 35S Loss of floral organ identity [39]miR319 LA S.l La-1 to 5 endo locus Precocious differentiation of leaf margins [49]miR319 TCP2 A.t mTCP2 35S Complementation of jaw-D [4]miR319 TCP4 A.t mTCP4 endo/35S Seedlings patterning defects [4]miR319 TCP4 A.t soj3, 6, 8, 18 endo locus Suppression of jaw-D [51]miR390 via TAS3 ARF3 A.t ARF3:ARF3mut endo Accelerated phase change [86]miR390 via TAS3 ARF3 A.t 35S:ETTmAB 35S Leaf morphology defects, accelerated phase change [87]m X35S

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iR824 AGL16 A.t AGL16m1, AGL16m2 2

bbreviations used: A.t, Arabidopsis thaliana; N.s, Nicotiana sylvestris; Z.m, Zea mais; S5S promoter, endo: endogenous promoter, endo locus: mutation in the endogenou

ated individually for each miRNA/target couples and can be crucialo understand the biological significance of miRNA-based repres-ion. At least three types of regulatory functions are supported byxperimental data for miRNAs (Fig. 3). Some miRNAs are competento define the spatial expression pattern of their targets: miRNA andarget are then expressed on adjacent non-overlapping domainsFig. 3A). Other miRNAs prevent excessive variations in the pat-ern and expression level of their targets. This buffering functionccurs for miRNA and target genes sharing overlapping expres-ion domains (Fig. 3B). Finally a third possible miRNA function ishe temporal regulation of target gene accumulation. This kind ofegulation involves opposite gradients of miRNA and target genesccumulation up to a predetermined target threshold inducingevelopmental transition (Fig. 3C).

.1. Spatial restriction

miRNAs following the clearing hypothesis [67], control develop-ental processes by depleting the tissue where they accumulate

rom their target, thereby promoting cell fate transition or dif-erentiation (Fig. 3A). These miRNAs have a complementaryccumulation pattern with their targets, and the miRNA repres-ion is necessary for the spatial restriction of target accumulation.his function is usually proposed when the target expression pat-ern expands in the absence of miRNA regulation (Fig. 3A). Spatialestriction was proposed for the action of miR165/166 in the reg-

lation of PHB in Arabidopsis and RLD1 in maize (Table 1). Thoseenes are polarly expressed on the adaxial side of the leaf. Inontrast, in situ hybridization in maize and Arabidopsis revealedhe accumulation of miR165/166 on the adjacent abaxial domain68,69]. Supporting this observation, the absence of miR166 regula-

matui

Increased rate of stomata in high order complexes [49]

num lycopersicum. 35S: promoter from the Cauliflower Mosaic Virus, 2X35S: doubles, AlcA: ethanol inducible promoter.

ion in Rld1-0 and phb-1D allows the ectopic accumulation of theseenes on the adaxial domain (Fig. 3A) [60,69]. These experimen-al evidences strongly support the patterning action of miR166/165.ocalization of the miRNA accumulation by in situ hybridizations not always an easy task as Arabidopsis sections seem to be

ore recalcitrant to miRNA in situ hybridization than other species70]. Alternatively other strategies can be used to determine theegulatory impact of a given miRNA. Such strategies have beensed to study miR167 regulation of the AUXIN RESPONSE FACTORenes, ARF6 and ARF8. These genes act redundantly to control theevelopment of reproductive structures (Table 1) [46]. miR167 over-xpression phenocopies arf6 arf8 double mutant, and leads to malend female sterility [46]. Plants expressing a miR167 resistant ver-ion of ARF6 (mARF6) are defective in integument growth leadingo poor pollen tube guidance, defective support of embryo growthnd sterility [46]. This observation demonstrates the important rolef miR167 in the orchestration of male and female reproductiveevelopmental programs. In situ hybridizations showed preferen-ial accumulation of the ARF transcripts in the funiculus duringvule development. In contrast miRNA-resistant forms mARF6 andARF8 are expressed in the funiculus and their expression domain

ctopically extends to ovule integuments (Table 2) [46]. The broaderccumulation of mARF6 and mARF8 was confirmed by comparinghe accumulation of translational fusions ARF6:GUS and ARF8:GUSith miRNA-resistant versions [46]. Finally, the study of the pro-oter activity for four members of the miR167 family showed a

utual exclusion between the expression of MIR167a and MIR167b

nd accumulation of their targets [46]. This study demonstrateshe essential patterning role of miR167. A similar approach wassed to study miR159 activity. The phytohormone Gibberellin (GA)

nfluences a variety of developmental processes [71]. GAMYB are

D. Garcia / Seminars in Cell & Developmental Biology 19 (2008) 586–595 591

Fig. 3. Regulatory roles of miRNAs. (A) Spatial restriction of target accumulation. The graph illustrates the accumulation of the miRNA target in domains A, where the miRNAis not present, and accumulation of the miRNA in domain B depleted from the target. The scheme gives the example of PHB regulation by miR166 in Arabidopsis leaf. In thepresence of miRNA regulation, target accumulation is restricted to the adaxial domain (domain A). In the miRNA-resistant mutant phb-1D leaf polarity is changed and ectopicexpression of PHB is seen throughout the leaf in domain A and B (adapted from refs. [60,68]). (B) Buffering function. The graph shows reduction and stabilization of targetexpression level in presence of miRNA regulation. The scheme represents miR164 regulation of CUC2 in floral meristem (M). In wild type, miR164 accumulates in the meristemand boundary regions, where it overlaps with CUC2. In miR164abc triple mutant (no miRNA regulation), expression of CUC2 is increased in boundary regions, sparse ectopice n. Thet meristp of miRi

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xpression can also be detected (adapted from refs. [78,79]). (C) Temporal regulatioarget overtime. The scheme represents the regulation of LA by miR319 in a tomatoresent in mature leaves (ml) leading to the accumulation of the target. In absence

n P0 and P1 (adapted from ref. [50]). P0 and P1 (leaf primordia 0 and 1).

A-specific transcriptional regulators that bind GA-response ele-ents to control GA activated genes. miRNAs of the miR159 family

re regulated by GA and target the GAMYB genes, MYB33 and MYB6538]. Overexpression of miR159 affects flowering time in short days,nthers developments and leads to male sterility [38,40], a pheno-ype reminiscent of that exhibited by myb33 myb65 double mutant72]. Developmental defects including hyponastic leaves and steril-ty appear in transgenic plants expressing the miRNA-resistantersion mMYB33 and in the double mutant miR159ab [72,73]. Theseefects are abolished in the quadruple mutant miR159ab myb33yb65, conclusively demonstrating the role of miRNA-based regu-

ation of MYB genes for the occurrence of these phenotypes [73], andhe essential role of miR159 for plant reproduction. The comparisonf the translational fusion MYB33:GUS and mMYB33:GUS, shows thectopic accumulation of mMYB33 throughout young seedlings. Thisbservation is confirmed in a double mutant with reduced miR159ctivity (miR159ab) in which the MYB33:GUS shows an expressionattern reminiscent of mMYB33 in wild type [72,73]. These studiesighlight the role of miR159 in the spatial restriction of MYB geneso the anthers.

.2. Buffering function

In addition to spatial restriction, some miRNAs function to bufferhe accumulation level of a target on a transcriptionally definedxpression domain. This action prevents fluctuations and accu-

dflma

graph shows a gradient of miRNA expression inducing an opposite gradient of itsem. miR319 is expressed at early stages, with no target detected. The miRNA is notNA regulation (La-2 mutant) the expression of the target is occurring prematurely

ately controls the expression level of targeted genes (Fig. 3B).xperimental data collected independently support this mode ofction for miR164. miR164 controls auxin signal transduction path-ays. This miRNA is induced by auxin treatment and controls the

xpression of NAC domain transcription factors involved in the inte-ration of the auxin signal [41]. Overexpression of miR164 leads tohe downregulation of CUP SHAPED COTYLEDON1 (CUC1), 2 (CUC2)nd the NAC family gene NAC1, and induce organ fusion resemblinghose found in the cuc1 cuc2 double mutant as well as reduced lat-ral rooting reminiscent of the nac1 mutant [37,39–41]. Transgenicxpression of miRNA-resistant versions of these genes underscoreshe relevance of the repression of individual genes. Expression ofesistant 5mCUC1 indicates a role in determination of floral organumber [39]. The CUC2g-m4 version is insensitive to miRNA cleav-ge and induces distinct phenotypes including an increase in leaferration, phylotaxis and carpel fusion defects [74,75]. In contrasthe NAC1m resistant version specifically affects root developmenthowing an increased lateral root formation [41]. These phenotypesre reminiscent of abnormalities induced by auxin distributionefects [76,77], and illustrate the role of miRNAs in auxin signalingathways.

Transcripts under the control of miR164, CUC1 and CUC2, areetected by in situ hybridization in the boundary domain betweenoral primordia and the floral meristem (Fig. 3B) [78]. The accu-ulation level of these genes is increased in single miR164c as well

s triple miR164abc mutants but their expression domain is not

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92 D. Garcia / Seminars in Cell & Dev

ramatically changed [78,79]. This observation indicates that theajor role of miR164 is to control the accumulation level of CUC

enes and not their spatial distribution. However in the absencef miR164 the precision of the CUC expression domain is alterednd some ectopic accumulation can be observed. This is an indica-ion that the miRNA function is also important to adjust potentialUC1 and CUC2 promoter leaking, ensuring the precision of theirxpression pattern [79]. Besides, during vegetative developmenthe transcriptional activity of miR164 genes and CUC2 overlaps ineaf sinus [74]. The experimental manipulation of MIR164 and CUC2xpression suggests that miR164 acts in a quantitative manner toontrol the intensity of CUC2 expression determining the severityf leaf serration pattern [74]. A similar buffering role has been pro-osed for miR156 regulation of SPL9 that determines the timingf leaf initiation [80]. The expression domains of miR156 and SPL9re largely overlapping and the expression of SPL9 in plants witheduced miR156 activity increases, but does not accumulate ectopi-ally. This observation suggests that similarly to miR164, miR156xerts a quantitative control on the accumulation of SPL9 [80].inally, co-expression has been reported for miR168 and AGO1 [81],hose correct interaction is crucial for plant development [82].uring development the buffering function of miRNAs is critical torevent the fluctuation of target gene expression level and ensuresevelopmental robustness, a function also described for animaliRNAs [83,84].

.3. Temporal regulation

Similar to the role of some miRNAs for developmental timingn C. elegans, some plant miRNAs also function as developmentallocks. Multiple examples show that miRNAs can efficiently controlevelopmental timing in plants. This function often relies on prop-rties of the miRNA target to induce, at a tightly defined threshold,he transition to the next developmental phase.

In Arabidopsis, the targeting of AP2-like genes by miR172 isontrolling the transition from vegetative to reproductive phase35,36]. The progressive accumulation of miR172 during vegeta-ive development induces a gradual decrease in the abundancef its targets. When the target reaches a critical threshold, it pro-otes flowering [35]. This mode of action seems to be conserved inaize, in which miR172 controls vegetative phase change through

he repression of the AP2-like gene glossy15 (gl15). Lauter et al.howed that the expression level of gl15 determines the lengthf the juvenile phase. The increase in miR172 accumulation pro-ressively represses gl15 during juvenile development. When a gl15hreshold is reached, the apparition of adult traits is promoted [85].

Flowering time is also controlled by the miR156 family. Overex-ression of miR156 leads to the repression of transcription factorsf the SBP family, extends juvenile development and delays flow-ring time [40,45]. In contrast, the deletion of the miRNA bindingite in the 3′ UTR of SPL3, SPL4 and SPL5 promotes vegetative phasehange and floral induction [45]. The quantification of miR156 andPL3 accumulation during leaf development indicates an oppositeattern, with initial high level of miR156 correlating with low SPL3

evel and a progressive inversion at later stages. Moreover the tem-oral accumulation pattern of SPL3 is disrupted in miRNA-resistantersions. These experiments demonstrate that miR156 repressesoral induction by controlling the temporal expression of SPL3 [45].imilarly, in maize the extended juvenile development of the het-rochronic mutant Corngrass1 (cg1) results from overexpression of

iR156 [48]. Interestingly miR172 is reduced in Cg1 suggesting that

he ratio of miR156 relative to miR172 is a key determinant to set uphe timing of juvenile to adult transition in plants [48].

The study of dominant La-2 mutant in tomato, demonstrates theole of miR319 in the timing of morphogenesis versus differentiation

scaob

ental Biology 19 (2008) 586–595

f leaf margins [50]. The early differentiation phenotype of La-2 isue to a precocious accumulation of the LA transcript in the mutant.he accumulation of miR319 and LA shows opposite gradients in theourse of leaf development. The role of miR319 in this context is toeduce the early expression of LA, repressing differentiation andxtending the duration of leaf morphogenesis [50] (Fig. 3C).

TAS3 ta-siRNAs are targeting ARF2, ARF3 and ARF4 [20]. A ta-iRNA-resistant version of ARF3 (mARF3) induces an early transitiono adult phase during vegetative development, reminiscent oflants impaired for the production of trans-acting siRNAs [86,87].AS3 is produced from three independent loci, TAS3a, b and c, buthe phenocopy of mARF3 in the tas3-1 mutant demonstrates thatAS3a has a predominant role in phase change [28]. In addition,he study of double mutants between asymmetric leaves 1 (as1)nd mutants impaired in the production of ta-siRNAs revealed theole of ARF3 targeting by TAS3, in the establishment of adaxial cellate during leaf development [88]. TAS3 accumulation has not beeneported to follow a gradient and its role seems more related to theetermination of the threshold of ARF genes instead of the controlf the developmental clock itself [87]. The advantage of using a reg-lation based on ta-siRNA instead of a direct miRNA regulation inhis process remains unclear. One difference between miRNAs andiRNAs is that miRNAs appear to act in a cell autonomous manner89], whereas siRNAs have the potential to move from cell-to-cell90]. Whether ta-siRNAs are able to act non-cell autonomously hasot been demonstrated for endogenous loci, but the experimen-al mimic of ta-siRNAs are indeed non-cell autonomous [91]. Thus,

ovement of TAS3 ta-siRNAs could generate spatial gradients thatight be important for TAS3 function in phase change or leaf polar-

ty.The miRNAs controlling the temporal accumulation of their tar-

et have two specific features: they are co-expressed with theirarget, and the miRNA and target accumulate overtime as oppositeradients (Fig. 3C). The examples presented in this part illustrate themportance of temporal rather than spatial control of gene expres-ion by miRNAs for plant development. miRNAs operating throughhis mode determine the gradual accumulation of their target tonsure the correct developmental timing and act as developmentallocks.

. Subfunctionalization of miRNAs

Many conserved miRNAs are present in the genome as geneamilies, and functional redundancy is seen among family mem-ers. These redundant functions probably explain the relativelyare descriptions of developmental phenotypes for miRNA knockut mutants [28,41,74,78,92,93]. Nevertheless, the study of miRNAnock out mutants allows the access to a different layer of miRNAunction, the specialization of miRNA family members (Table 3).his miRNA subfunctionalization can rely on alternative mecha-isms presented in the following paragraph.

.1. miR164 family

The miR164 family comprises three loci, miR164a, b and c. Therst miRNA KO mutant described is the early extra petal 1 (eep1)utant, in which the control of petal number is disturbed. Theutant contains a transposon insertion upstream the miR164c

ocus, which reduces its accumulation, consequently, CUC1 andUC2 transcripts overaccumulate. The study of eep1 outlines the

pecific function of miR164c during flower development [78]. Inontrast a KO mutant for miR164a exhibits different abnormalitiesnd induces an accentuated leaf serration pattern [74]. The rolef the miRNA repression of CUC2 in this phenomenon is revealedy the abnormal serration pattern of the miRNA-resistant version

D. Garcia / Seminars in Cell & Developmental Biology 19 (2008) 586–595 593

Table 3miRNA and ta-siRNA KO mutants

Family Locus name Mutant name Species Phenotypes

miR159 miR159a miR159b miR159ab A.t Pleiotropic developemntal defects including hyponastic leaves and sterility [73]miR164 miR164c eep1 A.t Flower defects [78]miR164 miR164b miR164b A.t Increased lateral rooting [41]miR164 miR164a miR164a A.t Increased lateral rooting [41], increased leaf serration [74]miR169 BL bl-1 P.h Floral homeotic conversion [92]mmT

A ; Z.m,

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AS3 TAS3a tas3-1 A.t

bbreviations: A.t, Arabidopsis thaliana; A.m, Antirrhinum majus; P.h, Petunia hybrida

UC2g-m4. Moreover, CUC2 and miR164a are coexpressed in leafinus, and miR164a phenotype can be specifically complementedy cuc2 but not cuc1 mutation. The experimental manipulation ofUC2 and miR164a levels suggests that leaf serration depends onhe relative amount of the miRNA and its target [74]. A differenttudy shows that in addition to these defects, mutants in miR164and miR164b, have increased lateral root formation due to excessiveccumulation of NAC1 [41]. This similarity highlights the redun-ancy between these two loci in the control of root initiation [41].inally the study of the triple mutant miR164abc and different dou-le mutant combinations suggests that the three loci are equally

nvolved in the control of the phylotactic pattern, probably throughhe repression of CUC2 [79].

.2. miR159/319 family

The microRNAs of the miR159/miR319 family are related inequence but repress two independent sets of genes, the MYB andCP transcription factors respectively. MYB and TCP genes harborlosely related miRNA binding sites. The specificity of miRNA/targetnteraction has been studied in detail and relies on two distinct

echanisms. Overexpression of miR319 demonstrates that thisiRNA has the required biochemical properties to induce the cleav-

ge of MYB genes in vivo. The exclusive targeting of MYB genes byiR159, in wild type conditions, is in fact due to a higher dose of thisiRNA. In contrast, overexpression of miR159 does not affect the

xpression of TCP genes, in this case sequence differences ensurehe specificity to avoid that high expression of miR159 interfere withargets of miR319 [51]. This study is the first to point out sequence-ased specialization of miRNA of the same family. There is a greatotential for this kind of specialization as 30% of conserved miRNA

amilies vary at two nucleotides position or more between mem-ers [51].

In addition, the selective overexpression of individual miRNAoci can lead to a gradation in the developmental phenotypesbserved. The overexpression of miR319a leads to strong miR319ccumulation and strong phenotypes. In contrast, miR319c onlynduces weak miRNA accumulation and phenotypes [51]. A similarituation occurs for miR167 overexpression, for which among theour predicted miR167 genes only miR167a recapitulates the arf/arf8ouble mutant phenotype when overexpressed [46]. A possibility ishat these differences underlie unequal efficiency in processing ofhe stem loop by DCL1. Redundancy can result in the loss of process-ng efficiency of certain miRNA locus, without any developmentalonsequences. This point has to be taken in account when studyingiRNA families in addition to the transcriptional activity of pro-oter regions, the efficiency of stem loop processing might play a

ole in the potential of miRNAs for subfunctionalization.

. Perspectives

Our understanding of RNA silencing mechanisms provided greatools to the entire scientific community through the use of RNA

aalum

Floral homeotic conversion [92]Perturbed sex determination and meristem function [93]Accelerated phase change [28]

Zea mais. miRNA and tasi-RNA knock out phenotypes.

nterference (RNAi) for directed gene silencing in a variety of plantpecies. The recent advances in understanding miRNA and ta-siRNAction gave birth to interesting new alternatives for plant biology,hrough the use of artificial-miRNAs (amiRs). What are the advan-ages of amiRs over conventional hairpin based RNAi? First, despiteNAi [94], amiRs have not been observed to induce systemic spreadf RNA silencing, and do not act outside their expression domain95,96]. This cell autonomy of amiRNAs provides the ideal system topatially control downregulation of target genes with inducible andissue specific promoters. Moreover in contrast to classical RNAi,miRs produce single discrete small RNA specie, allowing a betterredictability of the targets. The use of amiRs allows successful tar-eting of single genes as well as multigenic gene families in a varietyf plants including Arabidopsis, non-model species and crop plants95–97]. In addition, similarly to the production of ta-siRNAs, whentranscript is subjected to a double targeting by artificial-miRNAs,

t can induce the production of secondary siRNAs and a short-rangeilencing signal [91]. This strategy could as well be an alternativeo RNAi allowing a control over the establishment of the mobileilencing signal. A major aspect of these methods is that they canasily be adapted in non-model crop plants to control traits of agro-omic interest. For these reasons, amiRNAs will probably becomen unavoidable silencing strategy in the near future.

The study of the phosphate starvation induced miRNA miR399,ed to the identification of a new mechanism of miRNA inhibi-ion termed ‘‘target mimicry’’. This inhibition is achieved by theon-coding RNA INDUCED BY PHOSPHATE STARVATION1 (IPS1). IPS1ontains a non-cleavable miR399 site, sequester the miR399 andvoid its action [98]. This property of the IPS1 transcript can bexperimentally manipulated to inhibit the function of unrelatediRNAs, by changing the sequence of the non-cleavable miRNA site

80,98]. This strategy could be very useful to identify the regulatoryunction of large miRNA families for which the study of multiplenock out is difficult. The use of tissue specific promoters driv-ng the expression of such artificial ‘‘target mimics’’ also provides

powerful tool to inactivate entire miRNA families in a spatiallyontrolled fashion that could be adapted to plants of agronomicnterest.

One of the next challenges in plant miRNA biology is to dis-over the functions of non-conserved or recently emerged miRNAs.heir identification has been slowed down by their low expressionevel and the conservation bias implemented in miRNA identifica-ion software. Nevertheless, the use of deep sequencing techniquesllowed a preliminary evaluation of their diversity [21,99]. Thetudy of miR824, a recently evolved miRNA conserved in the Bras-icaceae, shows its role in the formation of high order stomatalomplexes [49]. This example demonstrates the potential of theseiRNAs to influence plant development. In many cases, transcript

ccumulation for predicted targets of the non-conserved miRNAsre not changed in mutants of the miRNA pathway. This observationed to the idea that a high proportion of these miRNAs have no reg-latory functions [99]. This possibility was recently challenged foriR834, for which the predominant mode of action was revealed to

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e translational repression [54]. It would be interesting to investi-ate the regulatory potential of other ‘‘young’’ miRNAs that are notnducing transcript cleavage, to determine if translational repres-ion represents their preferential mode of action.

cknowledgements

I would like to thank Olivier Voinnet, Rebecca Schwab, Patrickchard, Peter Brodersen and Fabrice Michel for helpful discussionsnd critical reading of the manuscript; Shahinez Garcia for helpith the formatting of the tables and references; the editor Frederger and the anonymous reviewer for constructive comments onhe review. DG is supported by a postdoctoral fellowship from theU integrated project SIRROCO (LSHG-CT-2006-037900).

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