R756 Dispatch

Plant development: Two sides to organ asymmetryAndrew Hudson

Three gene families have been identified which interactto polarize plant lateral organs. The results suggest thatorgan polarity is initially determined by a signal fromthe shoot tip which specifies adaxial organ identity andresults in repression of abaxial identity, therebyaligning the polarity of organs with the stem.

Address: Institute of Cell and Molecular Biology, University ofEdinburgh, Edinburgh, EH9 3JH, UK.E-mail: andrew.hudson@ed.ac.uk

Current Biology 2001, 11:R756–R758

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The polarity of plant shoots and flowers is obvious in theirdirection of growth and in the morphology of their lateralorgans, such as leaves and floral organs. Leaves tend to beflattened perpendicular to the stem axis and may show anasymmetric distribution of cell types, with tissues spec-ialised for light harvesting on the ‘adaxial’ side — facingthe shoot tip — and those specialised for gas exchange onthe ‘abaxial’ side. Similar adaxial–abaxial polarity can beseen in floral organs and cotyledons. Recent studies [1–4]have revealed three families of genes which act to promoteeither adaxial or abaxial organ fate. The results suggestthat adaxial fate is dependent on signals from the shootapex which activate proteins needed to repress expressionof abaxial genes.

Experiments in the 1950s revealed that leaf asymmetrycould be disrupted by inserting a barrier into the shootapical meristem, on the adaxial side of a group of leafinitial cells [5,6]. The resulting leaf primordium grew withradial symmetry and lacked adaxial cell types, suggestingthat a signal from the centre of the meristem promotedadaxial organ identity and that abaxial identity occurred bydefault. A modern interpretation would predict there aregenes that specify adaxial fate early in organ development,in response to a signal in the shoot apical meristem, andthat repress genes that promote abaxial organ fate.

A family of regulatory genes fitting these criteria hasnow come to light in Arabidopsis. It was initially revealedby the phabulosa-1d (phb-1d) mutation, which causes dose-dependent adaxialisation of all lateral organs [7]. Thissemi-dominance is consistent with PHB activity promot-ing adaxial fate and phb-1d being a gain-of-function muta-tion that causes ectopic activity. The recent isolation byMcConnell et al. [1] of PHB has provided considerablesupport for this view. They found that PHB encodes amember of a small subfamily of homeodomain–leucine

zipper transcription factors — the HD-ZIP III proteins —which are know to bind specific DNA target sequences[8]. The other two members of this family in Arabidopsis— PHAVOLUTA (PHV) [1,8] and REVOLUTA (REV) [9,10]— have similar expression patterns to PHB [1,10,11], sug-gesting related roles. All three genes are normally expressedin the shoot apical meristem, uniformly within the organinitials. But their expression becomes restricted to anadaxial domain of each organ primordium as it initiatesfrom the shoot apical meristem, and later expression is con-fined to developing vascular tissues. PHB and PHV showan equivalent expression in embryos — initially through-out the apical region of the globular embryo and then onlyin the shoot apical meristem, an adaxial domain of eachdeveloping cotyledon and developing vascular cells [1,10].

Analysis of phb-1d and equivalent phb mutations suggeststhe basis for their semi-dominance. PHB and the otherHD-ZIP III proteins contain a so-called START domain.This motif is present in a range of functionally diverse pro-teins and in some has been found to bind a lipid ligand[12]. The best characterised version of this motif, in thehuman steroidogenic acute regulatory (StAR) protein, formsa hydrophobic pocket that binds cholesterol and transportsit through the outer mitochondrial membrane [13]. Fiveindependent phb mutations were found to alter PHB’sSTART domain: two cause an in-frame insertion of thesame 10 or 11 amino acids near the protein’s amino termi-nus, while the others substituted glutamic acid for glycineat the adjacent position. Semi-dominant phv mutations,which have a similar adaxialising effect, were found to causethe equivalent substitution in PHV. One simple explana-tion for the effects of these mutations is that binding a lipidligand in adaxial cells activates PHB and PHV proteins.Amino acid insertions or substitutions within the STARTdomain might therefore render the proteins constitutivelyactive, thereby causing them to specify adaxial identityectopically. Further support for regulation of this kind hascome from the finding that ectopic expression of the wild-type PHB gene had no effect, presumably because distrib-ution of the putative ligand remained limiting.

Analysis of phb-1d expression also proved informative.Transcripts were found in the shoot apical meristem andorgan initials, as in the case of wild-type PHB, but theydid not become restricted to adaxial cells as organ primor-dia initiated. Because phb mutations are likely to affectprotein activity, this suggested that active PHB promotesaccumulation of PHB RNA. Such autoregulation, coupledwith the ability of activated PHB protein to promoteavailability of its ligand, could provide a mechanism by

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which adaxial fate is maintained in growing primordia. Italso suggests that PHB RNA levels may reflect the con-centration of the activating ligand. PHB transcripts areabundant in the centre of the shoot apical meristem andshow graded expression in organ primordia, with highestlevels adaxially. Such a pattern suggests the existence of amorphogen which forms a gradient from the centre to theperiphery of the shoot apical meristem and specifiesadaxial fate in a concentration-dependent manner. It isalso consistent with the loss of adaxial fate observed inleaf initials surgically isolated from the centre of the shootapical meristem, which is proposed to be the source of themorphogen. Although it is appealing to speculate that thepotential lipid ligand of PHB might be this morphogen,the ligand might alternatively be produced as a secondmessenger within cells that have been activated by a dif-ferent signal.

The higher levels of PHB expression detected in shootapical meristem cells adaxial to organ initials are furthersuggestive of reverse signalling from organs to the shootapical meristem. Because the related REV gene is neededto promote axillary shoot apical meristem formation [11,14],and phb-1d mutants have larger shoot apical meristemsthan wild-type [7], the three HD-ZIP III genes mightfunction redundantly to promote shoot apical meristemactivity. Such roles should be revealed in plants carryingmultiple loss-of-function mutations of PHV, PHB or REV.

The HD-ZIP III genes are not alone in promoting adaxialcell fate in higher plants. For example, PINHEAD (PNH),which encodes a potential transcription elongation factor,is expressed in a similar domain to PHB and promotesadaxial fate and shoot apical meristem activity redundantlywith the related AGONAUTE1 gene [15,16]. It should nowbe possible to test how this role is related to that of theHD-ZIP III genes.

On the other side of the organ, two Arabidopsis genefamilies promote abaxial fate. These include all fivemembers of the YABBY (YAB) family, which encode likelytranscription factors. [17–19]. Each organ expresses at leasttwo YAB genes, first in internal organ initials and then inan abaxial domain once primordia initiate. Although thispattern suggested a role in promoting abaxial fate, leavesof plants mutant for the two most similar YAB genes,FILAMENTOUS FLOWER (FIL) and YAB3, showed alter-ations to abaxial cell types that were considerably less severethan in plants carrying the phb-1d allele. Eshed et al. [2]therefore proposed that additional genes must act inde-pendently to promote abaxial fate, and that these might beidentified by mutations that enhance yab mutant pheno-types. Their approach led to identification of theKANADI1 (KAN1) gene, which had also been found inde-pendently by Kerstetter et al. [3]. A further screen for

enhancers of the kan1 phenotype identified additionalgenes, including KAN2 [4].

The recent isolation of KAN1 and KAN2 has shown thatthey encode two of the three Arabidopsis members of aprotein subfamily implicated in transcriptional regulation.In wild-type plants, KAN1 expression is detected in anabaxial domain of each organ primordium, consistent witha role in promoting abaxial fate [3]. KAN1 expression inthe embryo is established in a domain that includes theabaxial part of the developing cotyledons and periphery ofthe hypocotyl, the apparent complement of the PHB andREV domain. This embryonic domain of expression alsoprovides further evidence for a relationship between theabaxial side of an organ and the peripheral stem.

The enhanced mutant phenotypes produced by combiningkan and yab mutations suggested that the genes areexpressed independently. Loss of KAN2 activity has noeffect in a wild-type background [3], while kan1 mutationsalone have subtle and semi-dominant effects on leaf andcarpel polarity [4]. Organs of plants mutant for both KAN1and KAN2 show more severe adaxialisation — leaves arenarrow, contain adaxial cell types abaxially and produceadaxial outgrowths consisting of abaxial and marginal celltypes. Additional loss of YAB activity in the kan1 kan2 filyab3 quadruple mutant causes further loss of abaxial fate,so organs approached those of the phb-1d mutant in pheno-type. Residual differences were proposed to result from theactivity of other genes, possibly including the remainingYAB or KAN family members [4].

Interaction between cells with adaxial and abaxial iden-tities has been suggested to induce the lateral growth ofleaves that flattens them perpendicular to the stem axis[20]. The expression of FIL in the abaxial outgrowth ofkan1 kan2 mutant leaves, and the loss of these outgrowthswith additional loss of FIL and YAB3 activity, led Eshedet al. [4] to propose that the outgrowth resulted from juxta-position of domains of high and low YAB gene expression.This is consistent with the observed correlation of theboundaries between high and low (or no) YAB expressionwith outgrowth of the normal leaf blade, and with thereduction in blade growth that results from uniform YABexpression [17].

How the KAN and YAB genes interact is not yet clear,partly because of genetic redundancy. Enhancement ofyab mutant phenotypes by kan mutations suggests thatKAN and YAB genes act independently. Similarly, theabaxialising effect of ectopic KAN1 or KAN2 expressionis not dependent on YAB3 and FIL activity [4].. However,the abaxial expression domain of FIL is reduced in organsof kan1 kan2 mutants [17], consistent with KAN genespromoting YAB gene expression. Similarly, uniform

expression of either KAN1 or KAN2, unlike ectopic YABexpression, appears sufficient for complete abaxialisationof cotyledons [4].

Several other genes that promote abaxial organ fate werealso identified by genetic enhancement of yab or kanmutant phenotypes. These included PICKLE (PKL), whichis likely to encode part of a chromatin remodelling complexinvolved in the repression of target genes [2,21]. Oneexplanation for the involvement of PKL, which is con-sistent with its role in repressing shoot apical meristem-promoting homeobox genes in organs [21], is that itmaintains organ fate and therefore the correct activity oforgan polarity genes.

Adaxial and abaxial organ fates appear mutually exclusive:a gain of adaxial identity in phb mutants occurs at theexpense of abaxial identity, whereas ectopic KAN geneexpression has the opposite effect. This suggests thatadaxial and abaxial genes might repress each other’s expres-sion. Although current evidence has been limited byredundancy within gene family, available data generallysupport this view. Thus, the domain of PHB and REVexpression in wild-type embryos appears complementaryto that of KAN1 [1,4]; the YAB gene FIL is not expressedin severely adaxialised leaves of phb-1d [17]; and PHV andREV show ectopic abaxial expression in kan1 kan2 fil yab3mutants [4]. An organ initial cell can express both HD-ZIPIII and YAB genes, however, suggesting that additionalfactors that are specific to organ primordia must be neededfor mutual repression to occur.

What might be the significance of mutual repression?Current evidence supports the view that activation ofHD-ZIPIII proteins is the primary step in determinationof organ polarity, and repression of YAB and KAN genes isa secondary consequence. Subsequent repression of HD-ZIPIII proteins by KAN (and possibly YAB) genes mightthus serve to reinforce the distinction between adaxialand abaxial domains. Reinforcement might also involveabaxial cells signalling to adaxial cells to promote theiridentity, as suggested by the mild adaxial defects of kan1mutants [3]. In this scenario, abaxial fate represents theabsence of adaxial specification. The available evidencedoes not, however, rule out the possibility that abaxialfate is itself specified independently, for example bysignals from the periphery of the shoot apical meristem.In this case, mutual repression could act to partition pri-mordium cells to the two alternative fates, as well as toreinforce the distinction between them. Current evidencealso cannot determine the extent to which HD-ZIP IIIgenes directly promote adaxial fate, rather than repress-ing activity of abaxial genes (and vice versa). It has,however, provided the tools to allow these outstandingissues to be investigated.

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