ORIGINAL ARTICLE

Yue Sun Æ Qingwen Zhou Æ Wei Zhang Æ Yanlei FuHai Huang

ASYMMETRIC LEAVES1, an Arabidopsis gene that is involvedin the control of cell differentiation in leaves

Received: 3 May 2001 / Accepted: 21 June 2001 / Published online: 10 November 2001� Springer-Verlag 2001

Abstract During leaf development, the formation ofdorsal-ventral and proximal-distal axes is central to leafmorphogenesis. To investigate the genetic basis ofdorsoventrality and proximodistality in the leaf, wescreened for mutants of Arabidopsis thaliana (L.) Heynh.with defects in leaf morphogenesis. We describe here thephenotypic analysis of three mutant alleles that we haveisolated. These mutants show varying degrees ofabnormality including dwarfism, broad leaf lamina, andaberrant floral organs and fruits. Genetic analysisrevealed that these mutations are alleles of the previouslyisolated mutant asymmetric leaves1 (as1). In addition tothe leaf phenotypes described previously, these allelesdisplay other phenotypes that were not observed. Theseinclude: (i) some rosette leaves with petiole growth un-derneath the leaf lamina; (ii) leaf vein branching in thepetiole; and (iii) a leaf lamina with an epidermis similar tothat on the petiole. The mutant phenotypes suggest thattheASYMMETRIC LEAVES1 (AS1) gene is involved inthe control of cell differentiation in leaves. As the first stepin determining a molecular function for AS1, we haveidentified theAS1 gene using map-based cloning. TheAS1gene encodes a MYB-domain protein that is homologousto the Antirrhinum PHANTASTICA (PHAN) and maizeROUGHSHEATH2 (RS2) genes.AS1 is expressed nearlyubiquitously, consistent with the pleiotropic mutantphenotypes. High levels of AS1 expression were found intissues with highly proliferative cells, which further sug-gests a role in cell division and early cell differentiation.

Keywords Arabidopsis Æ ASYMMETRIC LEAVES1Cell differentiation Æ MYB protein Æ Proximodistalaxis Æ Vascular patterning

Abbreviations DIC: differential interference con-trast Æ EMS: ethyl methanesulfonate Æ Ler: Landsbergerecta Æ SEM: scanning electron microscopy Æ SSLP:simple sequence length polymorphism

Introduction

In higher plants, all post-embryonic leaves derive fromsmall populations of mitotic cells, the shoot apicalmeristem (SAM). At the tip of the SAM, the meristem‘central zone’ (CZ), which is characterized by a slow rateof cell division, replenishes meristematic cells and givesrise to peripheral cells (Sylvester et al. 1996). Leaforganogenesis occurs in a lateral region of relatively highmitotic index called the meristem ‘peripheral zone’ (PZ)(Scanlon 2000). Leaf founder cells are subsequentlyrecruited in the PZ, and initiate outgrowth of leaf pri-mordia and simultaneously establish polarity along theproximodistal and dorsoventral axes.

A number of mutants involved in SAM formation(Barton and Poethig 1993; McConnell and Barton 1995;Laux et al. 1996) and leaf development (Berna et al.1999; Serrano-Cartagena et al. 1999; Ori et al. 2000;Byrne et al. 2001) have been identified and characterizedin details. Mutations in the SHOOT MERISTEMLESS(STM) gene in Arabidopsis thaliana result in the lack of aSAM, indicating that STM is essential for embryonicSAM formation (Barton and Poethig 1993). The STMgene encodes a member of the maize KNOTTED1 classof homeodomain proteins (KNOX) (Long et al. 1996).This class of proteins plays a very important role inregulating SAM development, and its orthologs arewidely found in many plants (Matsuoka et al. 1993;Kerstetter et al. 1994; Lincoln et al. 1994; Hareven et al.1996; Tamaoki et al. 1997). STM transcripts aredetectable in SAM cells, but not in those that will form

Planta (2002) 214: 694–702DOI 10.1007/s004250100673

Y. Sun and Q. Zhou contributed equally to this work

Y. Sun Æ Q. Zhou Æ W. Zhang Æ Y. Fu Æ H. Huang (&)Shanghai Institute of Plant Physiology,Chinese Academy of Sciences, 300 Fenglin Road,Shanghai 200032, ChinaE-mail: hhuang@iris.sipp.ac.cnFax: +86-21-64042385

H. HuangShanghai Research Center of Life Sciences,Chinese Academy of Sciences, 320 Yueyang Road,Shanghai 200031, China

the next leaf primordium (P0 initials) (Long et al. 1996).Ectopic expression of KNOX genes results in the devel-opment of lobed leaves and in the formation of ectopicmeristems in some dicot species (Sinha et al. 1993; Lin-coln et al. 1994; Chuck et al. 1996; Hareven et al. 1996).All of the studies described above suggest that the down-regulation of KNOX gene expression is essential fornormal leaf initiation and development.

Recent studies on leaf development led to the iden-tification of another class of genes, including PHAN inAntirrhinum (Waites and Hudson 1995), RS2 in maize(Schneeberger et al. 1998), and AS1 in Arabidopsis(Byrne et al. 2000). Mutant characterizations revealedthat the leaves, bracts and petal lobes of phan showvarying degrees of reduction in adaxial tissues, indicat-ing that PHAN is required for establishing adaxial cellidentity (Waites and Hudson 1995). Mutations in theRS2 locus cause a range of pleiotropic phenotypicchanges, which are sensitive to genetic background andenvironmental conditions (Schneeberger et al. 1998).These include dwarfism, leaf twisting, disorganized dif-ferentiation of the blade-sheath boundary, aberrantvascular patterning, and the generation of semi-bladelessleaves. Mutations in the AS1 locus result in a broad andweakly wrinkled lamina in vegetative and cauline leaves(Serrano-Cartagena et al. 1999), wider sepals and curledpetals (Ori et al. 2000), and the mutant leaf lamina insome as1 alleles is subdivided into lobes (Byrne et al.2000). However, a detailed phenotypic analysis of theas1 mutant at the cellular level has not been reported.PHAN, RS2 and AS1 genes have been isolated, and theyall encode a MYB transcription factor, and have a highdegree of sequence similarity to each other (Waites et al.1998; Timmermans et al. 1999; Tsiantis et al. 1999;Byrne et al. 2000). Immunolocalization and in situhybridization experiments demonstrated that KNOXproteins are ectopically accumulated in phan, rs2 and as1mutants, providing direct evidence that KNOX genes aredown-regulated by these genes in leaf initials (Waiteset al. 1998; Timmermans et al. 1999; Tsiantis et al. 1999;Byrne et al. 2000).

To understand better the regulatory mechanisms inleaf initiation and development, we carried out a large-scale screening ofArabidopsis leaf mutants and obtained anumber of mutants with abnormal leaves (Sun et al. 2000).Here, we report the characterizations of three new as1alleles, as1-101, as1-102 and as1-103, and describe as1phenotypes that were not observed previously. We foundthat the AS1 gene is involved in the control of cell differ-entiation in leaves. In addition, we also report the iden-tification of the AS1 gene by map-based cloning, and thesequence analysis for five different as1 alleles.

Materials and methods

Plant materials and plant growth

The as1 mutants of Arabidopsis thaliana (L.) Heynh., which are inthe Landsberg erecta (Ler) genetic background, were isolated from

ethyl methanesulfonate (EMS)-induced populations. The identifi-cation of as1-101 and as1-102 was reported previously (Sun et al.2000) and as1-103 was isolated in this work. Mutants as1-101, as1-102 and as1-103 were each backcrossed to the wild-type Ler four,three and two times, respectively, before phenotypic analyses.Additional as1 alleles, CS146 (as1-1) and N321 (as1-14), were ob-tained from the Ohio State University Arabidopsis Stock Centerand Nottingham Arabidopsis Stock Centre, respectively. Arabid-opsis was grown according to our previous conditions (Chen et al.2000).

Microscopy

Plant tissues were fixed in FAA (5% formalin, 6% acetic acid, 62%ethanol, by vol.) at room temperature overnight, and then clearedwith Herr’s fluid (85% lactic acid:chloral hydrate:phenol:cloveoil:xylene, 2:2:2:2:1, by wt.). Vascular patterns were viewed with aZeiss dissecting microscope using the dark-field setting. Differentialinterference contrast (DIC) analysis of leaf tissues was according toYang et al. (1999), using a Zeiss light microscope. Scanning elec-tron microscopy (SEM) was performed according to the methoddescribed by Chen et al. (2000).

Map-based cloning and sequencing of AS1

Based on the preliminary map position for AS1, fine-structuremapping of the as1 locus was performed by the analysis of an F2mapping population from a cross between as1-101 and the poly-morphic Columbia (Col) ecotype. Simple sequence length poly-morphism (SSLP) and single nucleotide polymorphism (SNP) wereidentified either by searching the Internet (http://arabidopsis.org/cereon/index.html), or by sequencing Ler genomic DNA in theregions of interest. Marker design based on the SSLP and SNPsequences was according to the methods described by Bell andEcker (1994) and Newton et al. (1989), respectively. DNA prepa-ration and polymerase chain reaction (PCR) were performed ac-cording to our previous report (Li et al. 1999). For sequencing ofthe putative AS1 gene in as1 mutants, the PCR-amplified genomicfragment from each allele was cloned into the EcoRI site of thepGEM7Z(+) vector (Promega). Two independent subclones fromeach as1 allele were sequenced, and the sequences were subse-quently aligned with that from the Arabidopsis Genome Initiativedatabase to identify nucleotide changes.

DNA and RNA gel blot analyses,and complementation of AS1

DNA and total RNA were isolated (Huang et al. 1995; Li et al.1999), gel-separated and blotted using a standard protocol(Sambrook et al. 1989). The hybridization was performed at55 �C. For complementation of AS1, a 1.1-kb genomic DNAcontaining the entire AS1 coding region was PCR-amplified fromwild-type plants and sequenced. This DNA fragment was theninserted into a binary T-DNA vector pMON530 (Monsanto),downstream from a 35 S promoter. This construct, 35 S::AS1,was introduced into as1-101 mutant plants by Agrobacterium-mediated transformation.

Results

Mutant isolation and genetic analysis

In the course of screening Arabidopsis mutants withdefects in leaves, three mutants from different EMSmutagenesis experiments were isolated with a similarleaf phenotype: some of the rosette leaves had a

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lotus-leaf-like structure with the petiole growing fromunderneath the leaf lamina. These monogenic recessivemutants, which were subsequently demonstrated to beallelic, were named ll2-1 to ll2-3 for lotus leaf. Sincesome rosette leaves of ll2 plants were also similar tothose of as1 (Redei 1965; Serrano-Cartagena et al. 1999),we crossed ll2-1 to an as1 mutant, as1-1, for the allelismtest. The result showed that ll2 and as1 are allelic;therefore, we renamed these mutants as1-101, as1-102,and as1-103, respectively. Since these three as1 mutants

Fig. 1A–D Phenotypes ofwild-type and as1-101 mutantplants of Arabidopsis thaliana.A Wild-type (left) and as1-101(right) plants at the same stage.B A wild-type rosette leaf (left)and as1-101 rosette leaves(middle and right). Note thatsome of the rosette leaves fromthe first pair display a lotus-leafstructure (right).C Wild-type (left) and as1-101(right) flowers. D Wild-type(left) and as1-101 (right) sili-ques. Bars = 0.5 cm (A),0.25 cm (B), 1 mm (C, D)

Fig. 2A–F Vascular patterns of wild-type and as1-101 mutantplants of A. thaliana. A Wild-type (left) and as1–101 (right)cotyledons. Arrow indicates a proximal vein that is not fullydeveloped. B One of the first pair of rosette leaves in a wild-typeplant (left) and an as1-101 mutant (right). Note that this leaf doesnot show a lotus-leaf structure; the arrow indicates veins thatbranch in the as1 petiole. C A later-appearing rosette leaf in thewild type (left) and in an as1-101mutant (right). The DIC images inthe lower panels are enlargements of the regions indicated by thearrows. D Wild-type (left) and as1-101 (right) cauline leaves.E Wild-type (left) and as1-101 petals (right). F Wild-type (left) andas1-101 sepals (right). Bars = 1 mm (dark-field images), 50 lm(DIC images in C)

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had overall similar phenotypes, we focused the mutantanalysis only on as1-101, except where otherwise noted.

Phenotypes of as1 plants

To test what developmental stages are affected by the as1mutation, we analyzed the phenotypes of as1 plants usinglight microscopy. Compared with wild-type plants(Fig. 1A, left), as1 plants are dwarfish (Fig. 1A, right).Instead of the petiole growing from the proximal end as inwild-type leaves (Fig. 1B, left), petioles of some of the firsttwo rosette leaves in as1 grow from underneath the leaflaminae (123/740 in as1-101, 3/598 in as1-102, and 1/144in as1-103; Fig. 1B, right). Although the later-appearingrosette leaves do not show this lotus-leaf structure, they

are all broad at the proximal end, displaying a heart-shaped architecture (Fig. 1B, middle). as1 has relativelysmall flowers with early opening floral buds (Fig. 1C,right), and some gynoecia consist of three (3/42) or four(1/42) carpels. In addition, growth of sepals of as1 isimpaired, and elongation of the petals and stamens isdelayed relative to the gynoecium, which is consistentwith the observations in the previously identified as1-1mutants (Ori et al. 2000). The surface of as1 siliques isrough (Fig. 1D, right), and carpel valves hardly dehisce atfruit maturation. In addition, some lateral inflorescencesdo not show the accompanying cauline leaves (10/38).The pleiotropic nature of as1 mutants indicates that theAS1 gene is involved in multiple developmental processes.

Vascular patterns of the as1-101 mutant

To characterize in more detail the abnormalities of theas1 mutant, we analyzed as1 vascular patterns in leavesas well as in sepals and petals. Most wild-type cotyle-dons have similar vascular patterns, as shown in Fig. 2A(left). In as1 cotyledons, however, development of lateralveins seems incomplete, with only one vascular loop (45/46; Fig. 2A, right). In the first rosette leaf of wild-typeplants, lateral veins were visible only in the lamina(Fig. 2B, left), whereas vein branching in the as1 mutantoccurs more proximally, appearing in the petiole(Fig. 2B, right). In petioles of later-appearing rosetteleaves (Fig. 2C), vein branching was seen in both wild-type and as1 petioles. However, veins in as1 mutant

Fig. 3A–N SEM of epidermal cells on wild-type and as1-101rosette leaves of A. thaliana. A The proximal part of a third-leaflamina on the adaxial side of a wild-type plant. B Close-up of A,showing two types of epidermal cell. C A petiole from a wild-typerosette leaf. D Close-up of C. E The adaxial side of the proximalpart of a third-leaf lamina from an as1-101 plant. F Close-up of E,showing the long, narrow epidermal cells. G A petiole from an as1-101 rosette leaf. H Close-up of G; note that the epidermal cells ofthe as1-101 petiole have a very similar structure to those found onthe proximal part of the as1-101 leaf lamina (F). IWild-type abaxialepidermis at the equivalent of the adaxial region shown in A. JClose-up of I, showing the midvein. K Close-up of I, showing theirregularly shaped epidermal cells neighboring the midvein. L Theas1 abaxial epidermis at the equivalent of the adaxial region shownin E. M Close-up of L, showing the as1 midvein. N Close-up of L,showing the as1 epidermal cells near the midvein. Bars = 100 lm

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petioles are much thicker than those in the wild type,mimicking the thicker lateral veins in wild-type laminae.The tracheary elements in as1 petioles are dramaticallyincreased (Fig. 2C, right, and the lower panel) as com-pared with normal tracheary elements in the wild type(Fig. 2C, left and the lower panel). Compared withcauline leaves in the wild type (Fig. 2D, left), most as1cauline leaves are lobed (54/57; Fig. 2D, right). In ad-dition, the number of small veins that appear at later leafdevelopmental stages is reduced in both as1 rosette andcauline leaves (Fig. 2B–D). The aberrant vascular sys-tem is also present in floral organs, sepals and petals(Fig. 2E, F). The vascular patterning in sepals and petalsis consistent with that in rosette and cauline leaves in theas1 mutant, where thick veins appear more proximally(Fig. 2B–D, right). Analysis of the vascular system ofthe as1mutant suggests that AS1 function is required fornormal cell differentiation, which is essential for thespecification of proximodistality in plant organs.

Epidermal cell differentiation

To investigate further the function of AS1 in celldifferentiation, we analyzed leaf epidermal patterns in

the as1 mutant by SEM. Figure 3A shows the adaxialepidermis at the proximal end of a wild-type leaf lamina.Figure 3B is an enlargement of Fig. 3A in the denotedregion, showing that the wild type has at least two dis-tinctive cell types: elongated cells of the midvein and theirregularly shaped epidermal cells that cover most of thelamina. Figure 3C, D shows that most of the epidermalcells on a wild-type petiole have an elongated structure.In the leaf region equivalent to that shown in Fig. 3A,the as1 rosette epidermis (Fig. 3E) contains only onetype of cell, which is long and narrow. These cells(Fig. 3F) resemble the epidermal cells on wild-type pet-ioles in terms of cell patterns (Fig. 3D), and are verysimilar to the epidermal cells on as1’s own petiole(Fig. 3G, H). More-distal epidermal cells appear to bethe same in wild-type and as1 plants (data not shown).Interestingly, the abaxial epidermal cells of the leaflamina are similar in the wild type (Fig. 3I–K) and as1mutant (Fig. 3L–N) in terms of cell type, although theirregularly shaped epidermal cells in as1 have asmoother surface. The SEM results provide furtherevidence that the AS1 function is required for celldifferentiation.

Cloning and sequence of AS1

To isolate the AS1 gene, we crossed the homozygousas1-101 to the polymorphic strain Columbia, and self-pollinated the F1 plants to generate an F2 mappingpopulation. Linkage analysis with SSLP markersshowed that the AS1 locus was mapped to the proximalarm of chromosome 2, between SSLP markers AthBIO2and nga361 (Fig. 4A). Recombination analysis wasperformed by using AthBIO2 for 1,150 F2 mutants andby nga361 for 441 F2 mutants. The resulted recombi-nants were further analyzed by a number of SSLP

Fig. 4A, B Molecular identification of the A. thaliana ASI gene.A Fine structure mapping of the ASI locus. Five putative genes(denoted by boxes) were identified between markers S2071-1 andD2071-3. The open box indicates the predicated MYB-domainprotein. B Nucleotide and protein sequences of the ASI codingregion. The stop codon at position 1102 is indicated by an asterisk(*). The A-to-G and T-to-C nucleotide substitutions resulting inpremature stop codons in asl 1-14, asl-101, asl-102 and asl-103 areindicated above the sequence. An additional A-to-G nucleotidesubstitution was also found in asl-103, but this change may notcontribute to the mutant phenotypes. The position of a G that hasbeen deleted in an X-ray-induced asl allele, asl-1, is denoted (#)Conserved MYB-domain sequences are underlined

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markers and allele-specific markers, and the AS1 locuswas narrowed to a 24-kb genomic DNA region betweenan SSLP marker S2071-1 and an allele-specific markerD2071-3 (Fig. 4A). Annotation of this region identifiedfive putative genes (http://www.ncgr.org/cgi-bin/ff?acc-loc=AC004684). Among them, one gene encoding aMYB-domain protein was identified with a high se-quence similarity to those of the Antirrhinum proteinPHAN and the maize protein RS2. Since the heart-shaped rosette leaf in the phan mutant is similar to thatin as1, we amplified and sequenced genomic fragmentsthat contained the entire AS1 coding sequence from

as1-101, as1-102, as1-103, as1-1 and as1-14, respectively.Sequencing results showed that all these DNA fragmentscontained nucleotide deletion or substitutions that re-sulted in premature stop codons (Fig. 4B).

To further confirm by complementation that we hadidentified the correct gene, we transformed the as1-101mutant with a complementation construct. This con-struct allows the 35 S promoter to drive a DNA se-quence that encodes the Arabidopsis MYB-domainprotein. The transgenic T1 plants were examined forcomplementation of the as1-101 phenotypes. Ten inde-pendent transgenic lines were obtained and examinedclosely, and nine of these lines showed complete rescueof most as1-101 phenotypes, including leaf shapes, plantstature (Fig. 5B; for comparison, see Fig. 5A, D), andflower shapes (data not shown). One transgenic line,however, showed phenotypes that were similar overall tothose of other transgenic lines except that the first trueleaf had a lotus-leaf structure (Fig. 5C). These resultsindicate that the AS1 gene indeed encodes this MYB-domain protein.

Pattern of AS1 expression

In an attempt to link the molecular data on theAS1 locusand the phenotype of as1 mutants, we examined AS1copy number and AS1 expression in different wild-typetissues by DNA and RNA gel blot analyses. A 789-bpDNA fragment immediately downstream of the con-served MYB sequence was isolated by a convenient

Fig. 4 (Contd.)

Fig. 5A–D Transgenic complementation of the as1 mutant ofA. thaliana. A Wild-type Ler plant. B A T1 transgenic plant,T35AS1, showing a wild-type-like phenotype. C A T1 transgenicplant, T35AS9, showing a partially complemented phenotype withone rosette leaf having the lotus-leaf structure (arrow). D An as1-101 mutant plant. All plants shown were 3-weeks old and grown inthe same conditions. Images are at the same magnification.Bar = 5 mm

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HindIII digestion, and was used as a probe. Hybridiza-tion using wild-type genomic DNA at 55 �C revealed onlyone band (Fig. 6A), indicating that AS1 is a single-copygene in the Arabidopsis genome. RNA gel blot analysiswith the same probe detected a 1.3-kb band of AS1mRNA. AS1 was expressed in all plant tissues examined:roots, stems, leaves, flowers and siliques, with preferentialexpression in young and immature plant tissues. In thedeveloped rosette and cauline leaves, AS1 had very lowexpression, while in young siliques, about 6 days afterpollination, AS1 expression was strong (Fig. 6B).

Discussion

We have identified the AS1 gene in Arabidopsis andfound that it encodes a MYB-domain protein, which is

consistent with the recent result shown by Byrne et al.(2000). This gene, previously named AtMYB91, hasbeen characterized as one of the members of the R2R3-MYB gene family by a systematic search forMYB genesin Arabidopsis (Kranz et al. 1998). The predicted AS1protein contains 367 amino acids with a 106-amino-acidN-terminal MYB domain consisting of two MYB-likerepeats. The MYB-domain and C-terminus share a highsequence similarity to those of PHAN and RS2, indi-cating that AS1 is a homolog of PHAN in Antirrhinumand RS2 in maize (Waites et al. 1998; Timmermans et al.1999; Tsiantis et al. 1999). AS1 expression was found inmost plant tissues, and seemed to be developmentallyregulated. AS1 was expressed in roots, cotyledons, andflowers, very weakly expressed in developed rosettes,cauline leaves and stems, strongly expressed in floralbuds, and very strongly expressed in young siliques. Thepreferential expression of AS1 in immature plant organssuggests a role in controlling cell division and differen-tiation.

The lotus-leaf structure associated with our newlyisolated as1 alleles was not reported for the previouslyidentified as1 alleles (Serrano-Cartagena et al. 1999;Byrne et al. 2000; Ori et al. 2000). This structure mayrepresent more-severe as1 alleles, but more likely, is abackground-specific phenotype. First, we did not see anylotus-leaf structure associated with as1-14, a previouslyidentified as1 allele that is in the Enkheim-2 geneticbackground, although our sequencing data revealed thatAS1 mutations in as1-101 and as1-14 are very close.Second, according to our sequencing data, as1-102 andas1-103 should be weaker alleles than as1-101; however,both mutations give rise to a lotus-leaf structure that isindistinguishable from that associated with as1-101. Thenotable phenotypic difference between as1-101 and theweaker alleles as1-102 and as1-103 is that the formerproduced a higher frequency of lotus leaves. Back-ground-specific phenotypic differences were also seen inRS2 mutations, indicating a common characteristic inthis MYB-protein class.

The phenotypes of as1 resemble those of phanmutants in some aspects, such as the broader and heart-shaped rosette leaves, but the needle-like leaves that arepresent in phan mutants were not seen in mutants withthe newly isolated as1 alleles, nor reported for the pre-vious as1 alleles (Serrano-Cartagena et al. 1999; Ori et al.2000). The most prominent as1 phenotype is the ab-normal proximodistality in leaves, and similar defectswere also reported for phan and rs2 (Schneeberger 1998;Waites et al. 1998). Maize leaf consists of sheath tissueproximal to the blade tissue. A disorganized cell patternappears along the proximodistal axis of rs2 leaves. Forexample, in rs2-twd, the mutation causes a transforma-tion of the distal leaf blade into the proximal sheathtissue (Schneeberger 1998). However, another rs2 allele,rs2-R, gives rise to separate sectors of sheath and auriclecells running up into the blade (Schneeberger 1998). Inthe as1 mutant, the epidermal cells of the distal laminahad a similar pattern to those of the proximal petiole,

Fig. 6A, B DNA and RNA gel blot analyses. A Arabidopsis LerDNA was prepared and digested separately with EcoRI andHindIII. B Expression of AS1 in different wild-type tissues asindicated. Total RNAs were prepared from soil-grown plants.Upper panel Hybridization results. Lower panel The same amountsof total RNA, separated in a 1.3% agarose gel and stained withethidium bromide. Lengths of DNA fragments in kilobases areindicated on the left

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and the thicker lateral vein branching occurred in peti-oles, both of which reflect a disorganized proximodis-tality similar to that of the rs2 mutants.

For the establishment of dorsoventral and proximo-distal axes in plant organs, normal cell differentiation isessential. We hypothesize that AS1 has a function tocontrol the spatial and temporal differentiation of cells inleaves, and that loss of AS1 activity causes the differ-entiation of certain cell types at the wrong time or place.The aberrant vascular system in the as1 mutant mayresult from a slowed course of cell differentiation, so thatthe cell differentiation processes in leaves are prolonged.However, during cell differentiation in as1 leaves, thetime period for cell elongation and maturation is lesschanged. In the end, as1 displays incomplete lateral veinsin cotyledons and reduced small veins in the leaf laminabecause cells loose the competency to make differenttissues while developmentally aging (Sachs 1969). Thethicker lateral veins seen in the as1 petiole may be due tothe same mechanism. The delayed vascular differentia-tion in as1 cannot match the cell elongation in the petioleduring later leaf developmental stages. Another possi-bility is that the cells that should differentiate to deter-mine their fates in as1 fail to interpret the positionalsignals. This could lead to the formation of mosaic leavesin as1: lamina and petiole share similar epidermal cells.Cell differentiation is a complex biological process, andmany genes may function in this process. Our resultsshowed that although the adaxial epidermal cells areabnormally differentiated, the epidermal cells at theequivalent abaxial location of a leaf lamina have a sim-ilar pattern to that in the wild type. On the adaxial sideof a lamina, the abnormal cell pattern is usually found atthe proximal end, while the distal epidermal cell patternis normal. All these results indicate that cell differentia-tion in different regions of a leaf requires different genefunctions, and AS1 is one of those genes that are in-volved in the control of cell differentiation.as1 mutations exhibit substantial aberrations in vas-

cular patterning. Maize rs2 mutant leaves also show asignificant alteration in vascular patterns, ranging froman increased number of intermediate veins to the pres-ence of multiple midribs (Schneeberger et al. 1998). It iswell documented that the plant hormone auxin acts asan inductive signal for the development of vascular el-ements in the shoot (Shinninger 1979; Sachs 1991), andthe results of rs2 phenotypic analysis suggested thatauxin homeostasis might be perturbed in rs2 mutants(Schneeberger et al. 1998). Although Schneeberger et al.(1998) have mentioned the auxin-transport defects in rs2mutants, it is not clear yet if the same defects are presentin the Arabidopsis as1 mutants. It must be of interest todetermine the interrelationship between developmentalpathways: gene expressions regulated by the MYBtranscription factor and the coordination in the actionof plant hormones.

Acknowledgements The authors thank C. Chen, X. Xu, M. Zeng,the Ohio State University Arabidopsis Stock Center, and the Not-

tingham Arabidopsis Stock Centre for providing mutant seeds,J. Mao for assistance with light and electron microscopy, D. Luofor allowing us to share experimental equipment, H. Ma for helpfuldiscussions and comments on the manuscript, and the ShanghaiAcademy of Life Sciences for partial financial support. This workwas supported by the State Key Program of Basic Research (973),G1999011605, and a National Distinguished Young ScholarAward to H. Huang.

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