PEAPOD regulates lamina size and curvaturein ArabidopsisDerek W. R. White*

AgResearch, Private Bag 11008, Palmerston North, New Zealand

Edited by Enrico Coen, John Innes Centre, Norwich, United Kingdom, and approved July 5, 2006 (received for review May 25, 2006)

Although a complex pattern of interspersed cell proliferation andcell differentiation is known to occur during leaf blade develop-ment in eudicot plants, the genetic mechanisms coordinating thisgrowth are unclear. In Arabidopsis, deletion of the PEAPOD (PPD)locus increases leaf lamina size and results in dome-shaped ratherthan flat leaves. Siliques are also altered in shape because of extralamina growth. The curvature of a �ppd leaf reflects the differencebetween excess growth of the lamina and a limitation to theextension capacity of its perimeter. Excess lamina growth in �ppdplants is due to a prolonged phase of dispersed meristematic cell(DMC) proliferation (for example, the meristemoid and procam-bium cells that form stomatal stem cells and vascular cells, respec-tively) during blade development. The PPD locus is composed oftwo homologous genes, PPD1 and PPD2, which encode plant-specific putative DNA-binding proteins. Overexpression of PPDreduces lamina size by promoting the early arrest of DMC prolif-eration during leaf and silique development. Therefore, by regu-lating the arrest of DMC proliferation, the PPD genes coordinatetissue growth, modulate lamina size, and limit curvature of the leafblade. I propose a revised model of leaf development with twocell-cycle arrest fronts progressing from the tip to the base: theknown primary front, which determines arrest of general cellproliferation, followed by a secondary front that involves PPD andarrests DMC division.

cell proliferation � coordinated growth � leaf development

Despite a life spent in one position, higher plants are able toadapt readily to environmental changes by altering the size

of their lateral organs, such as leaves. However, changing the sizeof the flat, oval-shaped leaf blade of a eudicot plant is not atrivial task; it involves coordinating complex patterns of cellularproliferation, differentiation, and expansion (1, 2), all in theabsence of cell migration (3).

The lamina of a eudicot plant leaf is initially formed by generalproliferative cell division. Subsequently, during leaf develop-ment, a front of cell-cycle arrest moves from the tip to the base(1, 4, 5). Immediately behind this arrest front, there is a gradientof cellular differentiation. Although most of the cells begin todifferentiate and enlarge, including those contributing to thelamina margins and trichomes, there are also meristematic cellsthat undergo division, resulting in the formation of specific celltypes within each cell layer (1). For example, in the epidermis,meristemoids (6) are recruited from meristemoid mother cellsand undergo a limited number of asymmetric divisions to formstomatal guard and stomatal-lineage ground cells (7). In asimilar manner, procambial cells are recruited in the mesophylltissue layer and divide to form and extend the vascular tissuenetwork of the leaf (1). These cells are defined here as dispersedmeristematic cells (DMCs). Each type of DMC proliferates foronly a limited duration, and the temporal patterns of DMCcell-cycle arrest differ between tissues. For example, during leafdevelopment, the arrest of epidermal meristemoid cell divisionoccurs before procambium cell-cycle arrest (1).

Recent studies using mutant plants have identified a number ofgenes that influence the pattern and maintenance of cell prolifer-ation during leaf development. Some of these genes have positive

roles in regulating cell proliferation. Included in this category aregenes such as AINTEGUMENTA (ANT), ARGOS, ANGUSTIFO-LIA (AN3), ERECTA, GROWTH-REGULATING FACTOR 5(GRF5), JAGGED (JAG), STRUWWELPETER (SWP), andSWELLMAP (SMP1), which act to prolong the proliferative celldivision phase during organ development (8–16). There are alsogeneral negative regulators of cell proliferation and organ growth,such as the interaction of AUXIN RESPONSE FACTOR 2 (ARF2)with ANT (17), and the BLADE ON PETIOLE (BOP) genes, whichrepress the transcription of JAG (18). One of the key aspects oflamina development is the coordination of cell proliferation and cellexpansion, which are required to promote leaf flatness. The An-tirrhinum majus gene CINCINNATA (CIN) determines the shapeand progression of the general cell-cycle arrest front during leafdevelopment, and in the cin mutant, excess growth in the marginsresults in crinkly leaves with negative curvature (19). In Arabidopsis,the microRNA-mediated regulation of class II TCP genes similar toCIN is also required to prevent excess margin growth in leaves (20).

However, despite these advances in our understanding of thegenetic elements controlling the proliferative phase during leafdevelopment, genes regulating the arrest of DMC proliferationhave yet to be identified. Indeed, it has not been clear whatcontribution, if any, DMC proliferation makes to plasticity inlamina size. Here, I present information about the geneticcontrol of DMC proliferation in developing plant organs ob-tained from the characterization of peapod (ppd), an Arabidopsismutant with altered lamina size and curvature.

ResultsThe ppd Mutant Phenotype. The effect of a ppd mutation (�ppd)induced in the Landsberg erecta (Ler) ecotype by fast-neutrontreatment was particularly obvious in leaves and siliques. In �ppdplants, leaf and cotyledon laminae were larger, and leaves had adome-shaped, positive Gaussian curvature, in contrast to flatWT leaves (Fig. 1 A–C). Siliques of the �ppd mutant wereshorter, f lattened, and wider at the top and had undulations inthe seedpod walls, compared with the smooth, narrow, cylindri-cal shape of WT siliques (Fig. 1 D and E). There was also areduction in the branching of trichomes on �ppd leaves, to tworather than the three to four branches of WT.

The larger lamina area observed in the leaves and cotyledonsof �ppd plants was due to increases in both length and width(Table 1). For cotyledons, this increase in growth occurredwithout affecting shape or flatness. However, mature leaves of�ppd plants were more oval in shape because of an alteredlamina length�width ratio and could not be flattened withoutintroducing cuts in the margin because of their positive curva-ture. The area of �ppd leaf blades was �30–50% greater than

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Freely available online through the PNAS open access option.

Abbreviations: DMC, dispersed meristematic cell; Ler, Landsberg erecta; PPD, PEAPOD;T-DNA, transfer DNA; DAG, days after germination.

*E-mail: derek.white@agresearch.co.nz.

© 2006 by The National Academy of Sciences of the USA

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that of WT leaves, but perimeter length was similar (Table 1).The positive curvature of �ppd leaves thus appears to be due toadditional growth of the lamina exceeding the extension capacityof the perimeter of the blade. The extra leaf growth of �ppdplants appeared to have no detectable effect on mesophyllpalisade cell size, spacing, or the general anatomy of tissues in thelamina (Fig. 6, which is published as supporting information onthe PNAS web site). However, there was a more extensivevascular network in the cotyledons of �ppd plants than WTplants (4.93 � 0.53 areoles per cotyledon for �ppd versus 3.97 �0.32 for WT) (Fig. 7, which is published as supporting informa-tion on the PNAS web site). The number of stomata per area inthe abaxial epidermis of leaves was also greater (137%) in �ppdplants than in WT plants.

The PPD Locus Has Two Plant-Specific Homologs. The �ppd mutationwas backcrossed five times to Ler before being used for geneticanalysis. Although reduced trichome branching of the �ppdallele was recessive, silique width and leaf curvature aspects ofthe phenotype were weakly semidominant (Fig. 1D and Table 1).PPD was mapped to the bottom arm of chromosome 4 by usingmolecular markers (21) and colocation of the trichome branch-ing gene, FRC3 (At4g14750) (22, 23). PCR analysis demon-strated that the �ppd allele is an �60-kb deletion encompassing12 predicted genes, from At4g14700 to At4g14760, includingFRC3. Within the deleted region, there is a duplicated genomicsegment containing two interspersed homologous gene pairs,At4g14710�At4g14716 and At4g14713�At4g14720 (Fig. 2A). Al-though the At4g14710�At4g14716 homologs are members of awider gene family, there are no genes similar to At4g14713�

At4g14720 elsewhere in the Arabidopsis genome. Homozygoustransgenic plants with transfer DNA (T-DNA) insertions ineither At4g14713 or At4g14720 had the weak semidominant ppdsilique width (Table 1) and leaf curvature phenotype but hadWT trichome branching, whereas plants with insertions inflanking genes (At4g14700, At4g14716, At4g14730, At4g14740,and At4g14760) were WT. The phenotypes of mutant alleles�ppd, ppd1-1, ppd1-2, and ppd2-1 indicate that PPD is a complexlocus made up of two genes, designated here as PPD1(At4g14713) and PPD2 (At4g14720). Furthermore, the influenceof PPD gene copy number on lamina size, illustrated by com-paring the silique widths of plants with 0, 1, 2, or 4 copies of aPPD gene (Table 1), suggests that PPD1 and PPD2 are individ-ually haplo-insufficient. The similarity of silique dimensions forboth homozygous T-DNA insertion lines (ppd1-1�ppd1-1 andppd2-1�ppd2-1) and the heterozygote between them indicatesthat PPD1 and PPD2 are homologs of equivalent function.

Genomic transgenes of either of the PPD genes rescued thelamina size and curvature mutant phenotype of �ppd (but not thereduced trichome branching component), confirming that PPD1and PPD2 are redundant. Of the 80-plus transgenic plants obtainedfrom transformation with PPD1 or PPD2, most were WT inappearance. For example, of 26 �ppd��ppd::PPD1 transgenicplants examined in detail, 20 were WT in leaf and silique dimen-sions, 2 had siliques similar in width to �ppd�� heterozygotes, and4 had smaller cotyledons and leaves and shorter, narrower siliquesthan WT. Quantitative expression analysis on two of these smallgenotypes (designated �ppd��ppd::PPD1-OE) demonstrated thatthey had elevated levels of PPD mRNA (Fig. 4E). The cotyledon,leaf, and silique dimensions of representative complemented�ppd��ppd::PPD1 and �ppd��ppd::PPD1-OE plants are given inTable 1. Silique dimensions of complemented �ppd��ppd::PPD1plants were similar to those of WT plants, as were cotyledon andleaf sizes (data not shown). Leaves of PPD1-OE plants were flat,smaller, and more rounded than those of the WT, and the siliqueswere reduced in both width and length. There was also a reducedvascular network pattern in the cotyledons of PPD1-OE plants(2.31 � 0.54 areoles per cotyledon versus 3.97 � 0.32 for WT) anda reduction in the number of stomata on the abaxial leaf surface(65%) relative to that found in WT plants.

PPD1 and PPD2 have nine exons and encode 314-aa and316-aa proteins, respectively, with 84% identity. The predictedPPD proteins each have a central putative DNA-binding se-quence, termed a ZIM motif (24), and a unique �50-aa N-terminal domain spanning amino acids 14–63 (Fig. 2B). Proteinswith homology to this combination of a conserved N-terminal‘‘PPD’’ domain and a central ZIM motif that were identified bysearching databases were found in the lycophyte Selaginellamoellendorffii, the conifer Picea sitchensis, and in a wide varietyof eudicot genera, including the basal eudicot Aquilegia. How-ever, PPD proteins appear to be absent from rice and othergrasses.

PPD Regulates the Arrest of DMC Proliferation During Lamina Devel-opment. To investigate the cause of increased lamina growth inthe �ppd mutant, I compared the appearance of epidermal cellsin the leaves and siliques of mutant and WT plants at stages ofWT leaf and silique development when meristemoids had ceaseddivision (Fig. 3 A and C). WT plants had the expected patternof pavement and stomata guard cells (7), but in the leaf andsilique epidermis of �ppd mutant plants, clusters of small cells,some with the isometric shape characteristic of meristemoids,were interspersed amongst the enlarged pavement cells (Fig. 3 Band D). A transgene marker of cell-cycle progression, aCYCB1;1::GUS fusion (1), was used to compare the extent ofDMC proliferation in developing leaves and siliques of �ppdmutant and WT plants (Fig. 3 E and F). In the leaf and carpelepidermal tissues examined, cyclin activity was expressed in foci

Fig. 1. Phenotype of the �ppd mutant. (A) Phenotypes of WT (Left) and�ppd mutant (Right) plants. (B) Side view of WT Ler (Left) and �ppd (Right)seedlings. (C) Abaxial view of WT (Upper) and �ppd (Lower) mature first leaf.(D) Silique shapes: WT Ler (Left), �ppd (Right), and heterozygote (Center). (E)Inflorescences of WT (Left) and �ppd (Right). (Scale bars: A, 10 mm; C and D,5 mm).

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corresponding to the appearance of meristemoid cells (see Fig.8, which is published as supporting information on the PNASweb site). In WT plants, meristemoid cell cycling in the abaxialleaf epidermis was arrested 12 days after germination (DAG),whereas in the �ppd mutant, this phase was extended beyond 20DAG. Similarly, meristemoid cell proliferation in the outerepidermis of the valve of the �ppd mutant was prolonged intomore advanced developmental stages, well after cell-cycle arresthad occurred in the silique valves of WT plants (Fig. 3F). Thearrest of cell cycling in the procambium cells of the developingvasculature of �ppd mutant leaves was also delayed (Fig. 8) andcontinued after division of meristemoid cells in the epidermishad stopped. Therefore, despite a prolonged phase of prolifer-ation in the leaf of �ppd mutant plants, DMC division eventuallystops, and the order of this arrest in different tissues is the sameas in WT plants.

To determine whether prolonged cell division in the �ppdmutant occurred throughout lamina development or was con-fined to the DMC phase, I compared the position of the generalproliferative cell-cycle arrest front in the leaves of WT, �ppdmutant, and transgenic PPD1-OE plants. If the loss of PPDfunction increases the duration of all cell proliferation duringlamina development, then the general cell-cycle arrest front(primary arrest front) should be delayed, and the size of thegeneral proliferation zone should be increased. However, if theeffect of PPD gene expression is only on the zone of DMCproliferation, the tip-to-base advance of the primary arrest frontshould remain unaltered by either PPD loss of function or PPDoverexpression. Because the position of this arrest front was notaffected by altered expression of the PPD genes (Fig. 9, which ispublished as supporting information on the PNAS web site), it

appears that the additional leaf and silique lamina growth of�ppd mutant plants was solely due to delayed DMC cell-cyclearrest. The CYCB1,1::GUS transgene was also used to determinethe effect of PPD1 overexpression on meristemoid cell prolif-eration in leaves and siliques (Fig. 3F). At leaf and siliquedevelopmental stages where both WT and �ppd mutant plantshad a high cyclin index, meristemoid cell cycling in the epidermisof PPD1-OE transgenic plants was reduced by �80–90%.

The Expression Pattern of PPD Coincides with DMC Proliferation inDeveloping Leaves and Siliques. To establish where PPD genes areexpressed relative to the zone of proliferating DMCs, I carriedout RNA in situ hybridizations and examined the expressionpattern of a PPD1 promoter::GUS (�-glucuronidase) transgenein developing lateral organs (Fig. 4 A–D). During leaf devel-opment, PPD expression was first detected at the tip of thedeveloping leaf, distal to the general proliferative cell-cyclearrest front and coincident with the initiation of trichome andmargin cell development (Fig. 4 A and D Inset). This expressionappeared to exist initially in all cells in the newly formed DMCzone and was not restricted to meristemoid cells. Subsequently,during leaf development, the pattern of PPD expression fol-lowed the tip-to-base progression of the general proliferativearrest front before becoming restricted to developing vasculartraces and eventually declining in expression in the samepattern as the arrest of procambial cell division. PPD1 geneexpression during the development of silique valves alsocoincided with the occurrence of meristemoid cell prolifera-tion. PPD1::GUS expression occurred throughout the valvesduring developmental stages 15 and 16 and declined in moremature siliques (Fig. 4C).

Table 1. Influence of PPD gene copy number and expression level on Arabidopsis mature cotyledon, leaf, and silique dimensions

Genotype Ecotype Length, mm Width, mm Length�width Area, mm2

Perimeter,mm

PEAPOD genecopy no.

Cotyledons

��� Ler 1.99 � 0.2 1.99 � 0.2 1.00 3.04 � 0.5 6.48 � 0.5 4�ppd��ppd Ler 2.64 � 0.3 2.55 � 0.3 1.04 5.69 � 0.9 8.56 � 0.7 0TG �ppd��ppd::PPD1-OE Ler 1.72 � 0.2 1.52 � 0.3 1.13 2.55 � 0.5 5.92 � 0.6 1TG

Leaf 1 lamina

��� Ler 7.77 � 0.9 6.69 � 0.7 1.16 40.58 � 6.7 22.48 � 2.1 4�ppd��ppd Ler 10.67 � 1.1 7.96 � 0.7 1.34 53.22 � 8.5 22.55 � 2.1 0TG �ppd��ppd::PPD1-OE Ler 2.82 � 0.3 2.53 � 0.3 1.11 7.71 � 1.6 9.72 � 0.7 1TG

Leaf 4 lamina

��� Ler 15.84 � 0.9 10.51 � 0.6 1.51 126.46 � 11.3 41.23 � 2.9 4�ppd��ppd Ler 25.00 � 2.5 13.31 � 1.2 1.88 196.24 � 25.3 46.52 � 4.2 0TG �ppd��ppd::PPD1-OE Ler 5.25 � 0.5 4.27 � 0.4 1.23 18.90 � 3.8 14.86 � 1.4 1TG

Siliques

��� Ler 13.92 � 0.4 1.27 � 0.0 10.96 — — 4�ppd��ppd Ler 8.12 � 0.6 3.13 � 0.2 2.59 — — 0�ppd�� Ler 14.08 � 0.3 1.51 � 0.0 9.32 — — 2TG �ppd��ppd::PPD1 Ler 13.02 � 0.3 1.28 � 0.0 10.17 — — 1TGTG �ppd��ppd::PPD1-OE Ler 7.77 � 0.6 0.73 � 0.1 10.64 — — 1TG��� Col-0 16.01 � 0.5 0.84 � 0.1 19.05 — — 4�ppd��ppd Col-0 10.69 � 0.7 2.20 � 0.2 4.86 — — 0ppd1-1�ppd1-1 Col-0 16.24 � 0.3 1.18 � 0.1 13.76 — — 2ppd2-1�ppd2-1 Col-0 16.33 � 0.3 1.17 � 0.0 13.96 — — 2PPD1�ppd1-1; PPD2�ppd2-1 Col-0 16.30 � 0.3 1.17 � 0.0 13.93 — — 2ppd1-1��ppd; PPD2��ppd Col-0 15.28 � 0.4 1.28 � 0.1 11.94 — — 1

Values are the mean (�SD) of measurements from at least 30 cotyledon, 14 leaf, and 30 silique samples. TG, transgenic; TG �ppd��ppd::PPD1, �ppdtransformed with At4g14713, WT phenotype, and PPD expression levels; TG �ppd��ppd::PPD1-OE, �ppd transformed with PPD1 overexpression; ppd1-1�ppd1-1, SALK�057237 T-DNA insertion homozygote; ppd2-1�ppd2-1, SALK�142698 T-DNA insertion homozygote; PPD1�ppd1-1; PPD2�ppd2-1, ppd1-1�ppd2-1heterozygote; ppd1-1��ppd; PPD2��ppd, PPD2 hemizygote; —, not applicable.

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DiscussionI report the identification of a gene regulating the cell-cyclearrest of a diverse group of stem cell-like meristematic cellsduring lamina development in Arabidopsis. Alterations solely tothe duration of DMC proliferation can account for the effects ofPPD gene expression on leaf morphogenesis (Fig. 5). In theabsence of the PPD genes, leaf DMC proliferation is prolongedand lamina tissue outgrows the extension capacity of the margincells, changing the blade from flat to dome shaped. The eventualdecline of DMC division in the laminae of �ppd mutant plantssuggests that either DMC proliferation is intrinsically limited orthat other cell-cycle arrest mechanisms are involved. Conversely,overexpression of PPD reduces the duration of the DMC pro-liferation zone and thus the number of cells in the lamina,resulting in smaller, f lat leaves. The lack of negative curvaturein the leaves of PPD-OE plants is consistent with expansion ofthe margin cells being determined largely by the amount ofgrowth of the lamina. If so, then reduced lamina growth wouldresult in a flat blade, and only increased lamina growth, beyondthe expansion capacity of the margin cells, would result in alteredleaf curvature. An explanation for the shortened siliques of �ppdmutant plants is less obvious. During the development of WTsiliques, cell-cycle arrest occurs before a phase of proximal–distal longitudinal cell expansion, when the length of the septummay be established (25). It is possible that the presence ofinterspersed small cells in �ppd plants at this phase of siliquedevelopment may physically limit this expansion. Once the phaseof longitudinal expansion has ceased, the shortened length of theseptum in �ppd mutant plants may be set. Subsequent prolongedDMC proliferation would then cause the excessive lateral growthand seedpod wall undulations observed in the mutant.

The seeming lack of PPD genes in the grasses may be becausethe pattern of cell proliferation during the development of grassleaves is quite different from eudicot and other vascular plants.In grasses, cell proliferation is confined to a narrow band oftissue located at the base of the leaf, and specialized cells, suchas stomatal guard cells, have a more condensed cell lineage thanin eudicots (26, 27). Therefore, PPD genes may only be requiredto regulate more extensive DMC proliferation during laminadevelopment.

I propose a model in which there are two separate cell-cyclearrest fronts progressing from the tip to the base during leafdevelopment: a primary front that determines arrest of generalcell division in the primordium, followed by a secondary frontthat involves PPD and arrests DMC proliferation. The shape andprogression of the primary arrest front is known to be influencedby class II TCP genes, CIN in Antirrhinium (19) or TCP2 andTCP4 in Arabidopsis (20), and it has been suggested that theextent of cell proliferation in the primordium may be regulatedby a balance between the antagonistic activities of class I and IITCP genes on the expression of cyclin and ribosomal proteingenes (28). Other genes, such as ANT, ARGOS, AN3, GRF5,JAG, SMP, and SWP, which promote cell division during leafdevelopment, also seem to act on proliferation in the primor-dium (8–16). However, it is has not been established whether anyof these genes also influences cell proliferation in the DMC zone.

Fig. 2. Genomic structure of the PPD locus (A) and predicted amino acidsequences of proteins encoded by the Arabidopsis PPD genes (B). (A) Filledtriangles indicate the position of T-DNA insertions. (B) Alignment of thepredicted amino acid sequences of PPD proteins encoded by At4g14713(PPD1) and At4g14720 (PPD2). Gray shading indicates similar residues. Aprotein domain that is highly conserved in PPD homologues is marked by anoverline; dotted underlining identifies a putative ZIM DNA-binding motif.

Fig. 3. Variations in meristemoid cell proliferation in the epidermis ofdeveloping leaves and siliques. (A–D) Replica SEM of the abaxial epidermalsurface of WT (A) and �ppd leaf 1 (B) at 18 DAG. SEM of the outer surface ofsilique valves of WT (C) and �ppd (D) at developmental stage 17. (Scale bars:40 �m.) (E and F) Comparison of the frequency of CYCB1;1::GUS-expressingmeristemoid cells in WT (gray bars) and �ppd mutant (black bars) plants atdifferent stages of leaf (E) or silique (F) development.

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In this model, the arrest of DMC proliferation is regulatedindependently from the arrest of cell division in the primordium.Overlapping patterns of PPD1 expression and DMC prolifera-tion suggest a possible feedback mechanism whereby DMCsinduce PPD gene expression until PPD protein reaches a thresh-old level that, in turn, restricts DMC recruitment. Hence, a singlearrest mechanism, mediated by means of PPD, might act tocoordinate the limitation of DMC proliferation. This type ofinteraction would provide a mechanism for varying the size of aleaf blade distal to the primary arrest front while maintaininglamina anatomy and flatness. It will be interesting to test this

model by examining the interactions between the PPD genes andthose promoting the maintenance of cell proliferation during leafdevelopment.

Materials and MethodsPlant Materials and Growth Conditions. Arabidopsis thaliana (L.)Heynh ecotype Ler was used as WT. The mutant designated�ppd was identified during a screen of �3,500 M2 plants grownfrom a fast-neutron-mutagenized population of ecotype Lerobtained from Lehle Seeds (Round Rock, TX). Plants weregrown in a temperature-controlled glasshouse at a continuous21°C or in a controlled environment cabinet at 23°C in 16-hlight�8-h dark cycles.

Morphological and Cellular Analyses. Dimension measurements offully expanded cotyledons and first and fourth leaves collectedat the floral bolt stage were made by flattening the organsbetween two microscope slides, scanning to produce a digitalimage, and then calculating length, width, area, and perimeter byusing an image analysis program (ImageJ). Because of theircurvature, leaves of the �ppd mutant could not be flattenedwithout cutting the margins. Mature silique dimensions weredetermined by using a digital micrometer. To detect the influ-ence of the ppd mutation on cell proliferation, a CYCB1,1::GUSreporter gene (29) was introgressed into WT and mutant back-grounds. Histochemical detection of GUS activity was carriedout as described by Donnelly et al. (1). The frequency ofmeristemoid cells with GUS activity in the abaxial epidermis ofleaves and the outer epidermis of carpel valves at differentdevelopmental stages was assessed across the midpoint of thelength of the lamina. The total cell number and the number ofmeristemoid cells with GUS activity in a medial-lateral micro-scopic (�40) traverse across the leaf were counted and expressedas a cyclin index (number of cells with GUS activity�number oftotal cells � 100). The cyclin index was determined for at least15 samples for each leaf or silique developmental stage exam-ined. Silique developmental stages 17.1–17.3 were defined as theprogressively older siliques formed before a stage-17 silique (30).

SEM. To prepare leaf surface replicas for SEM analysis, medialtransverse segments dissected from the first leaves of WT andppd mutant seedlings (at 18 DAG) were coated on the abaxialsurface with polyvinylsiloxane dental impression material tomake a mold. The leaf tissue was removed and replaced withSpurr’s resin. The positive resin replica was polymerized, de-tached from the mold, and then sputter-coated with gold forSEM examination.

Genetic Analysis and Mapping of the ppd Locus. The �ppd mutantwas backcrossed five times to WT Ler plants. Inheritance of �ppdwas determined in BC5 self-progeny by scoring leaf curvatureand trichome branching and measuring silique width. The map-based identification of the PPD locus is described in SupportingMethods, which is published as supporting information on thePNAS web site.

T-DNA Insertion Mutants. Plant lines containing T-DNA inser-tions in the coding regions of annotated genes within the ppddeletion region were identified by searching the TAIR Arabi-dopsis web site (www.arabidopsis.org). Insertion lines forAt4g14700 (SA LK�140010, SA LK�104400), At4g14713(SALK�149924, SALK�057237), At4g14716 (SALK�119327),At4g14720 (SALK�14698), At4g14730 (SALK�046652),At4g14740 (SALK�013371), and At4g14760 (SALK�003809)were obtained from the Arabidopsis Biological Resource Cen-ter (Columbus, OH).

Fig. 4. Analysis of PPD1 expression pattern and the influence of alteredexpression levels on lamina cell division. (A and B) In situ localization of PPD1RNA in longitudinal sections of WT (A) or �ppd (B) seedlings (at 6 DAG) probedwith PPD1. (C and D) Expression pattern of PPD1 promoter::GUS in theinflorescence (C Left), developing stage-15 silique (C Right), vegetative leaves(D Left), and leaf primordia (D Right) of WT plants. Arrow indicates theposition of developing trichomes. (E) Relative expression levels of PPD1 in WTLer, �ppd::PPD1, and �ppd::PPD1-OE plants as monitored by real-time quan-titative RT-PCR. C#2, �ppd::PPD1 plant with WT phenotype; C#19 and C#26,independent �ppd::PPD1 transgenic plants with small leaf�silique pheno-types. Each bar represents the mean of three replicates relative to ornithinetransfer carboxylase. (F) Comparison of the meristemoid cyclin index of de-veloping leaves and siliques of WT, �ppd, and PPD1-OE plants. Leaves weresampled at 9 DAG (black bars), and siliques were sampled at carpel develop-mental stage 15 (gray bars).

Fig. 5. Schematic illustration of the effect of PPD expression levels on DMCproliferation, leaf curvature, and size. Solid black represents the primordiumcell proliferation zone. The DMC proliferation zone is shown in speckled gray;regions without cell division are white.

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Cloning and Gene Constructs. For DNA and RNA manipulations,standard molecular biology techniques were used (31). TheAt4g14713 and At4g14720 genes were amplified by PCR fromWT Col-0 ecotype genomic DNA by using Platinum TaqDNApolymerase high fidelity (Invitrogen, Auckland, New Zealand)and gene-specific primers for sequences at the end of the 3� UTRand �1.5 kb upstream of the beginning of the 5� UTR. Con-struction of pPPD1, pPPD2, and a PPD1 promoter::GUS reportergene are described in Supporting Methods. Constructs weretransformed into Arabidopsis by the floral dip infiltrationmethod (32). Transgenic plants were confirmed by PCR analysiswith a combination of transgene-specific and T-DNA primers.For �ppd complementation experiments, the plants were alsotested for the absence of genes in the deleted region and for themutant reduced trichome branching phenotype.

In Situ Hybridization. Tissues for in situ hybridization were fixedwith 4% (wt/vol) paraformaldehyde�4% (vol/vol) DMSO andpassed through a graded butanol series before embedding inParaplast Plus wax (BioLab, Auckland, New Zealand). Sections(10 �m) were affixed to polylysine-coated slides. Probe prepa-ration is described in Supporting Methods. Slides were takenthrough prehybridization treatments according to the protocol

of Drews and Okamura (33) and hybridized overnight at 45°C.The slides were washed and prepared for immunodetection byfollowing the protocol of Roche (Manheim, Germany).

Real-Time Quantitative RT-PCR. Total RNA was extracted frominflorescence tips dissected from WT Ler and three individual�ppd::PPD1 complemented transgenic plants (C no. 2, C no. 19,and C no. 26) by using an RNeasy Plant Mini Kit (Qiagen,Valencia, CA). First-strand cDNA was synthesized from totalRNA by using an oligo(dT) primer and SuperScript II reversetranscriptase (Invitrogen). Quantitative RT-PCR assays for therelative level of PPD1 (At4g14713) transcript were performed ona MyIQ color real time PCR instrument (Bio-Rad, Auckland,New Zealand) by using ornithine transfer carboxylase as aninternal control. Primer sequences and PCR amplification con-ditions are given in Supporting Methods. The comparative CTmethod (34) was used to determine relative levels of expression.

I thank Roy Meeking, Ruth Cookson (AgResearch), and DouglasHopcroft (HortResearch, Palmerston North, New Zealand) for technicalassistance; David Smyth (Monash, Melbourne, Australia) for theCYCB1,1::GUS transgenic line; and John Bowman, Toshi Foster, andcolleagues for comments on the manuscript. This work was funded byRoyal Society of New Zealand Marsden Fund Grant AGR304.

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