The REVOLUTA gene is necessary for apical meristem development and for limiting cell divisions in the leaves and stems of Arabidopsis thaliana

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1 Development 121, (1995) Printed in Great Britain The Company of Biologists Limited The REVOLUTA gene is necessary for apical meristem development and for limiting cell divisions in the leaves and stems of Arabidopsis thaliana Paul B. Talbert, Haskell T. Adler, David W. Parks* and Luca Comai University of Washington, Department of Botany, Box , Seattle, WA , USA *Present address: Camellia Forest Nursery, 125 Carolina Forest Rd, Chapel Hill, NC, SUMMARY The form of seed plants is determined by the growth of a number of meristems including apical meristems, leaf meristems and cambium layers. We investigated five recessive mutant alleles of a gene REVOLUTA that is required to promote the growth of apical meristems and to limit cell division in leaves and stems of Arabidopsis thaliana. REVOLUTA maps to the bottom of the fifth chromosome. Apical meristems of both paraclades (axillary shoots) and flowers of revoluta mutants frequently fail to complete normal development and form incomplete or abortive structures. The primary shoot apical meristem sometimes also arrests development early. Leaves, stems and floral organs, in contrast, grow abnormally large. We show that in the leaf epidermis this extra growth is due to extra cell divisions in the leaf basal meristem. The extent of leaf growth is negatively correlated with the development of a paraclade in the leaf axil. The thickened stems contain extra cell layers, arranged in rings, indicating that they may result from a cambium-like meristem. These results suggest that the REVOLUTA gene has a role in regulating the relative growth of apical and non-apical meristems in Arabidopsis. Key words: meristem, branching, leaf growth, Arabidopsis, REVOLUTA INTRODUCTION Elaboration of the plant body pattern depends primarily on the proper regulation of cell division versus cell differentiation at the growth sites, called meristems. In seed plants apical growth is carried out by the apical meristems. Although structurally identical, shoot apical meristems differ ontogenetically. A primary shoot apical meristem originates during embryogenesis and becomes the apex of the primary shoot. Secondary shoot apical meristems develop later on the sides of the primary shoot and form lateral shoots. In many seed plants, radial growth of the shoot is conferred by the cambium, a cylindrical meristematic layer in the shoot body. Growth of lateral leafy organs (i.e. leaves, petals, etc.) occurs from transient meristems formed on the flank of the apical meristem. Root growth occurs from analogous apical and cambial meristems. At present we understand very little of the regulation and interaction of these different types of meristems. Morphological and developmental studies suggest that different meristems interact. An example is provided by the development of axillary meristems. These meristems form at repeated positions along the shoot, known as nodes, that are separated by sections of stem called internodes. A node typically bears one or more leaves, each of which contains a meristem in its axil that can form a branch shoot. Axillary meristems are thought usually to originate directly from the shoot apical meristem as detached meristematic cells in the axils of the leaf primordia (Garrison, 1955). However, in some cases axillary meristems appear to arise from the leaf primordium rather than from separate meristematic cells (Majumdar, 1942; Irish and Sussex, 1992). Pioneering surgical studies by Snow and Snow (1942) showed that the formation of an axillary shoot was dependent on the subtending leaf primordium and inhibited by the apical meristem. These results suggest that axillary meristems may form in response to positional information set up by the action of opposing morphogenetic gradients. Genetic or molecular data supporting this model are currently lacking. The flowering plant Arabidopsis thaliana has proved useful for the molecular genetic analysis of developmental problems. Arabidopsis has a basal rosette of vegetative leaves and an erect branched inflorescence. The branching pattern of the inflorescence is most easily described in terms of the phytomer concept. A phytomer (Galinat, 1959) or metamer (White, 1984) is a reiterated module typically composed of an internode and a node with its leaf and axillary branch. Many variations of this basic pattern occur, such as the suppression of leaves or branches, or their modification into structures specialized for assorted reproductive or vegetative functions. Three types of phytomers have been described in the primary shoot of Arabidopsis (Schultz and Haughn, 1991; Fig. 1A). All three are arranged in a spiral phyllotactic pattern. Type 1 comprises the basal rosette. Each type 1 phytomer has an extremely short internode, a rosette leaf, and an axillary meristem that may form a branch. Type 2 comprises the basal portion of the flowering stalk. These phytomers have long internodes, cauline leaves and axillary branches. The first axillary

2 2724 P. B. Talbert and others branch originates in the uppermost leaf axil and others develop basipetally through the type 2 and 1 phytomers (Alvarez et al., 1992; Hempel and Feldman, 1994). In both of these phytomer types, a second branch (accessory branch) may later develop between the axillary branch and its subtending leaf (Fig. 1A). Type 3 phytomers constitute the fertile terminal portion of the stalk, known as the main florescence (Weberling, 1989). Each type 3 phytomer has an intermediate-length internode, no leaf and a lateral flower. As observed by Goethe (1790), flowers are specialized shoots with floral organs in the place of leaves. Thus the flowers of Arabidopsis can be viewed as floral branches which are serially homologous with the axillary branches. While the floral branches have determinate growth and are composed of special phytomer types (i.e. the whorls of four sepals, four petals, six stamens and two carpels), the indeterminately growing axillary and accessory branches repeat in part the pattern of phytomers found on the primary shoot and are therefore termed paraclades (Weberling, 1989). The type 2 phytomers of a firstorder paraclade give rise to second-order paraclades, and these give rise to higher-order paraclades (Fig. 1A). The type 3 phytomers of a paraclade make up a coflorescence (Weberling, 1989). The primary shoot distal to the rosette and the paraclades together make up the inflorescence. The study of Snow and Snow (1942) suggests that genes involved in axillary meristem development might also have roles in the primary apical meristem or in the development of leaves. We therefore speculated that mutations in Arabidopsis genes affecting apical meristems might be identifiable by changes in leaf morphology, as is true of the tomato gene lanceolata (Caruso, 1968), and screened for mutants accordingly. We describe here mutants defective in a gene, REVOLUTA (REV), that is necessary to promote the normal growth of apical meristems, including paraclade meristems, floral meristems and the primary shoot apical meristem. Simultaneously, REV has an opposing effect on the meristems of leaves, floral organs and stems, being necessary to limit their growth. Thus rev mutations reveal a novel and fundamental regulatory feature of plant body pattern elaboration. MATERIALS AND METHODS Plant growth conditions Plants were grown in 5.0-cm pots in the greenhouse in a buffered soil mix of 67% peat moss and 33% pumice. Natural sunlight was supplemented by 16 hours of illumination with 1000 W high pressure sodium lights. Plants were subirrigated with alternating solutions of Peters Peat- Lite Special ( ) or Peters Dark Weather Food ( ), both diluted to 100 ppm. Temperature control set points were at C during the day and 9-13 C at night. To assist uniform germination, wetted seeds were kept at 4 C for 2 days prior to planting. Because of the inherent variability of greenhouse conditions, all rosette paraclade 2p REV 3p A 3p 2p 3 primary shoot comparisons of wild-type and mutant phenotypes were made on cohorts of plants grown side-by-side under identical conditions. Cohort 1 was grown in May-July of 1994, cohort 2 in July-August 1994, cohort 3 in October-November 1994, cohort 4 in March-May 1994, and cohort 5 in October-December To achieve 100% germination and uniform growth, cohort 4 was germinated on growth medium containing 0.5 Murashige-Skoog salts (Gibco) under continuous illumination with Philips 40 W Cool White lights (75 µe/m 2 sec) and transplanted to soil on day 14 after sowing. Mutagenesis and allelism tests All five rev mutations were recovered from the M 2 progeny of M 1 seeds mutagenized with ethyl methanesulfonate (EMS). Alleles rev- 1, rev-2 and rev-4 were each recovered from an independent batch of M 1 seeds of ecotype Nossen-0 (No-0) mutagenized in 10 mm EMS for 17 hours. This dosage gave siliques with segregating M 2 embryolethals on approximately half of the M 1 plants. The rev-3 and rev-5 (=spitzen-1; Alvarez, 1994) alleles were induced in the Columbia ecotype and generously supplied by Laura Conway and David Smyth, respectively. Allelism of rev-1 and rev-2 was determined by their noncomplementation in multiple reciprocal crosses. Further complementation tests were carried out by pollinating a marked er ttg rev-2 stock with a rev-3/rev, rev-4 /rev-4, or rev-5/rev-5 parent. Individuals that were Rev but not Er or Ttg made up half (15/32), all (7/7), and all (23/23), respectively, of the progeny in these three crosses. The rev- 1 allele was backcrossed to the wild type three times to eliminate any effects of additional mutations on the phenotype. The other alleles have been backcrossed once. Mapping Mapping of rev-1 used the polymerase chain reaction mapping markers DFR (Konieczny and Ausubel, 1993) and nga129 (Bell and 2 2p cauline leaf * rosette leaf cauline paraclade accessory paraclade 1 rosette leaf B rev primary shoot * * * * cauline paraclade * cauline leaf Fig. 1. Schematic representation of wild-type REV and mutant rev morphologies. (A) Wild type, showing phytomer types (1, 2 and 3) and branching pattern. (B) rev, showing reduced growth of primary shoot, paraclades and flowers, and increased growth of leaves and stems., flowers; 2p, second-order paraclade; 3p, third-order paraclade; *, axil lacking a paraclade.

3 Meristem development in Arabidopsis 2725 Ecker, 1994) on chromosome 5. DFR maps to 61.5 mu (AAtDB version 3-4) and nga129 maps to approximately 86.4 mu (6.6 mu below m435; Bell and Ecker, 1994). A rev-1 mutant (No-0 ecotype) was crossed to wild-type Landsberg erecta and phenotypically Rev F 2 individuals were scored. We also mapped REV with respect to the morphological marker lfy (Weigel et al., 1992). LFY maps to 89.5 mu on the distal tip of chromosome 5 (AAtDB version 3-4). Rev F 2 individuals from a rev-1 lfy-6/+ cross were progeny tested for segregation of lfy rev F 3 double mutants (Adler et al., unpublished data). Map distances were corrected for double cross-overs using the Kosambi mapping function (Koornneef and Stam, 1992), and the 95% confidence interval was computed using the two tails of the binomial distribution (Woolson, 1987). Measurements and cell counts Measurements of plant organ lengths were made with either a Measy dial caliper (Max Mägerle, Sen., Switzerland), a calibrated Zeiss reticle or a standard metric ruler. Leaf areas in cohort 4 were determined by harvesting leaves and tracing their outlines. The outlined areas were measured using a Kurta digitizing tablet and SigmaScan software (Neff and Van Volkenburgh, 1994). Epidermal peels of the adaxial leaf surfaces were made using Revlon nail strengthener and transparent tape. The peels were affixed to microscope slides and three patches of cells from different parts of each leaf were traced in a camera lucida, avoiding the midrib. Cells in each patch were counted and areas of the patches were determined (Neff and Van Volkenburgh, 1994). The mean cell sizes of the three patches of each leaf were averaged to get the mean cell size for that leaf. Similar results were obtained when the three patches were grouped as a single patch. Unpaired two-tailed Student s t-tests on measurement sets were conducted using the Statview Student statistical software package. The P value for these tests is the probability that the true means of the two genotypic populations are identical. Determination of the region of leaf growth was made by marking growing leaves from cohort 5 with dots of Sumi black calligraphy ink (Yasutomo & Co.) placed 1-2 mm apart along the midrib and measuring subsequent growth relative to the fixed dots. Anatomical sections Plant material for sectioning was fixed in formalin-aceto-alcohol, dehydrated through an increasing tertiary butyl alcohol series and embedded in Paraplast X-Tra (Oxford Labware) paraffin (Johansen, 1940). Serial 10 µm sections were cut on a Spencer microtome and mounted on microscope slides with Haupt s adhesive (Johansen, 1940). Paraffin was dissolved in xylene and the slides were hydrated through a decreasing ethanol series before staining 1-2 minutes in 0.25% toluidine blue, or in safranin O and fast green FCF essentially as described by Johansen (1940) except for the following: picric acid and ammonia were omitted from the washes; 0.25 g of fast green was dissolved in a mixture of 100 ml each of methyl cellosolve, absolute ethanol and methyl salicylate; and clearing was in a mixture of 50% methyl salicylate, 25% absolute ethanol, and 25% xylene. Sections were photographed with a Nikon Microphot FX using Fujicolor 100 film. RESULTS rev alleles and mapping We recovered three independent mutants with a syndrome consisting of revolute (downwardly curled) leaves, reduced branching and many infertile flowers from the M 2 self-progeny of the No-0 ecotype plants mutagenized with EMS. The first mutant was backcrossed to the wild type and its mutant phenotype segregated as a simple recessive mutation in the F 2 generation (160 wild type: 52 mutant; χ 2 = 0.02; P = 0.9), indicating that the mutation is likely to be a loss-of-function. Complementation tests showed that the mutations defined a single locus that we call REVOLUTA represented by three alleles (rev-1, rev-2 and rev-4). Two EMS-induced alleles (rev-3 and rev-5) were available in the Columbia ecotype. The rev-1, rev-2 and rev-4 alleles all had indistinguishable phenotypes. The rev-3 and rev-5 mutants were more and less severely affected than these alleles, respectively, but it is not yet clear whether these differences are due to the properties of the latter alleles or to modifying factors from the mutageneses or ecotype. The phenotype of rev-1 was stably transmitted through three backcrosses with only a slight reduction in severity and this allele was therefore selected for careful description. REV showed linkage to the fifth chromosome markers DFR (11 recombinants/32) and nga129 (13 recombinants/64). Six of the 11 recombinants for DFR were also recombinant for nga129, suggesting that REV is distal to nga129. This placed REV approximately 21 mu from nga129 at approximately 108 mu (99 mu actual location 124 mu) on the bottom tip of chromosome 5 (Koornneef and Stam, 1992). This map location was verified by REV showing tight linkage to LFY (2 recombinants/33). This second experiment placed REV approximately 6.1 mu from the LFY locus. Because the nga129 data suggest that REV is distal to LFY, this experiment gives a map position for REV of approximately 95.6 mu (90 mu actual location 110 mu) on the bottom tip of chromosome 5. Overview of the Rev vegetative phenotype The rev mutations were pleiotropic and strongly affected both vegetative and reproductive shoot development (Fig. 1B). In contrast, no differences from wild-type REV controls were discerned in the roots of rev-1 and rev-2 mutants up to 2-3 weeks of age. Older roots have not been examined. We will first describe the effects of the rev-1 mutation on the vegetative phytomers (types 1 and 2) before describing its effects on the reproductive phytomers (type 3). The rev-1 mutation caused overgrowth of both rosette and cauline leaves. The rosette leaves of rev-1 plants were not readily distinguishable from wild-type No-0 leaves prior to bolting. As bolting began, however, the youngest rosette leaves became abnormally large and distorted or uneven in shape as they matured. This overgrowth was even more dramatic in the cauline leaves. The latter were longer and narrower than wildtype cauline leaves, had rolled-under margins, and curved downward along their longitudinal axes (Fig. 2A,B). Both the leaves and the primary shoots of rev-1 mutants were often darker green than those of wild type. Paraclades frequently failed to develop in both rosette and cauline leaf axils (Fig. 2B). The axils instead appeared empty, contained thin filamentous structures usually bearing branched trichomes, or bore leaves with or without a visible supporting stem (Fig. 2C,E-G). Paraclade formation is reduced in rev-1 mutants Table 1 shows the mean numbers of vegetative nodes, leaves and paraclades on the primary shoots and first-order paraclades of REV and rev-1 plants from cohort 1. The rev-1 plants had an average of about one more rosette leaf and one less cauline leaf on the primary shoot than did the REV plants, but only the

4 2726 P. B. Talbert and others Fig. 2. Morphology of wild-type and rev-1 plants. (A) REV plant. (B) rev-1 plant. (C) REV cauline leaf axil with paraclade. (D) REV florescence apex. (E-G) rev-1 cauline leaf axils: (E) empty axil; (F) axillary filament; (G) axillary leaf. (H) rev-1 florescence apex with flowers and tapered filaments. (I) rev-1 florescence apex with clustered tapered filaments. (J) rev-1 tapered filaments subtended by knobs and a filament (arrow). (K) rev-3 arrested florescence apex. f, fertile flower; k, knobs; s; sterile flower: t; tapered filament.

5 Meristem development in Arabidopsis 2727 Fig. 3. Anatomy of cauline leaves and axils. (A-G) Longitudinal axillary sections. (A) Immature REV axil. (B) Near-mature REV axil. (C) Empty REV axil. (D-G) rev-1 axils with: (D) a leaf and terminal knob on a short stem; (E) a filament; (F) club-like projections; (G) trichomes. (H-K) Midlength leaf cross-sections. (H) REV leaf midvein. (I) rev-1 leaf midvein. (J) REV leaf margin. (K) rev-1 leaf margin. Bars, (A-D,F,H,I) 20 µm; (E,G,J,K) 100 µm. l, leaf; p, paraclade; s, stem.

6 2728 P. B. Talbert and others Table 1. Mean numbers of leaves and paraclades in wildtype REV and mutant rev-1 plants Structures REV rev-1 P value of t-test Primary shoots* Vegetative nodes Rosette leaves Cauline leaves Rosette paraclades Cauline paraclades Accessory rosette paraclades Accessory cauline paraclades First-order paraclades Vegetative nodes Cauline leaves Cauline paraclades Accessory cauline paraclades *Data are from the primary shoots of 13 REV and 13 rev-1 48-day-old plants (cohort 1). Data are from 55 REV and 30 rev-1 secondary shoots of cohort 1, comprised of approximately equal numbers of rosette and cauline paraclades. Table 2. Mean dimensions of leaves and internodes in REV and rev-1 plants P value Organ Dimension REV rev-1 of t-test Cotyledon blade* length (cm) st rosette leaf* length (cm) rd rosette leaf* length (cm) Longest rosette leaf length (cm) st cauline leaf length (cm) nd cauline leaf length (cm) st cauline leaf width (cm) nd cauline leaf width (cm) st cauline leaf area (cm 2 ) nd cauline leaf area (cm 2 ) st leaf epidermal cell area (µm 2 ) nd leaf epidermal cell area (µm 2 ) st cauline internode length (cm) st cauline internode diameter (mm) *Data from cohort 3, consisting of 17 REV and 20 rev-1 28-day-old plants. Data from cohort 2, consisting of 21 REV and 29 rev-1 37-day-old plants. Nearly identical mean lengths were made on a rev-3 cohort (data not shown). All cauline leaf measurements and internode length were measured on cohort 4, consisting of 12 REV plants and 14 rev-1 plants, on material harvested on day 49. See Materials and Methods and the text for further details. Internode diameter was measured just above the rosette on 53-day-old plants from cohort 1. Table 3. Growth in the basal portion of leaves Measurement REV rev-1 Length of first cauline leaf, day 21 (mm) Length of basal region*, day 21 (mm) Percentage of total leaf length in basal region, day Increase in total leaf length, day (mm) Increase in length of basal region, day (mm) Percentage of total increase occurring in basal region *The basal region is the arbitrary region of the leaf that is proximal to all ink dots placed on the expanding 21-day-old leaves (cohort 5) to serve as position markers. leaf on the primary shoot than did the REV plants, but only the latter difference was significant at the 95% confidence level. On the primary shoots of REV plants, paraclades developed from the axils of 100% of the cauline leaves and 72% of the rosette leaves. In sharp contrast, the primary shoots of rev-1 mutants formed paraclades in 61% of cauline leaf axils and in 18% of rosette leaf axils (Table 1). Accessory paraclades were visible in the axils of about 85% of the REV cauline leaves, but in only about 4% of rosette leaf axils. No accessory paraclades were formed in rev-1 mutants. Rosette paraclades had more vegetative nodes than cauline paraclades in both genotypes (REV: 3.5 vs. 1.9, P = ; rev-1: 2.5 vs. 1.6, P = 0.002). Second-order paraclades normally developed in the cauline leaf axils of the REV first-order paraclades, and accessory paraclades developed in 41% of these axils. However, some of the vegetative nodes of REV first-order paraclades (10/151) developed second-order paraclades even though they failed to form a macroscopic leaf. Such second-order paraclades were subtended by putative vestigial leaf tissue, similar to that described by Hempel and Feldman (1994) on the primary shoots of Columbia and Landsberg erecta plants. Rarely (2/151 nodes), a second-order paraclade failed to develop in the axil of a cauline leaf on a first-order paraclade. Thus about 93% of the vegetative nodes on REV first-order paraclades bore cauline leaves and about 99% bore second-order paraclades. On the rev-1 first-order paraclades, all vegetative nodes had cauline leaves, but only 4/62 (6%) also bore second-order paraclades. A leaf, with or without a visible supporting stem, grew from 3 of the 80 rev-1 cauline leaf axils on the primary and secondary shoots. Data from other cohorts of rev-1, rev-2 and rev-3 plants generally confirmed the results shown in Table 1, except that the number of accessory branches in wild-type plants was generally less and the rev mutants in these cohorts had even lower rates of paraclade formation, with the fraction of primary cauline leaf axils with paraclades ranging from 0% to 36% (data not shown). Decapitation of rev-1 plants We tested whether the failure of paraclade formation in rev-1 plants could be relieved by decapitation of the primary shoot, as might be expected if it resulted from extreme apical dominance. A strain of rev-1 (from a Landsberg erecta outcross) was used which typically forms no paraclades. Fourteen flowering rev-1 plants were decapitated just above the rosette on day 31, and 19 sibs were left undecapitated. 20 days later no rosette paraclades had formed on the decapitated plants, and only one rosette paraclade was present on the undecapitated plants. An analogous experiment with a normal rev- 1 strain also failed to detect a stimulation of paraclade formation by decapitation (data not shown). Anatomy of rev-1 axils To understand better the nature of the defect in rev-1 paraclade growth, we made serial longitudinal sections through wild-type and rev-1 cauline leaf axils. In a cauline leaf axil from a 17- day-old wild-type plant from cohort 2 (just prior to visible bolting), the developing paraclade appeared to arise from the base of the leaf (Fig. 3A). Procambium was differentiating in the leaf and primary shoot, and procambial strands were evident by their darker staining in the developing paraclade. In the leaf, vacuolation was more advanced in the abaxial cells

7 Meristem development in Arabidopsis 2729 than in the adaxial cells, as has been noted in other angiosperm species (Steeves and Sussex, 1989). On the edges of the leaves were heavily stained club-like stipules (data not shown). In a more mature (bolting) axil (Fig. 3B), vascular tissue was well developed in the leaf, stem and paraclade. Adaxial leaf cells were vacuolated; however, differentiation of the palisade layer and spongy mesophyll had not yet taken place. As mentioned above, some axils on wild-type first-order paraclades were found that lacked second-order paraclades. We sectioned four such mature axils and found all four lacked any apparent axillary bud or mound of meristematic cells (Fig. 3C). A cryptic meristem of a few cells lacking obvious organization could still be present. In contrast to the all-or-none development of paraclades in wild-type axils, rev axils frequently contained unusual intermediate structures. In 29 rev-1 cauline leaf axils, 3% of the axils bore a leaf (Fig. 2G), 24% bore thin filaments (Fig. 2F), and 62% of the axils appeared empty except for small bumps of tissue present in about half of them. In 44 rev-3 axils, 23% bore leaves, 23% bore filaments and 43% were empty or had small bumps. Seven rev-1 axils from bolting plants were chosen for sectioning. One axil contained a paraclade indistinguishable from a wild-type paraclade (data not shown), while another contained an apparent abortive paraclade consisting of a leaf and terminal knob separated from their subtending leaf by a short stem-like region (Fig. 3D). One axil contained a short filament about five cell layers in diameter (Fig. 3E). Four others contained one or more deeply staining club-shaped projections, often on a bulge of tissue in the position normally occupied by a paraclade (Fig. 3F). These were reminiscent of stipules, but were generally larger, more irregularly shaped, and were not located at the leaf edges, where normal stipules were present. These projections appear to correspond to the small axillary bumps visible macroscopically. Two of these axils also supported unbranched trichomes (Fig. 3G), which were not observed in wild-type axils. These unusual structures may indicate a premature differentiation of the presumptive rev-1 axillary meristem. The REV gene is required to limit cell division in leaves In contrast to the abortive development of paraclades, the leaves of rev-1 mutants grew abnormally large. The difference in leaf size between wild-type and rev-1 plants was not obvious in the earlier rosette leaves, but we measured significant size differences in the cotyledons and first and third leaves from cohort 3 (Table 2). Later leaves differed more dramatically: the mean length of the longest rosette leaf (ordinarily the youngest leaf) of rev-1 plants was about 39% longer than wild-type controls, and rev-1 cauline leaves became up to twice as long as their wild-type counterparts. To determine the timing of this excessive leaf growth, we measured the lengths of the first and second cauline leaves of wild-type and rev-1 plants in cohort 4, daily from first visible bolting on day 33 until the growth of the cauline leaves of all wild-type and most rev-1 plants had terminated on day 45. (Bolting in this cohort was delayed more than a week relative to other cohorts because seeds were germinated on plates and transplanted to soil on day 14.) Over the first week of measurement the average growth rate of the first cauline leaves was about 80% greater for rev-1 plants than for wild-type plants (5.8 mm/day vs. 3.2 mm/day), and rev-1 second cauline leaves grew more than twice as fast as their wild-type counterparts (Fig. 4A). Most wild-type leaves quit growing by day 43, but most rev-1 leaves continued to grow at least 2 days longer. To determine the respective contributions of cell expansion and increased numbers of cells to the excessive growth of rev- 1 cauline leaves, we harvested the first and second cauline leaves from cohort 4 on day 49 and compared the adaxial surface areas of REV and rev-1 leaves, as well as the average surface areas of the epidermal cells within these leaves (see Materials and methods). As shown in Table 2, both the first and second rev-1 cauline leaves had significantly larger surface areas (about 45% and 63% larger, respectively) than the wildtype controls. Despite these size differences, the average surface areas of individual epidermal cells from the rev-1 first cauline leaves and from wild-type first and second cauline leaves were not significantly different. The rev-1 second cauline leaf cells were smaller, perhaps indicating that not all of them had completed expansion by day 49. Both the first and second rev-1 cauline leaves must therefore contain more epidermal cells than their wild-type counterparts. We estimate the average number of epidermal cells on the adaxial surfaces of the first and second wild-type cauline leaves to be and , respectively. The corresponding estimates for rev-1 leaves are and epidermal cells. This implies that one function of the wild-type REV gene must be to limit cell division in the leaves. Cell division in rev-1 leaves is predominantly in the basal region of the leaf In an expanding wild-type leaf, cell division is confined to the basal region (Pyke et al., 1991). To determine if the extra growth in rev-1 leaves was also confined to the basal regions or spread over other parts of the leaves, expanding 21-day-old first cauline leaves of plants in cohort 5 were marked along their midvein with dots of ink. The positions of dots were determined on day 21 and again on day 56. Table 3 compares a representative REV leaf with a rev-1 leaf with a similar initial size and placement of dots. Both leaves had about 90% of their growth in the basal fifth of the leaf. Similar results with other leaves (data not shown) confirm that cell divisions in both REV and rev-1 leaves are confined to the basal regions of the leaves. rev-1 leaf size is diminished when paraclades form To see whether the overgrowth of leaves in rev-1 mutants is related to the failure to form paraclades, we compared the lengths of mature cauline leaves which subtended paraclades and those that lacked paraclades on 25 rev-1 plants from cohort 2. Although rev-1 cauline leaves were enlarged whether or not they subtended paraclades, the mean length of 11 first cauline leaves that lacked paraclades was 24% greater than the mean length of 14 first cauline leaves that subtended paraclades (6.8 cm vs. 5.5 cm, respectively; P = 0.014). Similarly, 15 second cauline leaves that lacked paraclades were 37% longer on average than 6 that had them (5.6 cm vs. 4.1 cm; P = 0.037). This suggests that the growth of a leaf is antagonistically related to the growth of the meristem in its axil. rev-1 leaf structure We cross-sectioned six REV and six rev-1 mature cauline leaves near their midlength. The wild-type leaves were 5 to 9

8 Length (cm) Length (cm) 2730 P. B. Talbert and others cell layers thick between vascular bundles while rev-1 leaves ranged from 6 to 12 cell layers. The rev-1 leaves also had more vascular elements in the midvein, with about 30 tracheary elements in the xylem versus about 15 in REV leaves (Fig. 3H,I). The leaf margins tapered in wild-type leaves (Fig. 3J) and some rev-1 leaves, but were blunt and thickened in other rev-1 leaves that had more cell layers and large intercellular spaces (Fig. 3K). The continued division of cells on the adaxial side of the leaf after vacuolation begins on the abaxial side may account for the thickening of leaf margins and the downward curling of rev-1 leaves. rev-1 cauline internodes are shortened and thickened The cauline leaves of rev-1 mutants were often lower on the primary shoot than wild-type cauline leaves. We measured the length of the first cauline internode (between the rosette and the first cauline leaf) on the bolting plants of cohort 4. Both wild-type and rev-1 internodes completed growth in about the same time interval, but rev-1 internodes became only slightly more than half as long as wild-type internodes on average (Fig. 4B). This suggests that the failure of cauline internode elongation may account for the average of one more rosette leaf and one less cauline leaf on rev-1 shoots compared to wildtype shoots (Table 1). The first cauline internodes of rev-1 plants were also 18% larger in diameter (Table 2). Despite this increased diameter, which was also seen in subsequent internodes (data not shown), the shoots of rev-1 mutants were weaker and tended to fall over earlier and more frequently than wildtype shoots. rev-1 shoots have extra cell layers that arise late in development To investigate the cause of thicker stems in rev-1 mutants, cross-sections of wild-type and rev-1 stems from cohort 2 were made just above the rosettes of 17-day-old plants (just prior to visible bolting) and 40-day-old plants (that had completed cauline leaf expansion). Individuals with six to eight procambial strands or vascular bundles were observed in both genotypes. In four REV and four rev-1 17-day-old plants protoxylem elements were visible but other tissues were poorly differentiated (Fig. 5A-D). A protodermal layer and about five cortical cell layers were found outside the procambial strands. The most obvious difference between the genotypes at this stage was that paraclades were present in the axils of wild-type rosette leaves but were frequently missing or reduced to filaments in rev-1 axils. The six stems of 40-day-old wild-type plants (Fig. 5E,F,I) differed from the younger stems primarily in cell enlargement and in the presence of well-differentiated tissues. The epidermal layer surrounded a cortex of about five layers of chlorenchyma. The vascular bundles contained metaphloem and heavily lignified metaxylem elements. Between the xylem and phloem were a few layers of small cells that may represent a fascicular cambium (Fig. 5I). Between and connecting the vascular bundles were a few cell layers of lignified (redstaining) interfascicular cells forming a continuous ring of sclerified tissue adjacent to the chlorenchyma. The stems of 40-day-old rev-1 plants (Fig. 5G,H,J) had a starkly contrasting anatomy. Inside the epidermis were several additional cortical layers not present in wild-type or younger A B Growth of cauline leaves Growth of first cauline internode REV rev-1 1st rev-1 2nd Days rev-1 stems, which included some very large parenchymatous cells, especially capping the phloem. The additional layers of chloroenchyma probably account for the darker color of rev-1 stems. Inside these extra layers the vascular bundles were broader and appeared to have more xylem and phloem cells. rev Days REV 1st REV 2nd Fig. 4. Growth of REV and rev-1 cauline leaves and internodes. (A) First and second cauline leaves. For simplicity, standard error bars are shown in only one direction. (B) First cauline internode.

9 Meristem development in Arabidopsis 2731 Fig. 5. Stem cross-sections immediately above the rosette. (A-D) Stems of 17-day-old plants: (A,B) REV, (C,D) rev-1. Some leaves were damaged by dissection. (E-J) Stems of 40-day-old plants: (E,F,I) REV, (G,H,J) rev-1. F and H are backlit. Bars, (A,C,E-H) 100 µm; (B,D,I,J) 20 µm. Arrowheads, protoxylem elements; arrows, cambium-like zone; c, cortex; e, epidermis; if, interfascicular region; l, leaf; p, paraclade; pc, procambial strand; pd: protoderm; ph: phloem; v, vascular bundle; x, xylem; *, axillary filaments. The interfascicular cells did not appear to be as heavily lignified as those in the wild type. This may account for the weaker stems of rev-1 mutants. Between the interfascicular cells and the cortical layers and between the xylem and phloem was a ring of many small cells in files resembling a cambial zone. We suppose that periclinal divisions in this zone gave rise to the extra layers of cells. Since the extra cell layers were not present in the 17-day-old rev-1 plants, in which the apical

10 2732 P. B. Talbert and others meristems had already initiated flowers, these layers must have arisen from cells that retained a capacity to divide at a considerable distance behind the apical meristem. Floral branch types in rev-1 florescences In addition to the striking effects rev-1 had on vegetative growth, it also dramatically affected the reproductive structures. In describing these rev-1 mutants, we will use the term floral branches to refer both to the flowers and to those abnormal structures lacking floral organs that occurred in the position of flowers. The phyllotaxy of the floral branches along rev-1 florescence axes was commonly irregular, and internode length was often reduced. This reduction, together with the shorter vegetative internodes, gave plants an overall shorter stature: the mean length of the primary shoot of 30 rev-1 plants in cohort 3 was 24.1 cm, whereas 22 REV plants had a mean length of 46.0 cm (P = ). The rev-1 floral branches exhibited a range of abnormalities which could be grouped into three branch types (Fig. 2D,H): fertile flowers that were larger than normal, sterile and usually misshapen flowers that lacked pistils, and tapered filamentous structures. Frequency of floral branch types The relative frequency of these three types of floral branches in an inflorescence varied widely even among siblings in the same cohort. Most rev-1 florescences bore some fertile flowers, especially on the first few floral nodes. In extreme cases, however, rev-1 florescences could consist almost entirely of closely spaced tapered filaments and have a brush-like appearance (Fig. 2I). Table 4 gives the relative frequencies of the different floral branch types for cohort 1. The number of fertile flowers per rev-1 main florescence ranged from 0 to 34 in this cohort. The overall fraction of fertile rev-1 flowers among the floral branches was about 32% for the main florescences and about 24% for the coflorescences, compared to over 90% for all florescences in the wild type. Although sterile flowers were observed in wild-type florescences, these were always wellformed, in contrast to the sterile flowers of rev-1 plants which lacked pistils. None of the tapered filaments seen in rev-1 florescences were observed in wild-type plants. Fertile flowers and seeds are enlarged in rev-1 plants We investigated in more detail the structures of the three floral branch types. Fertile rev-1 flowers generally had a complete set of floral organs and a normal shape, but the floral organs were consistently larger than those of wild-type flowers (Table 5). Sepals, petals and stamens from rev-1 flowers actively shedding pollen (stage 15 of Smyth et al., 1990) were found to be 37%, 58% and 28% longer, respectively, than wild-type controls. Since pedicels and pistils are actively expanding at stage 15, we compared mature REV and rev-1 pedicels and siliques just prior to dehiscence. Pedicels were about 28% larger in rev-1 compared to wild type. No difference was seen in REV and rev-1 silique length, but rev-1 siliques were 56% larger in diameter. These measurements suggest that the rev-1 floral organs experience an overgrowth analogous to that seen for leaves. We also measured the size of REV and rev-1 seeds from cohort 3. The rev-1 seeds were 15% longer than REV seeds (Table 5). Although we did not measure seed diameter, this appeared to be increased proportionally in rev-1 seeds. Table 4. Occurrence of floral branch types in REV and rev-1 plants P value Florescence Floral branch type REV rev-1 of t-test Main florescences Coflorescences Fertile flowers Sterile flowers Tapered filaments Fertile flowers Sterile flowers Tapered filaments The mean numbers of fertile flowers, sterile flowers and tapered filaments on 13 REV and 12 rev-1 main florescences and on 52 REV and 29 rev-1 coflorescences from 48-day-old plants in cohort 1 are shown. Table 5. Mean dimensions of floral organs and seeds in REV and rev-1 fertile flowers Dimension (mm) REV rev-1 P value of t-test Length of sepal* Length of petal* Length of long stamen* Length of mature silique Width of mature silique Length of long pedicel Length of seed *Lengths of one sepal, one petal and one of the four long stamens were measured from a stage 15 flower from each of 20 REV and 22 rev-1 plants from cohort 2 on day 38. Measurements on mature siliques and pedicels were made on three of the largest siliques and pedicels (ordinarily from the most basal floral branches on the main florescence except when these were sterile) from each of 12 REV and 12 rev-1 plants from cohort 1 on day 48. Five seeds were measured from basal siliques of each of 9 REV and 9 rev- 1 plants from cohort 3 on day 80. Sterile rev-1 flowers are missing floral organs The floral organs of the rev-1 sterile flowers ranged in size from as large as the fertile flowers to extremely dwarfed forms with incomplete development. The sepals in these sterile flowers usually were of varying sizes, giving an asymmetrical appearance to the flowers. The sepals sometimes bore branched trichomes, which are normally found only on leaves in wildtype plants. In both sterile and fertile rev-1 flowers, rare individual organs of either the first or second whorl were found with a hybrid identity that was part sepal and part petal. Dissection of 50 sterile flowers revealed that they were missing a variable number of floral organs in an acropetally increasing manner: the mean numbers of organs found were 2.1 sepals, 0.88 petals, 0.08 stamens and no carpels. Filamentous structures were found in 26% of the flowers. Two adjacent organs in a whorl were fused proximally in 6/50 sepal whorls, in 2/27 petal whorls, and 1/2 stamen whorls. One of the two organs involved in a fusion was usually filamentous or reduced in size. A similar dissection of 17 rev-4 flowers gave means of 3.0 sepals, 1.9 petals, 0.24 stamens and no carpels, with filamentous structures in 29% of the flowers. The acropetally increasing loss or reduction of floral organs suggests that the rev-1 and rev-4 floral meristems become exhausted prematurely. Tapered filaments and the rev-1 florescence apex The tendency to form incomplete flowers and incomplete floral

11 Meristem development in Arabidopsis 2733 organs appeared to reach its extreme in the third type of floral branch, the tapered filaments. These resembled pedicels that terminated development before forming any normal floral organs. Both tapered filaments and pedicels could be subtended by knobs somewhat resembling leaf bases (Fig. 2H-J) that are absent from wild-type pedicels. In addition to the knobs, the tapered filaments could also be subtended or flanked by additional filaments (Fig. 2J). The positions of these additional filaments are suggestive of homology to leaves and stipules. Some of the tapered filaments bore branched trichomes. This may indicate that the filaments had partial leaf identity; however, we have infrequently observed branched trichomes on wild-type pedicels. The phyllotaxy of the floral branches tended to be more irregular for the tapered filaments than for rev-1 pedicels (Fig. 2H), with the internode distance often severely reduced, as in the brush-like florescence in Fig. 2I. The excessive production of tapered filaments was followed by the premature termination of growth of the rev-1 florescence apex. Premature termination also occurred in other contexts that are not well defined. Sporadic rev-1 florescences terminated abruptly in a filament. In a significant fraction of rev-3 plants, apical growth terminated only a few nodes above the rosette in a cluster of primordia (Fig. 2K). A similar phenotype was observed in preliminary experiments on rev-1 plants grown at 29 C (unpublished data). These observations indicate that the REV gene has a role in the maintenance of the primary shoot apical meristem. To investigate the structures of the tapered filaments and the rev-1 primary apical meristem, we made sections through the apices of 17-day-old and 40-day-old REV and rev-1 plants from cohort 2. No difference in size or organization of the primary shoot apical meristem could be found between REV and rev-1 plants at day 17 (Fig. 6A,B). The densely cytoplasmic cells of the apical meristem stained deeply in both genotypes, as did the procambial strands. In contrast, the apices of the 40-day-old REV and rev-1 plants (Fig. 6C,D) had markedly different staining. The REV plant had a domed, deeply staining meristem surrounded by new floral primordia and developing flowers (Fig. 6C). The rev-1 plant had a more flattened apex with severely reduced staining and dramatically larger cells (Fig. 6D). The apex was surrounded by arrested primordia. We suggest that the rev-1 apical meristem cells had stopped dividing and were far along in cell enlargement compared to the dividing cells in the wild-type apex. We also sectioned some other arrested rev-1 apices. Arrested primordia and tapered filaments with their subtending knobs exhibited light staining (Fig. 6E), indicating a lack of meristematic and vascular tissues and suggesting that the development of the rev-1 floral primordia arrested quite early. DISCUSSION REV performs similar roles in vegetative and reproductive phytomers We have recovered mutations in a gene, REVOLUTA, which is necessary to regulate basic cell division patterns in Arabidopsis. The rev mutations are pleiotropic and affect all aerial parts of the plant. Although the effects of rev-1 on vegetative and reproductive structures appear to be quite different, they share some notable similarities when they are examined in light of the phytomer concept. Both the vegetative and reproductive phytomers of rev-1 mutants undergo excessive leaf or leaf-homologous growth and simultaneously have reduced branch structures. In the rev- 1 vegetative phytomers, leaves grow too large and formation of many paraclades is drastically reduced to club-shaped objects and filaments. In the type 3 phytomers rev-1 mutants grow knobs, filaments and stipule-like objects beneath their floral branches in the positions homologous to those of leaves and stipules in vegetative phytomers. The floral branches are often reduced to incomplete flowers or tapered filaments. Within the flower itself, floral organs are regarded as homologous to leaves, and rev-1 floral organs are enlarged like rev-1 leaves. Thus the same growth-altering processes appear to act on all phytomers. This contrasts with other known mutations affecting axillary meristem development such as the tomato mutants lateral suppressor (Malayer and Guard, 1964) and torosa-2 (Mapelli and Kinet, 1992), which affect branch formation in only a specific subset of vegetative phytomers and have no reciprocal effect on leafy organ growth. REV limits the growth of non-apical meristems At least two disparate aspects of the Rev phenotype, the overgrowth of leaves and the thickening of the stems, are due to the presence of extra cells. This is compelling evidence that REV is involved in limiting cell division. Extra cell divisions in rev-1 leaves are largely confined to the base of the leaf as in the wild type. The extra cell layers in rev-1 stems have no counterpart in greenhouse-grown wild-type plants, but are not due to a generalized deregulation of cell division. The apparent derivation of the extra cells from a cambium-like layer may reveal a potential for secondary growth in Arabidopsis stems. Secondary growth has been observed in the roots of wild-type plants (Dolan et al., 1993). A structure resembling a fascicular cambium is visible in wild-type stems. A cryptic interfascicular cambium may also be present in wild-type stems and both cambia may be deregulated in rev-1 mutants. If this is true, both leaf and stem growth in rev-1 plants may arise from the enhancement of normal meristematic activities. REV is required to maintain apical meristem growth Although rev-1 mutants have enhanced cell division in leaves and stems, they have a defect in apical (including floral) meristem activity that results in the premature termination of the rev-1 shoot apex and in the formation of abnormal or incomplete structures in place of paraclades and flowers. These incomplete structures imply the defect is in apical meristem organization or maintenance, in contrast to the failure to initiate meristematic growth that is seen in empty wild-type axils. There is a hierarchy to the probability of apical meristem failure: the primary apical meristem is least likely to fail, while first-order paraclades and flowers fail more frequently and the second-order paraclades fail even more frequently (Table 1). This suggests that the partitioning of some resource may influence the probability of meristem failure. Models of REV function What is the role of REV in maintaining apical meristems? It is unlikely that REV is required for general cell viability or

12 2734 P. B. Talbert and others Fig. 6. Longitudinal sections of primary florescence apices. (A,B) Apical meristems of 17-day-old plants: (A) REV, (B) rev-1. (C,D) Apices of 40-day-old plants: (C) REV, (D) rev-1. (E) Backlit rev-1 arrested apex. Bars, (A-D) 20 µm; (E) 100 µm. f, flower or floral primordium; kt, knobs and tapered filaments; m, apical meristem. division, since rev-1 leaves and stems grow excessively and rev-1 plants are vigorous. Although the effects of rev-1 mutations on growth resemble the pleiotropic actions of phytohormones, the particular suite of defects in rev-1 mutants are not easily interpretable in terms of the known actions of any one hormone. REV appears to regulate cell proliferation with opposing effects on apical versus non-apical meristems. Explaining these opposing effects is crucial to understanding REV function. One possibility is that REV controls the partitioning of nutrients or growth factors between apical and non-apical meristems, either by direct regulation of nutrient allocation or by indirect re-allocation to non-apical meristems caused by lack of competition for nutrients from the failed apical meristems. This model requires the unusual assumption that the formation of de novo stipules and knobs below floral branches is normally limited by nutritional partitioning. Another possibility is that REV directs meristematic cells to be incorporated into the axillary (or floral) meristem instead of contributing to the formation of leaves (or stipules, knobs and filaments). This possibility is particulary interesting because axillary meristems are clonally related to their subtending leaves in Arabidopsis (Furner and Pumfrey, 1992; Irish and Sussex, 1992). However, it is difficult to explain the overgrowth of cotyledons and floral organs by this model. A third possibility is that REV could promote or inhibit cell division according to the presence of cofactors that differ between apical and non-apical meristems. Finally, REV could control the production of or response to a morphogen necessary to maintain apical growth and to inhibit non-apical growth. Regardless of which model is correct, REV clearly has profound regulatory effects on the development of meristems. Together with other mutations affecting meristem growth, the rev mutations offer the exciting possibility of dissecting the processes involved in the regulation and interactions of meristems that result in plant morphogenesis.