The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis

The leaf organs of higher plants appear in succession around the periphery of the shoot apical meristem (SAM), which has the ability to self-perpetuate(Steeves and Sussex, 1989). The SAM can be divided into the central, peripheral and rib zones. The central zone contains a core of stem cells. The rib zone lies beneath the central zone and contributes to the differentiation of the vasculature and interior stem structures. The peripheral zone is located on the flanks of the SAM and provides founder cells that direct cell division and differentiation in both spatial and temporal manners to produce leaf primordia with three primary developmental axes: the proximodistal, dorsoventral and mediolateral axes(Steeves and Sussex, 1989;Tsukaya, 1998;Waites et al., 1998). The leaf organ is formed from the leaf primordium through a series of cell divisions,differentiation and expansion processes that are also controlled both spatially and temporally. Therefore, the intricate and precise mechanisms that regulate the timing, location and extent of cell division, differentiation and expansion contribute to leaf morphogenesis.

Arabidopis provides an excellent tool to genetically dissect developmental processes of the leaf organ owing to its simple and stable pattern formation. Each Arabidopsis leaf can be divided into a proximal petiole and a distal blade region. A key factor that contributes to the control of leaf morphogenesis in Arabidopsis is the spatial and temporal regulation of meristematic activity, which involves cellular differentiation. Class I knox genes are believed to play crucial roles in the specification of leaf primordia and in the control of leaf patterning through regulation of the meristematic activities of leaf cells. Class I knox genes include KNAT1 (for KNOTTED-like from Arabidopsis thaliana), KNAT2, KNAT6 andSHOOTMERISTEMLESS (STM) in Arabidopsis(Lincoln et al., 1994;Long et al., 1996;Semiarti et al., 2001),knotted1 and ROUGH SHEATH1 in maize(Becraft and Freeling, 1994;Jackson et al., 1994;Schneeberger et al., 1995),and Nicotiana tabacum Homeobox 15 in tobacco(Tamaoki et al., 1997). These genes are strongly expressed in the SAM but not in the incipient young primordia (Jackson et al.,1994; Long et al.,1996; Tamaoki et al.,1997; Nishimura et al.,1999; Sentoku et al.,1999). Suppression of class I knox gene expression in primordia and mature leaf organs is critical for the determination of leaf cell fate. Aberrant expression of class I knox genes triggers multiple abnormal developmental responses, including altered leaf development and, in extreme cases, formation of ectopic meristems(Matsuoka et al., 1993;Sinha et al., 1993;Lincoln et al., 1994;Schneeberger et al., 1995;Chuck et al., 1996;Hareven et al., 1996;Tamaoki et al., 1997;Sentoku et al., 2000). Furthermore, mutations of the Arabidopsis gene ASYMMETRIC LEAVES1 (AS1) and AS2, which produce lobed rosette leaves with leaflet-like organs on their petioles, result in aberrant expression of class I knox genes(Byrne et al., 2000;Ori et al., 2000;Semiarti et al., 2001). Abnormal expression of class I knox genes in leaf cells due to a lack of negative regulators, such as AS1 and AS2, leads to the aberrant regulation of meristematic activity and ectopic growths. AS1encodes a Myb protein domain and is a homologue of both the ROUGH SHEATH2 (RS2) of maize and the PHANTASTICA(PHAN) of Antirrhinum(Waites et al., 1998;Timmermans et al., 1999;Tsiantis et al., 1999;Byrne et al., 2000).AS2 encodes a novel protein with cysteine repeats (C-motif) and a leucine-zipper-like sequence (Iwakawa et al., 2002). In addition, LEAFY PETIOLE is thought to be involved in controlling cell division in the marginal meristem of leaves(van der Graaff et al.,2000).

In spite of considerable efforts to reveal the genetic and molecular mechanisms underlying cell fate determination in leaves, our understanding is still largely limited (Tsukaya,2002a; Tsukaya,2002b; Tsukaya,2002c). In this report, we describe the bop1-1 mutation that induces abnormal expression of class I knox genes. The mutation was originally identified by the phenotype of vigorous ectopic outgrowths along leaf petioles. Through characterization of this mutation, we suggest that BOP1 has a critical role in regulating meristematic activity in leaves by modulating expression of class I knox genes.

The Arabidopsis thaliana Landsberg erecta (Ler;CS20) ecotype and the as1-1 (CS3774), as2-2 (CS3118),stm-1 (CS8154) and bp-1 (CS30) mutants were obtained from the Arabidopsis Biological Resource Center (ABRC, Ohio State University, USA). The KNAT1::GUS transgenic plants (Col Background)were kindly provided Sarah Hake. For the phenotypic analyses, the seeds were imbided at 4°C in darkness for 3 days, and then sown on a mixture of vermiculite, peat moss and perlite (1:1:1, v/v) in a plant growth room(KOENCON, Seoul, Korea) with 16 hours of light and 8 hours of darkness daily,and with a day/night temperature cycle of 22°C/18°C.

Approximately 40,000 seeds of the ecotype Ler were mutagenised by soaking in a 0.33% solution of ethylmethane sulfonate (EMS) in 100 mM phosphate buffer (pH 7.0) for 8 hours, as described previously(Oh et al., 1996). The M₁ plants from the mutagenised seeds were grouped into 8 subpopulations and the progeny of self-fertilised M₁ plants from each group was harvested separately. A single M₂ plant that exhibited the bop1-1 phenotype was identified from more than 200,000 M₂ plants. A backcross was performed using bop1-1 mutant pollen and Ler gynoecia. Progenies of a single plant that were derived from four backcrosses to Ler were used for genetic and phenotypic analyses.

The bop1-1 mutation was initially mapped using the cleaved amplified polymorphic sequence (CAPS) markers(Konieczny and Ausubel, 1993). The bop1-1 mutant was crossed with the Columbia (Col) ecotype. DNA for mapping studies was prepared from 60 individual F₂ progenies with the mutant phenotype. In addition, we generated the bop1-1 tt5-1double mutant and examined the segregation ratio of the mutant phenotypes in 261 F₂ progenies that were derived from a cross between the double mutant and Col. The map distance was estimated using the Kosambi mapping function (Koorneef and Stam, 1992).

The vein patterns in cotyledons and rosette leaves of plants were examined as described previously (Hamada et al.,2000; Jun et al.,2002). Photographs were taken under dark-field microscopy.

Plant materials for histological analysis were fixed overnight at room temperature in a solution of 45% ethanol, 2.5% glacial acetic acid and 2.5%formaldehyde (v/v). The samples were then dehydrated by sequential 30-minute incubations in 50%, 60%, 70%, 80%, 90%, 95% and 99.5% (v/v) ethanol, followed by two incubations of 1 hour each in 100% (v/v) ethanol. The dehydrated samples were set in Technovit 7100 resin (Heraeus Kulzer, Wehrheim/Ts.,Germany) at room temperature, once in 50% (v/v) resin and twice in 100% resin. Serial 2-μm thick sections of the plant tissues were cut with a rotary microtome (MICROM International, Walldorf, Germany), and stained with 0.5%Methylene Blue and 0.5% borate for 30 seconds.

The samples used for scanning electron microscopy were prepared in the same way as those for the histological analysis. After 100% ethanol treatment, the pretreated samples were soaked overnight in a solution of amyl acetate(Aldrich). The material was then critical point dried in liquid CO₂, coated with gold and palladium at 10-20 nm thickness, and examined at an acceleration voltage of 10-20 kV using a scanning electron microscope (Model 1420; LEO Electron Microscopy Ltd., Cambridge, England).

RNA was prepared from 10-day-old plants grown on Gamborg's B-5 Medium(GIBCOBRL) using TRI REAGENT (MCA). The cDNA molecules were synthesised from total cellular RNA using 1 μg of oligo(dT) primer and a first-strand cDNA Synthesis Kit (Roche). The PCR conditions for each cycle were as follows: 0.5 minutes at 94°C, 0.5 minutes at 55°C, and 1.5 minutes at 72°C. PCR was performed over 31 cycles for the KNAT1, KNAT2 and KNAT6cDNAs and 26 cycles for the β-tubulin cDNA. The primers used for amplification of the β-tubulin cDNA(Kim et al., 1998), KNAT1,KNAT2 and KNAT6 cDNAs(Semiarti et al., 2001) were as described previously. The amplification of β-tubulin cDNA was used for normalization of the RT-PCR (Kim et al.,1998).

Plants were grown on Gamborg's B-5 Medium (GibcoBRL) in a culture room under the long day condition. Histochemical staining of the GUS activity was performed as described earlier (Jun et al., 2002).

We reciprocally crossed bop1-1 mutant plants with as1-1,as2-2, stm-1 and bp-1 mutants. The double mutants were identified as novel or additive phenotypes in the F₂ generation. The frequencies at which these plants arose were compatible with a Mendelian segregation ratio of 1:16 for two genetically unlinked recessive loci.

Identification of the bop1-1 mutation was based on its pronounced phenotype, i.e., the presence of ectopic organ outgrowths along the leaf petioles of rosette leaves (Fig. 1A,B). The ectopic outgrowths were observed, along with extensive lobe formation, on both sides of the petiole midvein. This phenotype was observed in all of the rosette leaves. The development of ectopic organs was also observed on the petioles and on the basal regions of cotyledons(Fig. 1C) and cauline leaves(Fig. 1D), respectively.

The mutant plants exhibited pleiotropic phenotypes other than leaf patterning. In the cotyledons and the first two leaves of the mutant, an extended, adventitious, petiole-like organ grew out from the base of the original petiole (Fig. 1B,E,F,)arrowheads; see Fig. 3 for the nature of these ectopic organs). The petiole region of the early rosette leaves of mutants frequently fused with the basal part of the petiole of a cotyledon or adjacent leaf(Fig. 1G). The leaves of the mutant exhibited extended longevity (Fig. 1H). The leaves of a 37-day-old mutant were still green and viable, whereas wild-type leaves had already turned yellow. The vegetative growth of mutant plants was extended compared to that of wild-type plants(Table 1). The average number of rosette and cauline leaves formed on the mutant plant was slightly lower than that of the wild-type plant (Table 1). Mutant flowers were often slightly distorted and had more variable numbers of sepals and petals, as evidenced by a larger standard error in these experiments (Fig. 1I,Table 2). The mutant flower also contained a slightly reduced number of stamens.

The F₁ offspring from crosses between mutant and wild-type plants had the wild-type phenotype. The F₂ offspring from these plants segregated at a Mendelian segregation ratio of 3:1 (123 wild type: 45 mutant; χ²=0.28, P>0.5) for the ectopic blade phenotype, demonstrating that the mutant phenotype was caused by a single recessive nuclear mutation. We named the mutation blade-on-petiole 1(bop1). The genetic locus of the bop1 mutation was mapped to the lower arm of chromosome 3 at approximately 7.49 cM below the AFCCAPS locus and 7.28 cM below the TT5 locus.

The leaf phenotypes of the mutant suggested that the mutation played a critical role in leaf development and patterning. Therefore, we initially focused our analyses on leaf development. When examined by scanning electron microscopy, the proximal parts of the cotyledons and the first leaf of wild-type plants had smooth surfaces without protrusions(Fig. 2A,C, respectively). In contrast, the adaxial side of the basal regions of the petioles ofbop1-1 cotyledons showed several outgrowths(Fig. 2B, arrowheads). Ectopic organogenesis was also observed on the first rosette leaf of the mutant(Fig. 2D). The ectopic organs(Fig. 2D, arrows) that formed on the adaxial side of the rosette leaf resembled leaf blades with flattened structures. These ectopic outgrowths appeared to possess dorsoventrality, with differential growth between the two surfaces of the blade-like structures, and trichome development on only one of the two surfaces. The abaxial side of the first rosette leaf of bop1-1 also developed ectopic outgrowths(Fig. 2F). However, the shapes of these ectopic outgrowths differed from those on the adaxial side. They never developed into blade-like structures, but remained as small,unidentifiable outgrowths. The development of ectopic outgrowths was much more pronounced on the third rosette leaf of a mutant than on the first rosette leaf (Fig. 2G,H). Importantly,numerous trichomes and leaf primordia were found in the proximal region of the leaf (Fig. 2H). Cauline leaves of wild-type plants appeared as smooth surfaces in the proximal region(Fig. 2I). In contrast, ectopic outgrowths on blade-like structures were observed at the base of the cauline leaves in the mutant (Fig. 2J). In addition, many filamentous outgrowths developed in the mutant on the abaxial and proximal sides of the cauline leaves(Fig. 2J).

The effect of the bop1-1 mutation was also examined at the cellular level. On both cotyledons and rosette leaves of 17-day-old mutant plants the ectopic organs were clearly seen on the adaxial surfaces(Fig. 3B,D, respectively). The cellular development of the ectopic outgrowths on the adaxial side resembled that of the leaf blade. Indeed, the ectopic outgrowths had flattened structures that resembled those of leaf blades. Methylene Blue staining revealed clear differences in cellular arrangements between the dorsal and ventral sides of the ectopic blade-like outgrowths. In addition, trichome development occurred only on the adaxial side of the ectopic outgrowth that formed on rosette leaves. The formation of secondary outgrowths on the primary ectopic outgrowths was observed frequently(Fig. 3D, arrows). Interestingly, we noticed that the development of ectopic outgrowths was associated with the formation of a new structure that resembled a vascular system (Fig. 3B,D,arrowheads).

The wild-type petiole has a single, round vascular system in its centre(Fig. 3A,C,E). In contrast, the vascular system of the mutant leaves had a flattened shape(Fig. 3B,D,F). The phloem-and xylem-like tissues were located on the abaxial and adaxial sides of the leaf,respectively (Fig. 3F), which is the arrangement in wild-type petioles(Fig. 3E). The flattened structure appeared to be due to the parallel development of many vascular bundles.

The newly formed, ectopic, adventitious petioles at the base of the original petioles of the cotyledons and the first two leaves ofbop1-1 (Fig. 1B,E,F,arrowheads) had features that were intermediate between stems and petioles. First, the vascular system of the ectopic organs had a eustelic arrangement of vascular bundles (Fig. 3G,H),which is typical for stem bundles (see Fig. 7C,D for comparison). Furthermore, a pith-like structure was observed in the region surrounding the central vascular system(Fig. 3G,H), which is another feature of stems (see Fig. 7C,Dfor comparison). In cross section the ectopic organs were rounder than the wild-type petioles but not as round as the wild-type stem. In addition, the surface was not as smooth as the wild-type stem(Fig. 3G,H; seeFig. 7C for comparison). We conclude that although the ectopic organ grows from the base of the original petiole, it is not a simple petiole.

The electron microscopic and histological analyses indicated that the ectopic organs of bop1-1 plants had some of the characteristics of true blades. To confirm this, we examined the venation of wild-type andbop1-1 leaves. The wild-type cotyledons had a very simple pattern of veins, with one central midvein (primary vein) and three or four lateral veins that branched from the midvein (Fig. 4A), as reported previously(Sieburth, 1999;Jun et al., 2002). The wild-type rosette leaves had a central midvein (primary vein), six to eight secondary veins that branched from the midvein, and numerous tertiary and quaternary veins that branched from the secondary and tertiary veins,respectively (Fig. 4B)(Nelson and Dengler, 1997;Sieburth, 1999). The cotyledons of bop1-1 plants had a typical venation pattern of wild-type cotyledons in the distal regions where no ectopic organogenesis was observed (Fig. 4C). The ectopic organ that formed on the proximal part of the mutant cotyledon developed a reticulated venation pattern that resembled that of wild-type leaf blades. However, the venation pattern was intermediate between those of cotyledons and rosette leaves of the wild type in terms of complexity: the ectopic organ in the mutant cotyledons had more complex venation than the wild-type cotyledon and less complex venation than wild-type rosette leaves. The ectopic organs that formed on the rosette leaves of the bop1-1 plants also developed extensively reticulated venation systems(Fig. 4D), which were similar to those seen in the blades of wild-type rosette leaves(Fig. 4B). However, unlike the wild-type leaves, the midvein systems in the proximal region of the mutant rosette leaves revealed an extensive vasculature with numerous parallel vein elements. The secondary lateral veins branched from the ectopic midveins to the ectopic outgrowths on the bop1-1 leaves.

These results show that ectopic outgrowths formed along the petioles ofbop1-1 mutants have the features of leaf blades in that they develop the higher order vascular system. The results also show that thebop1-1 mutation severely affects vein development in the proximal region of the midvein. This observation is consistent with the histological analysis of the petiole region of the mutant leaves (see above,Fig. 3B,D,F).

To identify the origin of the abnormal features described above, the lesions in the pattern and timing of expression of the mutant phenotypes were examined in early seedlings of the mutant. In 2-day-old seedlings, young and relatively undifferentiated cells with distinct nuclei were visible in both wild-type and bop1-1 cotyledons, particularly on the adaxial sides of cotyledons (Fig. 5A,B). At this time, there were no noticeable differences in cellular differentiation between the wild type and the mutant. In addition, scanning electron microscopic examination of the 2-day-old bop1-1 mutant cotyledons did not reveal structural differences on the surface of the cotyledon (data not shown). The first indication of the bop1-1 phenotype was recognized in 3-day-old seedlings (Fig. 5D,F). At this stage, most of the cells in the petiole of wild-type cotyledon had already differentiated (Fig. 5C,E). In contrast, the petiole of mutant cotyledon had clusters of young cells on the adaxial side (Fig. 5D,F). The differentiation status of these clustered cells was evident from the large nucleus and a lack of large vacuoles, which indicated that the cells were relatively undifferentiated. This feature was also observed in longitudinal sections (Fig. 5H). The mutant cotyledons had more undifferentiated cells along the longitudinal axis than the wild-type cotyledons. The bop1-1 cotyledonary cells near the rosette leaf primordia had large nuclei and were stained more strongly than the surrounding cells (Fig. 5F,H, arrowheads). At 6 days of age, the differences between wild-type and mutant seedlings became more evident(Fig. 5I,J). The wild-type cells had more extensive differentiation and a more developed vascular system than the mutant cells. The clusters of young cells in the mutant, however,continued to divide and subsequently produced ectopic outgrowths(Fig. 5J, arrowheads).

Taken together, the results show that the mutant petiole cells have ectopic meristematic activities that lead to the production of ectopic organs.

We investigated whether ectopic organs were formed on other parts of the mutant plants. Initially, we examined the floral organs, which are modified leaves. Arabidopsis has complete flowers, with four concentric whorls of floral organs, i.e., four sepals, four petals, six stamens and two fused carpels (Fig. 6A)(Smyth et al., 1990). In contrast to wild-type petals, filamentous organs of an unidentified nature developed at the base of mutant sepals(Fig. 6B,C). The development of these ectopic organs was more extensive in certain flowers(Fig. 6D, arrowheads). The petals of bop1-1 plants had some ectopic outgrowths in their proximal regions (Fig. 6F), in contrast to the smooth surfaces of wild-type petals(Fig. 6E). However, these outgrowths did not develop into the extensive outgrowths such as the ones observed in the mutant leaves.

Ectopic organ development was also found in the stem. The surface of the mutant stem, unlike the smooth surface of the wild-type stem(Fig. 7A), was rough in certain places along the longitudinal direction, and ectopic protrusions were evident in the areas of roughness (Fig. 7B). When the stems were sectioned, the eustelic bundle vasculatures were distinctively observed in the wild-type and mutant stems(Fig. 7C and D, respectively),although the number of vascular bundles was lower in the mutant stem. Furthermore, a few small ectopic outgrowths were observed on the outer surfaces (Fig. 7D,F), which was consistent with the results from electron microscopy. However, the ectopic growths on the stems were not as extensive as those on the leaves. The cellular morphology of the ectopic outgrowths on bop1-1 stems indicated that they were probably formed from outgrowths of cortical and/or epidermal cells. Trichomes often developed on these ectopic outgrowths(Fig. 7D, arrows), which indicated that the outer cells of the ectopic outgrowths resembled wild-type epidermal cells in some aspects.

The most pronounced phenotypic abnormalities observed in thebop1-1 mutant were the lobes on rosette and cauline leaves. These abnormalities are typical of Arabidopsis plants that ectopically express class I knox genes(Lincoln et al., 1994;Chuck et al., 1996,Ori et al., 2000;Semiarti et al., 2001;Iwakawa et al., 2002). Therefore, we examined the expression patterns of class I knox genes using RNA from 10-day-old seedlings. In the wild-type plants, the KNAT1,KNAT2, and KNAT6 transcript levels in cotyledons and leaves were low (KNAT2 and KNAT6) or undetectable (KNAT1)(Fig. 8A). This result was consistent with earlier reports (Lincoln et al., 1994; Dockx et al.,1995; Byrne et al.,2000; Semiarti et al.,2001). The expression levels of these genes in the cotyledons and rosette leaves of bop1-1 seedlings were significantly higher than those in wild-type seedlings, except in the case of the KNAT2 that showed only a slightly increased expression in the mutant rosette leaf. The expression levels of all of these genes were higher in the SAM than in leaves,as reported previously (Lincoln et al.,1994; Dockx et al.,1995; Byrne et al.,2000; Semiarti et al.,2001), but there were no notable differences between the wild-type and mutant SAM regions in the expression of these genes. STMtranscription was not detectable in our experiment in the wild-type or mutant cotyledons and rosette leaves (data not shown). To complement the above results, we analysed transgenic plants that expressed the β-glucuronidase(GUS) reporter gene under the control of the KNAT1 promoter. In wild-type plants, expression of KNAT1::GUS was observed in the shoot meristem and the upper part of hypocotyl(Fig. 8B), as reported previously (Ori et al., 2000). In wild-type cotyledons and leaves, weak GUS expression was only detected at the basal tip (Fig. 8C,D,arrows). In bop1-1 plants, KNAT1::GUS was expressed in a pattern similar to that in wild-type plants, but in more expanded regions(Fig. 8E). Examination of the expression pattern in single leaves of bop1-1 showed that, unlike in wild-type leaves, KNAT1::GUS was detected in the petioles of cotyledons and rosette leaves (Fig. 8F,G), suggesting that KNAT1::GUS was misexpressed in these regions in the bop1-1. Thus, at least for the KNAT1gene, the promoter activity observed through expression of the GUS reporter gene is consistent with the result of the RT-PCR analysis.

Our results suggested that the bop1-1 mutant has a lesion in controlling expression of class I knox genes. Several mutants are also known to have defects in the regulation of class I knox genes inArabidopsis (Byrne et al.,2000; Ori et al.,2000; Semiarti et al.,2001; Iwakawa et al.,2002).

AS1 and AS2 promote or maintain the differentiated state of leaf cells through negative regulation of class I knox genes(Byrne et al., 2000;Ori et al., 2000;Semiarti et al., 2001). A mutation in either AS1 or AS2 produces rumpled and lobed rosette leaves with leaflet-like organs on their petioles(Fig. 9A,B,E,F)(Tsukaya and Uchimiya, 1997;Ori et al., 2000;Semiarti et al., 2001).

The expression pattern of class I knox genes and the leaf phenotype of the bop1-1 mutant suggests that BOP1 plays a role similar to that of AS1 and AS2 in leaf development. Therefore, we examined the genetic interactions between BOP1 andAS1 and AS2 by constructing double mutants. None of thebop1-1 as1-1 and bop1-1 as2-2 double mutants showed a simple epistatic relationship. Instead, the bop1-1 as1-1 and bop1-1 as2-2 double mutants produced numerous leaflet-like structures along the fasciated leaf petioles (Fig. 9C,D,G,H). On the primary leaflet outgrowths of the double mutant,we observed smaller second- and third-degree leaflet outgrowths that made supercompound leaves. This phenomenon can be viewed as an enhancement of each mutant phenotype.

The other class I knox gene is STM. STM functions in the formation and maintenance of the SAM in Arabidopsis(Barton and Poethig, 1993;Endrizzi et al., 1996;Long et al., 1996). Thestm-1 mutant, a strong mutant allele, fails to establish a SAM: cells at the site of the presumptive SAM undergo terminal differentiation(Barton and Poethig, 1993). As a result, stm-1 plants lack all the organs that normally develop from the SAM. Instead, some adventitious, rescued leaves differentiate ectopically from a region below the fused cotyledons(Fig. 9I). The rescued leaves in stm-1 show a nearly wild-type appearance but with abnormal phyllotaxy. However, in the bop1-1 stm-1 double mutant, rescued leaves did not fully expand and were lobed(Fig. 9J). The whole plants showed severely fasciated morphology with a rough surface and many leaflet-like structures. These phenotypes, thus, appear more severe than those expected from a simple addition of the two mutant phenotypes.

BREVIPEDICELLUS (BP) encodes the homeodomain protein KNAT1(Douglas et al., 2002;Venglat et al., 2002). A loss-of-function mutation in BP (KANT1) is characterized by compact floral internodes, short pedicles and downward-pointing siliques(Fig. 9K). The vegetative rosette leaves of the bop1-1 bp-1 double mutant exhibited a lobed morphology characteristic for bop1-1. The reproductive shoot of the double mutant produced a compact inflorescence meristem, short pedicles and downward-oriented siliques, which are characteristic for bp-1(Fig. 9L). The phenotypes of the double mutant, thus, appear additive.

Arabidopsis has a typical simple leaf, which consists of a petiole and a blade. The developmental control of petioles is presumed to be important in the effective capture of light energy by ensuring that the leaf blades do not overlap. In order to produce this leaf shape, the cells on the proximal side of the leaf differentiate into petioles without producing blades or other organs. The most pronounced phenotype of the bop1-1 mutant was the ectopic development of outgrowths along the leaf petioles. This ectopic structure resembled the leaf blade in many aspects, since it had a blade-like shape with flattened growth pattern, dorsoventral growth differences on the adaxial and abaxial sides of the flattened outgrowths, and trichomes on the adaxial side. Furthermore, the ectopic structures possessed an extensive vasculature with a pattern similar to that of wild-type blades. Thebop1-1 mutation also caused ectopic development of a structure with intermediate features of petiole and stem on the base of the original petiole of the cotyledons and the first two rosette leaves.

These observations show that, following mutation of the BOP1 gene,the petiole cells do not undergo correct developmental specification and can be diverted towards other developmental fates. Thus, BOP1 may function to determine or maintain the fate of petiole cells.

The bop1-1 mutant plants developed extensive ectopic outgrowths in the petiole region as a result of defective cellular differentiation. Normally, petiole cells undergo cellular determination upon growth. However,in the mutant plants, some cells escaped determination and remained as clusters of young cells. These young cells continued dividing and produced ectopic outgrowths. Thus, the wild-type BOP1 gene is involved in the control of meristematic activity in the petiole region. However, the meristematic cells in the mutant petiole did not develop into a new shoot meristem. Instead, a leaflet-like structure appeared in the early developmental stages of the mutant cotyledons. This result appears to be consistent with previous reports that the formation of leaf primordia can be uncoupled from shoot meristem formation(Jackson et al., 1994;Long et al., 1996;Tamaoki et al., 1997;Nishimura et al., 1999;Sentoku et al., 1999).

Moreover, the ectopic outgrowth of leaf blades was not limited to the petiole regions of cotyledons and rosette leaves. Ectopic outgrowth of blades was also observed in cauline leaves that lacked the petiole. This means thatBOP1 is involved in mechanisms other than petiole cell differentiation. Indeed, the effect of the bop1-1 mutation was seen in all leaves, including cotyledons, rosettes and cauline leaves. A common feature found among all of these leaves in the mutant was that the proximal region produced ectopic blades. Thus, the bop1-1 defect in leaves may be explained in terms of the disruption of cellular differentiation along the proximodistal axis. In Arabidopsis, the leaves mature basipetally,from the tip to the base of the leaf (Pyke et al., 1991; Donnelly et al.,1999). Thus, cells of the proximal petiole region differentiate later and have a developmental competence that is different from that of distal blade cells. The combination of a less differentiated proximal region and bop1-1 mutation may have caused the mutant leaf phenotype to produce ectopic blades in the petioles of the cotyledons and rosette leaves and in the proximal regions of the cauline leaves. Developmental lesions were even observed in petals. Although the ectopic outgrowths in petals were not extensive, they appeared primarily in the proximal regions. Therefore, we speculate that BOP1 controls meristematic activity in a proximodistal manner in a broad range of leaf types.

The meristematic cells that developed on the petiole of the mutant had limited developmental fates and were not totipotent. The ectopic outgrowths that developed on the adaxial side of the petiole were leaf blades. The base of the original petiole in the mutant cotyledon and first two rosette leaves had some stem features, particularly with respect to the morphology of the vascular system. Other structures, such as a new shoot or inflorescence, did not develop.

Furthermore, the ectopic organs on the abaxial side of the mutant petiole had different characteristics than those on the adaxial side. While the outgrowths on the adaxial side of the petiole developed mainly into blades,those that formed on the abaxial side did not develop beyond small unidentifiable protrusions. This observation implies that the dorsoventrality of the petiole region of the mutant was maintained. This phenomenon was also observed in the ectopic blades that were generated on the adaxial side of the mutant petioles. The ectopic blades maintained dorsoventrality, as evidenced by trichome formation on the adaxial side only. The ectopic vascular system of the petiole also showed dorsoventrality, such that the xylem cells were oriented towards the adaxial side and the phloem cells were oriented towards the abaxial side. Thus, BOP1 appears to be minimally, if at all,involved in the control of dorsoventrality.

There were also some position-dependent differences in the developmental defects seen in the leaves. A clear example of this was the extended petiole-like region that occurred exclusively in the cotyledons and the first two leaves. The degree of ectopic organ formation also differed depending on the leaf position, which may be due to the heteroblastic nature of leaf development. In most plant species, including Arabidopsis, the leaves that form early in shoot development (juvenile leaves) are morphologically and physiologically different from the leaves that appear later (adult leaves)(Telfer et al., 1997;Kerstetter and Poethig, 1998;Tsukaya et al., 2000). Thus,the production processes for juvenile and adult leaves are modulated by different developmental programs(Kerstetter and Poethig,1998). The extended petiole-like outgrowth of the bop1-1mutant may have been caused by a combination of prolonged and ectopic meristematic activity that is unique to the developmental program of juvenile leaves.

As mentioned above, the mutation did not completely disrupt the developmental fate of the leaf cells, although it remains possible that this is a characteristic of this specific allele. Thus, although BOP1regulates the meristematic activity of leaf cells, it functions within a given developmental context that encompasses dorsoventrality, proximodistality and heteroblasty. In other words, BOP1 controls the developmental fate of leaf cells in association with other factors that are involved in leaf development.

Ectopic leaf blade formation on the petiole, which was the most prominent phenotype observed for the bop1-1 mutant, may be explained as follows. The mutation caused a disturbance in cellular specification along the proximodistal axis. The mutant petiole (essentially the proximal part of the leaf) displayed prolonged meristematic activity while escaping the differentiation program. Thus, these meristematic cells produced leaf blades by a developmental program that was specified in the leaf.

The mutant phenotypes of the bop1-1 plants was apparent in organs other than leaves, such as stems and floral organs. Ectopic outgrowths were observed in the proximal parts of petals and ectopic filamentous organs developed at the base of the sepals. Ectopic outgrowths were also observed on the surface of the stem. The morphology of these ectopic outgrowths did not resemble that of any wild-type organ, and they appeared to be simply unidentifiable overgrowths of cells. Thus, BOP1 also functions in the developmental control of organs other than leaves. The defects in these other organs are probably due to the ectopic meristematic activities of the mutant cells, as is the case in leaves.

Interestingly, the number of floral organs in mature flowers was different in bop1-1 plants than in wild-type plants. The morphological defects in bop1-1 mutant flowers mainly appeared in the organs of the first and second whorl. Therefore, BOP1 may have a role in the development of floral organs in addition to controlling the meristematic activities of lateral organs and stems.

Our results suggest that BOP1 is crucial for cellular differentiation by controlling meristematic activity in various organs and during certain developmental stages. How does BOP1 control this process?

The bop1-1 mutation led to a wide range of defects that were reminiscent of transgenic Arabidopsis lines that overexpress class Iknox genes (Lincoln et al.,1994; Chuck et al.,1996; Pautot et al.,2001). Expression of the three class I knox genes,KNAT1, KNAT2 and KNAT6, was increased in the cotyledons and rosette leaves of the mutant, in accordance with the phenotype. Thus,BOP1 is involved in controlling the expression of class Iknox genes in these organs. Plants with defective expression of class I knox genes exhibit developmental abnormalities along the proximodistal axis, such as displacement of the proximal features of leaves(stipule, sheath and petiole) to more distal tissues (blades)(Becraft and Freeling, 1994;Jackson et al., 1994;Lincoln et al., 1994;Schneeberger et al., 1995;Chuck et al., 1996;Tamaoki et al., 1997;Ori et al., 2000). These features are similar to those of the bop1-1 mutant. In fact it would seem that the distal lamina of the leaf is now displaced to more proximal regions. This leads us to suggest that the ectopic outgrowths inbop1-1 mutants are attributable to the failure of BOP1 to perform spatial and temporal regulation of class I knox genes. This proposition is supported by the synergistic genetic interaction of thebop1-1 mutation with the mutations in other knox gene regulators, AS1 and AS2.

The bop1 mutation was found to be recessive in terms of the ectopic outgrowth phenotype. Thus, the mutant phenotype is probably the result of a functionally defective wild-type protein. In this regard, BOP1may negatively regulate the expression of class I knox genes and the resulting meristematic activity of leaf cells. However, molecular analysis of the allele is needed to confirm this proposition.

Although BP is misexpressed in the bop1-1 mutant, the analysis of the bop1-1 bp-1 double mutant showed a rather simple additive genetic interaction. This suggests that misexpression of BPmay be neither responsible nor sufficient to induce the bop1-1phenotypes. One possible explanation would be that the lobed-leaf phenotypes of the bop1-1 mutant are caused by misexpression of class Iknox genes other than BP or by combined misexpression of three class I knox genes, KNAT1, KNAT2 and KNAT6,as has been suggested for the as1 and as2 mutants(Byrne et al., 2002). In this regard, it is notable that the bop1-1 stm-1 double mutant shows some degree of synergistic mutant phenotype.

The morphology of the leaf organs and the misregulation of class Iknox genes in the as1 and as2 mutants were similar to those found in the bop1-1 mutant. This indicates that these genes may function in related pathways that control leaf morphogenesis and regulate class I knox gene expression. Furthermore, the double mutants(bop1-1 as1-1 and bop1-1 as2-2) showed synergistic mutant phenotypes. Although it is difficult to precisely define genetic relationships based on the morphological phenotypes of the double mutants, it is clear that they interact both genetically and functionally to control leaf development. An interesting hypothesis is that both combinations of BOP1 andAS1 and/or BOP1 and AS2 control the expression of class I knox genes in a synergistic manner and thereby control leaf development.

The bop1 mutants had some leaf phenotypes that were not observed in the as1 or as2 mutants. Unlike the case of bop1,no lobed structures were observed on the cotyledons or the first two leaves ofas1 and as2 mutant plants at early stages of development(Tsukaya and Uchimiya, 1997;Ori et al., 2000;Semiarti et al., 2001). The occurrence of petiole-like outgrowths at the base of cotyledons and the first two leaves was also not observed in the as1 and as2 mutants. In addition, as1 se and as2 se double mutants had phenotypes that were very similar to those of KNAT1 overexpressers(Ori et al., 2000). In contrast, the bop1 mutation did not appear to interact with these mutation (data not shown). Thus, BOP1 may have functions that are distinct from those of AS1 and AS2 as well as the functions that are shared among them in controlling leaf development.

The known regulators of class I knox genes in higher plants include the RS2 of Zea mays, the AS1 gene ofArabidopsis and PHAN gene of Antirrhinum majus(Schneeberger et al., 1998;Byrne et al., 2000;Ori et al., 2000;Semiarti et al., 2001;Waites et al., 1998;Timmermans et al., 1999;Tsiantis et al., 1999;Byrne et al., 2000). The lesion in the rs2 and as1 mutants is characterized by alterations in their proximodistal identity(Schneeberger et al., 1995;Timmermans et al., 1999;Tsiantis et al., 1999;Ori et al., 2000). Theas1 mutants also exhibit some defects in the dorsoventral identity(Ori et al., 2000). Thephan mutation can be explained by defects in proximodistal and dorsoventral character (Waites and Hudson,1995; Waites et al.,1998). In addition to these genes, KANADY (KAN)also plays a major role in the promotion of abaxial cell fates(Kerstetter et al., 2001;Eshed et al., 2001). Inkan1 kan2 double mutants, ectopic outgrowths develop on their abaxial side. Although bop1-1 mutants developed some ectopic outgrowths in the abaxial side of the leaf, mostly in the proximal region, we suggest that the effect of the bop1-1 mutation on dosoventrality is minimal, if any. This is supported by the normal cellular and vascular arrangement and by trichome formation only on the adaxial side in ectopic young leaves.

The cotyledons of the bop1-1 embryos did not exhibit any distinct morphological defects prior to germination. Instead, the mutant cotyledons produced ectopic blades on their petioles only a few days after germination,at which stage cell expansion and differentiation were taking place in the wild-type petioles (Tsukaya et al.,1994). In addition, a clear difference in cellular morphology,i.e., the appearance of meristematic clusters of cells at the bases of cotyledonary petioles, was observed in 3-day-old seedlings of bop1-1. Although we cannot completely rule out the possibility that the gene functions at an earlier stage, it appears that the major effect of BOP1 is on post-embryonic development.

The data presented here strongly suggest that BOP1 is a key component in the differentiation of cellular states and in the specification of spatially and temporally regulated developmental patterns in plant leaves. Molecular analyses of the nature and function of the gene product should provide further insights into this important phenomenon.

We thank Sarah Hake for providing the KNAT1::GUS seeds and theArabidopsis Biological Resource Center for Ler, as1, as2,stm-1 and bp-1 seeds. We also thank S. I. Kim, K. Y. Yu, K. H. Suh and Y. O. Shin for plant care and help in the greenhouse. This work was supported by a grant from the Crop Functional Genomics Frontier Research Program of the Ministry of Science and Technology of Korea and in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan, by PRESTO, Japan, and by the Co-operative Research Program on Stress Tolerance of Plants of the National Institute for Basic Biology, Japan.