Continuous organ formation from the shoot apical meristem requires the integration of two functions: a set of undifferentiated, pluripotent stem cells is maintained at the very tip of the meristem, while their daughter cells in the periphery initiate organ primordia. The homeobox genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM) encode two major regulators of meristem formation and maintenance in Arabidopsis, yet their interaction in meristem regulation is presently unclear. Here, we have addressed this question using loss- and gain-of-function approaches. We show that stem cell specification by WUS does not require STM activity. Conversely, STM suppresses differentiation independently of WUS and is required and sufficient to promote cell division. Consistent with their independent and distinct phenotypic effects, ectopic WUS and STM activities induce the expression of different downstream target genes. Finally, the pathways regulated by WUS and STM appear to converge in the suppression of differentiation, since coexpression of both genes produced a synergistic effect, and increased WUS activity could partly compensate for loss of STM function. These results suggest that WUS and STM share labour in the shoot apical meristem: WUS specifies a subset of cells in the centre as stem cells, while STM is required to suppress differentiation throughout the meristem dome, thus allowing stem cell daughters to be amplified before they are incorporated into organs.
Postembryonic development of higher plants is characterized by the continuous formation of organs from the shoot apical meristem (SAM) (Steeves and Sussex, 1989). The SAM serves two main functions: in the central zone, a population of undifferentiated, pluripotent stem cells is maintained, and in the peripheral zone, lateral organ primordia are initiated. While all cells of the meristem dome remain undifferentiated until they are incorporated into organ primordia, only a specialized subset functions as long-term stem cells from which all cells of the shoot and its lateral organs are ultimately derived (Satina et al., 1940; Stewart and Dermen, 1970). These stem cells are located in three cell tiers at the very apex and coincide with the domain where the CLAVATA3 (CLV3) gene is expressed (Fletcher et al., 1999).
Genetic analysis in Arabidopsis has identified two major regulators of SAM formation and maintenance, the homeobox genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). In wus mutants the apical stem cells are unable to self-maintain (Laux et al., 1996; Mayer et al., 1998), whereas ectopic WUS expression can abolish organ formation at the SAM and induce expression of the putative stem cell marker CLV3 (Schoof et al., 2000). During embryogenesis, WUS mRNA can first be detected in the four inner apical cells of the 16-cell stage embryo and later becomes restricted to a small central cell group underneath the presumed stem cells in the outermost three cell layers. Thus, WUS expression appears to define an organizing centre whose activity establishes an apical group of long-term stem cells.
WUS expression is under negative control by the CLAVATA genes (CLV1, CLV2 and CLV3), which encode components of a presumed receptor-kinase signal transduction pathway (Clark et al., 1997; Jeong et al., 1999; Fletcher et al., 1999). In clv mutants, the SAM enlarges progressively by the accumulation of stem cells (Clark et al., 1993; Clark et al., 1995; Fletcher et al., 1999), and this enlargement appears to be a consequence of ectopic WUS expression in more apical and lateral cells in clv mutant SAMs (Schoof et al., 2000). This has led to a model in which stem cell maintenance is regulated by a negative feedback loop mediated by the WUS and CLV3 genes, with the organizing centre signalling to the apical neighbours to specify them as stem cells, which in turn signal back to restrict the size of the organizing centre (Brand et al., 2000; Schoof et al., 2000).
Loss-of-function mutations in the SHOOTMERISTEMLESS (STM) gene, which encodes a homeodomain protein of the KNOTTED class (Long et al., 1996) also result in a lack of a self-maintaining meristem. Instead of forming a SAM, the cells in the apex of stm mutant embryos appear to differentiate (Barton and Poethig, 1993; Endrizzi et al., 1996). In addition, stm mutant seedlings exhibit fusion of the cotyledon petioles, suggesting that STM fulfils two functions: it inhibits differentiation of the cells in the embryo apex and prevents outgrowth of the cells separating the cotyledon primordia in the periphery. Repression of differentiation by STM in the SAM primordium appears to occur mainly via repression of the MYB-related gene ASYMMETRIC LEAVES1 (AS1), since loss of AS1 function in an stm mutant background rescues SAM formation (Byrne et al., 2000). STM mRNA is expressed in the shoot meristem primordium from the globular embryo stage on, and postembryonically expression is found throughout the SAM, but is excluded from incipient organ primordia (Long et al., 1996).
Whether and how the regulatory pathways defined by WUS and STM interact in SAM formation and maintenance is presently unclear. However, several lines of evidence have been taken to suggest that WUS is a downstream target of STM in functional SAMs: wus mutations exacerbate the phenotype of weak stm loss-of-function alleles, while strong stm mutations are epistatic to wus (Endrizzi et al., 1996); STM exhibits dosage-sensitive interactions with the CLV genes (Clark et al., 1996), suggesting that STM and CLV may act antagonistically on common downstream targets, one of which could be WUS; although WUS expression is initiated correctly in stm mutants, it is not maintained in later embryo stages (Mayer et al., 1998). However, WUS expression is initiated earlier in embryogenesis than STM expression (Mayer et al., 1998; Long and Barton, 1998), arguing that at least in embryonic SAM formation there is no linear pathway with WUS downstream of STM.
To understand how the functions of WUS and STM are integrated in SAM regulation, we have analyzed their interactions, using a combination of loss- and gain-of-function approaches.
MATERIALS AND METHODS
Mutant lines, growth conditions and dexamethasone induction
The wild type used in all experiments was the Landsberg erecta (Ler) ecotype. The wus-1 mutant has been described previously (Laux et al., 1996; Mayer et al., 1998), as well as the stm5 mutant (Endrizzi et al., 1996). stm-5 carries a G to A transition of the first nucleotide of the third intron, which changes the conserved GA dinucleotide of the exon-intron boundary to AA and is predicted to prevent the intron from being spliced out. This would result in a translational stop after the addition of ten unrelated amino acids, causing a loss of the C-terminal half of the homeodomain (A. Haecker and T. L., unpublished). Plant growth conditions were as described previously (Laux et al., 1996). For dexamethasone induction, plants were sprayed with a solution of 5 μM dexamethasone (Sigma Aldrich; St. Louis, USA)/0.015% Silwet L-77 (OSi Specialties; Meyrin, CH) in tap water. For the mock treatment, 0.025% ethanol/0.015% Silwet L-77 in tap water was used, since the dexamethasone stock solution was 20 mM in 100% ethanol. Seedlings were harvested 2 days after induction.
Histology, scanning electron microscopy and GUS staining
Preparation of histological sections from LR-White embedded material, DAPI staining of seedlings and scanning electron microscopy were done as described previously (Laux et al., 1996; Schoof et al., 2000). GUS staining was performed as described previously (Schoof et al., 2000). In all cases, samples to be compared where stained for the same duration.
Construction of transgenes and plant transformation
For all misexpression experiments we used the pOpL two-component system, where a promoter of interest controls the expression of a synthetic transcription factor, LhG4 (Moore et al., 1998). The gene to be expressed is controlled by a synthetic promoter, pOp, which is specifically activated by LhG4. For the sake of simplicity, we will refer to plants, for example, of the genotype ANT::LhG4; pOp::STM as ANT::STM.
Generation of the pOp::WUS-pOp::GUS (MT72) transgenic line, as well as of ANT::LhG4 and CLV1::LhG4 lines was described before (Schoof et al., 2000).
For the pOp::STM construct, the STM coding region was isolated from pCGN1547:35S::STM (kindly provided by R. Williams) by digestion with BamHI and subcloned into pU-BOP (kindly provided by I. Moore) which had been digested with BamHI. The resulting pOp::STM fragment was excised from pU-BOP:STM by partial digestion with SacI and HindIII and subcloned into pBarM, a derivative of pGPTV-BAR (Becker et al., 1992), linearized with SacI and HindIII to yield plasmid MT153. For the pOp::STM-pOp::GUS construct, a pOp::GUS fragment was isolated from plasmid MT162 by digestion with EcoRI and inserted into plasmid MT153 to yield MT168.
For the 35S::WUS-GR construct, the WUS open reading frame was amplified using primers WUS5BAM (5′-AGT CGG GAT CCA CAC ACA TGG-3′) and WUS3BAM+2 (5′-GAG CGG ATC CAG ACG TAG CTC AAG AG-3′), digested with BamHI and subcloned into the BamHI site of pRS020 (kindly provided by R. Sablowski) which contains the coding sequence of the C terminus of the rat glucocorticoid receptor (GR), producing an N-terminal fusion of WUS to GR (MT141). The WUS fragment was sequenced to exclude amplification errors. The resulting WUS-GR fusion gene was inserted as an XbaI/SmaI-fragment into pBar35S (kindly provided by G. Cardon) to yield MT142.
Generation of the WUS::NLSGUS and CLV3::NLSGUS constructs have been described previously (Groß-Hardt et al., 2002).
All constructs were introduced into Agrobacterium strain GV3101 (pMP90) (Koncz and Schell, 1986) by electroporation. Arabidopsis wild-type plants were transformed by floral-dip (Clough and Bent, 1998).
KNAT1::GUS transgenic plants were kindly provided by S. Hake; the KNAT2::GUS line was obtained from J. Dockx and J. Traas, and the CycB1;1::CDBGUS line was a gift from J. Celenza. In this construct, the cyclin-destruction-box (CDB) of CycB1;1 is fused in frame to GUS, causing the protein to be degraded at the end of mitosis, allowing visualization of cell-cycle progression by staining for GUS activity.
In situ hybridization
For the KNAT1 riboprobe, the KNAT1 cDNA was amplified from reverse transcribed poly(A)+ RNA of Landsberg erecta seedlings using primers KNAT1-FOR (5′-TCT CTC GAG TCT TTA CTC ATC TGG G-3′) and KNAT1-REV (5′-AAA GGA TCC GTT GTA ACA AGA AAG C-3′). After digestion with XhoI and BamHI, the cDNA was inserted into pBluescript II KS–. The C-terminal part, containing the homeobox, was removed by digestion with XbaI and religation to yield ML343. For the antisense probe, ML343 was linearized with XhoI and transcribed with T7 RNA polymerase (Promega; Madison, USA) using a digoxigenin-labelling kit (Roche Diagnostics; Mannheim, Germany); for the sense probe, ML343 was linearized with XbaI and transcribed with T3 RNA polymerase (Promega; Madison, USA).
For the KNAT2 antisense riboprobe, plasmid pCKI-30 (kindly provided by J. Traas) which contains the full-length KNAT2 cDNA was linearized with XhoI and transcribed with T7 RNA polymerase; for the sense probe, pCKI-30 was linearized with HindIII and transcribed using SP6 RNA polymerase (Promega; Madison, USA).
For all comparisons of wild-type and mutant or transgenic seedlings, sections from plants of the two genotypes under study were hybridized on the same slides, and only those slides were included in the analysis that showed clear expression in the wild-type samples. Where expression is reported, this was observed in several serial sections. The numbers given for CLV1::WUS-expressing stm5 mutants refer only to those seedlings that contained an adventitious meristem.
We showed that no significant cross hybridization could occur between the KNAT2 antisense riboprobe and KNAT1 mRNA by a filter hybridization experiment that mimicked the conditions of in situ hybridization (data not shown).
Ectopic expression of STM in leaf primordia suppresses cell differentiation
Based on its expression pattern and loss-of-function phenotype, STM appears to maintain cells in an undifferentiated state, before they are incorporated into leaf primordia. To test whether STM was sufficient to suppress differentiation, we expressed STM ectopically in leaf primordia, using the pOpL two-component system (Moore et al., 1998; see Materials and Methods). The functionality of the STM transgene was confirmed by complementation of the meristem defect in stm5 homozygous mutants (Fig. 1A-D).
We expressed STM under the control of the AINTEGUMENTA (ANT) promoter, which shows a complementary expression pattern to that of STM, i.e. it is active in primordia of cotyledons and leaves (Elliott et al., 1996; Klucher et al., 1996). Staining for the activity of a linked ANT::GUS reporter gene confirmed expression of the transgenes in cotyledons and leaf primordia (Fig. 2D). ANT::STM-expressing plants showed cotyledon and leaf phenotypes of varying severity, depending on the individual STM target line used. The petioles of the cotyledons and of leaves were up to approximately threefold wider than in non-transgenic plants (Fig. 1E-H). Leaves were smaller than in wild type, and in the most extreme cases, were reduced to small finger-like structures (Fig. 1F, arrow). Their dorsoventrality was maintained, however, as judged from the development of trichomes only on the adaxial side of early vegetative leaves and their anisotropic growth, causing the leaves to bend over the SAM as they do in wild type. Furthermore, leaves of the transgenic plants developed lateral outgrowths from the leaf blade or petiole which was never observed in wild type (Fig. 1G,H).
Histological analysis showed that differentiation of leaf cells was suppressed in ANT::STM-expressing leaves compared to wild type. In the most severe cases we did not observe a vascular bundle in the finger-like structures at a time when wild-type petioles contained a well differentiated vascular strand (Fig. 1I,J). In addition, the cells throughout the leaf were small and cytoplasmically dense, resembling meristematic cells in contrast to the large, vacuolated differentiated cells of wild-type leaves (Fig. 1K,L).
Thus, STM is able to suppress cell differentiation in developing leaves and instead maintains the potential to form additional lateral outgrowths. These results support the reported phenotype of 35S::STM-expressing plants which have a stunted appearance with a disorganized shoot and leaf-like bulges that do not develop into mature leaves (Williams, 1998). However, the effects of ectopic STM expression in leaf primordia are relatively subtle compared to those of ANT::WUS expression, which entirely abolishes organ formation (Schoof et al., 2000).
STM induces the expression of KNAT genes and CycB1;1, but not stem cell identity
In order to molecularly characterize the effects of ectopic STM activity, we analyzed the expression of several candidate downstream genes in ANT::STM-expressing plants.
The formation of lateral outgrowths by ANT::STM-expressing leaves suggested that STM was able to promote cell proliferation when expressed in leaves. To test this, we examined the expression of the mitotic cyclin CycB1;1 using a promoter-GUS fusion. CycB1;1 is expressed shortly before and during mitosis and overexpression analysis suggests it may be a limiting factor for cell division, making it a suitable marker for mitosis and cell proliferation (Doerner et al., 1996; Mironov et al., 1999).
In 10-day old wild-type plants carrying a CycB1;1::CDBGUS reporter gene, GUS staining was restricted to the shoot meristem and young leaf primordia, but was absent from the expanding first pair of leaves (Fig. 2A). In ANT::STM; CycB1;1::CDBGUS seedlings the first pair of leaves became visible at the same time as in wild type, yet still showed GUS staining at 10 days after germination, in addition to staining in the shoot meristem with younger leaf primordia (Fig. 2B). In older ANT::STM-expressing leaves, ectopic GUS staining was most pronounced in the lateral outgrowths (Fig. 2C), consistent with our observation that these arose after the main leaf had already reached a certain size (data not shown). This result suggests that ectopic STM expression in cells of leaf primordia promotes their proliferation.
Since the leaf phenotype of ANT::STM-expressing plants was similar to the effects observed when either KNAT1 or KNAT2, two homeobox genes with potential regulatory functions in the shoot meristem, were overexpressed (Lincoln et al., 1994; Dockx et al., 1995; Chuck et al., 1996; Pautot et al., 2001), we addressed whether KNAT1 or KNAT2 was acting in one regulatory pathway with STM. Staining for a KNAT1::GUS reporter revealed ectopic expression in the vasculature of the cotyledons and in strongly affected leaves of ANT::STM-expressing seedlings (Fig. 2E,F), suggesting that ectopic KNAT1 expression can be activated by ectopic STM activity. Similarly, the KNAT2::GUS reporter showed ectopic staining in the vasculature of the cotyledons and in leaves of ANT::STM-expressing seedlings (Fig. 2G,H).
In contrast to KNAT1 and KNAT2, the stem cell marker CLV3 was not expressed ectopically in ANT::STM-expressing seedlings: using in situ hybridization CLV3 RNA was only detected in the apical stem cells of the shoot meristem, which was indistinguishable from wild type (Fig. 2I,J).
Thus, ectopic expression of STM in leaf primordia induces expression of two meristem genes and promotes cell proliferation, yet STM is not able to induce ectopic stem cell identity, based on expression of the presumed stem cell marker CLV3.
WUS induces ectopic stem cell identity, but not the expression of KNAT genes
To molecularly delimit the functions of STM and WUS, we aimed to test whether expression of the above marker genes could be induced by ectopic WUS activity in leaves, complementary to the analysis for STM. Since constitutive ANT::WUS expression completely suppresses leaf formation (Schoof et al., 2000), we used an inducible construct to produce leaves with ectopic WUS activity: we expressed a posttranslationally inducible form of WUS fused to the C terminus of the rat glucocorticoid receptor (GR) (see Sablowski and Meyerowitz, 1998) from the constitutive Cauliflower Mosaic Virus 35S promoter. Nuclear translocation of this fusion protein, and thus its potential to activate transcription, can be induced by addition of a GR-ligand such as dexamethasone. When germinated on dexamethasone-containing medium, 35S::WUS-GR seedlings are indistinguishable from 35S::WUS seedlings with suppressed differentiation, whereas in the absence of dexamethasone the transgene has no effect on plant development as has dexamethasone treatment of 35S::GR-expressing seedlings, indicating that the fusion protein behaves as predicted and that the effects observed are due to ectopic WUS activity (Fig. 3A; data not shown). We introduced GUS reporter genes for CLV3, KNAT1, KNAT2 and CycB1;1 into 35S::WUS-GR seedlings and analyzed GUS activity in 14-day old F1 seedlings that had been treated for 2 days with dexamethasone or with a control solution.
Dexamethasone induction of 35S::WUS-GR seedlings resulted in strong ectopic activation of the CLV3::NLSGUS reporter gene in cotyledons, leaves and hypocotyl, mainly associated with the vasculature (Fig. 3D), whereas uninduced siblings showed GUS staining exclusively in the apical stem cells of the SAM (Fig. 3B,C). Thus, WUS appears to be sufficient to induce aspects of stem cell identity de novo in differentiated tissue. Preferential induction close to the vasculature could either be due to predominant expression of the 35S promoter there (e.g. Chuck et al., 1996) or to a higher sensitivity of cells near the vasculature to WUS activity.
By contrast, expression of neither the KNAT1::GUS nor the KNAT2::GUS reporter genes could be induced ectopically by 35S::WUS-GR (Fig. 3E-H), indicating that WUS-GR is not able to activate expression from the KNAT1 and KNAT2 promoters.
In dexamethasone-induced 35S::WUS-GR seedlings carrying the CycB1;1::CDBGUS reporter, we occasionally detected ectopic staining in the first pair of leaves (5 out of 15 seedlings analyzed) which was never detected in uninduced seedlings of the same genotype (Fig. 3I; n=15). The ectopically stained cells were always associated with the vasculature.
In summary, WUS is sufficient to induce ectopic stem cell identity – as judged by CLV3 expression – and occasional ectopic cell divisions, but is not able to ectopically activate expression of KNAT1 or KNAT2. Taken together, these results suggest that ectopic expression of STM or WUS in leaf primordia activates distinct sets of downstream target genes.
Ectopic STM and WUS functions act independently of each other
To study how the activities of WUS and STM are interconnected, we analyzed whether the activity of one gene is required for the effects of ectopic expression of the other gene in leaf primordia.
To analyze whether STM might be a downstream target of WUS, we tested whether ectopic WUS expression could still repress organ formation in an stm5 mutant background. While ANT::WUS expression in a wild-type background produced an enlarged SAM in place of leaves immediately after germination, no effect of the transgene was observed in stm5 mutant seedlings up to 7 days after germination. However, thereafter ANT::WUS-expressing stm5 mutant seedlings formed a mass of small meristematic cells inside the fused cotyledon petioles that was indistinguishable from that observed in ANT::WUS-expressing wild-type seedlings (Fig. 4A,B,D,E). The relatively late effect in stm5 mutants compared to wild type appears to be due to the fact that the transgene is not expressed in stm5 mutants up to 7 days after germination, as judged from staining for the activity of a linked ANT::GUS reporter gene (data not shown), and expression only becomes detectable thereafter (Fig. 4C). By contrast, non-transgenic stm5 seedlings never produced a similar enlarged SAM, but formed adventitious leaves between the fused cotyledon petioles (Fig. 4F) (Endrizzi et al., 1996).
These observations indicate that suppression of leaf formation by ectopic WUS activity does not require STM and suggest that STM is not an essential downstream target of WUS.
In the converse experiment, we tested whether WUS might be a downstream target of STM. To do so, we analyzed whether WUS is required for the effects of ectopic STM activity by expressing ANT::STM in wus1 mutants. ANT::STM-expressing wus1 mutant plants exhibited a leaf phenotype that was indistinguishable from the effect of ANT::STM expression in a wild-type background (Fig. 4G-J), suggesting that WUS is not an essential downstream target of ectopically expressed STM. This finding was confirmed by analyzing the expression of a WUS::NLSGUS reporter gene in plants with ectopic STM activity. ANT::STM; WUS::NLSGUS plants showed GUS staining in a small central cell group in the shoot meristem, in a pattern that was indistinguishable from that in wild type (Fig. 4K-M), but they did not show ectopic GUS staining in the cells that expressed ANT::STM (compare with Fig. 2D). Thus, ectopic STM activity does not appear to induce expression from the WUS promoter.
Taken together these results indicate that ectopic WUS and STM activities function independently of each other.
Coexpression of WUS and STM produces synergistic effects
Their loss-of-function phenotypes indicate that both WUS and STM activities are essential for SAM function (Barton and Poethig, 1993; Endrizzi et al., 1996; Laux et al., 1996), yet our above experiments demonstrate that their functions are genetically independent. One interpretation of these findings is that the developmental pathways regulated by them ultimately converge on some downstream process. We thus asked whether ectopic WUS and STM functions act synergistically on some shared process and coexpressed both in developing cotyledons and leaf primordia. Except for a widening of the cotyledon petioles in ANT::STM-expressing plants, ectopic expression of either gene alone under the control of the ANT promoter leaves the cotyledons largely unaffected, although staining for the activity of a linked ANT::GUS reporter gene showed the transgenes to be expressed throughout embryonic cotyledon primordia (data not shown). By contrast, ANT::STM; ANT::WUS coexpressing seedlings, in which the presence of both transgenes was confirmed by PCR (data not shown), showed a novel phenotype which was clearly distinct from the effects of ectopic expression of either gene alone (Fig. 5A-D): they completely lacked cotyledon petioles and had fields of small cells extending from the apex into the lamina of the cotyledons. These cells strongly resembled the dense meristematic cells in the apex of ANT::WUS plants as judged from their appearance under the scanning electron microscope and in histological sections (Fig. 5E,F) and showed ectopic CLV3 expression (Fig. 5G,H).
Thus, simultaneous ectopic expression of WUS and STM produced a non-additive phenotype in that meristematic cells were induced in cotyledons which was not the case in plants expressing either gene alone. This suggests that in differentiated tissue both genes synergistically confer meristem cell identity.
Increased WUS activity can induce self-maintaining meristems in stm mutants, but not vice versa
We next asked whether similar to the results of the above ectopic coexpression experiment, the pathways activated by WUS and STM also converge in the regulation of SAM function. We therefore tested whether an increase of one gene’s activity in the SAM could compensate for the effects of a mutation in the other gene. For this purpose we expressed WUS or STM under the control of the CLV1 promoter in the respective other mutant.
First, we expressed CLV1::STM in wus1 mutants. Since the expression patterns of transgenic and endogenous STM roughly overlap, this would be expected to increase the STM expression level throughout the apex. Expression of the CLV1 activator line in wus1 mutant embryos was evident from its ability to rescue the mutant phenotype when combined with a WUS target line (Groß-Hardt et al., 2002) and was confirmed by staining for the activity of a linked CLV1::GUS reporter (Fig. 6A,B). The phenotype of CLV1::STM; wus1 plants was indistinguishable from that of non-transgenic wus1 mutants: shoot development in seedlings of both genotypes arrested after the formation of two to three leaves (Fig. 6E,F). 10 days after germination, we observed strong transgene expression in what are most likely adventitious meristems (Fig. 6D; see Laux et al., 1996). Despite this, no self-maintaining meristems could be formed in a wus1 mutant background, and CLV1::STM-expressing wus1 mutant plants showed the same ‘stop and go’ mode of development as non-transgenic wus1 mutants (Laux et al., 1996; data not shown). The leaves, however, showed the same wrinkled phenotype that was also observed in CLV1::STM-expressing wild-type plants and which appears to be due to weak expression of the transgene in leaves as judged by prolonged staining for the activity of the linked CLV1::GUS reporter gene (data not shown), confirming that in principle STM was active in wus mutants.
Thus, increasing STM expression in the shoot apex is not able to compensate for the shoot meristem defects of wus mutants.
Secondly, in the converse experiment, we analyzed the effects of CLV1::WUS expression in stm5 mutants. CLV1::WUS-expressing wild-type seedlings produce an enlarged meristem immediately after germination due to the enlarged WUS expression domain throughout the SAM (Fig. 6G-J) (Schoof et al., 2000). By contrast, 7 days after germination stm5 mutant seedlings carrying the CLV1::WUS transgene lacked a recognizable shoot meristem and were indistinguishable from non-transgenic stm5 mutant seedlings. That the CLV1 activator was expressed in stm5 mutants was demonstrated by its ability to rescue the mutant defect when combined with an STM target line (see above, Fig. 1A-D); however, even in combination with our strongest WUS target line, the resulting embryonic expression was only very weak as judged from staining for the activity of a linked CLV1::GUS reporter gene (data not shown). While such weak expression appears to be sufficient to rescue the wus1 mutant defect (Groß-Hardt et al., 2002), it is apparently unable to overcome the lack of STM activity during embryogenesis. After day 7, CLV1::WUS; CLV1::GUS-expressing stm5 mutant seedlings showed small clusters of GUS staining cells inside the fused cotyledon petioles and by day 12 after germination, 26 out of 40 seedlings had developed a conspicuous adventitious structure resembling a meristem surrounded by small leaf primordia (Fig. 6J,K,M,N). No similar structures were observed in any of 25 non-transgenic stm5 mutant seedlings 12 days after germination (Fig. 6L).
To analyze whether the induced structures were meristems, we examined them for expression of the meristem marker genes CLV3, KNAT1 and KNAT2 using in situ hybridization (see above). Both CLV1::WUS-expressing wild-type and stm5 mutant seedlings 10 or 14 days after germination showed strong CLV3 expression in the outermost cell layers across their enlarged meristems and the induced structures, respectively (Fig. 7A,B). By contrast, we could not detect CLV3 expression in any of 25 non-transgenic stm5 mutant seedlings 10 days after germination (data not shown). While we could not detect KNAT1 expression in the induced structures of 10-day old CLV1::WUS-expressing stm5 mutant seedlings (Fig. 7E,F; n=6; see Materials and Methods), by 14 days after germination the induced structures in CLV1::WUS-expressing stm5 mutant seedlings showed clear KNAT1 expression in small patches on the flanks and at their base close to the vasculature (Fig. 7G), similar to the pattern observed in meristems of CLV1::WUS-expressing and non-transgenic wild-type seedlings (Fig. 7C,D) (Chuck et al., 1996). Hybridization with a KNAT2 antisense riboprobe produced a similar result: While no KNAT2 expression could be detected in the induced structures of 10-day old CLV1::WUS-expressing stm5 mutant seedlings (Fig. 7I; n=11; see Materials and Methods), consistent weak staining was found at the flanks and base of the induced structures by 14 days after germination (Fig. 7J). CLV1::WUS-expressing wild-type seedlings showed virtually the same expression pattern for KNAT2 as found for KNAT1, i.e. at the periphery of the enlarged SAM and at the base of young leaf primordia (Fig. 7H).
Thus, the structures induced by CLV1::WUS expression in stm5 mutant seedlings showed expression of the three marker genes tested, suggesting that they represent meristems. However, these meristems never reached a size comparable to those formed by CLV1::WUS-expressing wild-type plants, as judged from staining for the activity of the linked CLV1::GUS reporter gene (Fig. 6O,P). Since the size of the cells in CLV1::WUS-expressing wild-type and stm5 mutant meristems appeared to be roughly equal (compare Fig. 7A and 7B), the reduced growth of the meristem in stm5 seedlings likely results from fewer cell divisions, rather than from reduced cell expansion. This suggests a critical requirement for STM in allowing amplification of meristem cells which cannot be compensated for by increased WUS activity.
In summary, CLV1::STM expression in wus mutants cannot compensate for the loss of WUS function. However, conversely expressing CLV1::WUS in stm mutants induces the formation of adventitious shoot meristems at a high frequency, although it cannot fully rescue the stm mutant defect. Thus, it appears that increasing WUS activity can at least partly compensate for the loss of STM function, suggesting a convergence of the pathways activated by WUS and STM in SAM regulation.
The WUS and STM homeobox genes are both essential for the same processes, formation and maintenance of a functional shoot meristem (Barton and Poethig, 1993; Endrizzi et al., 1996; Laux et al., 1996), yet it is unknown whether and how their functions are integrated in SAM regulation. To address this issue, we have analyzed their genetic interactions using a combination of gain- and loss-of-function experiments.
STM and WUS function in different pathways in shoot meristem regulation
Our results suggest that WUS and STM fulfil independent, yet complementary functions in SAM regulation, for the following reasons.
(1) When expressed ectopically in leaf primordia, the effects of WUS and STM are clearly distinct. WUS is sufficient to completely abolish organ formation, but has little, if any, stimulating effect on cell division, as evidenced both by its inability to efficiently induce expression of the mitotic marker gene CyclinB1;1 and by the low proportion of cells in S-phase in the enlarged central zone of CLV1::WUS-expressing meristems (M. L. and T. L., unpublished). By contrast, ectopic STM activity still allows organs to develop, but cell differentiation is suppressed and the cells continue to proliferate. This effect is strikingly similar to the phenotype of dominant mutations in knotted1, the maize ortholog of STM, whose misexpression in leaves leads to local overproliferation (Smith et al., 1992). At least on the basis of expression levels of the linked GUS reporter genes (Fig. 4B,I), these distinct effects do not appear to be due to strongly differing levels of transgene expression, suggesting that they reflect intrinsic functional differences between the two transcription factors.
(2) Ectopic expression of WUS and STM in leaf primordia induces the expression of distinct downstream target genes. WUS is able to induce expression of the presumed stem cell marker CLV3 even in differentiated organs, but does not activate KNAT1 or KNAT2 expression. By contrast, expression of the latter genes can be induced by ectopic STM activity, which has, however, no effect on CLV3 expression. The conclusion that, unlike WUS, STM thus does not appear to be directly involved in stem cell specification is further supported by our preliminary result that CLV3 expression is initiated in the apex of stm5 mutant embryos, and is lost only in late stages of embryogenesis when the apex differentiates (M. L. and T. L., unpublished).
(3) The gain-of-function phenotypes of ectopic WUS and STM expression in leaf primordia do not require the activity of the respective other gene, indicating that they function in independent genetic pathways.
(4) The shoot meristem defects of both WUS and STM loss-of-function mutants cannot be rescued by transgenic expression of the other gene: transgenic expression of STM in the apex is not able to compensate for the lack of self-maintaining stem cells in wus mutants. Conversely, even though WUS expression can induce the formation of meristems in stm mutants, these appear to grow significantly slower than the corresponding meristems in a wild-type background, suggesting that loss of STM function results in reduced proliferation of meristem cells and/or their premature differentiation. Thus, WUS and STM appear to fulfil distinct functions in shoot meristem regulation.
(5) Based on the synergistic effect of ectopically coexpressing both genes in leaf primordia and on the ability of WUS to partly compensate for loss of STM activity in the apex, the developmental pathways regulated by WUS and STM appear to converge, in that both genes suppress cell differentiation.
Integration of WUS and STM in shoot meristem maintenance
Our data suggest the following model for how the independent pathways regulated by WUS and STM are integrated to produce a self-maintaining meristem. In the central region of the meristem WUS-dependent signalling from the organizing centre specifies an apical stem cell niche whose residents act as long-term stem cells. STM is not directly involved in stem cell specification, but is required throughout the meristem dome to antagonize cell differentiation and allow meristem cells to proliferate. Thus, peripheral stem cell daughters are prevented from being prematurely incorporated into organ anlagen and can amplify cell numbers. STM appears to act by repressing AS1 expression and thus allowing expression of the homeobox genes KNAT1 and KNAT2 (Byrne et al., 2000). Local downregulation of STM expression in the periphery finally allows lateral organs to be formed.
The observations described here and in previous studies (Mayer et al., 1998; Fletcher et al., 1999) suggest a refinement of the classical histological zonation concept of the SAM (Steeves and Sussex, 1989). The centre of the shoot meristem, roughly equivalent to the central zone, is composed of an apical stem cell niche, whose residents express the CLV3 gene, and the underlying WUS-expressing organizing centre. The peripheral zone comprises a transition zone, where differentiation is repressed by STM, allowing the cells to amplify, and regions where STM expression is discontinued and organ primordia are initiated.
Similar to other stem cell systems (Potten and Loeffler, 1990), the amplification of cell numbers by the peripheral stem cell daughters may allow the long-term stem cells to divide only relatively rarely – for example only once per 14 initiated leaves in privet (Stewart and Dermen, 1970), while still ensuring a continuous supply of sufficient cells for organ initiation. This division of labour could in turn minimize the danger for stem cells of incurring mutations associated with DNA replication and chromosome segregation. As a large portion of the plant body is ultimately derived from a single stem cell (Stewart and Dermen, 1970), mutations in them would likely be more deleterious than mutations in their daughter cells which only give rise to a more limited part of the plant.
A critical number of cells and cellular competence appear to be required for shoot meristem initiation
Our results imply two important requirements for meristem formation. First, we found that a CLV1::WUS transgene can induce adventitious meristems at a high frequency in stm mutant seedlings, which is observed to a similar extent in stm clv double mutants (Clark et al., 1996). In both cases, the effect is likely due to WUS being expressed in an enlarged domain (Schoof et al., 2000). How might this lead to more frequent meristem initiation? One conceivable interpretation is that meristems can be formed as long as there are enough undifferentiated cells, no matter whether these are produced by increasing the size of the WUS expression domain – as in CLV1::WUS-expressing plants or in clv loss-of-function mutants – or by a small WUS-expressing region in combination with STM activity in a larger zone as in the wild-type apex. In contrast to WUS, STM on its own does not appear to be able to induce self-maintaining meristems in the absence of WUS function. This could either be due to a reduced potency of STM in suppressing differentiation compared to WUS or to its inability to induce stem cells, which are lacking in wus mutants, or to a combination of both. Differences between the two genes in their potency to suppress cell differentiation are suggested by the different severity of the effects caused by ectopic expression of WUS or STM in leaf primordia.
Evidence supporting the above hypothesis that formation of a stable SAM requires a critical number of undifferentiated cells has also been obtained by studying the STM ortholog KNOTTED1 in maize (Vollbrecht et al., 2000). The penetrance of the meristem defect in knotted1 mutant embryos is inversely correlated with the size of the meristem primordium in wild-type embryos of the respective genetic background, such that knotted1 mutants form meristems much more frequently in inbred lines with a large meristem primordium than in ones with a small meristem primordium.
Secondly, meristem initiation appears to depend on a competence of cells to switch to meristem identity, which they appear to gradually lose as they differentiate. While relatively undifferentiated cells in leaf anlagen can easily be respecified towards stem cell identity by WUS alone, the differentiated cells in cotyledons are no longer responsive to WUS alone. However, this block to switch to meristem identity can be overcome by the combined effects of WUS and STM, suggesting that a strongly reduced cellular competence can be compensated for by increased meristem promoting activity. This synergistic effect of coexpressing WUS and STM could have important biotechnological implications for adventitious meristem formation from differentiated cells, which could possibly be strongly enhanced by coexpression of WUS and STM orthologues.
In summary, the results presented here indicate that WUS and STM serve distinct functions in the SAM, regulation of stem cell identity and protection of meristem cells from premature differentiation, respectively, and support a division of labour between a slowly dividing set of long-term stem cells and a more rapidly proliferating population of stem cell daughters that only transiently function as initials, both of which are required for continuous organ formation from a self-maintaining meristem.
We would like to thank R. Williams for providing the pCGN1547:35S::STM construct, S. Hake, J. Traas and J. Celenza for providing KNAT1::GUS, KNAT2::GUS and CycB1;1::CDBGUS lines, respectively, V. Pautot and J. Traas for the KNAT2 plasmid and I. Moore for the components of the pOpL expression system. We are grateful to Andrea Bohnert for technical assistance and to Arp Schnittger and members of the Laux laboratory for helpful suggestions on the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft to T. L. and a PhD fellowship of the Boehringer Ingelheim Fonds to M. L.