Signaling In and Out

To build a plant requires strict control of stem cell populations as well as specification of appropriate cell fates once cells enter a differentiation program. It is now accepted that both intracellular and intercellular signaling play important roles in controlling cell division patterns and cell specification (Westhoff et al., 1998; Scheres, 2001). The molecular nature of this signaling, however, has long been elusive. Recent molecular and genetic studies have begun to reveal the signaling mechanisms that regulate cell differentiation in both shoot and root. In this review, we discuss recent advances in the areas of stem cell control in both vegetative and floral meristems as well as in pattern formation in roots. Our knowledge of the molecular mechanisms underlying these processes has been obtained primarily from Arabidopsis. Whether common mechanisms operate in other species awaits experimental proof by the isolation and characterization of orthologous genes. Schemes Depicting Wild-Type and Mutant Arabidopsis SAM Structures and Expression Patterns of Key Regulatory Genes. (A) Subdivision of SAM domains based on histological observations (left) and clonal relationships of cell layers (right). (B) SAM structure of loss-of-function wus mutants (left) and wild-type WUS gene expression (right) (Laux et al., 1996; Mayer et al., 1998). (C) SAM structure of strong loss-of-function stm mutants (left) and wild-type STM gene expression (right) (Barton and Poethig, 1993; Endrizzi et al., 1996; Long et al., 1996). In strong stm mutants, the region corresponding to the wild-type SAM is reduced to a small number of cells between the cotyledon petioles. Leaf primordia arise in the center of this region. (D) SAM structure caused by a loss-of-function mutation in CLV1, CLV2, or CLV3 (left) and gene expression patterns of CLV1 and CLV3 (right) (Clark et al., 1993, 1995, 1997; Kayes and Clark, 1998; Fletcher et al., 1999). CP, cotyledon petiole; CZ, central zone; LA, leaf anlagen; LP, leaf primordium; L1, L2, and L3, epidermal, subepidermal, and underlying layers, respectively; PZ, peripheral zone; RZ, rib zone; WT, wild type. Cells in the SAM also can be grouped according to their clonal relationships: the epidermal, subepidermal, and underlying layers (Figure 1A, right) (Satina et al., 1940; reviewed by Steeves and Sussex, 1989). Cells in the epidermal and subepidermal layers divide in a plane perpendicular to the layers (anticlinal division). These cells are ultimately incorporated into the epidermal and subepidermal layers of lateral organs (i.e., leaves and floral organs). Cells in the underlying layer divide in a more complex manner, and their daughter cells differentiate into the inner tissues of lateral organs as well as into the pith in the stem. Despite clear clonal distinctions between the three layers, their cell divisions are highly coordinated with each other, indicating an intimate intercellular communication that allows programmed development of organs with fixed shape and size. Maintaining a strict balance between the number of stem cells and the programmed differentiation of their progeny is critical to SAM function. In Arabidopsis, a stem cell population persists even after the transition from vegetative to reproductive growth, allowing the inflorescence SAM to produce an indeterminate number of flowers on its flanks. The maintenance of stem cells requires the WUSCHEL (WUS) homeodomain transcription factor. Loss-of-function wus mutants have defects in the SAM at all developmental stages (Laux et al., 1996; Mayer et al., 1998). The wus vegetative shoot apex is flat and lacks intensely stained cells typically found in the wild-type SAM (Figure 1B, left). Leaf primordia are formed slowly and fail to develop into mature leaves. At later stages, inflorescence stems emerge from the flank of the defective shoot apex as well as from leaf axils, giving a “wuschel” (tousled) appearance to the mutants. From these observations, it has been proposed that the primary role of WUS is to confer stem cell fate (Laux et al., 1996; Mayer et al., 1998). Expression analyses revealed that WUS mRNA is localized to a few cells underlying the stem cells (Figure 1B, right). Therefore, WUS controls stem cell fate in a non-cell-autonomous manner (Mayer et al., 1998). shoot-meristemless (stm) mutants exhibit defects in SAM formation/maintenance similar to those of wus (Barton and Poethig, 1993; Clark et al., 1996; Endrizzi et al., 1996). Plants with strong stm alleles fail to establish a SAM during embryogenesis (Figure 1C, left). Leaf primordia are formed occasionally between the fused cotyledon petioles, but they do not develop into typical rosette leaves. The STM gene encodes a putative transcription factor with a homeobox DNA binding domain (Long et al., 1996). STM is transcribed throughout the SAM except in the regions corresponding to incipient leaf primordia (Figure 1C, right). All of these features indicate that the primary function of STM is either to inhibit cells from entering differentiation programs or to promote cell proliferation in the center of the SAM (Endrizzi et al., 1996; Long et al., 1996). Predicted Signal Transduction Mechanism by the CLV Signaling Complex. CLV1 and CLV2 form a 185-kD heterodimer via thioester bonds on the plasma membrane. Their N-terminal Leu-rich repeat (LRR) regions are considered to be facing outside the cell to form a receptor domain, whereas the C-terminal kinase domain is located in the cytoplasm. Here, the LRR region of each CLV monomer is assumed to take a cylindrical form, based on three-dimensional modeling of plant-specific LRRs to a known LRR crystal structure (Kajava, 1998). Free CLV3 ligand likely forms a multimer, although its molecular nature is not known (a homodimer-like structure is assumed in this scheme). Upon binding of the CLV3 ligand to the CLV1/CLV2 receptor, the CLV1 kinase domain is phosphorylated (P), probably by other CLV1/CLV2 complexes. The phosphorylated kinase domain then is recognized by several protein molecules, including a Rho GTPase-related protein (Rop) and a kinase associated protein phosphatase (KAPP), forming a 450-kD complex. Rop is presumed to act via a mechanism analogous to the mitogen-activated protein kinase cascade. KAPP is anchored to the inner surface of the plasma membrane through its uncleaved signal peptide. KAPP negatively regulates the Rop-mediated signal transduction pathway, probably through its phosphatase activity. The scheme is drawn based on the publications by Stone et al. (1994)(1998), Williams et al. (1997), and Trotochaud et al. (1999)(2000). In both cauliflower and Arabidopsis, the CLV1 protein is present in two complexes of 185 and 450 kD (Trotochaud et al., 1999) (Figure 2). Convincing but indirect evidence indicates that the 185-kD complex is a disulfide-linked heterodimer of CLV1 and CLV2 (Trotochaud et al., 1999). The larger 450-kD complex includes the 185-kD complex, the CLV3 peptide, and at least two other noncovalently bound subunits: a kinase-associated protein phosphatase (KAPP) and a Rho GTPase–related protein (Rop) (Trotochaud et al., 1999, 2000). KAPP is likely to be a modulator of CLV1 kinase activity, whereas Rho may participate in the downstream signal transduction pathway analogous to the animal mitogen-activated protein kinase cascade (Hirt, 1997; Williams et al., 1997; Stone et al., 1998; Trotochaud et al., 1999). Assembly of the 450-kD complex requires the CLV3 peptide, because CLV1 was found exclusively in the 185-kD complex in strong clv3 mutants (Trotochaud et al., 1999). Furthermore, a CLV1/CLV2 receptor expressed on the yeast cell surface has been shown to bind CLV3 from a cauliflower extract (Trotochaud et al., 2000). All of these observations are consistent with a model in which the CLV3 peptide binds to and activates the 185-kD CLV1/CLV2 heterodimer through autophosphorylation, which then becomes a 450-kD complex that includes KAPP and Rop (Figure 2) (Trotochaud et al., 1999). In cauliflower, 76% of CLV3 was found in the 450-kD complex, whereas the remaining 24% was found in a 25-kD multimer (Trotochaud et al., 2000). It is not known, however, what CLV3 partner is in the 25-kD multimer or whether CLV3 binds to the CLV1/CLV2 receptor as a multimer or as a monomer. CLV1 and CLV3 are expressed in distinct regions of the SAM (Figure 1D, right). Although CLV3 mRNA accumulates specifically in the stem cells in the central zone, CLV1 is expressed in the center of the rib zone. The CLV1 expression domain overlaps that of CLV3 only slightly (Clark et al., 1997; Fletcher et al., 1999). This is consistent with the possible extracellular secretion of the CLV3 peptide (Fletcher et al., 1999): CLV3 may be secreted from the stem cells and perceived by the CLV1-expressing cells just below the stem cells. CLV2 is expressed in most organs and may have additional roles in other signaling pathways (Kayes and Clark, 1998; Jeong et al., 1999). Models of Stem Cell Regulation. (A) Stem cell maintenance in the vegetative and inflorescence meristems (Brand et al., 2000; Schoof et al., 2000). The size of the stem cell population is controlled by a regulatory loop between WUS and CLV. When the number of stem cells is increased, more CLV3 ligand is released from the stem cells, which is perceived by the CLV1/CLV2 receptor kinase in underlying layers. This results in fewer cells expressing WUS, thereby attenuating stem cell–promoting activity (left). In contrast, when the number of stem cells is decreased, less CLV3 ligand is released. Consequently, more cells start to express WUS, thereby promoting stem cell identity (right). (B) Termination of the stem cell population in a FM (Lenhard et al., 2001; Lohmann et al., 2001). In early flower primordia (stage 2, left), the meristem identity gene LFY is expressed throughout the primordia (Weigel et al., whereas WUS is expressed in the center to a stem cell population later developmental stages (Mayer et al., 1998). in LFY and WUS expression in the center of the flower development (stage WUS expression becomes by expression persists in the center of the primordia and floral in and et al., on this a mechanism a loop has been proposed stem cell maintenance (Figure the number of stem cells is in the central zone, more CLV3 peptide is which then the CLV1-expressing cells in the rib to WUS cells the number of stem cells. few stem cells are in the signaling by CLV3 be which to more cells expressing WUS and more stem cells et al., 2000). In WUS expression was from the in a similar to that of loss-of-function mutants, CLV3 WUS and CLV3 can signal cell layers. CLV3 signaling, this probably is by of the CLV3 peptide (Fletcher et al., 1999). The molecular mechanism the non-cell-autonomous of WUS is not it is not clear STM in to the regulatory on its expression pattern in the mature STM to the region of cells which WUS and CLV expression patterns are however, WUS expression can be as early as the when STM expression a SAM structure has been (Mayer et al., 1998). Furthermore, WUS STM expression in the requires the function of the other (Mayer et al., 1998). analyses have that although either the stm or the can be by mutation of the other, the wus mutation the defects caused by stm alleles (Clark et al., 1996; Endrizzi et al., 1996). These observations indicate that the signaling pathway and STM act at but are not in a their may on the developmental The flower stem cells at its it is a floral meristem and gene expression patterns indicate that the signaling pathway to stem cells in the wus flowers form of and but the central two are with a (Laux et al., 1996). This results from the of wus to a of stem cells to form the of and In contrast, the FM of mutants accumulates stem cells and to a flower with of floral (Clark et al., 1993, Kayes and Clark, 1998). WUS is expressed in a few cells in the center of the whereas CLV1 and CLV3 are expressed in the center and apex of the similar to their expression patterns in the SAM (Clark et al., 1997; Mayer et al., 1998; Fletcher et al., 1999). In the wild-type the of the stem cells to a population size in flower because the central of the FM is programmed to a number of The gene has been in this in to defects in floral flowers produce indeterminate of floral organs et al., 1989). This requires WUS, because flowers of wus mutants defects similar to those of wus mutants (Laux et al., 1996). Recent genetic analyses by two have a role in the of WUS (Lenhard et al., 2001; Lohmann et al., 2001). In the wild-type expression of both WUS and CLV3 as flower development and then by the primordia In the FM of mutants, both WUS and CLV expression long after floral development is (Lenhard et al., 2001; Lohmann et al., 2001). The of WUS by to be of CLV signaling, because in the FM of mutants, WUS expression is not only but et al., 2001). the expression of WUS in regions of the FM results in indeterminate formation in the corresponding floral region (Lenhard et al., 2001; Lohmann et al., 2001). All of these observations indicate a in the of WUS and in the of stem cell The of in stem cell the of expression is in the FM at the and is strong evidence that expression on WUS and on the floral meristem identity gene (Lenhard et al., 2001; Lohmann et al., 2001). The expression of WUS results in the transcription of a controlled by the regulatory region. This is consistent with the of WUS to form an stem cell population and an function by as shown by an indeterminate number of and expression and the indeterminate formation are when the are in a The of and was at the molecular in and in studies binding of LFY and WUS to the in the et al., Lohmann et al., 2001). a yeast expression that WUS and LFY act to even binding of the two was not in et al., 2001). on these observations, a signaling pathway stem cell has been proposed (Lenhard et al., 2001; Lohmann et al., (Figure In flower the signaling pathway a stem cell population in the WUS then activates transcription by binding to the The transcription of by WUS is by which is expressed in flower primordia but not in the WUS expression is to a small number of cells in the strong expression only at the center of the flower it and in flower WUS thereby the stem cell WUS and a loop in which WUS activates the transcription of which in WUS This signaling loop is similar to the regulatory loop in the important is that the pathway not however, that the pathway requires an as the Furthermore, in to the pathway, which in the SAM throughout plant the pathway to function only once in each Schemes of Wild-Type and Mutant Arabidopsis (A) et al., Cell are in the at shown in are in all (B) cells function as an center of the by the differentiation of cells et al., 1997; et al., 2000). (C) pattern of three Arabidopsis mutants, and et al., 1993; et al., 1996; et al., 2000; et al., 2000). The highly coordinated division of the cells the of evidence the of signaling in pattern formation et al., When a cell is an cell the and a The daughter cell then as a it a cell The daughter cell then giving to the cells in the and These cells have of as revealed by the of a similar by cells, by a corresponding cell fate was when an cell was et al., of cell fate is not to but to in et al. cell genetic of cells that been by from a of the after revealed that the cells in the both the and inner layers. those a pattern with or in the number of cell layers or in the number of cells in each Although may have the of cell this that the pattern is not even when cells in the meristem region are intercellular signaling is to the of the The cells can be of as to the stem cells of the The cells are a few cells in the center of the that a The to play an important role in activity. When a cell is the cells their to as stem cells start to differentiate or divide in a manner of their daughter cells et al., of cells also was when was on a that lacks cell that this function of the is of cell divisions et al., These observations indicate that the the identity of the cells by their differentiation (Figure similar was drawn from a in which cell differentiation was through of the expression of a kinase et al., 2000). The of the to inhibit cell divisions is similar to the function of the cells in the important however, is that cells to be whereas cells are a long Although a few have been to the formation and activity of the et al., 1998; et al., 2000; et al., putative signaling have been are the WUS and CLV This that cell division and the differentiation of shoot and are controlled by although both intercellular signaling to the stem cell stem cells may be by pathways that to have through genetic The plant cell division and differentiation as well as cell of its highly however, it has been to the role of in each developmental by genetic a the of to of the region et al., 1999). a gene fused to a a of was localized to the cells. This was either or in mutants of known signal and In mutants of the putative cell division and found in the in which the was more in pattern was when wild-type in the of The was to the and with the at the The in was by of cell fates and of cell division the cells in the of and as and lateral The and their daughter cells a of the Cell fate specification was not on the of but was by the of the to the et al., 1999). of cells as lateral cells has been and mutants et al., 2000). Although the gene has to be is likely to be in the cell fate pathway, because was in a and 1996). These observations a role in The pattern of cell layers in the mature is during early Upon the cells in the are and start a cell division that a pattern to that formed in the of experimental have that the pattern formation requires a of from mature cells to the cells. mutants with defective patterns have analogous defects in the and the mature et al., when intercellular communication between a cell and the more mature cells it is by the cell to divide et al., with a regulatory however, indicate that signaling is not of the (A) of mRNA protein and mRNA (right) in the et al., 1996; et al., 2000; et al., 2000; et al., 2001). (B) Mechanism of intercellular signaling in pattern formation of wild-type (left) and (right) et al., 2001). In wild-type the protein is in the and to to a probably through In the activates transcription that is the cell division of the daughter cells, in the of and layers. In the mature in the layer cell protein is to a cell by an as In intercellular signaling is considered to be by the of protein from the This results in the of layers because of divisions and the of cell fate in these layers. the cell are in is transcribed in the as well as in the and its daughter et al., 1996; et al., 2000). In contrast, is transcribed in the including and et al., 2000). This expression pattern indicates non-cell-autonomous of in two controls the differentiation of the is not is the division of the daughter cells, which do not Although the downstream of specification are is strong evidence that through to the division of the daughter cell et al., 2000; et al., 2001). is the mechanism of non-cell-autonomous it is on intercellular protein et al., 2001). Expression of an protein protein in the the control of the results in of the indicating that the protein is In these is localized to the of the the and the and its in to the and of the cells (Figure These cells to the are located in have their The of the protein was by the of to The in the mRNA and protein have to a model in which from the to a layer by its (Figure et al., 2001). to function in the cell in which the gene is transcribed intercellular not to be a of is a putative transcription factor and is localized to the of the it is likely that is in the control of downstream genes. of Wild-Type and (A) to (D) type. to (A) and of roots. indicates of cell indicates the of (B) and of indicates of indicates (C) and of indicate the specifically in the of wild-type whereas they are found in all layers between the and the of (D) and of mature the of In the wild-type is the strong whereas strong is found in the the cell are in protein is not to the but also is to the et al., 2001). with this the have layers of on not only the but also cell fate et al., 2001). is not to the but is found in most plant In Arabidopsis, have a similar pattern to that of the except that have two layers of of the layer found in the root. stems also have a similar except that the are the central pith common are the mechanisms that control and shoot is transcribed in the layers in and et al., 2000). and stems of mutants analogous pattern Furthermore, cell division in the early that the and cell et al., This results in a with two layers of the three et al., et al., 1998). In the of an layer results in an to to because the shoot that according to the thereby as a et al., 1998). similar in and roots. The of the have layers similar to those in roots. In these layers, the and expression also is in the and in a pattern similar to that in the and et al., 2001). All of these observations indicate that a common mechanism in in both and In to the and the formation of the requires more complex including specification of cell with as and (Figure In Arabidopsis, the number of is whereas cells are in from mature indicating that more two divisions take the progeny of each et al., 2000). of the gene results in a reduced number of cells in the of and and all cells differentiate into (Figure right). analyses have that controls cell divisions but not cell When cell divisions in are by the mutation the of the the of cell et al., et al., 2000). encodes a which has been shown to be to the receptor et al., 2001). is expressed in all cells in the as well as in the of the et al., 2000). Therefore, has been to extracellular on the cell surface and to a signal to the and 2001; et al., 2001). The signal is to act ultimately to promote cell allowing the differentiation of cell In the specification of cells that of other cell although differentiation becomes et al., 2000). Therefore, the has been to a to produce a number of cells that can cell other To is known cell fate is in the studies on leaf have that a role in pattern formation review, and 2001). The specification of cells just below the to signaling from the mature In contrast, studies that the pattern is by the review, 2000). These can be by the number of cells the through the or of function. studies based on and genetic analyses have the of in plant The molecular nature of however, has long been elusive. of the signaling are to emerge in developmental and of these are now at the molecular In shoot stem cell a to an important of the intercellular signaling pathway, whereas transcription factor to be The of a number of CLV3 expressed in a of organs that similar operate in other organs outside the SAM and 2001). the other a number of transcription have been to cell layers in the SAM and flower although the developmental of this is et al., et al., 1996; et al., 2000). The of intercellular signaling by protein in plant development be revealed in The characterization of processes has the of plant In Arabidopsis, are to be controlled primarily by which in on the of and 1998). only are expressed in cell but the are to cell et al., 1998; et al., 1998). Therefore, cell fate caused by to of the thereby This may act to a a the pattern to be the Arabidopsis at least et al., it is that in the plant is by a complex expression pattern of in with their intracellular protein genetic each of these to a of the role of in plant developmental It also may to an of the that control protein which be a of primary In the regulatory have been based on These now are a of a signaling pathway is more can be to specifically other in the signaling genetic are by the of the Arabidopsis and recent advances in the of analyses also can be to downstream of signaling In the few we a of the signaling that plant