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Plant development: local control, global patterning
475
Plant development: local control, global patterning Elliot M Meyerowitz Several different lines of inquiry have converged on the conclusion that the number and plane of cell divisions is under tight control in plant development. At the same time, there are new data which show that the pattern of cell division is less important in the formation of organ-scale plant patterns than was expected previously. This apparent paradox can be resolved by recognizing the role of cell-cell communication in the control of plant cell-division patterns.
Address Division of Biology 156-29, California Institute of Technology, 1201 East California Boulevard, Pasadena, California 91125, USA; e-mail: MeyerowitzE@starbasel .caltech.edu
Current Opinion in Genetics & Development 1996, 6:4?5-4?9 © Current Biology Ltd ISSN 0959-437X
Abbreviations CLV KN1 STM SUP
CLAVATA KNOTTED1 SHOOTMERISTEMLESS SUPERMAN
Introduction From the many themes that cot, ld be chosen from the unusually fruitfld past year of plant pattern formation research, the topic of cell division, information is among the most interesting. Cell division patterns have long been thought to be of key importance in the development of plants. As there is essentially no cell migration and only a limited range of cell death in higher plant development, the formation of organs of defined size and shape would appear to require very precise control of cell division. Whereas cell death occurs in a programmed fashion, as in animals, there is no known mechanism for removing individual dead cells: known examples of programmed cell death in higher plants either produce dead structures, such as xylem or autumn leaves, or result in dead regions, as in pathogen response [1"°,2]. Any multicellular organ in an organism can obtain its cells theoretically by a combination of cell division and cell migration into the organ and can trim its cell number by cell migration out of the organ and by cell death with subsequent cell removal. The single such mechanism used in plant organogenesis is cell division, indicating that its control must be critical. Cell division p a t t e r n is tightly c o n t r o l l e d Several papers have been published recently that show a fine degree of control of the pattern of cellular proliferation during flower development. These emphasize that precise control of planes and numbers of cell divisions are typical in floral organogenesis as well as in root development, where the precise patterns of division at the root meristem have been well characterized [3,4]. Three examples will
suffice: two involve analysis of cell lineage patterns in developing flowers. Vincent et al. [5°'] have marked clones of cells at different stages of snapdragon flower development by using a strain with a cold-activated endogenous transposable element; the element is in a gene for plant pigment biosynthesis. By using 1-day cold treatments, they were able to mark chines of cells initiated at different stages of flower development and to assess the developmental fate of the chines bv observations of pigmented regions in mature flowers. 'Fhev found that, in the earliest stages of flower deveh)pment, clones can cross whorl boundaries (whorls are the regions of developing flowers that give rise to organs of different types) and thus mark more than one type of organ. After the stage when expression of the B and C classes of organ-identity genes (which specify petal, stamen and carpel fate in flower development) is initiated--thus dividing the floral primordium into the concentric domains of regulatory gene activity that define the whorls--clones no longer include different organ types. This implies that cells at whorl boundaries, which separate domains of different organ identity, behave differently than other cells, perhaps because cells at whorl boundaries divide infrequently or in specific planes as a result of their proximity to cells expressing different organ-identity genes. A second set of mosaic experiments underlines the fact that floral organ development involves precise patterns of cell division. Bossinger and Smyth [6°°] have used transposition of a maize At-related transposon to activate, at random times in Azzzbidopsis flower development, a reporter [3-glucuronidase gene. Analysis of the resulting sectors demonstrated not only that the floral axes align with the earliest floral clones and thus that the earliest patterns of floral cell division coincide with the orientation of the mature flower but a l s o - - b y noting clone sizes and positions in each organ t y p e - - t h a t floral organs arise from small and well-defined numbers of cells, with typical orientations relative to the organs. These experiments again show that patterns of cells and cell divisions are regular in flower development, implying that control of cell division is an important aspect of floral organogenesis. A different type of experiment also shows that cell division patterns are highly regulated in the domains defined by floral organ identity genes and identifies one gene that may be involved in the establishment of clonal boundaries such as those observed in snapdragons. Sakai eta/. [7°°] have analyzed Arabidopsis flower development with loss of function alleles of the SUPEtL,IJAN (SUP) gene: a zinc-finger gene the mutant phenotype of which is an excess number of stamens (manifested as an enlarged
476 Pattern formation and developmental mechanisms
third whorl) and reduced size and n u m b e r of carpels (and therefore a reduced fourth whorl). T h e y have concluded that SUP may act in wild-type flowers to repress cell division in the third whorl, which normally gives rise to stamens and to activate division in the fourth whorl, where carpels develop. As S U P is expressed in the third whorl onl'> this implies a direct effect in repression of cell division there and a non-autonomous and opposite effect on cell division in the fourth whorl. Furthermore, SUP activation appears to require the activity of the B-function organ-identity genes that are active normally in whorls two and three. T h i s indicates that B-function genes not only define organ identity in the third whorl but also, by activating SUP, can influence the parameters of cellular proliferation in whorls three and four. T h e degree of this influence in wild-type flowers, however, must be questioned. Day et al. [8"] have used a B-flmction promoter attached to a diphtheria toxin gene to ablate the second and third whorls in Arabidopsis and tobacco flowers. T h e y found normal first whorls in both species and normal fourth whorl carpels in Arahidopsis. In tobacco, fourth whorl carpel d e v e l o p m e n t was delayed and abnormal, possibly indicating an effect of the second and third whorl ablation on fourth whorl cellular proliferation. T h e absence of such an effect in Arahidopsis demonstrates that the third whorl is not necessary for normal fourth whorl development. "I\vo possible explanations for this are that the first whorl can substitute for the third in signaling to the fourth whorl or that the fourth whorl in developing Arabidopsis flowers is affected only by the third whorl in the absence of SUP activity. Nevertheless, the mosaic and mutant studies together show that normal flower d e v e l o p m e n t is associated with regular and specifically controlled cell division patterns and the studies of SUP expression indicate that communication b e t w e e n different groups of cells may, at least in some circumstances, play a role in cell division control.
T h e s i g n i f i c a n c e of cell division p a t t e r n Cell division pattern is important A series of papers have, in a different way, underlined the importance of cell division control to organized plant development. T h e s e are all papers describing mutations that alter the n u m b e r of cell divisions in meristems, resuhing in the highly abnormal d e v e l o p m e n t of meristems and, subsequently, organs. Mutations of CLAVATAI (CLV1) and CLAVATA3 (CLI"3) increase cell n u m b e r in shoot apical meristems and in floral meristems [9,10,11"]. Each causes an irregular increase in leaf and floral organ n u m b e r and leads to the formation of abnormal structures. T h e s e two genes appear to define a pathway for cell division control in shoot meristems; double mutants homozygous for c/~,l and cl~,3 mutations appear no different from the single mutants, indicating that loss of function of either gene inactivates the same pathway. Furthermore, clvl/+ clv3/+ double heterozygotes
have a more striking mutant p h c n o t y p e when compared to either hetcrozygote alone, indicating that partial loss of function of each gene makes the plant sensitive to a reduction in the activity of the o t h e r - - a n o t h e r indication that they act in thc same pathway [11"1. Mutations in CI,F1 and CLI[¢ have cffects oppositc to those of mutations in the SHOOTilIERLYTbLIlI,bLYS ($7]1) gene, which codes for a homcobox protein relatcd to the products of the maize KNOTTF, D1 (K,VI) and RO~(;H S H E A T H 1 gencs [12"]. Ectopic activation of KNI or of ROUGH SH£'ATH1 causes abnormal cellular proliferation [13,14 "°] and both KN1 and S'I]l arc cxpressed nnrmalh: in the region of shoot and floral meristems whcre ccll division occurs [12",15]. Whereas a c o m p l e t e loss of S Z U function climinatcs shoot apical meristems, a partial loss of flmction [16"] sh(ms a continued requirement for S E l l product in mcristem cell division, as a partial loss of flmction mutant has flowers that lack central organs and has shoot meristcms that produce fewer leaves or flowers than in wild-type plants. c/z' stm double mutants display partial supprcssinn of the stm p h e n o t y p e and c/:, acts dominantly as a st;;; supprcssor. T h e dot, ble mutant also shows partial suppression of the c/t, p h e n o t y p c and, in this supprcssion, stm acts in a dominant t~shion. Again, the quantitative d c p e n d c n c e of the activity of these genes on each othcr indicates related (though opposing) roles in meristcm maintcnancc and growth. A flmrth gene, It'I.2Y(,WEI. [17"], can hc addcd to this set of interacting genes that regt, late ccll division in meristems. It:I(S'CHEL mutants lack a flmctional primary shoot apical meristcm (as with so;; mutants) but secondary mcristems develop cvcntuall\, fi)rming rosettes of leaves and somc inflorescence stems. T h c vegetative, inflorescence and floral meristcms in thcsc mutants terminate prematurely (causing absence of central organs in flowers), a result apparently of insufficient meristematic cell division; ~'uschd mutants thus resemble weak st;*; mutants. Mutations of IVI(S'CHF.I, are epistatic to clt, l mutations, indicating that HTS(:Hb~I, activity is required for the excess cell division occasioned by loss of (,'I,V activity and allowing, as one possibility, the p l a c e m e n t of IVUS'CHEL as a gene acting downstream of C/,I', with IVC(S'CHbJL required to promote cell division in mcristems and CLI 7 acting normally as a negative regulator of IV/'S. In this respect, IV{.{S'CHEL is not similar to ,YEll. It is possible thus to draw a pathway diagram that includes ()LV1, CLV3, SErf and WUSCHF1. activities, although there is too little information at present to make a unique pathwa> O n e piece of information that may help is the preliminary observation that CLV1 codes for a transmembrane receptor kinase (SE (]lark, RE \\qlliams, EM Meyerowitz, unpublished data), indicating that (7117 may be involved in a cell-cell communication part of this
Plant development: local control, global patterning Meyerowitz 477
pathway. C o m m u n i c a t i o n of cell division information may thus be transmitted by cell-cell contact or by diffusible substances; among the known diffusible substances that regulate plant cell division are lipo-chitooligosaccharides [18,19,20"1. Another possibility for commtinication is suggested by the observation that the maize KN1 protein and RNA, which is homologous with $7]11, can move from cell to celt in plant tissues [21"].
Cell division pattern is unimportant Concurrent with new papers which emphasize the fact that cell division is tightly controlled and that there are sexere d e v e l o p m e n t a l consequences of alterations in cellular proliferation, a series of papers have been published which indicate that major alterations in cell division patterns can easily be c o m p e n s a t e d for during plant organ d e v e l o p m e n t such that major changes in cell dixision patterns have only modest effects on nrgan size, organ shape, and cell fate. One e x a m p l e is the work of H e m e r l y eta/. [22°*], who have made a dominant negative version of the Arabidopsis (]dc2a kinase gene. T h e mutant form was designed to prevent entry into the plant cell c \ c l c and whcn introduced to the Arabidopsis g e n o m e it was apparently lethal: no transgenic plants with an active transgene could be recovered. T h e Arabidopsis gene was transfi)rmed successflilly into transgenic tobacco plants in ~ hich the (heternlogous) g e n t was expressed at a very low level. In these plants, the shoot apical meristem showed reduced cell n u m b e r and vegetative and floral organs also had far fewer cells than normal. T h e plants, however, were otherwise normal with all cell types present in appropriate positions, indicating that plant d e v e l o p m e n t is not very sensitive to a general reduction in cell division. T h i s is not at all similar to what is seen in mutants with partial function of SZ11 or loss of ItT'~S(,'HEL flinction, which also have reduced cell n u m b e r and redticed antounts of cell division in apical and floral meristems [ 1 6 " , 1 7 " ] . Additional examples of plant organs able to accommodate alterations in cell division patterns, in this case changes in the plane of cell division, are the Arabidopsis mutants that lack normal activity of the genes b~4SS 123], TONNEAU2 [24"] or 7'RA~\(FPARENT TESTA (;IABRO([S' [25] and the maize mutant altered in TANGLED [26 °°] activit3,. FAS,'S and TON:\'b;A~) mutants (FASS and T()NNEA~L) may be allelic [24"]) have abnormal and apparently random planes of cell division in the embryo. D e s p i t e a c o m p l c t c absence of the usual stereotvpcd cell division patterns of embryos and roots and of normally directed cell elongation, the m u t a n t plants (though short and misshapen) have normal structure and have normal cell types in appropriate positions in each organ. Plants mutant for T1L~L\(gPARENT TESTA GLABRO[(S' have abnormal size, shape and arrangement of cells in and near the root meristem but, remarkabl-y; have the normal precise arrangement of cells in the root itself, which is a product of meristematic cell divisions. Maize plants m u t a n t for TANGLED activity show abnormal planes of cell division
throughout lcaf d c v e l o p m e n t and lack the normal highl}~ ordered arrays of cells that usually characterize maize leaves; nonetheless, the leaves grow to normal shape and to half of normal size.
The paradox resolved? We thus have an apparently contradictory situation. Normal plant d e v e l o p m e n t is characterized by a tight control of the numbers and planes of cell division and some mutations that affect these parameters lead to monstrous structures. Other mutations and procedures that alter cell division drastically, however, have minimal effects on final structure. O n e recent set of e x p e r i m e n t s points to a possible resolution for this paradnx. Root meristems are normally more s t e r e o t y p e d in their structure than shoot apical meristems, with exact and reproducible numbers and pnsitions of cells in the meristem and in the cells of the root, which descend from the meristematic cells, van den Berg eta/. [27"] have used laser ablation to eliminate specific cells of several types from root meristems and have found that this removal caused new division patterns in adjacent cells, such that the missing cells were replaced. T h e replacements acquired the activities of the original cells and their descendants adopted fates (and patterns of division) appropriate to their new relative position in the meristem. This provides direct evidence that plant cells can sense the presence of their neighbors and can alter their cell division patterns and their fates when required. A way of resolving the apparent contradiction of the importance of cell division in plant d e v e l o p m e n t is thus to recognize that plant cells ' k n o w ' if and how their neighbors are dividing and that plant cells use this information to decidc w h e t h e r to divide and to plan their own plane of cell division. Some g e n e s - - t h o s e in which mutations lead to monstrous o v e r g r o w t h - - a r e parts nf a general plant machinery for cell division regulation in organ d e v e l o p m e n t . T h i s machinery acts locally by assessing cell division rates and planes of cell division in cells, then via a cell-cell communication mechanism directing cell division in adjacent (or nearby) cells. It thus provides a f e e d b a c k loop linking adjacent cells, acting in each cell to assess the division of neighbors then directing an appropriate response. O t h e r genes, such as the floral organ identity genes, interact with this machinery by regulating (e.g. repressing via SUP in one floral region) the rate of cell division in lncal domains. T h i s has a consequence on cell division in adjacent domains because the cell division/communication machinery signals from one cell to those nearby: as in the effect of SUP, activated in the third whorl, on cell division in the fourth whorl of d e v e l o p i n g flowers. T h i s leads to stable patterns of cell division in normal plants and to a remarkable degree of regulation when cell division patterns have been altered by mutations in genes whose products are not direct c o m p o n e n t s of the machinery.
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Pattern formation and developmental mechanisms
Mutations such as talzg/ed, transparent testa glabrous and tonneau2, sporadic failure of cell divisions as in the Cdc2a transgenics, and laser ablations of specific cells invoke the division/communication machinery. This allows for a degree of correction of the local deficit. A missing or misplaced cell is sensed by the machinery as an absence of c o m m u n i c a t e d cell division information and the f e e d b a c k loop alters s u b s e q u e n t divisions of surrounding cells to accommodate. T h i s sort of process must be occurring constantly in the growing parts of normal plants where these local interactions act to maintain the patterns and planes of cell division in meristems and d e v e l o p i n g organs. T h e s e patterns need not result from global controls, as has been proposed in the past [28-31] and may instead result entirely from local interactions.
Conclusions We are now beginning to identify the first c o m p o n e n t s of the communication machinery that allows cells to react to division of their neighbors. An open theoretical issue is to d e t e r m i n e what type of purely local cell division control (perhaps acting only b e t w e e n adjacent cells) can account for the global regulation of organ shape and size in plants. Are there a small n u m b e r of potential division patterns that cells switch b e t w e e n or a more flexible mechanism? An open question that can be addressed experimentally is how division and plane of division in one plant cell are perceived by neighboring cells and how the division and planes of division of the neighbors are then specified so that overall plant and organ shape does not d e p e n d on exact patterns of cell division. Analysis of additional mutations and cloning of the mutant genes are beginning to give us an answer.
Acknowledgements lkl~ laboratory's study of Arabidopsis development is supported b\ the United States Department of Ener~x; the United States National Institutes of Health, and the f 7nited States National Science Feundation.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: •
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Bossinger G, Smyth DR: Initiation patterns of flower and floral organ development in Arabidopsis thaliana. Development 1996, 122:1093-1102. Mosaic analysis reveals a coincidence of clones and both floral orientation and organ portions, revealing that regular patterns of cell division occur in the origin of Arabidopsis flowers and floral organs. Sakai H, Medrano U, Meyerowitz EM: Role of SUPERMAN in maintaining Arabidopsis floral whorl boundaries. Nature 1995, 378:199-203. This Arabidopsis gene, previously thought to be involved in whorl boundary establishment, is shown instead to be necessary for whorl boundary maintenance and to function by control of local cell divisions in floral whorls three and four, or by diffusion of third whorl products into fourth whorl cells. 7. .,,
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Long JA, Moan El, Medford JI, Barton MK: A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature 1996, 379:66-69. Molecular cloning of the Arabidopsis STM gene identifies it as a close relative of the maize KN1 homeobox gene, indicating that the loss-of-function phenotype of KN1 in maize might resemble stm mutant Arabidopsis and that the function of KN1 in vivo may be in meristem establishment and maintenance. 13.
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Clark SE, Jacobsen SE, Levin JZ, Meyerowitz EM: The CLAVATA and SHOOT MERISTEMLESS loci competitively regulate meristem activity in Arabidopsis. Deve/opment 1996, 122:1567-1575. Arabidopsis strains with mutations in clvl or cIv3 and either a complete or partial loss-of-function allele of stm were made. The phenotypes indicate that all three genes are involved together in a collection of processes that
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Plant development: local control, global patterning Meyerowitz
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17. ,e
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Plant development: local control, global patterning Elliot M Meyerowitz Several different lines of inquiry have converged on the conclusion that the number and plane of cell divisions is under tight control in plant development. At the same time, there are new data which show that the pattern of cell division is less important in the formation of organ-scale plant patterns than was expected previously. This apparent paradox can be resolved by recognizing the role of cell-cell communication in the control of plant cell-division patterns.
Address Division of Biology 156-29, California Institute of Technology, 1201 East California Boulevard, Pasadena, California 91125, USA; e-mail: MeyerowitzE@starbasel .caltech.edu
Current Opinion in Genetics & Development 1996, 6:4?5-4?9 © Current Biology Ltd ISSN 0959-437X
Abbreviations CLV KN1 STM SUP
CLAVATA KNOTTED1 SHOOTMERISTEMLESS SUPERMAN
Introduction From the many themes that cot, ld be chosen from the unusually fruitfld past year of plant pattern formation research, the topic of cell division, information is among the most interesting. Cell division patterns have long been thought to be of key importance in the development of plants. As there is essentially no cell migration and only a limited range of cell death in higher plant development, the formation of organs of defined size and shape would appear to require very precise control of cell division. Whereas cell death occurs in a programmed fashion, as in animals, there is no known mechanism for removing individual dead cells: known examples of programmed cell death in higher plants either produce dead structures, such as xylem or autumn leaves, or result in dead regions, as in pathogen response [1"°,2]. Any multicellular organ in an organism can obtain its cells theoretically by a combination of cell division and cell migration into the organ and can trim its cell number by cell migration out of the organ and by cell death with subsequent cell removal. The single such mechanism used in plant organogenesis is cell division, indicating that its control must be critical. Cell division p a t t e r n is tightly c o n t r o l l e d Several papers have been published recently that show a fine degree of control of the pattern of cellular proliferation during flower development. These emphasize that precise control of planes and numbers of cell divisions are typical in floral organogenesis as well as in root development, where the precise patterns of division at the root meristem have been well characterized [3,4]. Three examples will
suffice: two involve analysis of cell lineage patterns in developing flowers. Vincent et al. [5°'] have marked clones of cells at different stages of snapdragon flower development by using a strain with a cold-activated endogenous transposable element; the element is in a gene for plant pigment biosynthesis. By using 1-day cold treatments, they were able to mark chines of cells initiated at different stages of flower development and to assess the developmental fate of the chines bv observations of pigmented regions in mature flowers. 'Fhev found that, in the earliest stages of flower deveh)pment, clones can cross whorl boundaries (whorls are the regions of developing flowers that give rise to organs of different types) and thus mark more than one type of organ. After the stage when expression of the B and C classes of organ-identity genes (which specify petal, stamen and carpel fate in flower development) is initiated--thus dividing the floral primordium into the concentric domains of regulatory gene activity that define the whorls--clones no longer include different organ types. This implies that cells at whorl boundaries, which separate domains of different organ identity, behave differently than other cells, perhaps because cells at whorl boundaries divide infrequently or in specific planes as a result of their proximity to cells expressing different organ-identity genes. A second set of mosaic experiments underlines the fact that floral organ development involves precise patterns of cell division. Bossinger and Smyth [6°°] have used transposition of a maize At-related transposon to activate, at random times in Azzzbidopsis flower development, a reporter [3-glucuronidase gene. Analysis of the resulting sectors demonstrated not only that the floral axes align with the earliest floral clones and thus that the earliest patterns of floral cell division coincide with the orientation of the mature flower but a l s o - - b y noting clone sizes and positions in each organ t y p e - - t h a t floral organs arise from small and well-defined numbers of cells, with typical orientations relative to the organs. These experiments again show that patterns of cells and cell divisions are regular in flower development, implying that control of cell division is an important aspect of floral organogenesis. A different type of experiment also shows that cell division patterns are highly regulated in the domains defined by floral organ identity genes and identifies one gene that may be involved in the establishment of clonal boundaries such as those observed in snapdragons. Sakai eta/. [7°°] have analyzed Arabidopsis flower development with loss of function alleles of the SUPEtL,IJAN (SUP) gene: a zinc-finger gene the mutant phenotype of which is an excess number of stamens (manifested as an enlarged
476 Pattern formation and developmental mechanisms
third whorl) and reduced size and n u m b e r of carpels (and therefore a reduced fourth whorl). T h e y have concluded that SUP may act in wild-type flowers to repress cell division in the third whorl, which normally gives rise to stamens and to activate division in the fourth whorl, where carpels develop. As S U P is expressed in the third whorl onl'> this implies a direct effect in repression of cell division there and a non-autonomous and opposite effect on cell division in the fourth whorl. Furthermore, SUP activation appears to require the activity of the B-function organ-identity genes that are active normally in whorls two and three. T h i s indicates that B-function genes not only define organ identity in the third whorl but also, by activating SUP, can influence the parameters of cellular proliferation in whorls three and four. T h e degree of this influence in wild-type flowers, however, must be questioned. Day et al. [8"] have used a B-flmction promoter attached to a diphtheria toxin gene to ablate the second and third whorls in Arabidopsis and tobacco flowers. T h e y found normal first whorls in both species and normal fourth whorl carpels in Arahidopsis. In tobacco, fourth whorl carpel d e v e l o p m e n t was delayed and abnormal, possibly indicating an effect of the second and third whorl ablation on fourth whorl cellular proliferation. T h e absence of such an effect in Arahidopsis demonstrates that the third whorl is not necessary for normal fourth whorl development. "I\vo possible explanations for this are that the first whorl can substitute for the third in signaling to the fourth whorl or that the fourth whorl in developing Arabidopsis flowers is affected only by the third whorl in the absence of SUP activity. Nevertheless, the mosaic and mutant studies together show that normal flower d e v e l o p m e n t is associated with regular and specifically controlled cell division patterns and the studies of SUP expression indicate that communication b e t w e e n different groups of cells may, at least in some circumstances, play a role in cell division control.
T h e s i g n i f i c a n c e of cell division p a t t e r n Cell division pattern is important A series of papers have, in a different way, underlined the importance of cell division control to organized plant development. T h e s e are all papers describing mutations that alter the n u m b e r of cell divisions in meristems, resuhing in the highly abnormal d e v e l o p m e n t of meristems and, subsequently, organs. Mutations of CLAVATAI (CLV1) and CLAVATA3 (CLI"3) increase cell n u m b e r in shoot apical meristems and in floral meristems [9,10,11"]. Each causes an irregular increase in leaf and floral organ n u m b e r and leads to the formation of abnormal structures. T h e s e two genes appear to define a pathway for cell division control in shoot meristems; double mutants homozygous for c/~,l and cl~,3 mutations appear no different from the single mutants, indicating that loss of function of either gene inactivates the same pathway. Furthermore, clvl/+ clv3/+ double heterozygotes
have a more striking mutant p h c n o t y p e when compared to either hetcrozygote alone, indicating that partial loss of function of each gene makes the plant sensitive to a reduction in the activity of the o t h e r - - a n o t h e r indication that they act in thc same pathway [11"1. Mutations in CI,F1 and CLI[¢ have cffects oppositc to those of mutations in the SHOOTilIERLYTbLIlI,bLYS ($7]1) gene, which codes for a homcobox protein relatcd to the products of the maize KNOTTF, D1 (K,VI) and RO~(;H S H E A T H 1 gencs [12"]. Ectopic activation of KNI or of ROUGH SH£'ATH1 causes abnormal cellular proliferation [13,14 "°] and both KN1 and S'I]l arc cxpressed nnrmalh: in the region of shoot and floral meristems whcre ccll division occurs [12",15]. Whereas a c o m p l e t e loss of S Z U function climinatcs shoot apical meristems, a partial loss of flmction [16"] sh(ms a continued requirement for S E l l product in mcristem cell division, as a partial loss of flmction mutant has flowers that lack central organs and has shoot meristcms that produce fewer leaves or flowers than in wild-type plants. c/z' stm double mutants display partial supprcssinn of the stm p h e n o t y p e and c/:, acts dominantly as a st;;; supprcssor. T h e dot, ble mutant also shows partial suppression of the c/t, p h e n o t y p c and, in this supprcssion, stm acts in a dominant t~shion. Again, the quantitative d c p e n d c n c e of the activity of these genes on each othcr indicates related (though opposing) roles in meristcm maintcnancc and growth. A flmrth gene, It'I.2Y(,WEI. [17"], can hc addcd to this set of interacting genes that regt, late ccll division in meristems. It:I(S'CHEL mutants lack a flmctional primary shoot apical meristcm (as with so;; mutants) but secondary mcristems develop cvcntuall\, fi)rming rosettes of leaves and somc inflorescence stems. T h c vegetative, inflorescence and floral meristcms in thcsc mutants terminate prematurely (causing absence of central organs in flowers), a result apparently of insufficient meristematic cell division; ~'uschd mutants thus resemble weak st;*; mutants. Mutations of IVI(S'CHF.I, are epistatic to clt, l mutations, indicating that HTS(:Hb~I, activity is required for the excess cell division occasioned by loss of (,'I,V activity and allowing, as one possibility, the p l a c e m e n t of IVUS'CHEL as a gene acting downstream of C/,I', with IVC(S'CHbJL required to promote cell division in mcristems and CLI 7 acting normally as a negative regulator of IV/'S. In this respect, IV{.{S'CHEL is not similar to ,YEll. It is possible thus to draw a pathway diagram that includes ()LV1, CLV3, SErf and WUSCHF1. activities, although there is too little information at present to make a unique pathwa> O n e piece of information that may help is the preliminary observation that CLV1 codes for a transmembrane receptor kinase (SE (]lark, RE \\qlliams, EM Meyerowitz, unpublished data), indicating that (7117 may be involved in a cell-cell communication part of this
Plant development: local control, global patterning Meyerowitz 477
pathway. C o m m u n i c a t i o n of cell division information may thus be transmitted by cell-cell contact or by diffusible substances; among the known diffusible substances that regulate plant cell division are lipo-chitooligosaccharides [18,19,20"1. Another possibility for commtinication is suggested by the observation that the maize KN1 protein and RNA, which is homologous with $7]11, can move from cell to celt in plant tissues [21"].
Cell division pattern is unimportant Concurrent with new papers which emphasize the fact that cell division is tightly controlled and that there are sexere d e v e l o p m e n t a l consequences of alterations in cellular proliferation, a series of papers have been published which indicate that major alterations in cell division patterns can easily be c o m p e n s a t e d for during plant organ d e v e l o p m e n t such that major changes in cell dixision patterns have only modest effects on nrgan size, organ shape, and cell fate. One e x a m p l e is the work of H e m e r l y eta/. [22°*], who have made a dominant negative version of the Arabidopsis (]dc2a kinase gene. T h e mutant form was designed to prevent entry into the plant cell c \ c l c and whcn introduced to the Arabidopsis g e n o m e it was apparently lethal: no transgenic plants with an active transgene could be recovered. T h e Arabidopsis gene was transfi)rmed successflilly into transgenic tobacco plants in ~ hich the (heternlogous) g e n t was expressed at a very low level. In these plants, the shoot apical meristem showed reduced cell n u m b e r and vegetative and floral organs also had far fewer cells than normal. T h e plants, however, were otherwise normal with all cell types present in appropriate positions, indicating that plant d e v e l o p m e n t is not very sensitive to a general reduction in cell division. T h i s is not at all similar to what is seen in mutants with partial function of SZ11 or loss of ItT'~S(,'HEL flinction, which also have reduced cell n u m b e r and redticed antounts of cell division in apical and floral meristems [ 1 6 " , 1 7 " ] . Additional examples of plant organs able to accommodate alterations in cell division patterns, in this case changes in the plane of cell division, are the Arabidopsis mutants that lack normal activity of the genes b~4SS 123], TONNEAU2 [24"] or 7'RA~\(FPARENT TESTA (;IABRO([S' [25] and the maize mutant altered in TANGLED [26 °°] activit3,. FAS,'S and TON:\'b;A~) mutants (FASS and T()NNEA~L) may be allelic [24"]) have abnormal and apparently random planes of cell division in the embryo. D e s p i t e a c o m p l c t c absence of the usual stereotvpcd cell division patterns of embryos and roots and of normally directed cell elongation, the m u t a n t plants (though short and misshapen) have normal structure and have normal cell types in appropriate positions in each organ. Plants mutant for T1L~L\(gPARENT TESTA GLABRO[(S' have abnormal size, shape and arrangement of cells in and near the root meristem but, remarkabl-y; have the normal precise arrangement of cells in the root itself, which is a product of meristematic cell divisions. Maize plants m u t a n t for TANGLED activity show abnormal planes of cell division
throughout lcaf d c v e l o p m e n t and lack the normal highl}~ ordered arrays of cells that usually characterize maize leaves; nonetheless, the leaves grow to normal shape and to half of normal size.
The paradox resolved? We thus have an apparently contradictory situation. Normal plant d e v e l o p m e n t is characterized by a tight control of the numbers and planes of cell division and some mutations that affect these parameters lead to monstrous structures. Other mutations and procedures that alter cell division drastically, however, have minimal effects on final structure. O n e recent set of e x p e r i m e n t s points to a possible resolution for this paradnx. Root meristems are normally more s t e r e o t y p e d in their structure than shoot apical meristems, with exact and reproducible numbers and pnsitions of cells in the meristem and in the cells of the root, which descend from the meristematic cells, van den Berg eta/. [27"] have used laser ablation to eliminate specific cells of several types from root meristems and have found that this removal caused new division patterns in adjacent cells, such that the missing cells were replaced. T h e replacements acquired the activities of the original cells and their descendants adopted fates (and patterns of division) appropriate to their new relative position in the meristem. This provides direct evidence that plant cells can sense the presence of their neighbors and can alter their cell division patterns and their fates when required. A way of resolving the apparent contradiction of the importance of cell division in plant d e v e l o p m e n t is thus to recognize that plant cells ' k n o w ' if and how their neighbors are dividing and that plant cells use this information to decidc w h e t h e r to divide and to plan their own plane of cell division. Some g e n e s - - t h o s e in which mutations lead to monstrous o v e r g r o w t h - - a r e parts nf a general plant machinery for cell division regulation in organ d e v e l o p m e n t . T h i s machinery acts locally by assessing cell division rates and planes of cell division in cells, then via a cell-cell communication mechanism directing cell division in adjacent (or nearby) cells. It thus provides a f e e d b a c k loop linking adjacent cells, acting in each cell to assess the division of neighbors then directing an appropriate response. O t h e r genes, such as the floral organ identity genes, interact with this machinery by regulating (e.g. repressing via SUP in one floral region) the rate of cell division in lncal domains. T h i s has a consequence on cell division in adjacent domains because the cell division/communication machinery signals from one cell to those nearby: as in the effect of SUP, activated in the third whorl, on cell division in the fourth whorl of d e v e l o p i n g flowers. T h i s leads to stable patterns of cell division in normal plants and to a remarkable degree of regulation when cell division patterns have been altered by mutations in genes whose products are not direct c o m p o n e n t s of the machinery.
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Pattern formation and developmental mechanisms
Mutations such as talzg/ed, transparent testa glabrous and tonneau2, sporadic failure of cell divisions as in the Cdc2a transgenics, and laser ablations of specific cells invoke the division/communication machinery. This allows for a degree of correction of the local deficit. A missing or misplaced cell is sensed by the machinery as an absence of c o m m u n i c a t e d cell division information and the f e e d b a c k loop alters s u b s e q u e n t divisions of surrounding cells to accommodate. T h i s sort of process must be occurring constantly in the growing parts of normal plants where these local interactions act to maintain the patterns and planes of cell division in meristems and d e v e l o p i n g organs. T h e s e patterns need not result from global controls, as has been proposed in the past [28-31] and may instead result entirely from local interactions.
Conclusions We are now beginning to identify the first c o m p o n e n t s of the communication machinery that allows cells to react to division of their neighbors. An open theoretical issue is to d e t e r m i n e what type of purely local cell division control (perhaps acting only b e t w e e n adjacent cells) can account for the global regulation of organ shape and size in plants. Are there a small n u m b e r of potential division patterns that cells switch b e t w e e n or a more flexible mechanism? An open question that can be addressed experimentally is how division and plane of division in one plant cell are perceived by neighboring cells and how the division and planes of division of the neighbors are then specified so that overall plant and organ shape does not d e p e n d on exact patterns of cell division. Analysis of additional mutations and cloning of the mutant genes are beginning to give us an answer.
Acknowledgements lkl~ laboratory's study of Arabidopsis development is supported b\ the United States Department of Ener~x; the United States National Institutes of Health, and the f 7nited States National Science Feundation.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: •
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5. *.
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17. ,e
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