Shootmeristemless ties the knot

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Shootmeristemless ties the knot If ever there was a holy grail for plant developmental biologists, it would be understanding the meristem, the indeterminate group of cells whose derivatives elaborate the organs of the plant. Due to the action of meristems, plants can be considered as perpetually embryonic, and thus worthy of developmental analysis throughout their ontogeny. Meanwhile, the ~homeobox frenzy', which has permeated publications in the last decade 1-3has caused us to wonder what role these genes play in plant development. The recent publication in Nature by Barton and colleagues 4 on the cloning of the SHOOTMERISTEMLESS (STM) gene elegantly knots these areas of interest together. Meristems come in a number of different forms. The shoot apical meristem, which forms during embryogenesis, is responsible for the majority of the above ground growth, and axillary meristems, located where the leaf attaches to the stem, either reiterate the main shoot or form different types of shoots such as flowers. Within the shoot of certain plants, an internal meristem (cambium) is responsible for growth in width. Underground, the root apical meristem, which also arises during embryogenesis, produces the root, and lateral root meristems, which are born from internal root cells, form root branches. Given their different locations and functions, how do we define a meristem? Although histological and morphological criteria have been established for meristems 5, the geneticist dreams of a gene that by its mutant phenotype and pattern of expression serves as a functional marker for the meristem. One such gene is likely to be STM of Arabidopsis. Ascribing function from mutant phenotypes can be difficult when modifying factors, poor penetrance and pleiotropic effects interfere. Fortunately, plants carrying a null allele of STM display excellent penetrance and expressionS, and so gene function is easier to predict. The stm seedlings have fully functional root meristems and normal cotyledons, but no leaves. A few malformed leaves are produced from the hypocotyl, but the true leaves and flowers that should have arisen from the shoot apical meristem never form. If it is possible simply to define a plant embryo as containing certain pattern elements 7, then this mutation clearly lacks one of these elements. From their analysis of the stm phenotype, Barton and Poethig 6 suggest that STM is required for the shoot apical meristem to form. Function can also be surmised, albeit tentatively, from expression patterns and gain of function phenotypes. The knotted1 (knl) gene and related maize homeobox genes, such as rough sheath1, are expressed in shoot meristems and excluded from expression in leaves or floral parts 8-1°. Gain of function phenotypes occur when knl or © 1996 ElsevierScienceLtd

related genes are ectopically expressed in leaves. Finding that ectopic expression ofknl in tobacco produces meristems on leaves 11 led to the suggestion that knl plays a role in meristem function, either preventing differentiation or maintaining indeterminacy. The clincher needed in the argument was a loss of function phenotype. Fortunately, an Arabidopsis gene that was isolated by Medford using the knl homeobox as a hybridization probe mapped near STM. A collaboration between Barton and Medford proved that STM was in fact a knl-like gene. The expression patterns of STM corresponded precisely to expectations of the gene's function. STM is first detected in just a few cells of the embryo at mid-globular stage. These cells are located in a stripe between what will become the cotyledons. As the cotyledons grow out, the expression remains in that central notch. When leaf primordia form, expression is excluded from the leaves. Similar to results seen with the maize knl geneS,9 and a related Arabidopsis gene, knotted in Arabidopsis thaliana (KNAT1) ~2, STM expression disappears in a domain of the meristem in the position of the incipient leaf primordium, suggesting that its absence defines the position of the next leaf. The STM gene continues to be expressed in all shoot meristems, including the inflorescence and floral meristems. Thus, it is likely that STM has a role in floral development as well, perhaps triggering expression of certain floral homeotic genes or preventing expression of others. Understanding the requirement for STM at later stages of development should come from analysis of weak alleles or conditional gene expression. Is it possible to use STM to help determine when the meristem forms? If STM marks the meristem, then the meristem begins in the early stages of embryogenesis, before the cotyledons form. Yet Barton and Poethig suggest, based on the stm phenotype, that the shoot apical meristem forms after cotyledon formation 6. How do we resolve this dilemma? The expression patterns in maize and Arabidopsis have suggested that in order to make determinate organs, knl-like gene expression needs to be turned off. Since STM is not expressed in the presumptive cotyledons, the elaboration of cotyledons may not depend on STM gene expression. In contrast, the foliage leaves, which do not form in stm mutants, arise from cells that first express STM in the developing meristem. A similar scenario appears to exist in maize embryos, in which the cells that elaborate the cotyledon (scutellum) do not express knl (Ref. 13). Thus, the results suggest that the shoot apical meristem initiates in the globular stage of embryogenesis in Arabidopsis, but that the cells of the cotyledons are not its direct descendants.

Whether there are meristem-specific genes that act earlier than STM to specify- the cotyledons remains to be determined. It can also be asked whether STM distinguishes lateral primordia that are determinate, such as leaves and petals, from indeterminate primordia, such as floral meristems. The in situ hybridizations of Long et al.4 demonstrate that STM expression disappears from the flanks of the inflorescence meristem at an early stage of floral development, and then reappears when the floral meristems are distinct entities. Thus, STM treats the initiation ofprimordia equivalently and makes no distinction between indeterminate and determinate primordia until after their formation. The reappearance of STM in floral meristems may involve interactions with homeotic genes such as LEAFY or APETALA1 that are expressed early in floral meristem development14,1k It is interesting that STM and knl do not make a distinction between vegetative or floral meristems. They do, however, distinguish shoot from root meristems. Perhaps the knl gene family can be used as a tool to investigate evolution of the shoot meristem. For example, in ferns that have a distinctive apical cell in the shoot meristem, is the STM homolog only expressed in that single cell or in a larger domain? Hopefully, this gene family will prove to be the 'Rosetta stone' that the box cluster has turned out to be in animals. Acknowledgements

Thanks to K. Barton for sharing information on stm, and to members of my laboratory :for numerous stimulating conversations and suggestions on the manuscript. Sarah Hake Plant Gene Expression Center, UnitedStates Dept of Agriculture and University of California, 800 Buchanan St., Albany, CA 94710, USA References

1 McGinnis, W. et al. (1984) A conserved DNA sequence in homeotic genes of the Drosophila Antennapedia and bithorax complexes, Nature 308, 428-433 2 Slack, J.M.W., Holland, P.W.H. and G r a h ~ , C.F. (1993) The zootype and the phylotypic stage, Nature 361,490-492 3 Kenyon, C. (1994) If birds can fly, why can't we? Homeotic genes and evolution,/ Cell 78, 175-180 4 Long, J.A. et al. A member of the KNOTTED class of homeodomain proteins encoded by the SHOOTMERISTEMLESS gene of Arabidopsis, Nature (in press) 5 Kaplan, D.R. (1969) Seed development in downingia, Phytomorphology 19, 253-278 6 Barton, M.K. and Poethig, R.S. (1993) Formation of the shoot apical meristem in Arabidopsis thaliana: an analysis of development in the wild type and in the shoot meristemlessmutant, Development119, 823-831 March1996,Vol.1, No. 3

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7 Jfirgens, G. (1995) Axis formation in plant embryogenesis:cues and clues, Cell81, 467-470 8 Smith, L. et al. (1992) A dominant mutation in the maize homeobox gene, Knotted-I, causes its ectopic expression in leaf cells with altered fates, Development 116, 21-30 9 Jackson, D., Veit, B. and Hake, S. (1994) Expression of maize knotted1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot, Development 120, 405-413

10 Schneeburger, R. et al. (1995) Ectopic expression of the knox homeoboxgene rough sheath1 transforms cell fate in maize leaves, Genes Dev. 9, 2292-2304 11 Sinha, N., Williams, R. and Hake, S. (1993) Overexpression of the maize homeoboxgene, KNOTTED1, causes a switch from determinate to indeterminate cell fates, GenesDev. 7, 787-795 12 Lincoln, C. et al. (1994) A knotted-like homeobox gene in Arabidopsis is expressed in the vegetative meristem and dramatically

alters leaf morphologywhen overexpressed in transgenic plants, Plant Cell 6, 1859-1876 13 Smith, L.G., Jackson, D. and Hake, S. (1995) The expression of knotted1 marks shoot meristem formation during maize embryogenesis, Dev. Genet. 16, 344-348 14 Weigel, D. et al. (1992)LEAFY controls floral meristem identity in Arabidopsis, Cell 69, 843-859 15 Mandel, M.A.et al. (1992) Molecular characterization of the Arabidopsis floral homeoticgeneAPETALA1,Nature 360, 273-277

Fast flowering During the past few years, steady progress has been made towards understanding the genetic basis for the 'switch' from vegetative to floral development. However, two recent publications in the journal Nature appear to have set the stage for a long awaited revolution in the investigation of this developmental transition1~2.In these studies, the initiation of flowering has been tremendously accelerated in a variety of plant species by the ectopic expression of a single floral gene. Central to understanding the importance of these new results is an appreciation of the physiological and genetic experiments that have led to a predominately hierarchical view of the transition from vegetative to floral development. The modern era of research on this subject was driven by grafting experiments in which a florally induced plant could act as a catalyst for the induction of flowering in noninduced plants3% During the subsequent decades, research was focused largely on attempts to isolate biochemically a floral inducer molecule and a floral repressor molecule, 'florigen' and 'antiflorigen', respectively. A similar model for the regulation of the transition from vegetative to floral growth was also developed based on the idea that the switch was controlled by the level of nutrients, primarily sugars, available in the shoot apical meristem. The discovery that a wide variety of physiological and environmental treatments could induce flowering, and the failure to isolate florigen or antiflorigen, or to demonstrate a causal effect of increased nutrient levels in the shoot apex led to the proposal of a 'multifactorial' model for floral induction 6. This model proposed that there are multiple, interactive and overlapping developmental or physiological pathways that regulate the switch from vegetative to floral growth. Clearly, unraveling such a complex regulatory process by biochemical experiments alone presented a daunting task. Developmental biology and genetic experiments provided an important complemerit to the physiological approaches being used to understand the regulation of the floral transition. The developmental experiments led to the concept of the 'competence' of the shoot apical meristem to respond to regulatory signals produced in the leaf or root; 76

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flowering could only occur in situations where cells of the shoot apical meristem were able to respond properly to compounds that promote the transition from vegetative to floral development7. Thus, the 'developmental state' of the shoot meristem also became a critical component of any model to explain the regulation of the floral transition. Genetic analyses in several model systerns added another important level ofresolution to our understanding of the floral transition - traditional genetic mutant screens were used to identify genes that control the timing of the transition, or that regulate the establishment of floral meristem identity. In Arabidopsis thaliana, such transition mutations caused either late- or early-flowering phenotypes without changing shoot meristem identity (e.g. lateflowering mutations and early-flowering mutationsS), or changed the developmental fate of shoot meristems from floral identity to partial inflorescence meristem-identity (e.g. leafy, apetala 1, apetala 2 and cauliflower mutationsg-13). Floral meristem identity mutants are characterized by indeterminate growth and/or the production of bracts by lateral meristems that would normally develop as determinate flowers. A third class of Arabidopsis mutants that has shed light on the transition from vegetative to floral growth is represented by the mutations terminal flower I (tfll) 14,15 and terminal flower 2 (tfl2) (A. Sundas and D.R. Meeks-Wagner, unpublished). Mutations at either TFL1 or TFL2 cause both early flowering and the production of a terminal flower, whereas wild4ype plants normally develop an indeterminate inflorescence lacking any terminal differentiation. Genetic and molecular genetic experiments with mutant alleles of TFL1 and the floral meristem-identity genes LEAFY (LFY), APETALA 1 (AP1), APETALA 2 (AP2) and CAULIFLOWER (CAL) revealed a complex set of promotive and antagonistic interactions between the products of these genes, which regulate early events in the establishment of floral meristems in Arabidopsis 1~-18. These studies suggested that floral meristem development was prorooted by LFY, AP1 and CAL, and that mutations in both the LFY and AP1 genes were necessary to prevent the development

of obvious floral structures. Furthermore, the genetic experiments indicated that LFY, which encodes a novel protein that may act as a transcription factor lo, provides a function that is partially redundant with AP1 and CAL, which both encode MADS-box transcription factors12,19. Both AP1 and CAL appear to encode largely overlapping redundant functions. With these analyses, a simple hierarchical model for the floral transition emerged in which the inductive signals, encoded or processed by gene products identified as late- and earlyflowering mutations, would affect only shoot meristems competent to activate the floral meristem-identity genes (e.g. LFY, AP1 and CAL). Once activated, the floral meristem-identity gene products would, in turn, activate the floral target genes that control floral organ development. Recently, Weigel and Nilsson 1 analyzed the effect on plant development of ectopic expression of LFY by placing the LFY coding sequence under the regulatory control of the ubiquitous cauliflower mosaic virus (CaMV) 35S promoter, and introducing this construct into Arabidopsis, tobacco and aspen (a hybrid of Populus tremula and Populus tremuloides). In all of these species the 35S::LFY plants initiated flowering much earlier than the parental wild-type plants: both 35S::LFY Arabidopsis and 35S::LFY aspen plants precociously initiate flowering by the production of flowers from axillary meristems. Remarkably, 35S::LFY aspen plants form axillary flowers in just several months during regeneration in tissue culture, whereas the parental line initiates flowering after 8 to 20 years of growth. Thus, the LFY gene product is sufficient to trigger early flower development in these species. The shoot apical meristem, and all axillary meristems of 35S::LFY Arabidopsis and aspen plants terminate with the production of a flower. This 'phenocopy' of the terminal flower mutant phenotype supports the hypothesis that the TFL gene-products function to control shoot meristem development in part by regulating LFY activityIO, ~6-1s. Given the genetic redundancy that exists between the floral meristem-identity genes LFY, AP1 and CAL, it is interesting that ectopic expression of LFY can drive

© 1996 Elsevier Science Ltd