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Axial patterning in leaves and other lateral organs
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Axial patterning in leaves and other lateral organs John L Bowman The establishment of abaxial–adaxial polarity in lateral organs involves factors intrinsic to the primordia and interactions with the apical meristem from which they are derived. Recent molecular genetic studies have identified some of the genes that promote either adaxial or abaxial cell fates, with many of the genes encoding spatially localized transcription factors.
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Addresses Section of Plant Biology, University of California Davis, Davis, California 95616, USA; e-mail: [email protected] Current Opinion in Genetics & Development 2000, 10:399–404
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0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations CRC CRABS CLAW INO INNER NO OUTER rs2 rough sheath 2
Introduction Most lateral organs of vascular plants are polar in nature, exhibiting asymmetries in both their proximo-distal and adaxial–abaxial (dorsal–ventral) axes. For example, in angiosperm leaves, polarity in the abaxial–adaxial axis is evident in differences in the morphology and distribution of cell types in both the epidermis and mesophyll. Asymmetries in the proximo-distal axis are often manifested as a broad distal blade and a narrower, proximal petiole. The conspicuous polarity of lateral organs contrasts sharply with the radial symmetry of the evolutionarily more ancient stems. It is tempting to speculate that the evolution of polarity in lateral organs has permitted the generation of the diverse laminar structures seen in vascular plants. Although fully differentiated lateral organs have more than just two distinct populations of cell types, the establishment of tissue polarity in its simplest form only requires the generation of two populations of cells with distinct fates. The lateral organs of angiosperms are derived from cells recruited from the peripheral zone of the apical meristem [1,2]. Because lateral organs develop from the flanks of meristems, there exists a fundamental positional relationship between lateral organ primordia and the meristems from which they are derived (Figure 1). The adaxial side of the primordium is directly adjacent to the cells of the meristem, whereas the abaxial region of a primordium is at a distance from the meristem. Initially, lateral organ anlagen are composed of a small number of cells exhibiting uniform histology, and, as far as is presently known, homogeneous gene expression patterns. As lateral organ primordia become morphologically visible, however, polarity is evident both in terms of gene-expression patterns and morphological differentiation, indicating that polarity is established either at or prior to this time.
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Polarity of lateral organs. (a) Because lateral organs (lo) develop from the flanks of apical meristems (am), there exists a fundamental positional relationship between lateral organ primordia and the meristems from which they are derived. The adaxial side of the primordium (black) is directly adjacent to the cells of the meristem, whereas the abaxial region (gray) of a primordium is at a distance from the meristem. (b) Whereas lateral organs are often curved over the apical meristem early in their development, later expansion results in the adaxial surface being the top of the fully differentiated leaf and the abaxial surface the bottom. In many taxa, such as Arabidopsis and Antirrhinum, the midrib vasculature also exhibits a characteristic polarity with the xylem (X) towards the adaxial side and phloem (P) towards the abaxial side. In other taxa, such as Nicotiana, phloem is found on both sides of the xylem. (c) The radialized leaves of phantastica mutants are abaxialized, with abaxial epidermal cell fates around the entire circumference of the leaves [9]. (d) Conversely, the radialized leaves of phabulosa-1d mutants have ubiquitous adaxial epidermal cell fates [11]. In both cases, the vasculature may also reflect these polarity defects [9,11].
Establishment of abaxial–adaxial polarity Experiments in which incipient leaf primordia were separated by incisions from the shoot apical meristem support this timing of polarity establishment and suggest that communication between the apical meristem and leaf primordia is required for establishing polarity in the latter [1,2]. When anlagen were separated prior to primordium formation, the isolated primordia developed into
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radially symmetric, apparently abaxialized, organs — suggesting that the apical meristem could be the source for a signal required for proper abaxial–adaxial development of the leaf [1,2]. One interpretation is that signals emanating from the apical meristem promote adaxial cell fate and, in the absence of such signals, abaxial cell fate is the ‘default’ pattern of differentiation. That the establishment of the abaxial and adaxial domains occurs during the transition from leaf anlagen to leaf primordium is supported by the observation that older primordia can develop autonomously into phenotypically normal leaves [1,2]. Thus, as incipient lateral organ primordia develop from the flanks on the shoot apical meristem, factors both intrinsic and extrinsic to the primordia contribute to the specification of cells as either adaxial or abaxial. Several recent studies have shed light on the molecular genetic mechanisms by which polarity is established in lateral organs and these form the focus of my review. (For other aspects of leaf development, several recent excellent reviews are available [3–8]).
Cell–cell interactions help establish polarity The radially symmetric leaves of phantastica mutants in Antirrhinum have been interpreted as being abaxialized, suggesting that PHANTASTICA normally promotes adaxial cell identity [9]. On the basis of partial loss-of-function alleles in which ectopic abaxial–adaxial boundaries induce outgrowths of tissue, Waites and Hudson [9] proposed that a juxtaposition of abaxial and adaxial cell fates is required for lamina outgrowth, suggesting that signaling between the two distinct cell types induces polarized growth. Thus, in the absence of any adaxial tissue, the abaxialized organs of phantastica mutants develop as filamentous radially symmetric organs (Figure 1c). PHANTASTICA encodes a transcription factor of the MYB family and is expressed at the mRNA level throughout lateral organ anlagen and primordia, indicating post-transcriptional control in its promotion of adaxial cell fates [10]. Remarkably, when temperature-sensitive phantastica mutants are shifted to the non-permissive temperature, the apical meristem arrests, suggesting that proper development of lateral organ primordia is required for the maintenance of the apical meristem [10]. As lateral organs develop from cells recruited from the flanks of the apical meristem, perhaps lateral organ primordia signal the apical meristem to regenerate the corresponding peripheral zone. In the absence of such communication — possibly mediated by the adaxial tissues of the lateral organ primordia (see below) — the apical meristem fails to maintain its integrity. In contrast to the abaxialized lateral organs of phantastica mutants, phabulosa-1d mutants of Arabidopsis display an adaxialization of the lateral organs (Figure 1d) [11]. phabulosa-1d mutations are semi-dominant, with the adaxialization of lateral organs occurring in a dose-dependent manner [11]. Because of the semi-dominant nature of the phabulosa-1d allele, it is not clear whether PHABULOSA normally promotes either abaxial or adaxial cell
fates. Adaxialized lateral organs of phabulosa-1d homozygotes are filamentous and radially symmetric, consistent with the idea that a juxtaposition of adaxial and abaxial domains is required for lamina outgrowth. A striking feature of phabulosa-1d mutants is the development of axillary meristems, normally found only adaxially in the leaf axil, around the entire circumference at the base of the adaxialized leaves, suggesting that their formation is correlated with adaxial cell fate. phabulosa-1d mutants exhibit an enlarged apical meristem which led McConnell and Barton [11] to propose that there is a positive influence of adaxial cell fate on meristem formation. In other words, apical meristems produce lateral organs and the adaxial regions of the lateral organs, in turn, promote meristem formation. Thus, abaxialized lateral organs indirectly result in meristem arrest, whereas adaxialized lateral organs induce ectopic and prolific meristem formation.
YABBY gene family members promote abaxial identity In contrast to PHANTASTICA, and possibly PHABULOSA, whose function appears to be to promote adaxial identity, members of the YABBY gene family act in a redundant manner to specify abaxial cell fate in lateral organs [12••–14••]. The Arabidopsis YABBY gene family is composed of six members which likely encode transcriptional regulators [12••,13••,15••]. Several lines of evidence suggest that YABBY gene family members act to promote abaxial cell fate in lateral organs. First, each of the family members is expressed in a polar manner in one or more above-ground lateral organs. Three members — FILAMENTOUS FLOWER [FIL], YABBY2 and YABBY3 — are expressed in a polar manner in all lateral organs produced by the apical and flower meristems [12••,13••], whereas CRABS CLAW (CRC) [14••–16••] and INNER NO OUTER (INO) [17••] appear to be specialized members of the gene family in that their expression is restricted to the carpels and nectaries or outer integuments, respectively. Transcripts of the YABBY gene family members are detectable only in the abaxial domains of lateral organs when their primordia emerge and begin to differentiate from the apical meristem (Figure 2c). Second, when the fate of either adaxial or abaxial cells are altered by mutation, expression of the YABBY genes correlates with abaxial cell fate. For example, FIL expression is essentially abolished in the adaxialized lateral organs of phabulosa-1d mutants [12••]. Third, ectopic expression of family members in the adaxial regions of developing cotyledons and leaves is sufficient to cause adaxial epidermal tissues to differentiate with an abaxial cell fate (Figure 2) [12••–14••]. Fourth, loss of polar expression of YABBY genes results in loss of polar differentiation of cell types in lateral organs [12••]. Consistent with the hypothesis that FIL acts redundantly with YABBY2 and YABBY3 is the conspicuous lack of vegetative phenotype in fil loss-of-function mutants [18•–20•]. Furthermore, whereas loss of polar YABBY3 expression in the yabby3-1 promoter mutant has no apparent phenotypic consequence in an otherwise wild-type background, in fil yabby3-1 double mutants development of
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Figure 2 YABBY gene family members promote abaxial cell fates. Ectopic expression of members of the YABBY gene family in the adaxial regions of lateral organs is sufficient for adaxial epidermal cells to differentiate with abaxial fates [12··–14··]. The epidermal cells of (a) adaxial and (b) abaxial surfaces of cotyledons display distinct morphologies. The wild-type adaxial epidermis of cotyledons and leaves is characterized by a flat surface composed of uniformly sized cells and a low density of stomata. In contrast, the wild-type abaxial epidermis of cotyledons and leaves is characterized by an undulating surface, a high density of stomata and frequent large cells amongst smaller cells. Additionally, trichomes differentiate from only the adaxial surfaces of the first few leaves, but from both surfaces of subsequently produced leaves. (c) FILAMENTOUS (FIL) is initially expressed the lateral organ anlagen (of cotyledons, leaves, and floral organs) and becomes restricted to abaxial domains of lateral organs as they emerge [12··,13··]. (d) Constitutive expression of FIL, as in 35S::FIL plants, is sufficient to cause the adaxial epidermis of cotyledons to assume an abaxial fate [12··,13··]. ab, abaxial; ad, adaxial; am, apical meristem; an, anlagen.
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the vegetative part of the plant altered and most floral organs are radialized [12••]. Loss of the polar expression of both FIL and YABBY3 thus leads to loss of polar development of lateral organs. The radial nature of these lateral organs is consistent with the hypothesis that juxtaposition of abaxial and adaxial cell fates is required for lamina outgrowth [9]. Taken together, the primary function of YABBY gene family members is to promote abaxial cell fates in lateral organs produced by apical and flower meristems.
KANADI also promotes abaxial cell fates Although crc single mutants are not suggestive of a loss of carpel polarity [16••], double mutant combinations involving crc and either gymnos/pickle or kanadi result in development of adaxial tissues in abaxial positions in the carpel (Figure 3c,d) [14••]. As crc kanadi double mutants exhibit an accurate duplication of adaxial tissues, KANADI is likely to be involved in establishing abaxial cell fate in a pathway parallel to that of CRC [14••]. The loss of abaxial cell fates in crc kanadi carpels is restricted primarily to the medial regions despite CRC expression around their entire circumference. The lack of phenotype in the valves can be explained by redundancy with other members of the YABBY gene family that are also expressed in abaxial regions of the valves (Figure 3a–b) [12••,13••]. Thus it appears that at least two pathways promote abaxial cell fate: one mediated by members of the YABBY gene family and another mediated in part by KANADI [14••]. The relationship between these two pathways is presently not clear as the genes could act in a linear pathway, or alternatively, in parallel pathways with either common or distinct targets.
Current Opinion in Genetics & Development
Other players Several other genes have been implicated in either the establishment or maintenance of polarity in angiosperm lateral organs. For example, argonaute1 mutants of Arabidopsis produce lateral organs that could be interpreted as partially abaxialized [21,22••]. Genetic interactions between ARGONAUTE1 and PINHEAD (a.k.a. ZWILLE [23]) also imply a role for these genes in promoting adaxial cell fates [22••]. As both ARGONAUTE1 and PINHEAD encode similar proteins with overlapping expression patterns, it is proposed that they act redundantly [22••]. Paradoxically, whereas argonaute1 mutants exhibit a severe phenotype including abaxialization of lateral organs, AGRONAUTE1 is expressed throughout the lateral organ primordia and apical meristem [22••]. In contrast, PINHEAD mRNA is localized to the adaxial regions of lateral organ primordia and the apical meristem, but loss-of-function pinhead mutations cause either little or no loss of polarity in lateral organs [22••,23,24]. Although ARGONAUTE1 and PINHEAD display sequence similarity to translation initiation factors, their in vivo biochemical function is presently unclear. Because of the pleiotropic nature of argonaute1 and pinhead mutants, it may be that the corresponding proteins are involved in many developmental processes, of which polarity establishment in lateral organs is just one. Leaves lacking proper lamina expansion are produced in lam1 mutants of Nicotiana sylvestris [25]. These leaves display abaxial–adaxial polarity at their midrib, but blade tissue lacks adaxial cell types [26]. Utilizing periclinal
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Figure 3
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KANADI promotes abaxial cell fates. (a,b) Whereas carpels of crc single mutants do not exhibit obvious polarity defects [16··], carpels of crc kanadi double mutants display a duplication of adaxial (inside) tissues in abaxial (outside) positions [14··]. In crc kanadi carpels, septal tissue (sp) and placental tissue bearing ovules (ov) are found in both adaxial and abaxial positions. The loss of polarity is restricted to the medial domains of the carpels. This may be as a result of redundancy in YABBY gene function. (c,d) CRC is the only family member expressed in the medial regions of the carpel (arrow in [c]) [15··], whereas other family members, such as FILAMENTOUS (FIL), are expressed in the lateral regions (valves) of the carpel [12··,13··].
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chimeras, it was shown that signals produced in the L3 can restore adaxial tissues in the L2 but that these signals can act only over a short range [26]. These data suggest that LAM1 acts downstream of the establishment of polarity in lateral organs and may encode a component in the signaling pathway that mediates communication between the abaxial and adaxial domains. The development of grass leaves differs in several respects as compared to that of eudicot leaves [3,4] and the analysis of mutants altered in lateral organ polarity highlights some of the similarities and possible differences. In Zea mays, leafbladeless1 mutants produce leaves that are abaxialized to some extent and laminar outgrowths at ectopic abaxial–adaxial boundaries [27] suggest that lamina outgrowth in monocots may be similar to that proposed for dicots [9]. In contrast, loss of function alleles of rough sheath2 (rs2) the maize ortholog of PHANTASTICA, appear similar to gain-of-function alleles of the shoot apical meristem specific KNOTTED class I genes rather than to phantastica mutants, leading to the concept that PHANTASTICA and RS2 may have different functions in Antirrhinum and Zea leaves, respectively [28••,29••,30–32]. It was suggested specifically that RS2 could be involved in
establishing the proximal–distal axis rather than the abaxial–adaxial axis in developing leaves but the development of these two axes may be linked and one consequence of developing severely abaxialized lateral organs may be a concomitant loss of proximal–distal development. Analysis of orthologous genes in other species is required to clarify whether these phenotypic differences reflect fundamental variations in leaf development.
Conclusions: the generation of laminar structures The development of lateral appendages (e.g. wings and legs) in Drosophila can be thought of as an analogous situation to that of the specification of cell fate in plant lateral organs [33,34]. In the case of Drosophila appendages, two compartments — dorsal and ventral or anterior and posterior — are established in the imaginal discs by the activities of gene products that specify their fate. At the boundary of these two compartments, interactions between the juxtaposed dorsal and ventral (or anterior and posterior) cells result in the production of secreted factors that are produced at the boundary and act at a distance to specify cell fates in the dorsal and ventral compartments. The boundaries are thus termed ‘organizing centers’ because they are responsible for generating spatial patterns
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of differentiation at a distance [35]. The juxtaposition of dorsal and ventral cell fates is also required for the outgrowth of the laminar structure of the wing. Thus, the establishment of two juxtaposed compartments and interactions between the compartments to promote growth and differentiation are common themes in the development of lateral appendages in metazoans and lateral organs in plants. In the case of lateral organs in Arabidopsis, the YABBY and KANADI genes most likely play a primary role in the specification of the abaxial domain and several genes appear to be involved in promoting the establishment of the adaxial domain. A challenge for the future is to elucidate the mechanisms by which the abaxial and adaxial domains are established and to determine to what extent the boundaries of abaxial and adaxial domains act as organizing centers in the lateral organs of plants.
Acknowledgements Work in my lab is supported by the National Science Foundation, the Department of Energy, and the Beckman Young Investigator Program. I thank members of my laboratory for stimulating discussions and comments on this review.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
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Sussex IM: Morphogenesis in Solanum tuberosum L.: experimental investigation of leaf dorsoventrality and orientation in the juvenile shoot. Phytomorphology 1955, 5:286-300.
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Kaplan D: The monocotyledons: their evolution and comparative biology. VII. The problem of leaf morphology and evolution in the monocotyledons. Quart Rev Biol 1973, 48:437-457.
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Sylvester AW, Smith L, Freeling M: Acquisition of identity in the developing leaf. Annu Rev Cell Dev Biol 1996, 12:257-304.
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Kerstetter RA, Poethig RS: The specification of leaf identity during shoot development. Annu Rev Cell Dev Biol 1998, 14:373-398.
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Brutnell TP, Langdale JA: Signals in leaf development. Adv Bot Res 1998, 28:161-195.
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Pyke K, López-Juez E: Cellular differentiation and leaf morphogenesis in Arabidopsis. Crit Rev Plant Sci 1999, 18:527-546.
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Sinha N: Leaf development in angiosperms. Ann Rev Plant Physiol Plant Mol Biol 1999, 50:419-446.
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Waites R, Hudson A: phantasticatastica: a gene required for dorsiventrality of leaves in Antirrhinum majus. Development 1995, 121:2143-2154.
10. Waites R, Selvadurai HRN, Oliver IR, Hudson A: The PHANTASTICATASTICA gene encodes a MYB transcription factor involved in growth and dorsiventrality of lateral organs in Antirrhinum. Cell 1998, 93:779-789. 11. McConnell JR, Barton MK: Leaf polarity and meristem formation in Arabidopsis. Development 1998, 125:2935-2942. 12. Siegfried KR, Eshed Y, Baum SF, Otsuga D, Drews GN, Bowman JL: ·· Members of the YABBY gene family specify abaxial cell fate in Arabidopsis. Development 1999, 126:4117-4128. The cloning and expression patterns of FIL, YAB2, and YAB3 are presented along with gain-of-function phenotypes of FIL and YAB3. Each of the three genes is expressed abaxially in lateral organs and FIL and YAB3 can promote abaxial cell fates when ectopically expressed in adaxial positions. FIL is not expressed in adaxialized phabulosa-1d homozygotes. The authors propose members of the YABBY gene family specify abaxial cell fate in lateral organs.
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13. Sawa S, Watanabe K, Goto K, Kanaya E, Morita EH, Okada K: ·· FILAMENTOUS FLOWER, a meristem and organ identity gene of Arabidopsis, encodes a protein with a zinc finger and HMGrelated domains. Genes Dev 1999, 13:1079-1088. The cloning, expression patterns and gain-of-function phenotype of FIL are presented. FIL is expressed abaxially in all lateral organs and can promote abaxial cell fates when ectopically expressed in adaxial positions. The authors propose that FIL promotes abaxial cell fate in lateral organs. 14. Eshed Y, Baum SF, Bowman JL: Abaxial cell fate in the carpels is ·· established by two distinct mechanisms. Cell 1999, 99:199-209. Genetic interactions between crc, gymnos and kanadi are described as well as the cloning and expression pattern of GYMNOS. It is proposed that both CRC and KANADI promote abaxial cell-fate specification and that GYMNOS negatively regulates genes, promoting meristematic potential at the level of chromatin. 15. Bowman JL, Smyth DR: CRABS CLAW, a gene that regulates carpel ·· and nectary development in Arabidopsis, encodes a novel protein with zinc finger and helix-loop-helix domains. Development 1999, 126:2387-2396. The authors describe the cloning of CRC, the founding member of the YABBY gene family. The expression pattern of CRC in wild-type and floral homeotic mutant flowers is presented. The YABBY gene family is outlined with sequence comparisons. 16. Alvarez J, Smyth DR: CRABS CLAW and SPATULA, two Arabidopsis ·· genes that control carpel development in parallel with AGAMOUS. Development 1999, 126:2377-2386. The genetic interactions between crc, spatula and the ABC floral homeotic genes are described. This study demonstrates conclusively that genes promoting aspects of carpel development (e.g. CRC and SPATULA) are active independently of the ABC floral homeotic genes, and that most carpel tissues do not absolutely require AGAMOUS (C class) activity. This has implications for the evolution of flowers and their constituent organs. 17. ··
Villanueva JM, Broadhvest J, Hauser BA, Meister RJ, Schneitz K, Gasser CS: INNER NO OUTER regulates abaxial–adaxial patterning in Arabidopsis ovules. Genes Devel 1999 13:3160-3169. The cloning and expression pattern of INO is described. INO mRNA is detected only in the abaxial domain of the outer integument and loss-of-function ino alleles specifically affect the development of the abaxial regions of the outer integument. 18. Sawa S, Ito T, Shimura Y, Okada K: FILAMENTOUS FLOWER · controls the formation and development of Arabidopsis inflorescences and floral meristems. Plant Cell 1999, 11:69-86. See annotation [19·]. 19. Chen Q, Atkinson A, Otsuga D, Christensen T, Reynolds L, · Drews GN: The Arabidopsis FILAMENTOUS FLOWER gene is required for flower formation. Development 1999, 126:2715-2726. Phenotypic descriptions of fil single mutants are presented along with genetic interactions of fil and other mutations affecting establishment of floral meristems (see also [18·]). 20. Kumaran MK, Ye D, Yang W-C, Griffith M, Chaudhury AM, · Sundaresan V: Molecular cloning of ABNORMAL FLORAL ORGANS: a gene required for flower development in Arabidopsis. Sex Plant Reprod 1999, 12:118-122. The cloning of AFO, a.k.a. FIL, is presented along with a brief description of fil single mutants. 21. Bohmert K, Camus I, Bellini C, Bouchez D, Caboche M, Benning C: AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J 1998, 17:170-180. 22. Lynn K, Fernandez A, Aida M, Sedbrook J, Tasaka M, Masson P, ·· Barton MK: The PINHEAD/ZWILLE gene acts pleiotropically in Arabidopsis development and has overlapping functions with the ARGONAUTE1 gene. Development 1999, 126:1-13. Genetic interactions between PINHEAD and ARGONAUTE, and the cloning of PINHEAD are described. As both ARGONAUTE1 and PINHEAD encode similar proteins with overlapping expression patterns, it is proposed that they act redundantly. On the basis of phenotypes of lateral organs and the expression of PINHEAD in the adaxial regions of lateral organs (as well as meristems), the authors suggest that these genes promote adaxial cell fate (see also [21]). 23. Moussian B, Schoof H, Haecker A, Jürgens G, Laux T: Role of the ZWILLE gene in the regulation of central shoot meristem cell fate during Arabidopsis embryogenesis. EMBO J 1998, 17:1799-1809. 24. McConnell JR, Barton MK: Effect of mutations in the PINHEAD gene of Arabidopsis on the formation of shoot apical meristems. Dev Genet 1995, 16:358-366. 25. McHale NA: A nuclear mutation blocking initiation of the lamina in leaves of Nicotiana sylvestris. Planta 1992, 186:355-360.
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26. McHale NA, Marcotrigiano M: LAM1 is required for dorsoventrality and lateral growth of the leaf blade in Nicotiana. Development 1998, 125:4235-4243.
I homeobox genes in their leaves. It is proposed that the primary result of loss of function of RS2/PHANTASTICA is alterations in the proximo-distal axis. (See also [28••]).
27.
30. Schneeberger R, Tsiantis M, Freeling M, Langdale JA: The rough sheath2 gene negatively regulates homeobox gene expression during maize leaf development. Development 1998, 125:2857-2865.
Timmermans MCP, Schultes NP, Jankovsky JP, Nelson T: Leafbladeless1 is required for dorsoventrality of lateral organs in maize. Development 1998, 125:2813-2823.
28. Timmermans MCP, Hudson A, Becraft PW, Nelson T: ROUGH ·· SHEATH2: a myb protein that represses knox homeobox genes in maize lateral organ primordia. Science 1999, 284:151-153. The cloning of RS2, the Zea mays ortholog of PHANTASTICA, is described and it is shown that rs2 mutants display ectopic expression of Knotted class I homeobox genes in their leaves. It is proposed that the differences between rs2 and phantastica mutants might be caused by different effects of Knotted class I homeobox gene expression on developing leaves in the two species or that genetic redundancy may be masking aspects of gene function in Zea mays. (See also [29••]). 29. Tsiantis M, Schneeberger R, Golz JF, Freeling M, Langdale, JA: The ·· maize rough sheath2 gene and leaf development programs in monocot and dicot plants. Science 1999, 284:154-156. The cloning of RS2, the Zea mays ortholog of PHANTASTICA, is described and it is shown that rs2 mutants display ectopic expression of Knotted class
31. Freeling M, Hake S: Developmental genetics of mutants that specify knotted leaves in maize. Genetics 1985, 111:617-634. 32. Vollbrecht E, Veit B, Sinha N, Hake S: The developmental gene Knotted-1 is a member of a maize homeobox gene family. Nature 1991, 350:241-243. 33. Brook WJ, Diaz-Benjumea FJ, Cohen SM: Organizing spatial pattern in limb development. Annu Rev Cell Dev Biol 1996, 12:161-180. 34. Lawrence PA, Struhl G: Morphogens, compartments and pattern: lessons from Drosophila. Cell 1996, 85:951-961. 35. Meinhardt H: Cell determination boundaries as organizing regions for secondary embryonic fields. Dev Biol 1983, 96:375-385.
Axial patterning in leaves and other lateral organs John L Bowman The establishment of abaxial–adaxial polarity in lateral organs involves factors intrinsic to the primordia and interactions with the apical meristem from which they are derived. Recent molecular genetic studies have identified some of the genes that promote either adaxial or abaxial cell fates, with many of the genes encoding spatially localized transcription factors.
Figure 1
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Addresses Section of Plant Biology, University of California Davis, Davis, California 95616, USA; e-mail: [email protected] Current Opinion in Genetics & Development 2000, 10:399–404
am
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0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations CRC CRABS CLAW INO INNER NO OUTER rs2 rough sheath 2
Introduction Most lateral organs of vascular plants are polar in nature, exhibiting asymmetries in both their proximo-distal and adaxial–abaxial (dorsal–ventral) axes. For example, in angiosperm leaves, polarity in the abaxial–adaxial axis is evident in differences in the morphology and distribution of cell types in both the epidermis and mesophyll. Asymmetries in the proximo-distal axis are often manifested as a broad distal blade and a narrower, proximal petiole. The conspicuous polarity of lateral organs contrasts sharply with the radial symmetry of the evolutionarily more ancient stems. It is tempting to speculate that the evolution of polarity in lateral organs has permitted the generation of the diverse laminar structures seen in vascular plants. Although fully differentiated lateral organs have more than just two distinct populations of cell types, the establishment of tissue polarity in its simplest form only requires the generation of two populations of cells with distinct fates. The lateral organs of angiosperms are derived from cells recruited from the peripheral zone of the apical meristem [1,2]. Because lateral organs develop from the flanks of meristems, there exists a fundamental positional relationship between lateral organ primordia and the meristems from which they are derived (Figure 1). The adaxial side of the primordium is directly adjacent to the cells of the meristem, whereas the abaxial region of a primordium is at a distance from the meristem. Initially, lateral organ anlagen are composed of a small number of cells exhibiting uniform histology, and, as far as is presently known, homogeneous gene expression patterns. As lateral organ primordia become morphologically visible, however, polarity is evident both in terms of gene-expression patterns and morphological differentiation, indicating that polarity is established either at or prior to this time.
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Polarity of lateral organs. (a) Because lateral organs (lo) develop from the flanks of apical meristems (am), there exists a fundamental positional relationship between lateral organ primordia and the meristems from which they are derived. The adaxial side of the primordium (black) is directly adjacent to the cells of the meristem, whereas the abaxial region (gray) of a primordium is at a distance from the meristem. (b) Whereas lateral organs are often curved over the apical meristem early in their development, later expansion results in the adaxial surface being the top of the fully differentiated leaf and the abaxial surface the bottom. In many taxa, such as Arabidopsis and Antirrhinum, the midrib vasculature also exhibits a characteristic polarity with the xylem (X) towards the adaxial side and phloem (P) towards the abaxial side. In other taxa, such as Nicotiana, phloem is found on both sides of the xylem. (c) The radialized leaves of phantastica mutants are abaxialized, with abaxial epidermal cell fates around the entire circumference of the leaves [9]. (d) Conversely, the radialized leaves of phabulosa-1d mutants have ubiquitous adaxial epidermal cell fates [11]. In both cases, the vasculature may also reflect these polarity defects [9,11].
Establishment of abaxial–adaxial polarity Experiments in which incipient leaf primordia were separated by incisions from the shoot apical meristem support this timing of polarity establishment and suggest that communication between the apical meristem and leaf primordia is required for establishing polarity in the latter [1,2]. When anlagen were separated prior to primordium formation, the isolated primordia developed into
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radially symmetric, apparently abaxialized, organs — suggesting that the apical meristem could be the source for a signal required for proper abaxial–adaxial development of the leaf [1,2]. One interpretation is that signals emanating from the apical meristem promote adaxial cell fate and, in the absence of such signals, abaxial cell fate is the ‘default’ pattern of differentiation. That the establishment of the abaxial and adaxial domains occurs during the transition from leaf anlagen to leaf primordium is supported by the observation that older primordia can develop autonomously into phenotypically normal leaves [1,2]. Thus, as incipient lateral organ primordia develop from the flanks on the shoot apical meristem, factors both intrinsic and extrinsic to the primordia contribute to the specification of cells as either adaxial or abaxial. Several recent studies have shed light on the molecular genetic mechanisms by which polarity is established in lateral organs and these form the focus of my review. (For other aspects of leaf development, several recent excellent reviews are available [3–8]).
Cell–cell interactions help establish polarity The radially symmetric leaves of phantastica mutants in Antirrhinum have been interpreted as being abaxialized, suggesting that PHANTASTICA normally promotes adaxial cell identity [9]. On the basis of partial loss-of-function alleles in which ectopic abaxial–adaxial boundaries induce outgrowths of tissue, Waites and Hudson [9] proposed that a juxtaposition of abaxial and adaxial cell fates is required for lamina outgrowth, suggesting that signaling between the two distinct cell types induces polarized growth. Thus, in the absence of any adaxial tissue, the abaxialized organs of phantastica mutants develop as filamentous radially symmetric organs (Figure 1c). PHANTASTICA encodes a transcription factor of the MYB family and is expressed at the mRNA level throughout lateral organ anlagen and primordia, indicating post-transcriptional control in its promotion of adaxial cell fates [10]. Remarkably, when temperature-sensitive phantastica mutants are shifted to the non-permissive temperature, the apical meristem arrests, suggesting that proper development of lateral organ primordia is required for the maintenance of the apical meristem [10]. As lateral organs develop from cells recruited from the flanks of the apical meristem, perhaps lateral organ primordia signal the apical meristem to regenerate the corresponding peripheral zone. In the absence of such communication — possibly mediated by the adaxial tissues of the lateral organ primordia (see below) — the apical meristem fails to maintain its integrity. In contrast to the abaxialized lateral organs of phantastica mutants, phabulosa-1d mutants of Arabidopsis display an adaxialization of the lateral organs (Figure 1d) [11]. phabulosa-1d mutations are semi-dominant, with the adaxialization of lateral organs occurring in a dose-dependent manner [11]. Because of the semi-dominant nature of the phabulosa-1d allele, it is not clear whether PHABULOSA normally promotes either abaxial or adaxial cell
fates. Adaxialized lateral organs of phabulosa-1d homozygotes are filamentous and radially symmetric, consistent with the idea that a juxtaposition of adaxial and abaxial domains is required for lamina outgrowth. A striking feature of phabulosa-1d mutants is the development of axillary meristems, normally found only adaxially in the leaf axil, around the entire circumference at the base of the adaxialized leaves, suggesting that their formation is correlated with adaxial cell fate. phabulosa-1d mutants exhibit an enlarged apical meristem which led McConnell and Barton [11] to propose that there is a positive influence of adaxial cell fate on meristem formation. In other words, apical meristems produce lateral organs and the adaxial regions of the lateral organs, in turn, promote meristem formation. Thus, abaxialized lateral organs indirectly result in meristem arrest, whereas adaxialized lateral organs induce ectopic and prolific meristem formation.
YABBY gene family members promote abaxial identity In contrast to PHANTASTICA, and possibly PHABULOSA, whose function appears to be to promote adaxial identity, members of the YABBY gene family act in a redundant manner to specify abaxial cell fate in lateral organs [12••–14••]. The Arabidopsis YABBY gene family is composed of six members which likely encode transcriptional regulators [12••,13••,15••]. Several lines of evidence suggest that YABBY gene family members act to promote abaxial cell fate in lateral organs. First, each of the family members is expressed in a polar manner in one or more above-ground lateral organs. Three members — FILAMENTOUS FLOWER [FIL], YABBY2 and YABBY3 — are expressed in a polar manner in all lateral organs produced by the apical and flower meristems [12••,13••], whereas CRABS CLAW (CRC) [14••–16••] and INNER NO OUTER (INO) [17••] appear to be specialized members of the gene family in that their expression is restricted to the carpels and nectaries or outer integuments, respectively. Transcripts of the YABBY gene family members are detectable only in the abaxial domains of lateral organs when their primordia emerge and begin to differentiate from the apical meristem (Figure 2c). Second, when the fate of either adaxial or abaxial cells are altered by mutation, expression of the YABBY genes correlates with abaxial cell fate. For example, FIL expression is essentially abolished in the adaxialized lateral organs of phabulosa-1d mutants [12••]. Third, ectopic expression of family members in the adaxial regions of developing cotyledons and leaves is sufficient to cause adaxial epidermal tissues to differentiate with an abaxial cell fate (Figure 2) [12••–14••]. Fourth, loss of polar expression of YABBY genes results in loss of polar differentiation of cell types in lateral organs [12••]. Consistent with the hypothesis that FIL acts redundantly with YABBY2 and YABBY3 is the conspicuous lack of vegetative phenotype in fil loss-of-function mutants [18•–20•]. Furthermore, whereas loss of polar YABBY3 expression in the yabby3-1 promoter mutant has no apparent phenotypic consequence in an otherwise wild-type background, in fil yabby3-1 double mutants development of
Axial patterning in leaves Bowman
401
Figure 2 YABBY gene family members promote abaxial cell fates. Ectopic expression of members of the YABBY gene family in the adaxial regions of lateral organs is sufficient for adaxial epidermal cells to differentiate with abaxial fates [12··–14··]. The epidermal cells of (a) adaxial and (b) abaxial surfaces of cotyledons display distinct morphologies. The wild-type adaxial epidermis of cotyledons and leaves is characterized by a flat surface composed of uniformly sized cells and a low density of stomata. In contrast, the wild-type abaxial epidermis of cotyledons and leaves is characterized by an undulating surface, a high density of stomata and frequent large cells amongst smaller cells. Additionally, trichomes differentiate from only the adaxial surfaces of the first few leaves, but from both surfaces of subsequently produced leaves. (c) FILAMENTOUS (FIL) is initially expressed the lateral organ anlagen (of cotyledons, leaves, and floral organs) and becomes restricted to abaxial domains of lateral organs as they emerge [12··,13··]. (d) Constitutive expression of FIL, as in 35S::FIL plants, is sufficient to cause the adaxial epidermis of cotyledons to assume an abaxial fate [12··,13··]. ab, abaxial; ad, adaxial; am, apical meristem; an, anlagen.
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the vegetative part of the plant altered and most floral organs are radialized [12••]. Loss of the polar expression of both FIL and YABBY3 thus leads to loss of polar development of lateral organs. The radial nature of these lateral organs is consistent with the hypothesis that juxtaposition of abaxial and adaxial cell fates is required for lamina outgrowth [9]. Taken together, the primary function of YABBY gene family members is to promote abaxial cell fates in lateral organs produced by apical and flower meristems.
KANADI also promotes abaxial cell fates Although crc single mutants are not suggestive of a loss of carpel polarity [16••], double mutant combinations involving crc and either gymnos/pickle or kanadi result in development of adaxial tissues in abaxial positions in the carpel (Figure 3c,d) [14••]. As crc kanadi double mutants exhibit an accurate duplication of adaxial tissues, KANADI is likely to be involved in establishing abaxial cell fate in a pathway parallel to that of CRC [14••]. The loss of abaxial cell fates in crc kanadi carpels is restricted primarily to the medial regions despite CRC expression around their entire circumference. The lack of phenotype in the valves can be explained by redundancy with other members of the YABBY gene family that are also expressed in abaxial regions of the valves (Figure 3a–b) [12••,13••]. Thus it appears that at least two pathways promote abaxial cell fate: one mediated by members of the YABBY gene family and another mediated in part by KANADI [14••]. The relationship between these two pathways is presently not clear as the genes could act in a linear pathway, or alternatively, in parallel pathways with either common or distinct targets.
Current Opinion in Genetics & Development
Other players Several other genes have been implicated in either the establishment or maintenance of polarity in angiosperm lateral organs. For example, argonaute1 mutants of Arabidopsis produce lateral organs that could be interpreted as partially abaxialized [21,22••]. Genetic interactions between ARGONAUTE1 and PINHEAD (a.k.a. ZWILLE [23]) also imply a role for these genes in promoting adaxial cell fates [22••]. As both ARGONAUTE1 and PINHEAD encode similar proteins with overlapping expression patterns, it is proposed that they act redundantly [22••]. Paradoxically, whereas argonaute1 mutants exhibit a severe phenotype including abaxialization of lateral organs, AGRONAUTE1 is expressed throughout the lateral organ primordia and apical meristem [22••]. In contrast, PINHEAD mRNA is localized to the adaxial regions of lateral organ primordia and the apical meristem, but loss-of-function pinhead mutations cause either little or no loss of polarity in lateral organs [22••,23,24]. Although ARGONAUTE1 and PINHEAD display sequence similarity to translation initiation factors, their in vivo biochemical function is presently unclear. Because of the pleiotropic nature of argonaute1 and pinhead mutants, it may be that the corresponding proteins are involved in many developmental processes, of which polarity establishment in lateral organs is just one. Leaves lacking proper lamina expansion are produced in lam1 mutants of Nicotiana sylvestris [25]. These leaves display abaxial–adaxial polarity at their midrib, but blade tissue lacks adaxial cell types [26]. Utilizing periclinal
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Figure 3
(a)
KANADI promotes abaxial cell fates. (a,b) Whereas carpels of crc single mutants do not exhibit obvious polarity defects [16··], carpels of crc kanadi double mutants display a duplication of adaxial (inside) tissues in abaxial (outside) positions [14··]. In crc kanadi carpels, septal tissue (sp) and placental tissue bearing ovules (ov) are found in both adaxial and abaxial positions. The loss of polarity is restricted to the medial domains of the carpels. This may be as a result of redundancy in YABBY gene function. (c,d) CRC is the only family member expressed in the medial regions of the carpel (arrow in [c]) [15··], whereas other family members, such as FILAMENTOUS (FIL), are expressed in the lateral regions (valves) of the carpel [12··,13··].
(b) OV sp
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chimeras, it was shown that signals produced in the L3 can restore adaxial tissues in the L2 but that these signals can act only over a short range [26]. These data suggest that LAM1 acts downstream of the establishment of polarity in lateral organs and may encode a component in the signaling pathway that mediates communication between the abaxial and adaxial domains. The development of grass leaves differs in several respects as compared to that of eudicot leaves [3,4] and the analysis of mutants altered in lateral organ polarity highlights some of the similarities and possible differences. In Zea mays, leafbladeless1 mutants produce leaves that are abaxialized to some extent and laminar outgrowths at ectopic abaxial–adaxial boundaries [27] suggest that lamina outgrowth in monocots may be similar to that proposed for dicots [9]. In contrast, loss of function alleles of rough sheath2 (rs2) the maize ortholog of PHANTASTICA, appear similar to gain-of-function alleles of the shoot apical meristem specific KNOTTED class I genes rather than to phantastica mutants, leading to the concept that PHANTASTICA and RS2 may have different functions in Antirrhinum and Zea leaves, respectively [28••,29••,30–32]. It was suggested specifically that RS2 could be involved in
establishing the proximal–distal axis rather than the abaxial–adaxial axis in developing leaves but the development of these two axes may be linked and one consequence of developing severely abaxialized lateral organs may be a concomitant loss of proximal–distal development. Analysis of orthologous genes in other species is required to clarify whether these phenotypic differences reflect fundamental variations in leaf development.
Conclusions: the generation of laminar structures The development of lateral appendages (e.g. wings and legs) in Drosophila can be thought of as an analogous situation to that of the specification of cell fate in plant lateral organs [33,34]. In the case of Drosophila appendages, two compartments — dorsal and ventral or anterior and posterior — are established in the imaginal discs by the activities of gene products that specify their fate. At the boundary of these two compartments, interactions between the juxtaposed dorsal and ventral (or anterior and posterior) cells result in the production of secreted factors that are produced at the boundary and act at a distance to specify cell fates in the dorsal and ventral compartments. The boundaries are thus termed ‘organizing centers’ because they are responsible for generating spatial patterns
Axial patterning in leaves Bowman
of differentiation at a distance [35]. The juxtaposition of dorsal and ventral cell fates is also required for the outgrowth of the laminar structure of the wing. Thus, the establishment of two juxtaposed compartments and interactions between the compartments to promote growth and differentiation are common themes in the development of lateral appendages in metazoans and lateral organs in plants. In the case of lateral organs in Arabidopsis, the YABBY and KANADI genes most likely play a primary role in the specification of the abaxial domain and several genes appear to be involved in promoting the establishment of the adaxial domain. A challenge for the future is to elucidate the mechanisms by which the abaxial and adaxial domains are established and to determine to what extent the boundaries of abaxial and adaxial domains act as organizing centers in the lateral organs of plants.
Acknowledgements Work in my lab is supported by the National Science Foundation, the Department of Energy, and the Beckman Young Investigator Program. I thank members of my laboratory for stimulating discussions and comments on this review.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
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13. Sawa S, Watanabe K, Goto K, Kanaya E, Morita EH, Okada K: ·· FILAMENTOUS FLOWER, a meristem and organ identity gene of Arabidopsis, encodes a protein with a zinc finger and HMGrelated domains. Genes Dev 1999, 13:1079-1088. The cloning, expression patterns and gain-of-function phenotype of FIL are presented. FIL is expressed abaxially in all lateral organs and can promote abaxial cell fates when ectopically expressed in adaxial positions. The authors propose that FIL promotes abaxial cell fate in lateral organs. 14. Eshed Y, Baum SF, Bowman JL: Abaxial cell fate in the carpels is ·· established by two distinct mechanisms. Cell 1999, 99:199-209. Genetic interactions between crc, gymnos and kanadi are described as well as the cloning and expression pattern of GYMNOS. It is proposed that both CRC and KANADI promote abaxial cell-fate specification and that GYMNOS negatively regulates genes, promoting meristematic potential at the level of chromatin. 15. Bowman JL, Smyth DR: CRABS CLAW, a gene that regulates carpel ·· and nectary development in Arabidopsis, encodes a novel protein with zinc finger and helix-loop-helix domains. Development 1999, 126:2387-2396. The authors describe the cloning of CRC, the founding member of the YABBY gene family. The expression pattern of CRC in wild-type and floral homeotic mutant flowers is presented. The YABBY gene family is outlined with sequence comparisons. 16. Alvarez J, Smyth DR: CRABS CLAW and SPATULA, two Arabidopsis ·· genes that control carpel development in parallel with AGAMOUS. Development 1999, 126:2377-2386. The genetic interactions between crc, spatula and the ABC floral homeotic genes are described. This study demonstrates conclusively that genes promoting aspects of carpel development (e.g. CRC and SPATULA) are active independently of the ABC floral homeotic genes, and that most carpel tissues do not absolutely require AGAMOUS (C class) activity. This has implications for the evolution of flowers and their constituent organs. 17. ··
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