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Plant Development: The making of a plant hair
M. DAVID MARKS
M.DAIDMAKSPLNTDEELPMN
PLANT DEVELOPMENT
The making of a plant hair The identification of genes required for leaf-hair formation in the plant Arabidopsisthaliana is leading to the detailed dissection of a cell-differentiation pathway. An early event in the evolution of multicellular organisms was a division of labor among distinct cell types. In its most simple form, the division is between two cell types, as in the blue-green alga Anabena; this organism is composed of nitrogen-fixing cells that are separated from one another by non-fixing cells. As organisms became more complex, the number of cell types increased. Two important questions in biology today concern the generation of distinct cell types during the development of multicellular organisms. These are, first, how are the fates of different cell types determined? And second, how is a fate, once chosen, fulfilled? The development of the unicellular hairs (called trichomes) on the leaves of the plant Arabidopsis thaliana provides an excellent model system for studying these two fundamental processes [1]. Trichomes emerge from the epidermal surface of young developing leaves (Fig. la). On average, initiating trichomes are separated by three to four epidermal cells [2]. The first visible change in a trichome precursor is a cessation of cell division and marked lateral expansion (Fig. Ib). DNA replication continues in non-dividing precursors until three rounds of endoreduplication have been completed [2]. The precursors then begin to expand outwards, perpendicular to the leaf surface. Concomitantly, the enlarged nuclei migrate towards the tips of the emerging trichomes. After a short period of outward expansion, two protuberances form at the tips
of the nascent trichomes (Fig. lb). These protuberances define the focal points of additional cell expansion, which results in the formation of branches. During the expansion of the developing branches, another round of endoreduplication occurs and one or more additional branches are initiated [2]. As the trichomes approach full size, the smooth outer surface is replaced by a rough coat containing numerous papillae (Fig. a). The two key questions mentioned above can be asked of trichome development. The questions then become, first, how are trichome precursors selected from a field of apparently identical epidermal cells? And second, once trichome precursors are selected, how is their morphogenesis controlled? These questions can be addressed genetically because mutations that alter trichome development do not adversely affect plant viability. Under laboratory conditions, most Arabidopsis trichome mutants are indistinguishable, aside from their trichome phenotypes, from wild-type plants (Fig. 2). Over 20 different recessive mutations affecting various aspects of trichome development have been identified (for primary references, see [1,2]). In general, the mutations affect trichome development either at the initiation step or later during trichome morphogenesis. For example, mutations in either of the GLABROUS1 (CL1) or TRANSPARENT TESTA
Fig. 1. Scanning electron microscopy of developing leaf trichomes. (a) Micrograph of a young leaf with trichomes in various stages of development. White bar = 51 pm. The developmental stage of selected trichomes is indicated by the numbers 1-6: the least developed trichomes are labeled with a 1, and the most mature trichomes with a 6. (b) Higher magnification micrograph of developing trichomes. White bar = 12 pm. Arrows indicate expanding branches.
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Fig. 2. Low magnification photographs of the leaves of wild type and mutant Arabidopsis plants. (a)Wild type. (b) transparent testa glabra (ttg). (c)glabra3 (g13). (d)distorted2 (dis). (e) singed (sne). (f) stalkless (stl). GLABRA (TTG) genes lead to a complete loss of trichomes as illustrated by the ttg mutant shown in Figure 2b. Mutations in nineteen other genes result in trichomes with altered morphologies as illustrated by the mutants shown in Figure 2c-f. There is some overlap between the early and late events: mutations in two genes, GLABRA2 (GL2) and GLABRA3 (GL3) not only affect the shape of the trichomes but also appear to reduce the number of trichomes initiated. In addition, a weak GL1 allele promotes the initiation of a few trichomes, but many of the trichomes fail to develop fully 13]. Hiilskamp et al. [2] have recently identified at least ten new trichome mutants and have used a combination of approaches to assess the roles of the various trichome genes. The mutants were first divided into six groups according to their altered trichome phenotype. DAPI staining was used to estimate the DNA content in mutant trichomes to see if the mutations affected DNA endoreduplication. Mosaic analyses - in which plants are generated with tissues that are mosaics of wild-type and mutant cells - were conducted to determine if the products of the trichomes genes acted locally or over a distance. Finally, double trichome mutants were generated to test for epistasis (the effect of one mutation overriding that of another). The DAPI analysis revealed that the earliest detectable stage of trichome morphogenesis is blocked by mutations in GLI or TTG. Furthermore, it was found that the
mutant phenotypes of GL1 or TTG are epistatic to those of all other trichome genes tested. Plants mosaic for the gil mutation contained discrete sectors lacking hairs, suggesting that the product of the GLI gene acts locally. These results place TTG and GL1 at the beginning of the trichome-differentiation pathway. A gene of particular interest, TRIPTYCHON (TRY), appears to function at the next step in the pathway. Mutations in TRY result in the formation of clusters of trichomes. As the trichome clusters emerge at the same time, TRY may be involved in the establishment of a field of inhibition around a selected trichome precursor that normally prevents adjacent cells from becoming trichomes. Interestingly, the try mutation can suppress the phenotype of gl2 mutants, which normally exhibit a reduced number of initiated trichomes and the production of trichomes that expand laterally along the epidermal surface, as opposed to outwards from the surface. Mutations in the genes TRY, KAKTUS (KAK) and GL3 were found to change trichome size. Trichomes on gl3 mutant plants are smaller and less branched than the wild type, whereas those on try and kak mutants are larger and more branched than the wild-type. This change in size is paralleled by a change in nuclear size. The nuclei of the g13 mutants do not undergo the final round of endoreduplication that occurs when the nucleus migrates up into the stalk of the trichome. In
DISPATCH contrast, the nuclei of try and kak mutants undergo one extra round of endoreduplication. Eight different trichome mutations define genes that are required solely for the normal outgrowth of emerging trichomes. Mutations in these genes result in the formation of twisted and swollen trichomes, as in the distorted2 (dis) mutant shown in Figure 2d. Finally, mutations define three classes of trichome branch alterations as illustrated by the stalkless (stl) mutant shown in Figure 2f. Branching is usually a two-step process. Two primary branches are typically formed first, followed by the formation of a third branch. Mutations in the STICHEL (STI) gene eliminate branch formation resulting in single spiked trichomes. Mutations in ANGUSTIFOLIA (AN) undergo primary branching but prevent production of additional branches. Finally, mutations in STACHEL (STA) apparently inhibit the first round of branch formation but allow branches to form later in trichome development, resulting in the production of trichomes with a long stalk and two short branches. Most progress in the molecular characterization of the pathway has been made with the GL1 gene. GLI was the first gene to be isolated by the use of T-DNA tagging in plants (in which a mutation is created by insertion of the T-DNA of the Agrobacterium tutnefaciens Ti plasmid, and the disrupted gene is cloned using the adjacent T-DNA as a 'handle') [4]. Sequence analysis of the cloned GL1 gene revealed that it has extensive sequence similarity to Myb-type transcription factors [5]. In situ hybridization analysis has shown that the gene is expressed in fields of undifferentiated epidermal cells and in individual trichome precursors [6]. Interestingly, the sequences responsible for controlling this pattern of expression are located in the 3' non-transcribed region of the gene. The effects of ectopic expression of GLI in transgenic Arabidopsis plants has shown that more than GL1 expression is required to induce the differentiation of all cells to trichomes [5]. However, the ectopic GL1 expression did result in the initiation of a few trichomes on tissues, such as the cotyledons, that normally lack trichomes [7]. While the TTG gene has yet to be cloned, Lloyd et al. [8] have shown that the maize R gene can functionally replace TTG. This suggests that TTG may be a homologue of the maize R gene, which encodes a basic helixloop-helix type transcription factor. In maize, the R protein cooperates with a Myb-type transcription factor, C1, to promote the activation of genes required for
anthocyanin pigment formation [9]. Given the nature of the interaction between the C1 and R transcription factors, it is possible that GL1, a Myb-type transcription factor, may directly cooperate with TTG to activate the transcription of genes required for trichome morphogenesis. Recent genetic analyses that made use of plants ectopically expressing GL1 and/or R suggest that this is the case [7]. What genes might be the targets of TTG and GL1? Likely candidates are the genes defined by the downstream trichome mutations. Indeed, GL2 has recently been cloned, so it will be possible to test this hypothesis directly [10]. In conclusion, the various trichome mutations that have been identified mean that trichome development is one of the best dissected cell-morphogenic pathways in any multicellular organism. Molecular analysis of the genes defined by the trichome mutations will lead to a better understanding of the key processes that control cell fate and cell differentiation. References 1. 2. 3. 4. 5. 6. 7.
8. 9.
Marks MD, Esch IJ: Trichome formation in Arabidopsis as a genetic model for studying cell expansion. Curr Top Plant Biochem Physiol 1992, 11:131-142. Holskamp M, Misera S, lurgens G: Genetic dissection of trichome cell development in Arabidopsis. Cell 1994, 76:555-566. Esch II, Oppenheimer DG, Marks MD: Characterization of a weak allele of the GL 1 gene of Arabidopsis thaliana. Plant Mol Biol 1994, 24:203-207. Herman PL, Marks MD: Trichome development in Arabidopsis thaliana. II. Isolation and complementation of the GLABROUSI gene. Plant Cell 1989, 1:1051-1055. Oppenheimer DG, Herman PL, Esch J, Sivakumaran S, Marks MD: A Myb-related gene required for leaf trichome differentiation in Arabidopsisis expressed in stipules. Cell 1991, 67:483-493. Larkin JC, Oppenheimer DC, Pollock S, Marks MD: Arabidopsis Glabrous I gene requires downstream sequences for function. Plant Cell 1993, 5:1739-1748. Larkin JC, Oppenheimer DG, Lloyd A, Paparozzi ET, Marks MD: The roles of Glabrousl and Transparent Testa Glabra genes in the trichome development pathway of Arabidopsis thaliana. Plant Cell 1994, in press. Lloyd AM, Walbot V, Davis RW: Anthocyanin production in dicots activated by maize anthocyanin-specific regulators, R and C1. Science 1992, 258:1773-1775. Goff SA, Cone KC, Chandler VL: Functional analysis of the transcriptional activator encoded by the maize B gene: evidence for
direct functional interaction between two classes of regulatory proteins. Genes Dev 1992, 6:864-875. 10. Rerie B, Feldmann KA, Marks MD: The Glabra2 gene encodes a homeodomain protein required for normal trichome development in Arabidopsis thaliana. Genes Dev 1994, 8:1388-1399.
M. David Marks, Department of Genetics and Cell Biology and Department of Plant Biology, University of Minnesota, St Paul, Minnesota 55108-1095, USA.
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M.DAIDMAKSPLNTDEELPMN
PLANT DEVELOPMENT
The making of a plant hair The identification of genes required for leaf-hair formation in the plant Arabidopsisthaliana is leading to the detailed dissection of a cell-differentiation pathway. An early event in the evolution of multicellular organisms was a division of labor among distinct cell types. In its most simple form, the division is between two cell types, as in the blue-green alga Anabena; this organism is composed of nitrogen-fixing cells that are separated from one another by non-fixing cells. As organisms became more complex, the number of cell types increased. Two important questions in biology today concern the generation of distinct cell types during the development of multicellular organisms. These are, first, how are the fates of different cell types determined? And second, how is a fate, once chosen, fulfilled? The development of the unicellular hairs (called trichomes) on the leaves of the plant Arabidopsis thaliana provides an excellent model system for studying these two fundamental processes [1]. Trichomes emerge from the epidermal surface of young developing leaves (Fig. la). On average, initiating trichomes are separated by three to four epidermal cells [2]. The first visible change in a trichome precursor is a cessation of cell division and marked lateral expansion (Fig. Ib). DNA replication continues in non-dividing precursors until three rounds of endoreduplication have been completed [2]. The precursors then begin to expand outwards, perpendicular to the leaf surface. Concomitantly, the enlarged nuclei migrate towards the tips of the emerging trichomes. After a short period of outward expansion, two protuberances form at the tips
of the nascent trichomes (Fig. lb). These protuberances define the focal points of additional cell expansion, which results in the formation of branches. During the expansion of the developing branches, another round of endoreduplication occurs and one or more additional branches are initiated [2]. As the trichomes approach full size, the smooth outer surface is replaced by a rough coat containing numerous papillae (Fig. a). The two key questions mentioned above can be asked of trichome development. The questions then become, first, how are trichome precursors selected from a field of apparently identical epidermal cells? And second, once trichome precursors are selected, how is their morphogenesis controlled? These questions can be addressed genetically because mutations that alter trichome development do not adversely affect plant viability. Under laboratory conditions, most Arabidopsis trichome mutants are indistinguishable, aside from their trichome phenotypes, from wild-type plants (Fig. 2). Over 20 different recessive mutations affecting various aspects of trichome development have been identified (for primary references, see [1,2]). In general, the mutations affect trichome development either at the initiation step or later during trichome morphogenesis. For example, mutations in either of the GLABROUS1 (CL1) or TRANSPARENT TESTA
Fig. 1. Scanning electron microscopy of developing leaf trichomes. (a) Micrograph of a young leaf with trichomes in various stages of development. White bar = 51 pm. The developmental stage of selected trichomes is indicated by the numbers 1-6: the least developed trichomes are labeled with a 1, and the most mature trichomes with a 6. (b) Higher magnification micrograph of developing trichomes. White bar = 12 pm. Arrows indicate expanding branches.
© Current Biology 1994, Vol 4 No 7
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Current Biology 1994, Vol 4 No 7
Fig. 2. Low magnification photographs of the leaves of wild type and mutant Arabidopsis plants. (a)Wild type. (b) transparent testa glabra (ttg). (c)glabra3 (g13). (d)distorted2 (dis). (e) singed (sne). (f) stalkless (stl). GLABRA (TTG) genes lead to a complete loss of trichomes as illustrated by the ttg mutant shown in Figure 2b. Mutations in nineteen other genes result in trichomes with altered morphologies as illustrated by the mutants shown in Figure 2c-f. There is some overlap between the early and late events: mutations in two genes, GLABRA2 (GL2) and GLABRA3 (GL3) not only affect the shape of the trichomes but also appear to reduce the number of trichomes initiated. In addition, a weak GL1 allele promotes the initiation of a few trichomes, but many of the trichomes fail to develop fully 13]. Hiilskamp et al. [2] have recently identified at least ten new trichome mutants and have used a combination of approaches to assess the roles of the various trichome genes. The mutants were first divided into six groups according to their altered trichome phenotype. DAPI staining was used to estimate the DNA content in mutant trichomes to see if the mutations affected DNA endoreduplication. Mosaic analyses - in which plants are generated with tissues that are mosaics of wild-type and mutant cells - were conducted to determine if the products of the trichomes genes acted locally or over a distance. Finally, double trichome mutants were generated to test for epistasis (the effect of one mutation overriding that of another). The DAPI analysis revealed that the earliest detectable stage of trichome morphogenesis is blocked by mutations in GLI or TTG. Furthermore, it was found that the
mutant phenotypes of GL1 or TTG are epistatic to those of all other trichome genes tested. Plants mosaic for the gil mutation contained discrete sectors lacking hairs, suggesting that the product of the GLI gene acts locally. These results place TTG and GL1 at the beginning of the trichome-differentiation pathway. A gene of particular interest, TRIPTYCHON (TRY), appears to function at the next step in the pathway. Mutations in TRY result in the formation of clusters of trichomes. As the trichome clusters emerge at the same time, TRY may be involved in the establishment of a field of inhibition around a selected trichome precursor that normally prevents adjacent cells from becoming trichomes. Interestingly, the try mutation can suppress the phenotype of gl2 mutants, which normally exhibit a reduced number of initiated trichomes and the production of trichomes that expand laterally along the epidermal surface, as opposed to outwards from the surface. Mutations in the genes TRY, KAKTUS (KAK) and GL3 were found to change trichome size. Trichomes on gl3 mutant plants are smaller and less branched than the wild type, whereas those on try and kak mutants are larger and more branched than the wild-type. This change in size is paralleled by a change in nuclear size. The nuclei of the g13 mutants do not undergo the final round of endoreduplication that occurs when the nucleus migrates up into the stalk of the trichome. In
DISPATCH contrast, the nuclei of try and kak mutants undergo one extra round of endoreduplication. Eight different trichome mutations define genes that are required solely for the normal outgrowth of emerging trichomes. Mutations in these genes result in the formation of twisted and swollen trichomes, as in the distorted2 (dis) mutant shown in Figure 2d. Finally, mutations define three classes of trichome branch alterations as illustrated by the stalkless (stl) mutant shown in Figure 2f. Branching is usually a two-step process. Two primary branches are typically formed first, followed by the formation of a third branch. Mutations in the STICHEL (STI) gene eliminate branch formation resulting in single spiked trichomes. Mutations in ANGUSTIFOLIA (AN) undergo primary branching but prevent production of additional branches. Finally, mutations in STACHEL (STA) apparently inhibit the first round of branch formation but allow branches to form later in trichome development, resulting in the production of trichomes with a long stalk and two short branches. Most progress in the molecular characterization of the pathway has been made with the GL1 gene. GLI was the first gene to be isolated by the use of T-DNA tagging in plants (in which a mutation is created by insertion of the T-DNA of the Agrobacterium tutnefaciens Ti plasmid, and the disrupted gene is cloned using the adjacent T-DNA as a 'handle') [4]. Sequence analysis of the cloned GL1 gene revealed that it has extensive sequence similarity to Myb-type transcription factors [5]. In situ hybridization analysis has shown that the gene is expressed in fields of undifferentiated epidermal cells and in individual trichome precursors [6]. Interestingly, the sequences responsible for controlling this pattern of expression are located in the 3' non-transcribed region of the gene. The effects of ectopic expression of GLI in transgenic Arabidopsis plants has shown that more than GL1 expression is required to induce the differentiation of all cells to trichomes [5]. However, the ectopic GL1 expression did result in the initiation of a few trichomes on tissues, such as the cotyledons, that normally lack trichomes [7]. While the TTG gene has yet to be cloned, Lloyd et al. [8] have shown that the maize R gene can functionally replace TTG. This suggests that TTG may be a homologue of the maize R gene, which encodes a basic helixloop-helix type transcription factor. In maize, the R protein cooperates with a Myb-type transcription factor, C1, to promote the activation of genes required for
anthocyanin pigment formation [9]. Given the nature of the interaction between the C1 and R transcription factors, it is possible that GL1, a Myb-type transcription factor, may directly cooperate with TTG to activate the transcription of genes required for trichome morphogenesis. Recent genetic analyses that made use of plants ectopically expressing GL1 and/or R suggest that this is the case [7]. What genes might be the targets of TTG and GL1? Likely candidates are the genes defined by the downstream trichome mutations. Indeed, GL2 has recently been cloned, so it will be possible to test this hypothesis directly [10]. In conclusion, the various trichome mutations that have been identified mean that trichome development is one of the best dissected cell-morphogenic pathways in any multicellular organism. Molecular analysis of the genes defined by the trichome mutations will lead to a better understanding of the key processes that control cell fate and cell differentiation. References 1. 2. 3. 4. 5. 6. 7.
8. 9.
Marks MD, Esch IJ: Trichome formation in Arabidopsis as a genetic model for studying cell expansion. Curr Top Plant Biochem Physiol 1992, 11:131-142. Holskamp M, Misera S, lurgens G: Genetic dissection of trichome cell development in Arabidopsis. Cell 1994, 76:555-566. Esch II, Oppenheimer DG, Marks MD: Characterization of a weak allele of the GL 1 gene of Arabidopsis thaliana. Plant Mol Biol 1994, 24:203-207. Herman PL, Marks MD: Trichome development in Arabidopsis thaliana. II. Isolation and complementation of the GLABROUSI gene. Plant Cell 1989, 1:1051-1055. Oppenheimer DG, Herman PL, Esch J, Sivakumaran S, Marks MD: A Myb-related gene required for leaf trichome differentiation in Arabidopsisis expressed in stipules. Cell 1991, 67:483-493. Larkin JC, Oppenheimer DC, Pollock S, Marks MD: Arabidopsis Glabrous I gene requires downstream sequences for function. Plant Cell 1993, 5:1739-1748. Larkin JC, Oppenheimer DG, Lloyd A, Paparozzi ET, Marks MD: The roles of Glabrousl and Transparent Testa Glabra genes in the trichome development pathway of Arabidopsis thaliana. Plant Cell 1994, in press. Lloyd AM, Walbot V, Davis RW: Anthocyanin production in dicots activated by maize anthocyanin-specific regulators, R and C1. Science 1992, 258:1773-1775. Goff SA, Cone KC, Chandler VL: Functional analysis of the transcriptional activator encoded by the maize B gene: evidence for
direct functional interaction between two classes of regulatory proteins. Genes Dev 1992, 6:864-875. 10. Rerie B, Feldmann KA, Marks MD: The Glabra2 gene encodes a homeodomain protein required for normal trichome development in Arabidopsis thaliana. Genes Dev 1994, 8:1388-1399.
M. David Marks, Department of Genetics and Cell Biology and Department of Plant Biology, University of Minnesota, St Paul, Minnesota 55108-1095, USA.
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