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Plant morphogenesis: Designing leaves
Dispatch
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Plant morphogenesis: Designing leaves David Jackson
Leaf shape and architecture vary greatly throughout the plant kingdom, and even within an individual plant during different phases of growth. Now, the development of a compound leaf architecture in tomato has been shown to be associated with the expression of the knotted1 homeobox gene in leaf primordia. Address: Plant Gene Expression Center, USDA/ARS, University of California, Berkeley, 800 Buchanan Street, Albany, California 94710, USA. Current Biology 1996, Vol 6 No 8:917–919 © Current Biology Ltd ISSN 0960-9822
Leaves exhibit a wide range of form and function: as well as the flattened photosynthetic organs we are most familiar with, modifications of leaf morphology produce the rigid spines of cacti, the cup-shaped insect traps of pitcher plants, colorful flower parts such as petals and some of the tendrils used for attachment by climbing plants. One fundamental choice in leaf architecture is whether to be simple or compound (see Fig. 1a–c). Simple leaves can have simple shapes — such as round, oval or strap-like — or they can be lobed, like an oak leaf. In contrast, compound leaves are subdivided into smaller units called leaflets. Given the immense variation in leaf architecture, how do we define a leaf? The only defining feature may be that all leaves are initiated as dorsiventral or flattened primordia from the shoot apical meristem [1]. Despite its complexity, a compound leaf is considered to be a single leaf because it is initiated as a single primordium from the shoot apical
meristem; the leaflets are initiated from the leaf primordium later in development [2]. The shoot apical meristem is the group of indeterminate stem cells at the apex of the growing shoot that serves to initiate organs throughout the life of the plant [3]. In contrast, leaves usually have a restricted, or determinate, potential for growth (certain plants, such as some ferns, have indeterminate leaves, however). In flowering plants, the shoot apical meristem also initiates axillary shoot meristems that will become leafy branches or flowers, but axillary shoot primordia differ from leaf primordia because they are radially symmetrical. In a variety of plants with simple leaves, knotted1 (kn1) and closely related members of the kn1 homeobox gene family are expressed in the shoot apical meristem and in axillary meristems, but not in leaves (Fig. 2). In fact, kn1 expression is down-regulated in a group of cells on the flank of the meristem which will form the next leaf primordium. These observations suggest that the role of kn1 might be to maintain cells of the shoot apical meristem in an indeterminate state [4]. Support for this hypothesis came from studies of dominant Kn1 mutations in maize that cause ectopic expression of kn1 in leaf primordia. This ectopic expression is associated with the sporadic growths (‘knots’) that result from extra cell divisions in the leaf, perhaps indicating a reduction in determinacy in the leaf [4]. Hareven et al. [5] have discovered a possible new role for kn1 in the development of compound leaves. The compound leaf of tomato has about nine leaflets borne on a
Figure 1 (a)
(b)
(c)
(d)
© Current Biology 1996
Different leaf forms (architectures). (a) Simple leaf (sunflower). (b) Simple, lobed leaf (oak). (c) Compound leaf (tomato). (d) ‘Super
compound’ leaf from a tomato plant overexpressing the maize knotted1 gene (reproduced with permission from [5]).
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Current Biology 1996, Vol 6 No 8
Figure 2
(a)
(b) 2
Shoot apical meristem
1
(c)
*
Midvein
Midvein
Expression pattern of kn1 mRNA in maize and Tkn1 mRNA in tomato. (a) Expression of kn1 in the maize shoot apical meristem, shown in longitudinal section. Note that kn1 is not expressed in the leaf primordia, denoted 1 and 2 [1,5] and is down-regulated in a group of cells on the flank of the shoot apical meristem where the next leaf will initiate (marked *). (b) Transverse section through a maize leaf primordium; note the lack of kn1 expression. (c) Transverse section through a tomato leaf primordium; note that Tkn1 is expressed at the margins of the leaf primordium, where leaflets will initiate, and in the provascular strands. Leaf primordia are shown in green, vascular strands in mauve and kn1 expression in purple.
© Current Biology 1996
central midrib. Upon overexpression of the maize kn1 gene, however, the leaves become ‘super compound’, with each leaf having up to 2000 leaflets (Fig. 1d). The analysis by Hareven et al. [5] suggests that compound leaves develop by a mechanism fundamentally different from that in simple leaves, and that the kn1 gene may have been recruited to function in the compound developmental program. A major advantage of tomato for studying the control of leaf architecture is the large collection of mutants that have defects in this process [6]. Three recessive mutations — entire, potato leaf and trifoliate — reduce the compound nature of the leaf, although only one mutation, the semidominant Lanceolate (La), is known to convert the tomato leaf to a fully simple architecture. Another dominant mutation, Petroselinum (Pts), increases the compound nature of the leaf such that each leaflet is further subdivided. Overexpression of kn1 in either the potato leaf, trifoliate or Pts mutant backgrounds gives largely additive effects on leaf architecture. Interestingly, the simple leaf architecture of La plants is epistatic to the effects of the kn1 transgene; the double mutant in this case showed an alteration of leaf shape (lobing) but did not become compound [5]. Could one of the tomato leaf mutants correspond to the tomato homologue of the maize kn1 gene? For example, Pts has the phenotype expected for a kn1 overexpressor, and La could conceivably be a dominant-negative or antimorph allele of kn1. Previous dosage studies are consistent with the idea that La acts as an antimorph [7]. To address this question, a kn1-related gene was isolated from tomato and mapped. This gene, named Tkn1, did not map to the same location as La or Pts, although there are probably at least two other kn1-related genes in tomato [5,8,9]. However, Tkn1 did map close to entire, a gene characterized
by recessive mutants whose leaves appear simple because their leaflets are fused. Tkn1 cDNAs from entire plants had the same sequence as wild-type, although this does not rule out the possibility that the entire gene encodes Tkn1, as the effects of the mutation could be regulatory, affecting for example the domain of expression of the gene rather than its coding sequence. The normal expression pattern of Tkn1 is strikingly different from that of its putative homologues in either maize or Arabidopsis. In these plants, which have simple leaves, kn1 is normally expressed only in the shoot apical meristem and is down-regulated as a leaf is initiated (Fig. 2a,b) [4,8,10,11]. Tkn1, however, is expressed in both leaf primordia and the shoot apical meristem of tomato (Fig. 2c). Is the initiation of leaflets from the leaf primordium of a compound leaf therefore somewhat analogous to the initiation of leaves by the shoot apical meristem? Should the compound tomato ‘leaf’ be more accurately described as a shoot? Several observations refute this hypothesis. First, the tomato leaf is initiated as a dorsiventral structure, unlike shoots which are radially symmetrical at inception. Second, the leaflets of a tomato leaf are initiated basipetally — first near the tip and later towards the base of the leaf; organ initiation by shoots is in the opposite direction (acropetal). Third, the leaves of flowering plants are initiated in close association with an axillary meristem; the leaflets on a compound leaf do not have axillary meristems. Perhaps one should consider the tomato leaf as sharing characteristics of both shoot and leaf identities, an idea that has been proposed for other compound leaves [12]. What then is the role of kn1 homologues in the development of compound leaves? In tobacco or Arabidopsis — plants with simple leaves — kn1 overexpression causes the
Dispatch
leaves to become lobed but not compound [8,13,14]. Even in tomato, overexpression of kn1 in sepals (simple leaf homologues in the flower) or in the simple leaves of La mutant plants does not make these organs compound. Thus, kn1 overexpression appears to accentuate a developmental program specific for compound leaves. Presumably it does this by maintaining leaf cells in a developmental state in which they can respond to the ‘compound’ signal. An alternative possibility is that lobed and compound leaves might not be fundamentally different, but that compoundness is a severe version of lobing. Evidence to support this hypothesis comes from overexpression of kn1 or a kn1-like gene in simple-leaved plants: this induces lobed leaves which, like compound leaves, also have some shoot characteristics [13,14]. For example, stipules and meristems, normally found only at the base of the leaf, are produced ectopically at the bases of the lobes [14]. Elucidation of the role of Tkn1 in tomato leaf development awaits the isolation of loss-of-function mutations. In Arabidopsis, the shootmeristemless (stm) gene encodes the probable kn1 homologue [11]. Recessive stm mutants fail to initiate leaves as a result of a failure in either the initiation or the maintenance of the shoot apical meristem. It might therefore prove necessary to isolate weak alleles or to use conditional expression in order to allow leaf initiation to occur and then to test the role of Tkn1 in compound leaf development. The tomato experiments open up many interesting questions. For example, is Tkn1 down-regulated to allow initiation of a leaf and then reactivated in a group of cells at the margin of the leaf where the leaflets are initiated? Alternatively, Tkn1 expression may be constitutive during leaf initiation and early development in tomato. What are the mechanisms controlling the differential regulation of kn1 during the development of simple and compound leaves? As overexpression of kn1 is not sufficient to convert a simple leaf into a compound leaf, what are the other factors involved? Is kn1 normally expressed during the development of other compound leaves or of lobed leaves? What about fern leaves which differentiate acropetally, and so have even more similarities to shoots than do the compound leaves of tomato? Overexpression of kn1 in a number of species has elegantly demonstrated our abilities to manipulate leaf form, and grocery stores may one day carry designer vegetables resulting from such endeavors. However, further understanding of the underlying mechanisms controlling leaf architecture awaits the isolation of leaf-shape genes from compoundleaved plants like tomato and pea, and the determination of the interactions between these genes and kn1 in leaf primordia. Acknowledgements I thank members of the Hake lab, Seung Y. Rhee and James Keddie for discussion of this article.
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References 1. Kaplan DR: Comparative developmental evaluation of the morphology of unifacial leaves in the monocotyledons. Bot Jahrb Syst 1975, 95:1–105. 2. Dengler NG: Comparison of leaf development in normal (+/+), entire (e/e), and Lanceolate (La/+) plants of tomato, Lycopersicon esculentum ‘Ailsa Craig’. Bot Gaz 1984, 145:66–77. 3. Sussex I M: Developmental programming of the shoot meristem. Cell 1989, 56:225–229. 4. Smith L, Greene B, Veit B, Hake S: A dominant mutation in the maize homeobox gene, Knotted-1, causes its ectopic expression in leaf cells with altered fates. Development 1992, 116:21–30. 5. Hareven D, Gutfinger T, Parnis A, Eshed Y, Lifschitz E: The making of a compound leaf: genetic manipulation of leaf architecture in tomato. Cell 84:735–744. 6. Stevens AM, Rick CM: Genetics and breeding. In The Tomato Crop. Edited by Atherton JG, Rudick J. New York: Chapman and Hall; 1986. 7. Stettler RF: Dosage effects of the lanceolate gene in tomato. Am J Bot 1964, 51:253–264. 8. Lincoln C, Long J, Yamaguchi J, Serikawa K, Hake S: A knotted1-like homeobox gene in Arabidopsis is expressed in the vegetative meristem and dramatically alters leaf morphology when overexpressed in transgenic plants. Plant Cell 1994, 6:1859–1876. 9. Kerstetter R, Vollbrecht E, Lowe B, Veit B, Yamaguchi J, Hake S: Sequence analysis and expression patterns divide the maize knotted1-like homeobox genes into two classes. Plant Cell 1994, 6:877–1887. 10. Jackson D, Veit B, Hake S: Expression of maize KNOTTED-1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot. Development 1994, 120:405–413. 11. Long JA, Moan EI, Medford JI, Barton MK: A member of the KNOTTED class of homeodomain proteins encoded by the SHOOTMERISTEMLESS gene of Arabidopsis. Nature 1996, 379:66–69. 12. Lacroix CR, Sattler R: Expression of shoot features in early leaf development of Murraya paniculata (Rutaceae). Can J Bot 1994, 72:678–687. 13. Sinha NR, Williams RE, Hake S: Overexpression of the maize homeobox gene, KNOTTED-1, causes a switch from determinate to indeterminate cell fates. Genes Dev 1993, 7:787–795. 14. Chuck G, Lincoln C, Hake S: KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis. Plant Cell 1996, in press.
917
Plant morphogenesis: Designing leaves David Jackson
Leaf shape and architecture vary greatly throughout the plant kingdom, and even within an individual plant during different phases of growth. Now, the development of a compound leaf architecture in tomato has been shown to be associated with the expression of the knotted1 homeobox gene in leaf primordia. Address: Plant Gene Expression Center, USDA/ARS, University of California, Berkeley, 800 Buchanan Street, Albany, California 94710, USA. Current Biology 1996, Vol 6 No 8:917–919 © Current Biology Ltd ISSN 0960-9822
Leaves exhibit a wide range of form and function: as well as the flattened photosynthetic organs we are most familiar with, modifications of leaf morphology produce the rigid spines of cacti, the cup-shaped insect traps of pitcher plants, colorful flower parts such as petals and some of the tendrils used for attachment by climbing plants. One fundamental choice in leaf architecture is whether to be simple or compound (see Fig. 1a–c). Simple leaves can have simple shapes — such as round, oval or strap-like — or they can be lobed, like an oak leaf. In contrast, compound leaves are subdivided into smaller units called leaflets. Given the immense variation in leaf architecture, how do we define a leaf? The only defining feature may be that all leaves are initiated as dorsiventral or flattened primordia from the shoot apical meristem [1]. Despite its complexity, a compound leaf is considered to be a single leaf because it is initiated as a single primordium from the shoot apical
meristem; the leaflets are initiated from the leaf primordium later in development [2]. The shoot apical meristem is the group of indeterminate stem cells at the apex of the growing shoot that serves to initiate organs throughout the life of the plant [3]. In contrast, leaves usually have a restricted, or determinate, potential for growth (certain plants, such as some ferns, have indeterminate leaves, however). In flowering plants, the shoot apical meristem also initiates axillary shoot meristems that will become leafy branches or flowers, but axillary shoot primordia differ from leaf primordia because they are radially symmetrical. In a variety of plants with simple leaves, knotted1 (kn1) and closely related members of the kn1 homeobox gene family are expressed in the shoot apical meristem and in axillary meristems, but not in leaves (Fig. 2). In fact, kn1 expression is down-regulated in a group of cells on the flank of the meristem which will form the next leaf primordium. These observations suggest that the role of kn1 might be to maintain cells of the shoot apical meristem in an indeterminate state [4]. Support for this hypothesis came from studies of dominant Kn1 mutations in maize that cause ectopic expression of kn1 in leaf primordia. This ectopic expression is associated with the sporadic growths (‘knots’) that result from extra cell divisions in the leaf, perhaps indicating a reduction in determinacy in the leaf [4]. Hareven et al. [5] have discovered a possible new role for kn1 in the development of compound leaves. The compound leaf of tomato has about nine leaflets borne on a
Figure 1 (a)
(b)
(c)
(d)
© Current Biology 1996
Different leaf forms (architectures). (a) Simple leaf (sunflower). (b) Simple, lobed leaf (oak). (c) Compound leaf (tomato). (d) ‘Super
compound’ leaf from a tomato plant overexpressing the maize knotted1 gene (reproduced with permission from [5]).
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Current Biology 1996, Vol 6 No 8
Figure 2
(a)
(b) 2
Shoot apical meristem
1
(c)
*
Midvein
Midvein
Expression pattern of kn1 mRNA in maize and Tkn1 mRNA in tomato. (a) Expression of kn1 in the maize shoot apical meristem, shown in longitudinal section. Note that kn1 is not expressed in the leaf primordia, denoted 1 and 2 [1,5] and is down-regulated in a group of cells on the flank of the shoot apical meristem where the next leaf will initiate (marked *). (b) Transverse section through a maize leaf primordium; note the lack of kn1 expression. (c) Transverse section through a tomato leaf primordium; note that Tkn1 is expressed at the margins of the leaf primordium, where leaflets will initiate, and in the provascular strands. Leaf primordia are shown in green, vascular strands in mauve and kn1 expression in purple.
© Current Biology 1996
central midrib. Upon overexpression of the maize kn1 gene, however, the leaves become ‘super compound’, with each leaf having up to 2000 leaflets (Fig. 1d). The analysis by Hareven et al. [5] suggests that compound leaves develop by a mechanism fundamentally different from that in simple leaves, and that the kn1 gene may have been recruited to function in the compound developmental program. A major advantage of tomato for studying the control of leaf architecture is the large collection of mutants that have defects in this process [6]. Three recessive mutations — entire, potato leaf and trifoliate — reduce the compound nature of the leaf, although only one mutation, the semidominant Lanceolate (La), is known to convert the tomato leaf to a fully simple architecture. Another dominant mutation, Petroselinum (Pts), increases the compound nature of the leaf such that each leaflet is further subdivided. Overexpression of kn1 in either the potato leaf, trifoliate or Pts mutant backgrounds gives largely additive effects on leaf architecture. Interestingly, the simple leaf architecture of La plants is epistatic to the effects of the kn1 transgene; the double mutant in this case showed an alteration of leaf shape (lobing) but did not become compound [5]. Could one of the tomato leaf mutants correspond to the tomato homologue of the maize kn1 gene? For example, Pts has the phenotype expected for a kn1 overexpressor, and La could conceivably be a dominant-negative or antimorph allele of kn1. Previous dosage studies are consistent with the idea that La acts as an antimorph [7]. To address this question, a kn1-related gene was isolated from tomato and mapped. This gene, named Tkn1, did not map to the same location as La or Pts, although there are probably at least two other kn1-related genes in tomato [5,8,9]. However, Tkn1 did map close to entire, a gene characterized
by recessive mutants whose leaves appear simple because their leaflets are fused. Tkn1 cDNAs from entire plants had the same sequence as wild-type, although this does not rule out the possibility that the entire gene encodes Tkn1, as the effects of the mutation could be regulatory, affecting for example the domain of expression of the gene rather than its coding sequence. The normal expression pattern of Tkn1 is strikingly different from that of its putative homologues in either maize or Arabidopsis. In these plants, which have simple leaves, kn1 is normally expressed only in the shoot apical meristem and is down-regulated as a leaf is initiated (Fig. 2a,b) [4,8,10,11]. Tkn1, however, is expressed in both leaf primordia and the shoot apical meristem of tomato (Fig. 2c). Is the initiation of leaflets from the leaf primordium of a compound leaf therefore somewhat analogous to the initiation of leaves by the shoot apical meristem? Should the compound tomato ‘leaf’ be more accurately described as a shoot? Several observations refute this hypothesis. First, the tomato leaf is initiated as a dorsiventral structure, unlike shoots which are radially symmetrical at inception. Second, the leaflets of a tomato leaf are initiated basipetally — first near the tip and later towards the base of the leaf; organ initiation by shoots is in the opposite direction (acropetal). Third, the leaves of flowering plants are initiated in close association with an axillary meristem; the leaflets on a compound leaf do not have axillary meristems. Perhaps one should consider the tomato leaf as sharing characteristics of both shoot and leaf identities, an idea that has been proposed for other compound leaves [12]. What then is the role of kn1 homologues in the development of compound leaves? In tobacco or Arabidopsis — plants with simple leaves — kn1 overexpression causes the
Dispatch
leaves to become lobed but not compound [8,13,14]. Even in tomato, overexpression of kn1 in sepals (simple leaf homologues in the flower) or in the simple leaves of La mutant plants does not make these organs compound. Thus, kn1 overexpression appears to accentuate a developmental program specific for compound leaves. Presumably it does this by maintaining leaf cells in a developmental state in which they can respond to the ‘compound’ signal. An alternative possibility is that lobed and compound leaves might not be fundamentally different, but that compoundness is a severe version of lobing. Evidence to support this hypothesis comes from overexpression of kn1 or a kn1-like gene in simple-leaved plants: this induces lobed leaves which, like compound leaves, also have some shoot characteristics [13,14]. For example, stipules and meristems, normally found only at the base of the leaf, are produced ectopically at the bases of the lobes [14]. Elucidation of the role of Tkn1 in tomato leaf development awaits the isolation of loss-of-function mutations. In Arabidopsis, the shootmeristemless (stm) gene encodes the probable kn1 homologue [11]. Recessive stm mutants fail to initiate leaves as a result of a failure in either the initiation or the maintenance of the shoot apical meristem. It might therefore prove necessary to isolate weak alleles or to use conditional expression in order to allow leaf initiation to occur and then to test the role of Tkn1 in compound leaf development. The tomato experiments open up many interesting questions. For example, is Tkn1 down-regulated to allow initiation of a leaf and then reactivated in a group of cells at the margin of the leaf where the leaflets are initiated? Alternatively, Tkn1 expression may be constitutive during leaf initiation and early development in tomato. What are the mechanisms controlling the differential regulation of kn1 during the development of simple and compound leaves? As overexpression of kn1 is not sufficient to convert a simple leaf into a compound leaf, what are the other factors involved? Is kn1 normally expressed during the development of other compound leaves or of lobed leaves? What about fern leaves which differentiate acropetally, and so have even more similarities to shoots than do the compound leaves of tomato? Overexpression of kn1 in a number of species has elegantly demonstrated our abilities to manipulate leaf form, and grocery stores may one day carry designer vegetables resulting from such endeavors. However, further understanding of the underlying mechanisms controlling leaf architecture awaits the isolation of leaf-shape genes from compoundleaved plants like tomato and pea, and the determination of the interactions between these genes and kn1 in leaf primordia. Acknowledgements I thank members of the Hake lab, Seung Y. Rhee and James Keddie for discussion of this article.
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References 1. Kaplan DR: Comparative developmental evaluation of the morphology of unifacial leaves in the monocotyledons. Bot Jahrb Syst 1975, 95:1–105. 2. Dengler NG: Comparison of leaf development in normal (+/+), entire (e/e), and Lanceolate (La/+) plants of tomato, Lycopersicon esculentum ‘Ailsa Craig’. Bot Gaz 1984, 145:66–77. 3. Sussex I M: Developmental programming of the shoot meristem. Cell 1989, 56:225–229. 4. Smith L, Greene B, Veit B, Hake S: A dominant mutation in the maize homeobox gene, Knotted-1, causes its ectopic expression in leaf cells with altered fates. Development 1992, 116:21–30. 5. Hareven D, Gutfinger T, Parnis A, Eshed Y, Lifschitz E: The making of a compound leaf: genetic manipulation of leaf architecture in tomato. Cell 84:735–744. 6. Stevens AM, Rick CM: Genetics and breeding. In The Tomato Crop. Edited by Atherton JG, Rudick J. New York: Chapman and Hall; 1986. 7. Stettler RF: Dosage effects of the lanceolate gene in tomato. Am J Bot 1964, 51:253–264. 8. Lincoln C, Long J, Yamaguchi J, Serikawa K, Hake S: A knotted1-like homeobox gene in Arabidopsis is expressed in the vegetative meristem and dramatically alters leaf morphology when overexpressed in transgenic plants. Plant Cell 1994, 6:1859–1876. 9. Kerstetter R, Vollbrecht E, Lowe B, Veit B, Yamaguchi J, Hake S: Sequence analysis and expression patterns divide the maize knotted1-like homeobox genes into two classes. Plant Cell 1994, 6:877–1887. 10. Jackson D, Veit B, Hake S: Expression of maize KNOTTED-1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot. Development 1994, 120:405–413. 11. Long JA, Moan EI, Medford JI, Barton MK: A member of the KNOTTED class of homeodomain proteins encoded by the SHOOTMERISTEMLESS gene of Arabidopsis. Nature 1996, 379:66–69. 12. Lacroix CR, Sattler R: Expression of shoot features in early leaf development of Murraya paniculata (Rutaceae). Can J Bot 1994, 72:678–687. 13. Sinha NR, Williams RE, Hake S: Overexpression of the maize homeobox gene, KNOTTED-1, causes a switch from determinate to indeterminate cell fates. Genes Dev 1993, 7:787–795. 14. Chuck G, Lincoln C, Hake S: KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis. Plant Cell 1996, in press.