- Email: [email protected]
Unraveling the knots in plant development
[~EVII~WS
A combination of genetic and molecular approaches has succeeded in unraveling developmental mechanisms in animals such as Drosophila (reviewed in Ref. 1) and should prove equally fruitful in plants. Although many morphological mutants have been described in plants 2-5, only recently have any corresponding genes been cloned 6-9. The Knotted (Knl) locus in maize is defined by a number of dominant mutations that alter cell fate in the leaf. We isolated the Knl gene by virtue of a transposon at the locus that caused one of the dominant mutations 10. Our genetic and molecular studies of Knl mutations provide a framework for analysis of the wild-type gene product. Since Knl belongs to a family of transcription factor genes 11, mutation of the other members may also have a morphological effect, as has been found in Drosophila, where families of homeobox genes specify related developmental functions 12.
Unraveling the knots in plant development SARAH HAKE Homeobox genes, first discovered from studies of homeotic mutations in Drosophila, have recently been found in plants. The proteins encoded by homeobox genes thus join the ranks of other animal transcription factors that bave plant developmental counterparts, suggesting that even though plant and animal development are very differen~ regulatory mechanisms that direct development may be shared among all higher eukaryotes. The role of homeobox genes in plants remains elusive; nonetheless, gain-of-function mutations of one homeobox gene, Knotted, profoundly affect development. The phenotype suggests that ectopic expression of Knotted in leaves causes cells to take on alternative fates.
Normal maize development In all flowering plants, development is e l a b o r a t e d from a group of dividing cells known as the meristem (reviewed in Ref. 13). The meristem continually regenerates itself, and also produces organs from its flanks (Fig. 1). The development of the maize plant begins during embryogenesis, with the formation of shoot and root meristems, as well as five or six embryonic leaves initiated by the shoot meristem. After germination, 10-15 additional leaves arise from the meristem in alternate succession. The shoot is composed of repeating units, or nodes, each consisting of a leaf, internode and axillary bud. After generating a defined number of nodes, the vegetative meristem differentiates into an inflorescence meristem, which becomes the tassel (containing male flowers). The ears (female organs) are produced from axillary buds (Fig. 2). The maize leaf is composed of a blade and a sheath, the junction of which is demarcated by an epidermal fringe called the ligule (Fig. 3, top left). The first indications of the ligule region occur concomitantly
with the first visible differences between blade and sheath 14. At this stage, divisions occur throughout the leaf. As the ligule continues to differentiate, divisions are restricted to the base of the leaf. A similar wave of elongation and differentiation moves from the tip to the
FIGH The maize meristem. Scanning electron micrograph of a 6 day old meristem and first two leaf primordia. The outer leaves were removed to expose the meristem (M) and the youngest leaf primordia (LP). Bar, 10 p.m.
FIGM The mature maize plant (adapted from Ref. 46). The ligule region of one leaf is boxed.
TIG MARCH1992 VOL.8 NO. 3 ©1992 Elsevier Science Publishers Ltd (UK)
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i,i! FIGE
Maize leaves. Top left: The ligule region of a normal maize leaf (see box in Fig. 2); the ligule (L) separates the blade (upper) from the sheath (lower part). The midrib is in the center. Top right: Maize leaves divide, differentiate and elongate from the tip to the base. The blade (B), sheath (S) and internode (I) of a leaf are illustrated at successive stages (taken from Ref. 15). The black region indicates areas undergoing divisions. Bottom left: A transverse section through a maize leaf revealing a lateral vein (on left) and an intermediate vein (on right).
base (Fig. 3, top right). The mature sheath and blade are distinct in a number of characteristics, including epidermal cell shape 14, venation 15 and expression of photosynthetic enzymes (N. Sinha and S. Hake, unpublished). The maize leaf is also characterized by a series of veins that extend throughout the leaf length. The central midrib is flanked by several lateral veins, which in turn are interspersed with a number of lower order veins called intermediates (Fig. 3, bottom left). Effects o f K n l mutations o n leaf development K n l mutations primarily affect the ligule and lateral veins of the leaf blade, although some alleles also affect the sheath. The ligule of knotted leaves is often displaced from its normal position up into the blade, or is found ectopically in the blade (Fig. 4, left), or may be missing altogether. Several other dominant leaf
mutations in maize affect the ligular region 16, but the effect of K n l on vein morphology is unique. Knots result when groups of cells continue to divide and expand after surrounding cells have stopped growing. They are focused along lateral veins and occur sporadically, although there is an overall symmetry in the severity of the knots (Fig. 4, right). In addition to knots and ectopic ligule, lateral veins often assume sheath or ligule-region characteristics in histology, photosynthetic protein localization and lignin accumulation 17. The formation of knots can also be viewed as a sheath-like transformation, since sheath cells continue to divide and expand after blade cells have ceased growing 14,15 (Fig. 3, top right). Thus the phenotypic transformations of K n l mutants are such that leaf blade cells along the lateral veins adopt cell fates normally restricted to portions lower in the leaf: the sheath and the ligule.
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P~EVII~WS All the K n l mutations produce subtly different phenotypes. They differ in which leaves are affected, whether the ligule is grossly altered and whether knots are ubiquitous or localized. Trans-heterozygote phenotypes are often strikingly different from the homozygous phenotype of each allele, suggesting that the mutations interact synergistically.
Site of gene action Clonal analysis can be used to predict the site of gene action and timing of gene expression. Our clonal analysis employed a recessive albino mutation (lw) that is closely linked to the recessive wild-type allele of K n l . Loss of the chromosome arm carrying the FIG[] dominant K n l mutation and the Knottedleaf blades. i,eft: A close-up of a Knl-O leaf with ectopic ligule fringes (arrow) dominant (green) allele of the al- located in a vertical position along the lateral veins. Right: An arrow indicates knots bino mutation (Lw), by irradiation along a lateral vein. of heterozygous seedlings, procharacters occur in the inner layer, or there is a duced albino sectors that were wild type for K n l (Refs requirement for a product that can be made only in 18, 19; Fig. 5). We found that the genotype of the epidermis did not determine the presence of knots18; in the inner layer, and the other layers respond to the fact, only the innermost layer containing the vascular signal generated by this product. elements and the bundle sheath determines the mutant Northern blotting and protein localization data indiphenotype 19 (see Fig. 3, bottom left, for cross-section). cate that K n l is expressed in vegetative and floral In other words, the mutant gene is required only in meristems (L. Smith, B. Greene, B. Veit and S. Hake, the inner layer, even though all leaf layers participate unpublished). Knl protein is also localized to a subset of developing vascular bundles and many parenchyin knot formation. Similarly, while ectopic ligule fringes are formed from both the epidermis and the matous cells in the shoot apex region. No antibody adjacent mesophyl119, to cause this phenotype the staining is detected in normal leaf cells at any stage of mutant gene is required only in the innermost leaf development. In mutant leaves, sporadic ectopic exlayer. Either the earliest events leading to the mutant pression of K n l is observed in developing lateral
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Clonal analysis. 'A' represents the linked dominant alleles Knl-N2 Lw, 'a' represents the recessive alleles Knl lu'. Ceils of the genotype Aa are green and knotted. Albino sectors are produced after X-ray-induced chromosome breakage. TIG MARCH 1 9 9 2 VOL. 8 NO. 3
tm
[~EVIEWS
~ Kn1-0204
ATG
Knl-0174 (Mul)
~ / Kn1-0169 (Mu8)
TAG
ATG
TAG
FIGra The Knl-O mutation and derivatives. Knl-O results from a tandem duplication of 17 kb that includes the entire coding region. The exons are shown as boxes. The smaller vertical black bars and arrows indicate the extent of the duplication. Insertions immediately 5' to one transcription unit attenuate or abolish the mutant phenotype. Knl-0174 and Kn1-0169, resulting from insertion of a Mul and Mu8 element, respectively, show a mild, suppressible knotted phenotype. Kn1-0167 is almost completely normal in phenotype and contains a Mu7-1ike element; Kn1-0204 contains a 5 kb unknown insertion and is normal in phenotype22.
veins. Similarly, quantitative differences in K n l RNA are only discemible between mutant and wild-type leaf primordia. The expression in lateral veins of knotted leaves is consistent with the clonal analysis result that the innermost cell layer of the leaf (containing the vascular bundles) is the tissue focus of K n l mutations. We suggest that the inappropriate expression of K n l in the leaf results in the introduction of some signals that would normally contribute to the development of the meristem. The misplaced signals interact with mechanisms controlling normal differentiation in the leaf, such as ligule or sheath development, or cell division. However, because the meristem behavior is out of context with the surrounding cells, knots result or alternative fates ensue.
Dominant mutations: alterations to noncoding regions Dosage analysis, in which the dose of a chromosome region carrying a particular locus is varied, can be used to determine whether a dominant mutation results from a gain of function, loss of function, overexpression, or synthesis of a competing product. Our observation of ectopic protein expression in the leaf is consistent with previous dosage analysis, which demonstrated that K n l mutations are gain-of-function mutations 2°. Molecular characterization of 12 different K n l alleles revealed that the alterations are to noncoding portions of the gene, suggesting that the mutant phenotype results from incorrect expression of a normal gene product. For example, the original spontaneous mutation, Knl-O (Ref. 21), results from a tandem duplication of 17 kb that includes the entire coding region 22. Plants that have lost one copy of the repeat are normal, while addition of a third repeat exacerbates the mutant phenotype. Insertions into the junction of the repeats revert the mutant phenotype (Fig. 6), while reversion has never been associated with insertion into either gene copy. Thus, genetic and molecular data suggest that it is not the duplication o f sequences p e r se that causes the Knl-O mutation; rather, it is the novel juxtaposition of sequences 5' to the transcriptional start site of one of the repeated coding regions.
Other K n l mutations are caused by or associated with insertions into introns (Fig. 7). K n l - 2 F l l and eight K n l - m u m mutations result from insertion of a Ds2 element and either Mul or Mu8 elements within a 1 kb region in the third intron. These elements are all nonautonomous elements that transpose only in the presence of a particular autonomous element, Ac for Ds2 (Ref. 23) and MuR1 for Mu elements 24. The phenotypes of Kn1-2F11 and at least one of the K n l - m u m alleles are dependent on the presence of the autonomous element. For example, the mutant phenotype generated by the Ds2 insertion in Kn1-2F11 is very mild and poorly penetrant in the absence of Ac (Ref. 10). In the presence of Ac, the phenotype is strikingly augmented and the penetrance can be as high as 100%, depending on the particular Ac element. K n l - m u m 2 individuals are only knotted when the Mu elements are hypomethylated; individuals carrying methylated Mu elements are normal (S. Hake et al., unpublished). Methylation is correlated with a lack of Mu activity 25 and with the absence of the autonomous element 24. The possibility remains that the insertions alter proper splicing at Knl; however, no incorrectly spliced mRNAs have been detected. An alternative hypothesis is that the transposase encoded by the autonomous elements (Ac and MuR1) binds to the transposon residing at Knl, and hence interacts with regulatory elements at the K n l locus. Either the transposase has a long-distance effect on the promoter to enhance transcription ectopically, which may occur in the Om(1D)9 mutation in Drosophila ananassase 26, or it interferes with a silencer present in the intron. A precedent for the latter possibility is seen in Drosophila, where the product of the suppressor of Hairy-wing [Su(Hw)] gene interacts with gyps 9 elements of y2 (Ref. 27) or bithorax 28. The binding of Su(Hw) protein to the gypsy element, rather than the gypsy element itself, prevents enhancers in cis from activating transcription, thus producing a mutant phenotype. In the case of Knl, rather than blocking enhancers from activating K n l expression, binding of transposase may block silencers that normally repress the K n l gene, thereby allowing ectopic expression.
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[]~EVIEWS All the K n l mutations are dominant; no recessive alleles have been recovered. Either recessive alleles have no phenotype because the gene is redundant or they have a different phenotype, perhaps lethal. Expression of KnI in the meristem suggests that it may play an essential role. A deletion including KnI is not transmitted through the male, and causes embryonic lethality when uncovered by a much larger deficiency 22. Although the deletion is small genetically, it is difficult to determine whether Knl is the only gene deleted because there is repetitive DNA immediately adjacent to the 5' and 3' regions of the gene. Smaller deletions are at present being sought to determine the null phenotype.
Homeobox genes in plants
Knl-mum's Kn1-2F1I~
Knl.Z3
llll
Knl-mum's FIG[] Insertions that are associated with Knl mutations. Knl-2Fll is caused by a Ds2 insertion 1°. There are eight Knl-mum mutations so far, all resulting from the insertion of Mul or Mu8 elements within a 300 bp region. Knl-Z3 and Kn 1-N2 am spontaneous mutations associated with a 5 kb insertion and rDt, a nonautonomous element of the two-element Dt system 47, respectively. The exact position of the Knl-Z3 insert is not known.
The h o m e o b o x was first identified as a region of conserved nucleotide sequence in genes responsible for the Drosophila homeotic mutations Antennapedia and Ultrabithorax 29. The h o m e o b o x was subsequently found in other Drosophila genes controlling embryo development29, 3°. The function of the homeodomain, the sequence encoded by the homeobox, as a region responsible for sequence-specific DNA binding, is n o w well established31. The definition of the homeodomain has broadened as more genes cloned by methods other than cross-hybridization have been compared32. Of the 61 amino acids in the homeodomain, four are invariant and eight are highly conserved. Knl shares the four invariant amino acids and six of the highly conserved amino acids n. Using an antibody to Knl, we have detected the protein in nuclei, consistent with its probable function as a DNAbinding protein (L. Smith, B. Greene, B. Veit and S. Hake, unpublished). We have cloned a number of homeobox-containing genes in maize and other plants, using Knl as a hybridization probe. All these homeodomains share an extended region of similarity, consisting of 24 amino acids towards the amino terminus 11. A similarly positioned motif in Drosophila proteins contributes specificity to gene action33. Other plant h o m e o b o x genes that are very different from our Knl-like genes have been cloned using Drosophila homeobox sequences (Ref. 34; M. Schena and R. Davis, pers. commun.). The possibility exists that there are many different plant homeodomain proteins. Large numbers of regulatory genes may be required for the plasticity evident in plant development35. The maize h o m e o b o x genes that we have cloned all map to different chromosome regions and have different expression patterns (B. Lowe and S. Hake, unpublished). From map position and cosegregation data, at least two may correspond to k n o w n morphological mutants. In flies and mammals, related homeobox genes are organized in clusters along the chromosome; their physical order reflects the position in which they are expressed along the anteroposterior axis in the embryo36. The order is conserved between
Drosophila and mouse, suggesting that large regulatory regions as well as the coding regions are conserved. Either such families of related h o m e o b o x genes remain to be cloned in plants, or plants are different. Plants tolerate considerable genomic flux, such as aneuploidy and polyploidy 35, and the regulatory regions may have been condensed into essential components immediately adjacent to genes. In this way, large-scale rearrangements would not affect gene expression. Alternatively, the conserved arrangement of sequences may be necessary only in organisms with extensive embryogenesis. In plants, embryogenesis is brief, with the major portion of development and differentiation occurring progressively from the meristem. Given that plant development is a progressive process, at least two types of developmental mutations are expected: those that prevent the progression from one stage to the next, and those that alter the identities of organs. We can expect to find certain classes of mutants, such as those that never make a meristem37, or mutants that make a meristem but never elaborate any organs38. Mutations such as Teopod delay the transition from juvenile leaves to adult leaves 39, while other mutations such as indeterminate prevent the transition from a vegetative meristem to an inflorescence meristem 5. Examples of mutations that affect the transition from an inflorescence meristem to a floral meristem are floricaula in Antirrhinum 8, leafy in Arabidopsis 4° and branched silkless or ramosa in maize 41. Homeotic mutations are k n o w n that alter the identities of vegetative 2 and floral organs 4,42. With a broad but controversial interpretation of homeosis 43, mutations can be considered homeotic if they affect the identities of cells within an organ. Dominant Knl mutations could be considered homeotic in this sense because the fates of some cells within the leaf blade are altered, though considering the expression in the meristem, recessive Knl mutations may have an entirely different phenotype.
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[]~EVIEWS Goldberg, R., eds), pp. 41-62, Springer-Verlag
Sequence analysis of plant genes associated with morphological phenotypes reveals that the encoded products share features with known transcription factors 6-8,11,44. When these sequences and phenotypes are compared between diverged species, there is often striking similarity 45. Moreover, plant morphology can be altered in transgenic plants by the presence of transcription factors from heterologous species (N. Sinha, R. Williams and S. Hake, unpublished). Since mechanisms that regulate development appear to be shared by diverse plants, it may be possible to unravel plant development by combining the best of each plant system.
17 Sinha, N. and Hake, S. (1991) in Proc. 14thlnt. Conf. on Plant Growth Substances, Kluwer 18 Hake, S. and Freeling, M. (1986) Nature320, 621-623 19 Sinha, N. and Hake, S. (1990) Dev. Biol. 141,203-210 20 Freeling, M. and Hake, S. (1985) Genetics 111, 617-634 21 Bryan, A.A. and Sass, J.E. (1941) J. Hered. 32, 343-346
Acknowledgements I thank the members of the laboratory for their helpful comments on the manuscript and the opportunity to discuss their unpublished results. Thanks to T. Foster for the SEM, to the UC Berkeley EM facility, to Julie Mathern for help with figures, and to Ron Wells for editing and electronic preparation of the manuscript. The work has been supported by NSF grant DMB-8819325 and by the USDA. References 1 Akam, M. (1987) Development 101, 1-22 2 Marx, G.A. (1987) PlantMol. Biol. Rep. 5, 311-335 3 Stevens, M.A. and Rick, C.M. (1986) in The Tomato Crop (Atherton, J.G. and Rudich, J., eds), pp. 35-109, Chapman & Hall 4 Coen, E.S. (1991) Annu. Rev. Plant Physiol. PlantMol. Biol. 42, 241-279 5 Sheridan, W.F. (1988) Annu. Rev. Genet. 22, 353-385 6 Yanofsky, M.F. et al. (1990) Nature 346, 35-39 7 Sommer, H. et al. (1990) EMBOJ. 9, 605-613 8 Coen, E.S. etal. (1990) Cell63, 1311-1322 9 Marks, M.D. and Feldmann, K.A. (1989) Plant Cell 1, 1043-1050 10 Hake, S., Vollbrecht, E. and Freeling, M. (1989) ~ B O J . 8, 15-22 11 Vollbrecht, E., Veit, B., Sinha, N. and Hake, S. (1991) Nature 350, 241-243 12 Gehring, W.J. (1987) Science 236, 1245-1252 13 Sussex, I.M. (1989) Cell 56, 225-229 14 Sylvester, A.W., Cande, W.Z. and Freeling, M, (1990) Development 110, 985-1000 15 Sharman, B.C. (1942) Ann. Bot. 6, 245-281 16 Freeling, M. et al. (1988) in Spatial Regulation of Plant Genes (Plant Gene Research Vol. 5) (Verma, D.P. and
22 Veit, B., Vollbrecht, E., Mathern, J. and Hake, S. (1990) Genetics 125, 623-631 23 Fedoroff, N.V. (1989) Cell 56, 181-191 24 Chomet, D. et al. (1991) Genetics 129, 261-270 25 Chandler, V.L. and Walbot, V. (1986) Proc. NatlAcad. Sci. USA 83, 1767-1771 26 Tanda, S. and Corces, V.G. (1991) EMBOJ. 10, 407417 27 Corces, V.G. and Geyer, P.K. (1991) Trends Genet. 7, 86-90 28 Peifer, M. and Bender, W. (1986) EMBOJ. 5, 2293-2303 29 McGinnis, W. et al. (1984) Nature 308, 428-433 30 Laughon, A. and Scott, M.P. (1984) Nature310, 25-31 31 Hayashi, S. and Scott, M.P. (1990) Cel163, 883-894 32 Scott, M.P., Tamkun, J.W. and Hartzell, G.W., Ill (1989) Biochim. Biophys. acta 989, 2548 33 Gibson, G., Schier, A., LeMotte, P. and Gehring, W.J. (1990) Cell 62, 1087-1103 34 Ruberti, I., Sessa, G., Lucchetti, S. and Morelli, G. (1991) EMBOJ. 10, 1787-1791 35 Walbot, V. (1985) Trends Genet. 1, 165-169 36 Graham, A., Papalopulu, N. and Krumlauf, R. (1989) Cell 57, 367-378 37 Clark, J.K. and Sheridan, W.F. (1991) Plant Cell3, 935-951 38 Mayer, U. et al. (1991) Nature353, 402407 39 Poethig, R.S. (1988) Genetics 119, 959-973 4 0 Schultz, E.A. and Haughn, G.W. (1991) Plant Cell3, 771-781 41 Veit, B. et al. (1991) Development Suppl. 1, 105-111 42 Bowman, J.L., Smyth, D.R. and Meyerowitz, E.M. (1991) Development 112, 1-20 43 Sattler, R. (1988) Am.J. Bot. 75, 1606-1617 44 Oppenheimer, D.G. etal. (1991) Ce1167, 483-493 45 Coen, E.S. and Meyerowitz, E.M. (1991) Nature 353, 31-37 46 Galinat, W.C. (1959) Bot. Mus. Leafl. Harv. Univ. 19, 1-32 4 7 Brown, J.J. et al. (1989) Mol. Gen. Genet. 215, 239-244 S. HAKL~ IS IN THE PLANT GENE EXPRESSION CENTER~ USDA-ARS, DEPARTMENTOF PLANT BIOLOGY, UNIVERSITY OF CALIFORNIA, BERKELEY, 800 BUCHANAN STREET, ALnAN~, CA 94710, USA.
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TIG MARCH 1 9 9 2 VOL. 8 NO. 3
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A combination of genetic and molecular approaches has succeeded in unraveling developmental mechanisms in animals such as Drosophila (reviewed in Ref. 1) and should prove equally fruitful in plants. Although many morphological mutants have been described in plants 2-5, only recently have any corresponding genes been cloned 6-9. The Knotted (Knl) locus in maize is defined by a number of dominant mutations that alter cell fate in the leaf. We isolated the Knl gene by virtue of a transposon at the locus that caused one of the dominant mutations 10. Our genetic and molecular studies of Knl mutations provide a framework for analysis of the wild-type gene product. Since Knl belongs to a family of transcription factor genes 11, mutation of the other members may also have a morphological effect, as has been found in Drosophila, where families of homeobox genes specify related developmental functions 12.
Unraveling the knots in plant development SARAH HAKE Homeobox genes, first discovered from studies of homeotic mutations in Drosophila, have recently been found in plants. The proteins encoded by homeobox genes thus join the ranks of other animal transcription factors that bave plant developmental counterparts, suggesting that even though plant and animal development are very differen~ regulatory mechanisms that direct development may be shared among all higher eukaryotes. The role of homeobox genes in plants remains elusive; nonetheless, gain-of-function mutations of one homeobox gene, Knotted, profoundly affect development. The phenotype suggests that ectopic expression of Knotted in leaves causes cells to take on alternative fates.
Normal maize development In all flowering plants, development is e l a b o r a t e d from a group of dividing cells known as the meristem (reviewed in Ref. 13). The meristem continually regenerates itself, and also produces organs from its flanks (Fig. 1). The development of the maize plant begins during embryogenesis, with the formation of shoot and root meristems, as well as five or six embryonic leaves initiated by the shoot meristem. After germination, 10-15 additional leaves arise from the meristem in alternate succession. The shoot is composed of repeating units, or nodes, each consisting of a leaf, internode and axillary bud. After generating a defined number of nodes, the vegetative meristem differentiates into an inflorescence meristem, which becomes the tassel (containing male flowers). The ears (female organs) are produced from axillary buds (Fig. 2). The maize leaf is composed of a blade and a sheath, the junction of which is demarcated by an epidermal fringe called the ligule (Fig. 3, top left). The first indications of the ligule region occur concomitantly
with the first visible differences between blade and sheath 14. At this stage, divisions occur throughout the leaf. As the ligule continues to differentiate, divisions are restricted to the base of the leaf. A similar wave of elongation and differentiation moves from the tip to the
FIGH The maize meristem. Scanning electron micrograph of a 6 day old meristem and first two leaf primordia. The outer leaves were removed to expose the meristem (M) and the youngest leaf primordia (LP). Bar, 10 p.m.
FIGM The mature maize plant (adapted from Ref. 46). The ligule region of one leaf is boxed.
TIG MARCH1992 VOL.8 NO. 3 ©1992 Elsevier Science Publishers Ltd (UK)
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i,i! FIGE
Maize leaves. Top left: The ligule region of a normal maize leaf (see box in Fig. 2); the ligule (L) separates the blade (upper) from the sheath (lower part). The midrib is in the center. Top right: Maize leaves divide, differentiate and elongate from the tip to the base. The blade (B), sheath (S) and internode (I) of a leaf are illustrated at successive stages (taken from Ref. 15). The black region indicates areas undergoing divisions. Bottom left: A transverse section through a maize leaf revealing a lateral vein (on left) and an intermediate vein (on right).
base (Fig. 3, top right). The mature sheath and blade are distinct in a number of characteristics, including epidermal cell shape 14, venation 15 and expression of photosynthetic enzymes (N. Sinha and S. Hake, unpublished). The maize leaf is also characterized by a series of veins that extend throughout the leaf length. The central midrib is flanked by several lateral veins, which in turn are interspersed with a number of lower order veins called intermediates (Fig. 3, bottom left). Effects o f K n l mutations o n leaf development K n l mutations primarily affect the ligule and lateral veins of the leaf blade, although some alleles also affect the sheath. The ligule of knotted leaves is often displaced from its normal position up into the blade, or is found ectopically in the blade (Fig. 4, left), or may be missing altogether. Several other dominant leaf
mutations in maize affect the ligular region 16, but the effect of K n l on vein morphology is unique. Knots result when groups of cells continue to divide and expand after surrounding cells have stopped growing. They are focused along lateral veins and occur sporadically, although there is an overall symmetry in the severity of the knots (Fig. 4, right). In addition to knots and ectopic ligule, lateral veins often assume sheath or ligule-region characteristics in histology, photosynthetic protein localization and lignin accumulation 17. The formation of knots can also be viewed as a sheath-like transformation, since sheath cells continue to divide and expand after blade cells have ceased growing 14,15 (Fig. 3, top right). Thus the phenotypic transformations of K n l mutants are such that leaf blade cells along the lateral veins adopt cell fates normally restricted to portions lower in the leaf: the sheath and the ligule.
T1G MARCH1992 VOt. 8 NO. 3
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P~EVII~WS All the K n l mutations produce subtly different phenotypes. They differ in which leaves are affected, whether the ligule is grossly altered and whether knots are ubiquitous or localized. Trans-heterozygote phenotypes are often strikingly different from the homozygous phenotype of each allele, suggesting that the mutations interact synergistically.
Site of gene action Clonal analysis can be used to predict the site of gene action and timing of gene expression. Our clonal analysis employed a recessive albino mutation (lw) that is closely linked to the recessive wild-type allele of K n l . Loss of the chromosome arm carrying the FIG[] dominant K n l mutation and the Knottedleaf blades. i,eft: A close-up of a Knl-O leaf with ectopic ligule fringes (arrow) dominant (green) allele of the al- located in a vertical position along the lateral veins. Right: An arrow indicates knots bino mutation (Lw), by irradiation along a lateral vein. of heterozygous seedlings, procharacters occur in the inner layer, or there is a duced albino sectors that were wild type for K n l (Refs requirement for a product that can be made only in 18, 19; Fig. 5). We found that the genotype of the epidermis did not determine the presence of knots18; in the inner layer, and the other layers respond to the fact, only the innermost layer containing the vascular signal generated by this product. elements and the bundle sheath determines the mutant Northern blotting and protein localization data indiphenotype 19 (see Fig. 3, bottom left, for cross-section). cate that K n l is expressed in vegetative and floral In other words, the mutant gene is required only in meristems (L. Smith, B. Greene, B. Veit and S. Hake, the inner layer, even though all leaf layers participate unpublished). Knl protein is also localized to a subset of developing vascular bundles and many parenchyin knot formation. Similarly, while ectopic ligule fringes are formed from both the epidermis and the matous cells in the shoot apex region. No antibody adjacent mesophyl119, to cause this phenotype the staining is detected in normal leaf cells at any stage of mutant gene is required only in the innermost leaf development. In mutant leaves, sporadic ectopic exlayer. Either the earliest events leading to the mutant pression of K n l is observed in developing lateral
X-RAYS
BLOCK OF MERISTEMATIC CELLS
"~
FIGE!
Clonal analysis. 'A' represents the linked dominant alleles Knl-N2 Lw, 'a' represents the recessive alleles Knl lu'. Ceils of the genotype Aa are green and knotted. Albino sectors are produced after X-ray-induced chromosome breakage. TIG MARCH 1 9 9 2 VOL. 8 NO. 3
tm
[~EVIEWS
~ Kn1-0204
ATG
Knl-0174 (Mul)
~ / Kn1-0169 (Mu8)
TAG
ATG
TAG
FIGra The Knl-O mutation and derivatives. Knl-O results from a tandem duplication of 17 kb that includes the entire coding region. The exons are shown as boxes. The smaller vertical black bars and arrows indicate the extent of the duplication. Insertions immediately 5' to one transcription unit attenuate or abolish the mutant phenotype. Knl-0174 and Kn1-0169, resulting from insertion of a Mul and Mu8 element, respectively, show a mild, suppressible knotted phenotype. Kn1-0167 is almost completely normal in phenotype and contains a Mu7-1ike element; Kn1-0204 contains a 5 kb unknown insertion and is normal in phenotype22.
veins. Similarly, quantitative differences in K n l RNA are only discemible between mutant and wild-type leaf primordia. The expression in lateral veins of knotted leaves is consistent with the clonal analysis result that the innermost cell layer of the leaf (containing the vascular bundles) is the tissue focus of K n l mutations. We suggest that the inappropriate expression of K n l in the leaf results in the introduction of some signals that would normally contribute to the development of the meristem. The misplaced signals interact with mechanisms controlling normal differentiation in the leaf, such as ligule or sheath development, or cell division. However, because the meristem behavior is out of context with the surrounding cells, knots result or alternative fates ensue.
Dominant mutations: alterations to noncoding regions Dosage analysis, in which the dose of a chromosome region carrying a particular locus is varied, can be used to determine whether a dominant mutation results from a gain of function, loss of function, overexpression, or synthesis of a competing product. Our observation of ectopic protein expression in the leaf is consistent with previous dosage analysis, which demonstrated that K n l mutations are gain-of-function mutations 2°. Molecular characterization of 12 different K n l alleles revealed that the alterations are to noncoding portions of the gene, suggesting that the mutant phenotype results from incorrect expression of a normal gene product. For example, the original spontaneous mutation, Knl-O (Ref. 21), results from a tandem duplication of 17 kb that includes the entire coding region 22. Plants that have lost one copy of the repeat are normal, while addition of a third repeat exacerbates the mutant phenotype. Insertions into the junction of the repeats revert the mutant phenotype (Fig. 6), while reversion has never been associated with insertion into either gene copy. Thus, genetic and molecular data suggest that it is not the duplication o f sequences p e r se that causes the Knl-O mutation; rather, it is the novel juxtaposition of sequences 5' to the transcriptional start site of one of the repeated coding regions.
Other K n l mutations are caused by or associated with insertions into introns (Fig. 7). K n l - 2 F l l and eight K n l - m u m mutations result from insertion of a Ds2 element and either Mul or Mu8 elements within a 1 kb region in the third intron. These elements are all nonautonomous elements that transpose only in the presence of a particular autonomous element, Ac for Ds2 (Ref. 23) and MuR1 for Mu elements 24. The phenotypes of Kn1-2F11 and at least one of the K n l - m u m alleles are dependent on the presence of the autonomous element. For example, the mutant phenotype generated by the Ds2 insertion in Kn1-2F11 is very mild and poorly penetrant in the absence of Ac (Ref. 10). In the presence of Ac, the phenotype is strikingly augmented and the penetrance can be as high as 100%, depending on the particular Ac element. K n l - m u m 2 individuals are only knotted when the Mu elements are hypomethylated; individuals carrying methylated Mu elements are normal (S. Hake et al., unpublished). Methylation is correlated with a lack of Mu activity 25 and with the absence of the autonomous element 24. The possibility remains that the insertions alter proper splicing at Knl; however, no incorrectly spliced mRNAs have been detected. An alternative hypothesis is that the transposase encoded by the autonomous elements (Ac and MuR1) binds to the transposon residing at Knl, and hence interacts with regulatory elements at the K n l locus. Either the transposase has a long-distance effect on the promoter to enhance transcription ectopically, which may occur in the Om(1D)9 mutation in Drosophila ananassase 26, or it interferes with a silencer present in the intron. A precedent for the latter possibility is seen in Drosophila, where the product of the suppressor of Hairy-wing [Su(Hw)] gene interacts with gyps 9 elements of y2 (Ref. 27) or bithorax 28. The binding of Su(Hw) protein to the gypsy element, rather than the gypsy element itself, prevents enhancers in cis from activating transcription, thus producing a mutant phenotype. In the case of Knl, rather than blocking enhancers from activating K n l expression, binding of transposase may block silencers that normally repress the K n l gene, thereby allowing ectopic expression.
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[]~EVIEWS All the K n l mutations are dominant; no recessive alleles have been recovered. Either recessive alleles have no phenotype because the gene is redundant or they have a different phenotype, perhaps lethal. Expression of KnI in the meristem suggests that it may play an essential role. A deletion including KnI is not transmitted through the male, and causes embryonic lethality when uncovered by a much larger deficiency 22. Although the deletion is small genetically, it is difficult to determine whether Knl is the only gene deleted because there is repetitive DNA immediately adjacent to the 5' and 3' regions of the gene. Smaller deletions are at present being sought to determine the null phenotype.
Homeobox genes in plants
Knl-mum's Kn1-2F1I~
Knl.Z3
llll
Knl-mum's FIG[] Insertions that are associated with Knl mutations. Knl-2Fll is caused by a Ds2 insertion 1°. There are eight Knl-mum mutations so far, all resulting from the insertion of Mul or Mu8 elements within a 300 bp region. Knl-Z3 and Kn 1-N2 am spontaneous mutations associated with a 5 kb insertion and rDt, a nonautonomous element of the two-element Dt system 47, respectively. The exact position of the Knl-Z3 insert is not known.
The h o m e o b o x was first identified as a region of conserved nucleotide sequence in genes responsible for the Drosophila homeotic mutations Antennapedia and Ultrabithorax 29. The h o m e o b o x was subsequently found in other Drosophila genes controlling embryo development29, 3°. The function of the homeodomain, the sequence encoded by the homeobox, as a region responsible for sequence-specific DNA binding, is n o w well established31. The definition of the homeodomain has broadened as more genes cloned by methods other than cross-hybridization have been compared32. Of the 61 amino acids in the homeodomain, four are invariant and eight are highly conserved. Knl shares the four invariant amino acids and six of the highly conserved amino acids n. Using an antibody to Knl, we have detected the protein in nuclei, consistent with its probable function as a DNAbinding protein (L. Smith, B. Greene, B. Veit and S. Hake, unpublished). We have cloned a number of homeobox-containing genes in maize and other plants, using Knl as a hybridization probe. All these homeodomains share an extended region of similarity, consisting of 24 amino acids towards the amino terminus 11. A similarly positioned motif in Drosophila proteins contributes specificity to gene action33. Other plant h o m e o b o x genes that are very different from our Knl-like genes have been cloned using Drosophila homeobox sequences (Ref. 34; M. Schena and R. Davis, pers. commun.). The possibility exists that there are many different plant homeodomain proteins. Large numbers of regulatory genes may be required for the plasticity evident in plant development35. The maize h o m e o b o x genes that we have cloned all map to different chromosome regions and have different expression patterns (B. Lowe and S. Hake, unpublished). From map position and cosegregation data, at least two may correspond to k n o w n morphological mutants. In flies and mammals, related homeobox genes are organized in clusters along the chromosome; their physical order reflects the position in which they are expressed along the anteroposterior axis in the embryo36. The order is conserved between
Drosophila and mouse, suggesting that large regulatory regions as well as the coding regions are conserved. Either such families of related h o m e o b o x genes remain to be cloned in plants, or plants are different. Plants tolerate considerable genomic flux, such as aneuploidy and polyploidy 35, and the regulatory regions may have been condensed into essential components immediately adjacent to genes. In this way, large-scale rearrangements would not affect gene expression. Alternatively, the conserved arrangement of sequences may be necessary only in organisms with extensive embryogenesis. In plants, embryogenesis is brief, with the major portion of development and differentiation occurring progressively from the meristem. Given that plant development is a progressive process, at least two types of developmental mutations are expected: those that prevent the progression from one stage to the next, and those that alter the identities of organs. We can expect to find certain classes of mutants, such as those that never make a meristem37, or mutants that make a meristem but never elaborate any organs38. Mutations such as Teopod delay the transition from juvenile leaves to adult leaves 39, while other mutations such as indeterminate prevent the transition from a vegetative meristem to an inflorescence meristem 5. Examples of mutations that affect the transition from an inflorescence meristem to a floral meristem are floricaula in Antirrhinum 8, leafy in Arabidopsis 4° and branched silkless or ramosa in maize 41. Homeotic mutations are k n o w n that alter the identities of vegetative 2 and floral organs 4,42. With a broad but controversial interpretation of homeosis 43, mutations can be considered homeotic if they affect the identities of cells within an organ. Dominant Knl mutations could be considered homeotic in this sense because the fates of some cells within the leaf blade are altered, though considering the expression in the meristem, recessive Knl mutations may have an entirely different phenotype.
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[]~EVIEWS Goldberg, R., eds), pp. 41-62, Springer-Verlag
Sequence analysis of plant genes associated with morphological phenotypes reveals that the encoded products share features with known transcription factors 6-8,11,44. When these sequences and phenotypes are compared between diverged species, there is often striking similarity 45. Moreover, plant morphology can be altered in transgenic plants by the presence of transcription factors from heterologous species (N. Sinha, R. Williams and S. Hake, unpublished). Since mechanisms that regulate development appear to be shared by diverse plants, it may be possible to unravel plant development by combining the best of each plant system.
17 Sinha, N. and Hake, S. (1991) in Proc. 14thlnt. Conf. on Plant Growth Substances, Kluwer 18 Hake, S. and Freeling, M. (1986) Nature320, 621-623 19 Sinha, N. and Hake, S. (1990) Dev. Biol. 141,203-210 20 Freeling, M. and Hake, S. (1985) Genetics 111, 617-634 21 Bryan, A.A. and Sass, J.E. (1941) J. Hered. 32, 343-346
Acknowledgements I thank the members of the laboratory for their helpful comments on the manuscript and the opportunity to discuss their unpublished results. Thanks to T. Foster for the SEM, to the UC Berkeley EM facility, to Julie Mathern for help with figures, and to Ron Wells for editing and electronic preparation of the manuscript. The work has been supported by NSF grant DMB-8819325 and by the USDA. References 1 Akam, M. (1987) Development 101, 1-22 2 Marx, G.A. (1987) PlantMol. Biol. Rep. 5, 311-335 3 Stevens, M.A. and Rick, C.M. (1986) in The Tomato Crop (Atherton, J.G. and Rudich, J., eds), pp. 35-109, Chapman & Hall 4 Coen, E.S. (1991) Annu. Rev. Plant Physiol. PlantMol. Biol. 42, 241-279 5 Sheridan, W.F. (1988) Annu. Rev. Genet. 22, 353-385 6 Yanofsky, M.F. et al. (1990) Nature 346, 35-39 7 Sommer, H. et al. (1990) EMBOJ. 9, 605-613 8 Coen, E.S. etal. (1990) Cell63, 1311-1322 9 Marks, M.D. and Feldmann, K.A. (1989) Plant Cell 1, 1043-1050 10 Hake, S., Vollbrecht, E. and Freeling, M. (1989) ~ B O J . 8, 15-22 11 Vollbrecht, E., Veit, B., Sinha, N. and Hake, S. (1991) Nature 350, 241-243 12 Gehring, W.J. (1987) Science 236, 1245-1252 13 Sussex, I.M. (1989) Cell 56, 225-229 14 Sylvester, A.W., Cande, W.Z. and Freeling, M, (1990) Development 110, 985-1000 15 Sharman, B.C. (1942) Ann. Bot. 6, 245-281 16 Freeling, M. et al. (1988) in Spatial Regulation of Plant Genes (Plant Gene Research Vol. 5) (Verma, D.P. and
22 Veit, B., Vollbrecht, E., Mathern, J. and Hake, S. (1990) Genetics 125, 623-631 23 Fedoroff, N.V. (1989) Cell 56, 181-191 24 Chomet, D. et al. (1991) Genetics 129, 261-270 25 Chandler, V.L. and Walbot, V. (1986) Proc. NatlAcad. Sci. USA 83, 1767-1771 26 Tanda, S. and Corces, V.G. (1991) EMBOJ. 10, 407417 27 Corces, V.G. and Geyer, P.K. (1991) Trends Genet. 7, 86-90 28 Peifer, M. and Bender, W. (1986) EMBOJ. 5, 2293-2303 29 McGinnis, W. et al. (1984) Nature 308, 428-433 30 Laughon, A. and Scott, M.P. (1984) Nature310, 25-31 31 Hayashi, S. and Scott, M.P. (1990) Cel163, 883-894 32 Scott, M.P., Tamkun, J.W. and Hartzell, G.W., Ill (1989) Biochim. Biophys. acta 989, 2548 33 Gibson, G., Schier, A., LeMotte, P. and Gehring, W.J. (1990) Cell 62, 1087-1103 34 Ruberti, I., Sessa, G., Lucchetti, S. and Morelli, G. (1991) EMBOJ. 10, 1787-1791 35 Walbot, V. (1985) Trends Genet. 1, 165-169 36 Graham, A., Papalopulu, N. and Krumlauf, R. (1989) Cell 57, 367-378 37 Clark, J.K. and Sheridan, W.F. (1991) Plant Cell3, 935-951 38 Mayer, U. et al. (1991) Nature353, 402407 39 Poethig, R.S. (1988) Genetics 119, 959-973 4 0 Schultz, E.A. and Haughn, G.W. (1991) Plant Cell3, 771-781 41 Veit, B. et al. (1991) Development Suppl. 1, 105-111 42 Bowman, J.L., Smyth, D.R. and Meyerowitz, E.M. (1991) Development 112, 1-20 43 Sattler, R. (1988) Am.J. Bot. 75, 1606-1617 44 Oppenheimer, D.G. etal. (1991) Ce1167, 483-493 45 Coen, E.S. and Meyerowitz, E.M. (1991) Nature 353, 31-37 46 Galinat, W.C. (1959) Bot. Mus. Leafl. Harv. Univ. 19, 1-32 4 7 Brown, J.J. et al. (1989) Mol. Gen. Genet. 215, 239-244 S. HAKL~ IS IN THE PLANT GENE EXPRESSION CENTER~ USDA-ARS, DEPARTMENTOF PLANT BIOLOGY, UNIVERSITY OF CALIFORNIA, BERKELEY, 800 BUCHANAN STREET, ALnAN~, CA 94710, USA.
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