Molecules and morphology: comparative developmental genetics of ...

Pl. Syst. Evol. 259: 199–215 (2006) DOI 10.1007/s00606-006-0419-8

Molecules and morphology: comparative developmental genetics of the Brassicaceae J. L. Bowman Section of Plant Biology, University of California Davis, Davis, California, USA Received September 9, 2005; accepted November 13, 2005 Published online: June 19, 2006 Ó Springer-Verlag 2006

Abstract. For approximately 20 years Arabidopsis has been a model system to investigate developmental and physiological questions in plant biology, leading to the identification of genes and genetic systems involved in many processes. Extending ideas arising from knowledge of developmental genetic systems in Arabidopsis to other species of the Brassicaceae will require the application of genomics technologies developed in Arabidopsis and the establishment of additional genetic systems and resources in other species. Morphological variation in all plant organs, as well as in growth habit, mating systems, and physiology are represented in the breadth of Brassicaceae species offering ample opportunity to investigate the molecular basis of morphological evolution in this family. In addition, the frequent recent hybridization events in Brassica and Arabidopsis facilitate study of this pervasive force in the evolution of all plants. Key words: Brassicaceae, evo-devo, morphological evolution, Lepidium.

Introduction While many papers end with a token paragraph referring to the evolutionary implications with regards to their gene of interest, little is actually known about how changes in

gene regulation and function give rise to changes in morphology and physiology of plants within the Brassicaceae. Plants within this family are largely temperate herbs, although as always in biology, there are several exceptions. Nonetheless, most plant developmental and physiological processes can find potential ‘model’ taxa within the family. In this review I focus on some recent research using Brassicaceae species in an effort to understand the molecular bases of morphological evolution, and in addition, highlight some areas holding promise. However, this review is in no manner intended to be comprehensive. For several centuries it has been straightforward to assign species to the Brassicaceae due to the near universality of flower architecture, but the subdivision of the family into tribes and subtribes and even generic delineations have often been enigmatic. However, the recent application of molecular systematics has enabled the establishment of generic boundaries and intrageneric relationships have been defined for many taxa (reviewed in Koch et al. 2003). This has led to the realization that many morphological characters on which previous systematic relationships were based are in fact homoplasious, thus implying developmental processes

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J. L. Bowman: Molecules and morphology in the Brassicaceae

controlling fruit, cotyledon and leaf shapes are extremely plastic (e.g. Koch et al. 2003). The repeated evolution of specific morphologies could be due to convergent or parallel evolution. Convergence refers to the origin of similar traits that have a different developmental basis, whereas parallelism refers to the origin of similar traits via the same developmental alterations, although the line between these distinctions are blurred when referring to changes in different genes within the same developmental pathways. Given the large variation in physiology and in leaf and fruit shape within Brassicaceae there should be ample genetic material in which to investigate the molecular bases of the changes in morphology over evolutionary time. In the past, many evo-devo studies were limited to examining differences in gene expression patterns of orthologous genes in species with differing morphologies. However, these studies are limited in that one can only point to correlations, and cannot draw conclusions about causality. Thus, it is desirable to establish systems in which genetics can be utilized in conjunction with the genomic resources available in Arabidopsis to identify the specific allelic changes that have contributed to morphological evolution. Such studies have been extremely powerful in other plant systems such as Mimulus, Petunia and maize (Bradshaw et al. 1998, Doebley 2004, Schemske and Bradshaw 1999, Stuurman et al. 2004). In these cases, species (or in the case of maize, morphological variants within the species) differing in morphology were intercrossed and quantitative trait mapping in subsequent generations has led to the identification of loci involved in specific developmental processes. While the identity of genes is not yet known for most cases in Mimulus and Petunia, it is anticipated that advances in genomic resources in these species will allow the identification of alleles associated with changes in floral development imposed by selection mediated by pollinator preference (Bradshaw et al. 1998, Schemske and Bradshaw 1999, Stuurman et al. 2004).

In cases where it is not feasible to generate populations segregating for the alleles of interest, an alternative transgenic approach can provide functional data. Complementation of Arabidopsis loss-of-function mutants with genes isolated from other Brassicaceae species enables assessment of conservation of gene function. Transformation protocols for other Brassicaceae species (see below) should facilitate such approaches by enabling the transfer of genes between closely related species differing in the trait of interest. In such studies it would be useful to have loss-of-function alleles in the target species; these could be generated by two approaches. First, a traditional chemically induced mutagenesis of the species of interest would be extremely informative if the species is a diploid. Second, loss-of-function alleles induced by siRNA or microRNA mediated repression of gene expression could also be generated. The generation of such genetic resources will allow evolutionary studies to move into an era of functional analyses of specific alleles that contribute to the generation of diversity. Morphological development Inflorescence architecture. The domestication of Brassica oleracea has resulted in the selection of several morphological variants with altered plant architecture, such as cabbage, kale, Brussels sprouts, broccoli, and cauliflower. In cauliflower (Brassica oleracea var. botrytis L.) the morphology of the curd suggested that meristems that were normally specified as flower meristems, behaved instead as inflorescence meristems, such that the curd represents a proliferation of inflorescence meristems (Sadik 1962). A similar phenotype in Arabidopsis produced when the activities of both the APETALA1 and CAULIFLOWER genes are compromised provided candidate genes to be investigated in Brassica oleracea var. botrytis (Bowman et al. 1993, Kempin et al. 1995, Mandel and Yanofsky 1995). An investigation of the genetics of the curding phenotype in Brassica oleracea utilizing


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Fig. 1. Morphology of flowers and inflorescences. (A–D) Lepidium hyssopifolium (A, C, D) and Lepidium fasciculatum (B) flowers have reduced petals and only two medial stamens. (E–H) Lepidium oxytrichum flowers have very reduced petals and four medial stamens. (I–L) Lepidium phlebopetalum flowers exhibit the canonical Brassicaceae floral ground plan with four sepals, four petals, six stamens (four medial and two lateral), and two fused carpels. (M) Inflorescence meristems of Lepidium aschersonii differentiate directly into a thorn (t). (N–P) In species, e.g. Lepidium phlebopetalum (J–L) and Phlegmatospermum cochlearinum (N–P) with long styles, during development carpel height lags behind that of the stamens as compared to those with short styles (e.g. L. oxytrichum). Hirsuteness of the ovary also varies considerably, with Phlegmatospermum cochlearinum having dense trichomes, L. oxytrichum sparse trichomes and L. hyssopifolium and L. phlebopetalum being glabrous

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landraces from Italy confirmed that alleles of APETALA1 and CAULIFLOWER determine the extent and nature of curding growth in this species (Smith and King 2000). Similar candidate gene approaches might reveal the genetic basis of other Brassica oleracea morphological variants. For example, loss-of-function alleles of the TCP transcription factor CINCINNATA in Antirrhinum majus result in extra marginal growth in developing leaves (Nath et al. 2003), a phenotype reminiscent of that of kale. However, while the phenotypes of Brassica oleracea variants are dramatic, they are unlikely to be competitive in natural habitats due to their lower fitness levels. Most Brassicaceae species have indeterminate racemic inflorescences that are significantly elongated in a process termed bolting. However, at least three independent occurrences of ‘rosette flowering’, where flowers are elevated due to pedicel growth rather than inflorescence stem elongation, evolved within the Brassicaceae (Yoon and Baum 2004). One interpretation of the inflorescence morphology in these species is the primary inflorescence meristem is indeterminate, but does not bolt, whereas the axillary meristems, which usually produce indeterminate inflorescences, instead are determinate and only produce a solitary flower. This phenotype, in conjunction with precocious flowering in at least one of the species, Ionopsidium acaule, suggests that the regulation of LEAFY (LFY) or its targets may be altered, since ectopic expression of LFY in Arabidopsis displays some aspects of these phenotypes (Weigel and Nilsson 1995). Loss-of-function alleles of LFY result in a partial conversion of flowers into inflorescences, whereas gain-offunction alleles have the converse phenotype of conversion of inflorescence meristems into flower meristems (Schultz and Haughn 1991, Weigel et al. 1992, Weigel and Nilsson 1995). To assess whether changes in the regulation of LFY or in alleles of LFY itself are related to rosette flowering, complementation of Arabidopsis lfy loss-of-function mutants with genomic clones of LFY from three rosette flowering species was assessed (Yoon and Baum 2004).

The LFY gene from Ionopsidium acaule complemented Arabidopsis lfy mutants, restoring a normal Arabidopsis morphology. Since LFY is expressed in the shoot apical meristem in Ionopsidium acaule, it was proposed that changes upstream of LFY are responsible for rosette flowering in Ionopsidium acaule (Shu et al. 2000, Yoon and Baum 2004). In considering the morphology of this rosette flowering species, one hypothesis is that LFY is activated in the primary inflorescence meristem and in axillary meristems in response to environmental conditions that promote flowering and that the axillary meristems are competent to respond to LFY, but the primary inflorescence meristem is not competent, perhaps due to continued activity of TERMINAL FLOWER which could repress LFY activity (Alvarez et al. 1992, Bowman et al. 1993, Shannon and MeeksWagner 1991). In contrast with Ionopsidium acaule, LFY genes from two other species Idahoa scapigera and Leavenworthia crassa, induced some aspects of rosette flowering when introduced into an Arabidopsis lfy mutant background, suggesting that changes in LFY itself may have contributed to changes in inflorescence architecture (Yoon and Baum 2004). However, the conversion from an elongated raceme to rosette flowering was far from complete indicating that other factors are required. Another variation of inflorescence architecture is seen in Lepidium aschersonii, where, after the production of a small number of flowers, inflorescence meristems terminally differentiate into thorns (Hewson 1981; Fig. 1M). The loss of indeterminacy in the shoot apical meristems may also be due to ectopic activation of genes, such as LFY, that lead eventually to terminal differentiation of meristems in which they are expressed. Similar inflorescence phenotypes are also observed in three New Guinean species of Lepidium (Hewson 1982, van Royen 1964). While the flowers of many families of angiosperms are subtended by bracts, those of the Brassicaceae are mostly ebracteate. However, when observed with molecular markers a cryptic bract is evident subtending


floral meristems in Arabidopsis and changes in gene expression patterns in Arabidopsis allow development of bracteate flowers (Long and Barton 2000). Gain-of-function alleles of JAGGED, a gene likely involved in promoting growth of all lateral organs, have flowers subtended by bracts (Dinneny et al. 2004, Ohno et al. 2004). Conversely, loss-of-function alleles of LFY result in bracteate flower production (Schultz and Haughn 1991, Weigel et al. 1992). In a simple model, LFY could be responsible for excluding JAGGED expression from the cryptic bract resulting in a lack of growth promotion, and the eventual incorporation of these cells into the pedicel or flower proper. However, the control of JAGGED, or other factors promoting bract development, is multi-factorial since loss-of-function alleles of other genes, such as UNUSUAL FLORAL ORGANS, FILAMENTOUS FLOWER, also result in the development of filamentous structures that could be interpreted as a loss of suppression of bracts (Chen et al. 1999, Levin and Meyerowitz 1995, Sawa et al. 1999, Siegfried et al. 1999, Wilkinson and Haughn 1995). Flowers. The basic floral ground plan of Brassicaceae is remarkably well conserved with four sepals, four petals, six stamens, and two carpels (Figs. 1I–L, 2A). However, within Lepidium (about 200 species worldwide), more than half of the species have only two or four stamens, and in most of these species, petals are rudimentary (Fig. 1A-H). In species with two stamens, both are medial, whereas in species with four stamens they are all medial or there are two medial and two lateral. When stamens were reduced in number, the reduction was evident at the time of primordia initiation, whereas when petals were reduced, the primordia initiated but failed to continue to develop (Figs. 1–2, Bowman and Smyth 1998). Most traditional classifications of Lepidium relied on similarities in morphology, such as floral structures and fruit and embryo shape, along with geographical distribution to ascertain subgeneric relationships (Hewson 1981,

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Thellung 1906). However, molecular data do not support such groupings. The incongruence of species relationships based on two separate molecular markers, one of which is maternally inherited (cpDNA) and the other biparentally inherited, but with a potential for concerted evolution (ITS), and the large number of polyploid species suggest that the evolutionary history of Lepidium is reticulate (Bowman et al. 1999, Mummenhoff et al. 2001, Mummenhoff et al. 2004). Indeed, a phylogenetic study using a single-copy nuclear gene, PISTILLATA provided a framework for reconstructing the reticulate evolution of Lepidium, suggesting multiple independent hybridization events (Lee et al. 2002). Basal lineages within the genus are diploid and generally have the canonical Brassicaceae floral ground plan, and in contrast, the derived lineages are mostly allopolyploids with a large proportion having a reduced flower structure (Lee et al. 2002). Morphological similarities could have arisen via convergent evolution, or alternatively, introgression of morphological traits via allopolyploid hybridization (Rieseberg 1995). The patterns of hybridization and floral morphology in Lepidium support the hypothesis that the preponderance of derived species with reduced floral forms could have been produced by introgression by way of allopolyploid speciation (Lee et al. 2002). In this scenario, if the alleles conferring a reduced flower structure were dominant, either genetically or epigenetically, the trait would be predominant amongst allopolyploid species. Interspecific hybrid analyses support the hypothesis that alleles conferring a reduced flower trait are dominant. In an interspecific cross between L. oleraceum and L. hyssopifolium, alleles responsible for the absence of lateral stamens are dominant to those for their presence. F2 and F3 analyses suggested that the patterns of stamen distributions are genetically inherited, with the distribution of phenotypes in the F2 and F3 suggesting a multigenic origin (Lee et al. 2002). Another hybrid derived from L. virginicum and L. pseudohyssopifolium, both of which are

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Fig. 2. Morphology of floral organs and fruits. (A–B) Thickenings at the bases of the stamen filaments contribute to the posture of the stamens in Arabidella nasturtium flowers and may promote outcrossing. Nectaries in species with six stamens usually develop as a ring around the circumference of the receptacle within nectar glands at the abaxial bases of each of the stamens (B). (C–E) Outgrowths on the medial (C, E) and lateral (D) filaments of stamens in Alyssum linifolium may also contribute to mechanically restricting self-fertilization. (F) Lepidium species with four medial stamens, such as L. oxytrichum, have nectar glands between the medial stamens. A reduced petal (p) is dwarfed by the nectar gland (n). (G) Lepidium species with two medial stamens, such as L. fasciculatum, have nectar glands flanking the medial stamens. In this case the nectar glands (n) and petals (p) are of similar size. (H–I) In general, in species with fruits flattened laterally (I; L. phlebopetalum) the development of the gynoecium is asymmetric (H; L. fasciculatum) such that flattening is visible immediately following inception of carpel development. (J–K) In contrast, in species (e.g. Alyssum linifolium) in which fruits are flattened medially (K) most, if not all, of the asymmetric growth occurs subsequent to fertilization, with ovaries pre-fertilization appearing radial (J). (L) Other variations include post-genital fusion of sepals such as in Stenopetalum lineare


allopolyploid and lack lateral stamens, also supports the idea of allelic dominance of absence of lateral stamens since the parental species have different evolutionary genomic histories (Lee et al. 2002). Since Lepidium species with reduced flower structures are often self-fertilizing and are successful colonizing species, and allopolyploidization is one mechanism to increase the gene pool to avoid the deleterious outcome of inbreeding, it was speculated this may have been a factor for promoting hybridization events during the rapid radiation of the genus in the Americas and Australia. Given that allelic differences govern variation in floral structures, what genes might be involved in the process of floral reduction? Since the variations involve petals and stamens, it was suggested that genes specifically involved in organ formation, identity and growth of those two whorls might have been altered (Bowman and Smyth 1998, Endress 1992). The plausible candidates are the B class genes, APETALA3 and PI, and genes that regulate B class gene activity (Bowman et al. 1991, Goto and Meyerowitz 1994, Jack et al. 1992, Parcy et al. 1998). However, mapping studies using interspecific hybrids did not implicate these genes in stamen reduction, and furthermore Lepidium africanum (whose flowers have two stamens and reduced petals) versions of these genes complemented the corresponding Arabidopsis mutants (Lee et al. 2002). While reductions in stamens and petals are highly correlated, they can be genetically separated, implying genetic control of organ growth that is independent or downstream of B class activity. Considering their roles in moderating the growth of lateral organs and meristem in other angiosperms, TCP class transcription factors are obvious candidates for regulating organ development in Lepidium flowers (Cubas et al. 1999). Despite the conservation of Brassicaceae floral ground plans, except within Lepidium, a brief survey of Brassicaceae flowers provides ample variation in every floral organ. For example, modification of stamen filaments in

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Arabidella nasturtium and Alyssum linifolium both result in stamen positions that may facilitate outbreeding (Fig. 2A–E). Petals are reduced in Lepidium species, such that in L. fasciculatum the petals are similar in size to the nectary glands and in L. oxytrichum the petals are dwarfed by the nectary glands (Fig. 2F–G). The positioning of nectary glands is correlated with stamen position and number (Bowman and Smyth 1998). Six stamen species have the typical Brassicaceae nectary with glands at the abaxial base of each stamen (Fig. 2B). In contrast, species with two stamens have two nectary glands flanking each stamen, and species with four medial stamens have a single nectary gland between each pair of medial stamens (Fig. 2F–G). Floral organs other than the carpels are not congenitally fused in Rosid species, however, postgenital fusion can occur, as in the sepals of Stenopetalum lineare (Fig. 2L). Some additional variations in carpel development are described in the section about fruits. With respect to mating systems, in the Brassicaceae the ancestral condition is selfincompatible, implying that self-compatibility in Arabidopsis thaliana is derived. However, when the S-locus genes from Arabidopsis lyrata are expressed in some ecotypes, but not others, of Arabidopsis thaliana self-compatibility can be restored, suggesting that the effects of the recent loss of self-incompatibility continue to be detectable in the genome as the required components decay differentially in different lineages (Nasrallah et al. 2004). Furthermore, evidence of positive selection at the SCR locus to fixation of pseudogenes in Arabidopsis thaliana is evident, suggesting a recent selection for self-compatibility (Shimizu et al. 2004). As more downstream components of the self-incompatibility system are identified, the mechanisms of the many independent losses within the Brassicaceae can be investigated. As this has been the subject of several recent reviews, it will not be further elaborated upon here (Charlesworth and Vekemans 2005, Fobis-Loisy et al. 2004, Hiscock and McInnis 2003, Nasrallah 2002).

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Fruits. In contrast to the near invariability of flower architecture within the Brassicaceae, fruit shape and structure varies enormously within the family, relating directly to the ecology of seed dispersal. Brassicaceae gynoecia, from which fruits are derived, are composed of two fused carpels, the ovary being divided into two locules separated by a false septum. In many species, such as Arabidopsis, the fruit dehisces when ripe with the two valves separating from the remaining replum. Each locule may harbor from one to many (>30) seeds. Classically, Brassicaceae fruits have been divided into siliques and siliculas. A silique is defined as a dry, dehiscent, elongated fruit, having two valves that fall away leaving a central partition. A silicula is similar, except that it is ovate rather than being elongated and is no longer twice its width. From a developmental perspective the difference between the two types may merely reflect differential growth during fruit elongation. Most Brassicaceae fruits are flattened laterally such that the medial septum divides the fruit along its short axis, for example Lepidium, Capsella, and to a lesser extent Arabidopsis. In contrast, some genera, for example Alyssum, have fruits expanded along the medial plane such that the septum divides the fruit along its long axis, such that subsequent to dehiscence a large disc-shaped replum remains (Fig. 2K). In species whose fruits are flattened laterally, an expansion in the lateral direction is evident during the early stages of carpel development and is conspicuous at anthesis (Fig. 1H–I). In contrast, fruits that are expanded medially experience most or all of the asymmetric growth post fertilization (Fig. 1J–K). From a developmental perspective, this categorization may be a more biologically relevant system of fruit classification. Regardless, even these developmental distinctions may be highly homoplasious as fruit shape has been shown to be highly plastic within genera with several instances of convergent evolution of fruit shapes (Al-Shehbaz et al. 2002, Koch and Mummenhoff 2001, Koch et al. 1999, Koch et al. 2003, Mummenhoff et al. 1997, Mummenhoff et al. 2005).

Soon after the rediscovery of Mendel’s law, modified ratios were observed when following the inheritance of morphological traits in many organisms. The classic example of duplicate or redundant gene function was described by Shull (1914) in Capsella bursa-pastoris (Shepherd’s purse) whose fruits are normally heart shaped. A variant was discovered in the fields in Germany in which fruit shape was instead an elongated silique reminiscent of fruit shape in Arabidopsis and Brassica. When these variants were bred with the normal variety of Capsella bursa-pastoris, the elongated variant segregated in 1 in 16 of the F2 progeny indicating that loss of function of two unlinked genes was responsible for the mutant phenotype. In this case the redundancy is likely due to Capsella bursa-pastoris being an autotetraploid. Unfortunately, seeds of the variant are no longer available precluding the determination of the molecular lesion giving rise to the change in fruit shape. However, a combination of forward genetics and genome sequencing (Joint Genome Institute, United States Department of Energy) of the closely related diploid species, Capsella rubella, should provide insight into this and other developmental and physiological processes. While little is known about the genes responsible for variation in fruit development within the family, ectopic expression of multiple classes of genes in Arabidopsis fruits can mimic some of the variation seen amongst Brassicaceae species. Constitutive expression of a gene encoding a cytochrome P450, CYP78A9, causes Arabidopsis fruits to attain a more ovate shape, essentially converting the Arabidopsis silique into a silicula by lateral expansion, the effect being enhanced in an apetala2-1 mutant background (Ito and Meyerowitz 2000). The biochemical function of CYP78A9 is unknown, but may be involved in the production of a secondary compound that acts as a growth factor or signal. APETALA2 encodes a transcription factor involved in several developmental processes including specification of floral organ identity, development of the seed coat, and the transition to flowering, and at


present, the genetic interaction between CYP78A9 and APETALA2 is enigmatic. In a similar manner constitutive expression of at least five members of a small gene family, the DEVIL (DVL) genes, encoding polypeptides of approximately fifty amino acids results in changes in fruit shape in Arabidopsis (Wen et al. 2004). In most cases the resulting phenotypes are fruits that are expanded laterally, often locally at apical, central or basal regions, such that ‘horns’ or ‘wings’ develop with the resulting fruit shape mimicking that found in other Brassicaceae species. While loss-of-function alleles have not been informative thus far, at least some of the genes are expressed in the flowers suggesting they may have endogenous roles in fruit development. In this scenario, the encoded polypeptides would act as growth factors promoting localized cell division or cell expansion. In the gain-offunction DVL alleles all lateral organs are affected, being shorter and more ovate than those of wild-type plants. A similar phenotype is observed in plants with gain-of-function alleles of ROTUNDIFOLIA4 (ROT4), another member of the DVL/ROT4 gene family (Narita et al. 2004). Similar to the variation in fruit shape, other characteristics of carpel and fruit development have evolved convergently multiple times. For example, style length varies from very short (Fig. 1D, H) to as long or longer than the height of the ovary (Fig. 1L, P) and in species with long styles, ovary development is reduced in height from an early stage (Fig. 1J–K, N–O). This developmental difference might influence Agrobacterium mediated transformation efficiency when plants are infected via the ovary (Bechtold et al. 2003, Desfeux et al. 2000) due to the ovary becoming enclosed by post-genital fusion earlier in species with long styles. Three other Brassicaceae species (Arabidopsis lasiocarpa, Brassica napus, Raphanus sativa), all with short styles, have been reported to be transformed by the ‘floral dip’ method (Curtis and Nam 2001, Tague 2001, Wang et al. 2003). In this regard, species in which the ovary remains open for an extended developmental

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time may be amenable to ‘floral dip’ transformation. This phenotype may be at its most extreme in taxa within the related family Resedaceae, where the carpels are incompletely fused even at anthesis. Candidate genes for other characters of fruit development, such as hirsuteness (Fig. 1O–P) and dehiscence, whether indehiscent or explosive, have already been identified in Arabidopsis thaliana. For example, relative activities of the gibberellic acid and cytokinin hormone signaling pathways influence whether trichomes develop on the ovary wall in Arabidopsis (Greenboim-Wainberg et al. 2005). Arabidopsis genes directing several aspects of fruit dehiscence have been recently reviewed by Dinneny and Yanofsky (2005) and will not be discussed further here. As there appears to be convergent evolution in each of the developmental processes discussed above, there is ample opportunity to investigate whether the morphological convergence is also reflected at the level of molecular mechanisms. Leaves. Leaves can vary enormously in shape and size between species. This variation is often extended to leaves on a single plant, which may exhibit differences in leaf morphology in an age or environment dependent manner. While nearly any genus within the Brassicaceae would provide ample examples of variation in leaf morphology, this is illustrated in Fig. 3 showing nineteen species of Lepidium seedlings with a wide spectrum of leaf morphologies. The most basal lineage in the genus has strap-like leaves, but this phenotype is not necessarily ancestral, with comparisons to potential outgroups needed to clarify this issue. Based on current phylogenies of the genus, where most basal Eurasian lineages have entire leaves, dissected leaves likely evolved independently multiple times, and entire leaves are likely derived in other lineages (e.g. L. oleraceum). While changes in expression of Class I KNOX genes may be correlated in some cases, it is not causal in all (Bharathan et al. 2002), but with the plethora of potential candidate genes affecting leaf morphogenesis (Piazza et al. 2005), genetic and genomic

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Fig. 3. Leaf shape evolves rapidly, as evidenced by the varied leaf shapes exhibited by Lepidium species. (A) L. campestre (B) L. perfoliatum (C) L. ruderale (D) L. sativum (E) L. africanum (F) L. pseudohyssopifolium (G) L. oxycarpum (H) L. monoplocoides (I) L. oxytrichum (J) L. peregrinum (K) L. bonariense (L) L. densiflorum (M) L. meyenii (N) L. squamatum (O) L. lasiocarpum (P) L. virginicum (Q) L. oleraceum (R) L. divaricatum (S) L. hyssopifolium

approaches will be required to identify alleles responsible for changes in leaf morphology over evolutionary time. Hybridization Hybridization has played a major role in shaping the extant species of flowering plants, with a large proportion of angiosperm species hypothesized to be products of recent polyploidization events, either autopolyploidization or more frequently allopolyploidization. Indeed, it may be that all angiosperm species may have undergone cycles of polyploidy during their evolution. Polyploids often exhibit novel phenotypes not evident in their diploid progenitors, and many of these traits, such as time to flowering, pest resistance, organ size, apomixis, and stress tolerance, are likely to

have adaptive significance. The Brassicaceae family is no exception to this trend with a large proportion of polyploid species. Recently, our knowledge of the molecular bases of the phenotypic effects of hybridization has been advanced by investigations into both natural and re-synthesized hybrids (for recent reviews see: Adams and Wendel 2005, Birchler et al. 2005, Chen et al. 2004, Grummt and Pikaard 2003, Levy and Feldman 2004, Osborn et al. 2003, Pires et al. 2004). Both epigenetic changes in gene expression and gene loss have been observed immediately following hybridization, providing ample variation on which selection could act. For example, homeologs may be silenced or activated relative to their expression levels in progenitor species or gene conversion events can replace one allele with another as in the case of rRNA genes.


Subfunctionalization can occur with the functions of the progenitor gene divided amongst the two homeologs following hybridization, providing a rapid mechanism for maintenance of the two duplicate genes. In each of these cases the alterations in epigenetic gene regulation may be repeatable, as in the case of rRNA genes, or stochastic, with independent resynthesized hybrids exhibiting different outcomes and the changes can be stable over evolutionary time scales. Finally, hybridization events may lead to extensive genome remodeling and wide-scale reactivation of transposable elements, which in turn can result in stable genetic changes. Within the Brassicaceae, two hybridization events have received the majority of attention: (I) the allotetrapolid Brassica napus (derived from B. oleracea and B. rapa [Palmer et al. 1983]) and (II) the allotetrapolid Arabidopsis suecica (derived from A. thaliana and A. arenosa [Comai et al. 2000; Mummenhoff and Hurka 1994, 1995; O‘Kane et al. 1997]). In both cases, naturally occurring polyploids and multiple independent newly synthesized polyploids are available for comparison (Chen et al. 2004, Pires et al. 2004). Genome wide gene expression analyses utilizing the Arabidopsis thaliana microarrays have been performed with the Arabidopsis suecica complex providing evidence for most of the mechanisms of gene expression changes outlined above (Chen et al. 2004). While many examples of changes in gene expression during hybridization events have been documented, little is known yet about their adaptive significance in nature. One obvious trait under selection is flowering time, and in the case of resynthesized Brassica napus polyploids differential expression of FLC homeologs correlates significantly with differences in flowering time between lines (Pires et al. 2004). Due to the polygenic nature of the genetic control of flowering time, changes at additional loci are also likely to implicated. It is hypothesized that such dynamic changes in gene expression provide phenotypic variation on which natural selection can act following

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hybridization events, with time to flower a trait that would be selected upon in the generation immediately following hybridization. Similar analyses of other traits segregating in the newly synthesized polyploids will undoubtedly facilitate identification of other genes contributing to the phenotypic plasticity following hybridization. Another conspicuous segregating phenotype in both sets of resynthesized polyploids is a variation in leaf shape, another trait that can be adaptive and evolve rapidly (e.g. Chen et al. 2004; Fig. 3). Even closely related species, such as L. hyssopifolium (Fig. 3Q) and L. oleraceum (Fig. 3S), differ considerably in their leaf architecture. An interspecific cross between these two species reveals that leaf shape is under polygenic control, although both species are allotetraploid species confounding the genetic analysis (Lee et al. 2002). As mentioned previously, interspecific crosses of Lepidium species led to the hypothesis that alleles conferring a reduced floral ground plan were semidominant, and the preponderance of these alleles in Lepidium species could be due to dispersal via hybridization (Lee et al. 2002). Given the extent of genetic and epigenetic reprogramming occurring following hybridization, it is not an unreasonable hypothesis that the semi-dominant alleles may be epialleles and epigenetically maintained. Phylogenetic footprinting facilitates identification of regulatory sequences While most coding regions of genes are highly conserved across Brassicaceae species, noncoding regions vary relative to evolutionary distance enabling the identification of potential enhancer/silencer regions within promoters via phylogenetic footprinting (Colinas et al. 2002, Duret and Bucher 1997, Tautz 2000). Three recent studies have employed this method to identify sequences conserved in the promoters of four genes, CHALCONE SYNTHASE, APETALA3, AGAMOUS, and CRABS CLAW (Hong et al. 2003, Koch et al. 2001a, Koch et al. 2001b, Lee et al. 2005). In each case orthologous

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sequence comparisons were made between multiple species. Comparisons of the APETALA3 and CHALCONE SYNTHASE promoters, consisting of approximately 500 bases 50 to the transcription start site, from twenty-two species identified conserved elements, some of which have been experimentally tested for functionality, amidst sequences that are difficult or impossible to align (Hill et al. 1998, Koch et al. 2001b, Tilly et al. 1998). A similar situation was observed in comparisons of over 3500 bases of regulatory sequences of CRC from three species in that conserved sequences are flanked by sequences that cannot be aligned due to extensive divergence (Lee et al. 2005). In contrast, sequences of the second intron of AGAMOUS could be unambiguously aligned over their entire length of approximately 3000 bases (Hong et al. 2003). Since these studies spanned similar evolutionary distances it is not clear whether these differences are due to the sequences being intronic versus 50 of the transcription start site, or are due to gene specific conservation of sequences. Larger samples sizes are required to clarify these issues. Nonetheless, all studies were successful in identifying functional regulatory sequences within the promoters, confirming regulatory sequences identified in earlier ‘promoter bashing’ analyses as well as identifying additional potential regulatory elements. Thus, sequencing of Brassicaceae species of differing levels of evolutionary divergence from Arabidopsis facilitates identification of regulatory elements in Arabidopsis. Since Koch et al. (2001b) noted that the phylogenetic relationships of alignable promoter sequences parallels that of the coding regions, candidate species for large scale shotgun sequencing could be chosen based on these analyses. As an example of using phylogenetic footprinting to further developmental genetic analyses in Arabidopsis, the study of the CRABS CLAW (CRC) promoter provides an example (Lee et al. 2005). While only three species were analyzed, five conserved regions scattered over 3.5 kilobases of sequence 50 to the transcription start site were identified, and all were confirmed to be functional in regulating CRC

as only in combination was the CRC expression pattern recapitulated when these regions drove expression of a reporter gene and all were required for complementation when driving expression of a CRC cDNA. While the studies using more than twenty species for sequence comparisons were better able to identify potential specific enhancers, that the most important conserved region (of approximately 500 basepairs) identified in the CRC promoter was not easily subdivided suggests there may be functional significance to extended conserved sequences. Finally, it was noted that the Cauliflower Mosaic Virus TATA box was inefficient at interacting with the enhancer elements of the conserved regions of the CRC promoter, relative to the endogenous CRC TATA box and proximal promoter sequences, suggesting caution in interpreting results obtained with heterologous promoter elements. Phylogenetic footprinting studies should become standard practice once the genome sequences of Brassica oleracea, Capsella rubella and Arabidopsis lyrata become available (Joint Genome Institute, United States Department of Energy). Concluding remarks While identifying interesting morphological variation within the Brassicaceae is straightforward, identifying the alleles responsible for changes in morphology over evolutionary time will likely require both genetic and genomic approaches. For example, the ability to create gain- and loss-of-function alleles in the species of interest using microRNA and siRNA technologies should facilitate functional analyses of gene function. For this approach, candidate genes are required and might be identified using genome-wide expression analyses utilizing presently available genomics tools from Arabidopsis thaliana, and perhaps other Brassicaceae species in the future. In addition, standard forward genetic approaches may be used in species, such as Capsella rubella, that are diploid.


Where feasible, the application of forward genetics to identify alleles of interest is more powerful than reverse genetic approaches as it does not rely on knowledge of candidate genes. Such an approach has been successful in other angiosperm taxa as briefly mentioned in the introduction (Bradshaw et al. 1998, Doebley 2004, Schemske and Bradshaw 1999, Stuurman et al. 2004). In each of these cases the ability to create hybrids between species or variants within a species allowed the production of a mapping population, which could be used to identify genes by map position. In addition, the production of recombinant inbred and near-isogenic lines has facilitated the identification of specific alleles conferring specific morphological attributes. For example, when teosinte alleles of the TB1 gene are introgressed into maize they confer a branched phenotype typical of teosinte plants. In this case, the maize alleles exhibit evidence of selection during domestication, but similar molecular signatures are likely to be present at alleles selected upon by pollinator preference in Mimulus and Petunia. Thus, the establishment of additional genetic systems within the Brassicaceae focused on specific aspects of development of physiology, such as Thellungiella halophila for the study of salt tolerance (Inan et al. 2004, Taji et al. 2004), will lead to significant advances. Natural variation in Arabidopsis is the obvious place to initially search for phenotypes of interest since the genetic and genomic tools are unsurpassed in this species relative to other Brassicaceae species (reviewed in Koornneef et al. 2004, Maloof 2003). While variation within Arabidopsis thaliana represents only a small fraction of morphological variation present in the Brassicaceae, quantitative trait loci have been identified for flowering time, seed dormancy, leaf and flower size, light sensitivity, trichome density, inflorescence architecture, as well as for other traits. The subsequent identification of the corresponding genes will provide many more candidate genes for investigation in other Brassicaceae species to ask whether parallel or convergent

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evolution is occurring. There is a world of diversity waiting for us. I thank David Smyth for driving into the outback to help collect the Brassicaceae species presented in Figs. 1 and 2 and Bob Parsons and Sandy Floyd for comments of this manuscript. I thank Bob Parsons (LaTrobe University, Melbourne, Australia), Neville Scarlett (LaTrobe University, Melbourne, Australia), Klaus Mummenhoff (Universita¨t Osnabru¨ck, Germany), C. Gomez-Campo (Instituto Nacional de Investigaciones Agrarias, Madrid, Spain) and the USDA North Central Regional Plant Introduction Station (Ames, Iowa) for seed stocks. The Australian Research Council, The United States Department of Energy and the Beckman Foundation provided funding for Lepidium projects.

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Address of the author: J. L. Bowman (e-mail: [email protected]) Section of Plant Biology, University of California Davis, Davis, California 95616, USA.